PORTADA TESE final - Cristóbal Pérez web page

Señales sexuales y contaminación
por petróleo en un ave marina
Cristóbal Pérez Pérez
Tesis doctoral
Departamento de Ecoloxía e Bioloxía Animal
Universidade de Vigo
Departamento de Ecoloxía e Bioloxía Animal
Facultade de Bioloxía
Universidade de Vigo
Señales sexuales y contaminación por petróleo en un
ave marina
Memoria presentada por Cristóbal Pérez Pérez para optar al grado de Doctor
en Ciencias Biológicas
Vigo, Febrero 2009
VºBº del Director
Alberto Velando Rodríguez
Índice
ÍNDICE
Agradecimientos
1
Parte 1
Introducción
Las señales sexuales como indicadoras de calidad
ambiental
La contaminación por petróleo
Contaminación marina
El efecto del petróleo sobre los seres vivos
Hidrocarburos policíclicos aromáticos (HPAs)
Sustancias proxidantes y el sistema antioxidante
Biomarcadores de contaminación
Las señales sexuales dependientes de carotenoides
Características de los carotenoides
Función de los carotenoides
Importancia de la expresión de las coloraciones
dependientes de carotenoides
Mareas negras en la costa gallega
El Prestige
El Prestige y las aves marinas
La gaviota patiamarilla
7
7
8
8
9
10
11
12
13
13
14
16
17
18
19
21
Objetivos
25
Métodos
29
29
31
32
33
Resultados
37
Zona de estudio
Trabajo de campo
Trabajo de laboratorio
Análisis de las muestras
Coloración por carotenoides y estrés oxidativo
La disponibilidad de antioxidantes incoloros afecta a
la mancha roja que poseen las gaviotas patiamarillas
en el pico (anexo 1)
El petróleo afecta a la mancha roja del pico de la
gaviota patiamarilla (anexo 2)
La toxicidad del petróleo y la coloración por carotenoides
Evaluación de la exposición de las gaviotas al vertido
de petróleo del Prestige mediante el análisis sanguíneo
de los hidrocarburos policíclicos aromáticos (anexo 3)
Efectos subletales tras la exposición al petróleo en la
gaviota patiamarilla (anexo 4)
Efectos subletales y coloración tras la exposición
al petróleo en la gaviota patiamarilla (anexo 5)
37
39
43
46
48
I Índice
Discusión
49
Conclusiones
57
Bibliografía
59
Parte 2
Anexo 1: La disponibilidad de antioxidantes
incoloros afecta a la mancha roja que poseen las
gaviotas en le pico
Resumen
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References
75
77
77
80
83
87
90
90
Anexo 2: El petróleo afecta a la mancha roja del
pico de la gaviota patiamarilla
Resumen
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References
99
101
102
104
108
114
119
119
Anexo 3: Evaluación de la exposición de las
gaviotas al vertido depetróleo del Prestige mediante
el análisis sanguíneo de los hidrocarburos policíclicos
aromáticos
Resumen
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References
Supporting Information
II 131
133
133
136
138
143
148
148
153
Índice
Anexo 4: Efectos subletales tras la exposición al
petróleo en la gaviota patiamarilla
Resumen
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References
161
163
164
166
169
173
178
178
Anexo 5: Efectos subletales y coloración tras la
exposición al petróleo en la gaviota patiamarilla
Resumen
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References
187
189
190
191
193
194
197
197
Resumen
201
Abstract
209
III Agradecimientos
AGRADECIMIENTOS
Esta debe ser a parte máis complicada de escribir de todas, xa que posiblemente me
esquenza de alguén aínda que esta non é a miña intención. Para comezar os
agradecementos haberá que remontarse a anos atrás cando xa estaba un pouco
canso de mecerme ao compás do mar, naqueles embarques que me levaron a varias
partes do mundo e nos cales coñecín boa xente. Pero como dicía xa estaba canso así
que un día cando ía a clase de inglés atopaeime a Nacho. Falando con él, dalgún
xeito, animoume a que fixera os cursos de doutorado. Sen pensalo, como é a tónica
habitual en min, púxenme mans a obra movendo os fíos necesarios para inscribirme
nos cursos. Como tiña que facer un derradeiro embarque no Pacífico, pedínlle a
Maruxa e Puri que me fixeran o papeleo. Cando toquei terra en Perú lin nunha
mensaxe de correo electrónico que necesitaba un tutor para os cursos de doutorado,
obviamente non sabía que era necesario tal persoa, pero para a miña sorpresa na
mensaxe puña que Jorge facíase cargo de min. Cando voltei do embarque púxenme
en contacto con él, ofreceume realizar o traballo tutelado con miñocas que rechacei
non por despreciar ao marabilloso mundo da miñoca senon porque o que me
interesaba realmente era traballar con aves. Jorge díxome que Alberto estaba en
México e que esperaríamos a que voltara para ver a maneira de facer algo con aves.
Nesas primeiras semanas despois de voltar do embarque acontece o desastre do
Prestige algo que vai mudar a paixase galega e a vida de moita xente, entre a que me
incluo, durante unha boa tempada. Toca vestirse de branco, retirar chapapote e
coidar aves. Pasa o tempo e retorna Alberto que me propón facer un traballo con
gaivotas. Xa teño directores adoptivos dos cursos de doutorado! contento coma un
cuco marcho para as illas Cíes, no veleiro de Lua nada menos e na compaña de
David. Paso unha tempada coma un illán máis, xunto co persoal da Xunta e do
camping nas fins de semana. Pasan os días chovendo e traballando na colonia, pero
sae o sol e toca curso de doutorado nas illas. No curso coñezo a David e nunha
noite baixo os efectos dalgunha bebiba espiritosa acuño a nova definición de fitness.
Retorno ao laboratorio onde se acuña outra das máximas deste doutorado “coma
churros”, palabras que a día de hoxe aínda me poñen os pelos de punta. Coñezo os
que serán os meus compañeiros e amigos Manuel, Fernando, Domingo, Cristina,
María e Maigualida (gracias pola axuda, as festas e o brindis), outros están de paso
1
Agradecimientos
Gonzalo, Lucía, Aida, Julio, Juan e outros irán aparecendo Elisa, Emma, Iria, Elena,
Pablo. Ese ano toca facer PCRs que me levan primeiro a Pontevedra e logo a
Doñana onde finalmente aparecen as ansiadas bandas. Neses días acúñanse outras
tres palabras “mcd”, que serán miñas fieis compañeiras dende o comezo da tese ata o
día no que depositei esta memoria. Toca ademais facer inmunoglobulinas coa axuda
de África e o seu equipo: Dani, Susana, Elena, Silvia e Eva moitas gracias pola
axuda. Pasan os días e Alberto proponme traballar no proxecto Avemar. Proxecto
que nos levará a Alberto, Nacho, a miña nova compañeira Carmiña e a min a visitar
a costa e illas galegas onde nos pasaron cousas do máis variopintas dende o dialogo
de besugos coa 7ª lista en Ézaro, a visión de Alberto e Nacho enriba dunha pedra
da escolleira de Morás, ou a retórica dun cadro no Almacén entre outras. De
Carmiña non sei que dicir unha amiga jipi e currante alá onde as haia. Ese mesmo
ano coñezo a Manolo un bombeiro asturiano amante das aves. Chega o 2005 onde
Alberto propónme facer a tese, así que toca de novo facer os bartulos e irse para as
illas Cíes. Establezo a miña residencia durante case corenta días, alimentando,
expreitando, collendo e incluso falando coas gaivotas. Véñenme a axudar Carmiña 5
días e Julio 2, sempre vos agradecerei esa indispensable axuda. De novo toca porse a
bata e darlle duro a HPLC, coa indispensable axuda vía on-line de Marta todo sae
como a seda. Ese mesmo ano tamén lle toca sufrir a tese a Aurora, espero que algún
día vexas aos paxaros con outros ollos e non lle pases por riba co Yaris a moitas
máis gaivotas, moitas gracias polas fotos de París! Pasa o tempo e nace o
ConConLab, aparecen Antonio e José Manuel o equipo cormorán listo para a acción.
Un amante do tibetano e o outro amante de aventura, menudo equipaso! Dende
Sardinia aparece Ester, outra tola a que lles gustan as gaivotas e que nos fará
compaña durante uns meses, gracias por ensinarme italiano. Dende a capital
incorpórase Judith, unha todoterreo o mesmo se pasa tres meses nunha illa coma
todo o día diante do ordenador sen pestanexar. Dende Alicante (na extrema con
Murcia) José Carlos, un antigo anelador reconvertido a catador de licor café, amante
da festa nocturna e onde a pouco que se deixe perde o norte e o cinto dos
pantalóns. Dende Korea pasando por México Yeon, gran pescadora de chocos e
amante dos radiadores. Tamén dende Barcelona pasa brevemente polo grupo Rocío,
a tal para andar polos cantís vamos coma un pato fora da auga, lenta pero segura.
Vamos que o garito parece a ONU, a transformación e tal que sin darme conta
2
Agradecimientos
comezo a falar castelán. E pouco a pouco chega o día no que toca escribir estas
letras. Quen mo diría, agora si que vexo a luz ao final do tunel.
Así que toca agradecer a toda a xente que dalgún xeito me botou unha
man, sen vós este traballo nunca tería fin. Mención especial para Emilio e Pepín
directores do PN das illas Atlánticas e para todo o persoal da Xunta e despois de
Parques Nacionais destinados nas Cíes cos que compartín bos momentos. Diría os
vosos nomes pero sei que podo esquecer a alguén, e non é a miña intención, así que
vós ben sabedes quenes sodes. Moitas gracias.
Alberto, director, amigo e compañeiro teño que agradecerche todo o que
son neste mundo da ciencia. Non sei que verías en min para brindarme a
oportunidade de facer esta tese pero dende logo se cheguei ata aquí foi gracias a túa
paciencia.
Por suposto Jorge e Nacho, profesores amigos e bos conselleiros parte de
culpa de que sexa Dr. tamén e vosa. Puri e Maruxa, que me axudastes dende o
primerio momento, Maruxa de tiquismiquis nadiña gracias polas correccións. Os
meus amigos dende os máis achegados Jacobo, Berto e Rober ata as últimas
incorporacións Marco, Ani, Cesar, Patri, Pucho, Mabel, Pablo, Chiara Carlos, Cris,
Ricardo e un longo etc.. isto é máis difícil cun 9c!
Un agradecemento de última hora, moitas gracias Nacho pola impresión
dos Cds.
E por suposto a miña familia pais, avós e irmáns que coma sempre non
sabedes a que me adico (é moi difícil de explicar e as veces nin eu mesmo o sei),
pero que estades para o que faga falta.
A todos, moitas grazas!
3
Parte 1
Introducción
INTRODUCCIÓN
La presente tesis pretende validar el empleo de las señales sexuales para su uso
como indicadoras de calidad ambiental. Así, por primera vez se aborda esta
hipótesis, propuesta por Geoffrey Hill en 1995, desde un punto de vista integrador
y utilizando la gaviota patiamarilla (Larus michahellis) como modelo de estudio. Esta
especie es adecuada para validar esta hipótesis ya que presenta coloraciones
conspicuas dependientes de carotenoides, que se emplean como señal sexual y
porque, las poblaciones gallegas de esta especie de gaviota estuvieron recientemente
expuestas a un evento catastrófico de contaminación por petróleo provocado por el
accidente del buque petrolero Prestige, lo que permite poner a prueba la hipótesis en
un caso real tras un impacto agudo de contaminación ambiental.
Las señales sexuales como indicadoras de calidad ambiental
Las señales sexuales son todos aquellas señales que aumentan la probabilidad de
apareamiento, bien porque aumentan la capacidad para atraer a las posibles parejas
(por ejemplo la cola del pavo real) o bien porque sirven para repeler a posibles
rivales (como por ejemplo la coloración de los faisanes). Así, estos caracteres se
mantienen y se expanden mediante la selección sexual, es decir, mediante su efecto
sobre el éxito de apareamiento (Darwin 1871). La expresión de estas señales a
menudo transmite, a sus posibles parejas u oponentes, una información honesta de
la calidad del individuo portador de la señal (Grafen 1990, Anderson 1994). Esta
honestidad se basa en el hecho de que su mantenimiento acarrea un coste asociado,
el cual sólo se lo pueden permitir los animales de mayor calidad, evitando así el
engaño por parte de animales de menor condición; idea conocida como el
“principio del handicap” (Zahavi 1975, Zahavi y Zahavi 1997). Sin embargo, la
información que expresan estas señales, así como los mecanismos que subyacen a su
expresión, han generado una gran controversia en el mundo científico y han sido
objeto de intensas investigaciones en las últimas décadas.
Por otra parte, se ha observado que estas señales poseen una alta plasticidad
fenotípica, así su expresión depende mucho de las condiciones ambientales y es
particularmente sensible a la cascada de mecanismos fisiológicos que se producen
durante los episodios de estrés (Hill 1995, Buchanan 2000). En este contexto, se ha
7
Introducción
sugerido que estas señales honestas, dependientes de condición, podrían ser útiles
para medir la calidad ambiental ya que probablemente representen la suma de las
presiones ambientales a las que se enfrentan los animales (Hill 1995). Así, como
propuso G. Hill en 1995, es necesario identificar especies cuyas señales sexuales
podrían servir como indicadores de calidad ambiental y llevar a cabo experimentos
para ver como las perturbaciones ambientales específicas afectan a la expresión de
estas señales. De esta forma, este investigador sugirió que para el desarrollo de este
tipo de estudios se tenga en cuenta que:
1.
Las especies de estudio deben estar expuestas necesariamente a
perturbaciones ambientales y deben poseer señales que puedan servir
como indicadores de estas perturbaciones.
2.
Es necesario llevar a cabo experimentos controlados en estas especies.
3.
Tiene que demostrarse la aplicabilidad del uso de las señales sexuales como
indicadoras de perturbación ambiental.
La contaminación por petróleo
Contaminación marina
Las causas de los derrames de petróleo (o hidrocarburos extraídos directamente de
formaciones geológicas en estado líquido), y derivados en el medio marino son muy
diversas e incluyen por ejemplo: accidentes durante las operaciones de transferencia
de petróleo a petroleros, fugas en las cañerías de las refinerías, roturas en los
tanques de almacenamiento de crudo, lavado de tanques de almacenamiento en el
mar, colisiones de barcos y, por último, el hundimiento de petroleros
(http://www.cedre.fr/index_gb.html). El mayor interés por parte de la sociedad es
sobre aquellos derrames que se producen en ambientes costeros; bien sea porque
afectan a zonas de uso público y a la socioeconomía de la zona contaminada (Clark
et al. 1997, Miraglia et al. 2002, García-Pérez 2003) o bien porque los medios de
comunicación le dan gran difusión, normalmente con escenas dramáticas.
Desde la década de los años 60 hasta la actualidad se han producido en el
mundo unos 140 derrames de hidrocarburos, de los cuales la mayor parte se
encuentran en Europa (Figura 1). Este tipo de contaminación ambiental va a
provocar una serie de efectos negativos en los seres vivos que desarrollan su ciclo
8
Introducción
vital en las zonas afectadas por estos contaminantes (Peterson 2001, Freire y
Labarta 2003, Amat et al. 2006).
Figura 1. Localización de los derrames de petróleo (o derivados) en el mundo desde la
década de los años 60. Se observa que la mayor parte de los desastres se han producido en
Europa.
El efecto del petróleo sobre los seres vivos
Una vez ocurrido un derrame de petróleo, este llega a la franja costera en forma de
lo que se conoce como marea negra, la cuál va a producir una serie de efectos
deletéreos en los organismos de las áreas afectadas. Así, por una parte, los
hidrocarburos pueden producir, a muy corto plazo, efectos letales en los
organismos, lo que resulta en mortalidades masivas en las áreas afectadas, como las
que ocurren con muchas aves marinas después de una marea negra (Piatt et al. 1990,
Clark et al. 1997, Peterson et al. 2003, Samuel et al. 2008). Por otra parte, los
organismos que sobreviven quedan expuestos a los componentes persistentes y
bioacumulativos de los hidrocarburos, lo que conlleva efectos subletales que se
manifiestan a largo plazo (Teal et al. 1992, Golet et al. 2002, Alonso-Alvarez et al.
2007, Culbertson et al. 2008). La exposición a la contaminación por hidrocarburos
provoca en los animales, entre los que se incluye el hombre, desde daños
fisiológicos y genotóxicos, cuando hablamos a nivel de los individuos (Golet al.
2002, Harvey et al. 1999, Laffon et al. 2006), hasta daños ecológicos, cuando
hablamos a nivel del medio (Golet et al. 2002, Peterson et al. 2003, Martínez-Abraín
9
Introducción
2006). Entre los compuestos más persistentes y bioacumulativos de los
hidrocarburos, se encuentran los hidrocarburos policíclicos aromáticos (HPA).
Hidrocarburos Policíclicos Aromáticos (HAPs)
Estas moléculas son un gran grupo de compuestos orgánicos formados por dos o
más anillos aromáticos (bencénicos) que tienen relativamente baja solubilidad en
agua pero son altamente lipofílicos (McElroy et al. 1989). Pueden tener un origen
pirrolítico, resultado de la combustión incompleta de la materia orgánica;
petrogénico, consecuencia de derrames de hidrocarburos; o diagénico, por procesos
geoquímicos de aromatización de la materia sedimentaria (Page et al. 1999).
Los HPAs provocan una serie de efectos negativos en los organismos,
entre los que se encuentran daños hematológicos (Anselstetter y Heimpel 1986);
patológicos que llevan a la deshidratación de los ejemplares expuestos al
contaminante (Balseiro et al. 2005), irritación de las mucosas (Jones et al., 1997),
perdida de peso corporal (Bouquegneau et al. 1997); daños tóxicos que afectan al
hígado y riñones (Golet et al. 2002, Alonso-Alvarez et al. 2007); daños genotóxicos
que afectan a los cromosomas somáticos (Custer et al. 2000); efectos
inmunosupresivos (White et al. 1985) y daños inmunológicos (Auffret et al. 2004).
Así, los HAPs, una vez ingeridos por los organismos, son absorbidos en el
intestino y luego transportados a través de la sangre hasta el hígado, donde una serie
de sistemas enzimáticos los modificarán estructuralmente para favorecer su
eliminación. Entre estos sistemas enzimáticos se incluyen el sistema enzimático de
fase I y de fase II (Meador et al. 1995; Ramos y García 2007).
•
Sistemas enzimáticos de fase I: Su papel principal es convertir a los HPAs en
especies más reactivas y, por tanto, más susceptibles a entrar en las rutas
metabólicas. En la mayoría de los casos, este objetivo lo consiguen con la
introducción de un átomo de oxígeno en la molécula del HPA. Dentro de
este grupo destacan las monooxigenasas dependientes del citocromo P450.
•
Sistemas enzimáticos de fase II: Su papel principal consiste en aumentar la
solubilidad de los HPA y, por tanto, favorecer su excreción al medio
externo. Normalmente, actúan sobre sustancias que han sido modificadas
por el metabolismo de fase I, conjugándolas con pequeñas moléculas de
naturaleza hidrosoluble, tales como el ácido glucurónico, el glutatión o el
10
Introducción
sulfato. Dentro de esta categoría se incluyen las glutatión S-transferasas
(GST), la aspartato aminotransferasas (AST), las UDPglucuronosil
transferasas (UGT) y las epóxido hidrolasas (EH).
La acción de estos sistemas enzimáticos van a generar diferentes sustancias
prooxidantes conocidas como especies reactivas del oxígeno (EROs)
Sustancias prooxidantes y el sistema antioxidante
Las especies reactivas del oxígeno son especies altamente reactivas procedentes de
la reducción parcial de la molécula de O2. Las principales EROs son el radical anión
superóxido (O2-), el peróxido de hidrógeno (H2O2) y el radical hidróxilo (OH·). Los
efectos dañinos que las EROs provocan en la célula son numerosos y afectan a todo
tipo de biomoléculas. Entre ellos destacan la peroxidación lipídica, la inactivación
enzimática y la formación de aductos del ADN (Livingstone 1991, Lewis 2002). El
organismo posee mecanismos de defensa contra estas sustancias, así, para
contrarrestar el exceso de EROs activa sus sistemas antioxidantes, evitando de esta
forma que se produzca el estrés oxidativo o desequilibrio entre las sustancias
prooxidantes y las antioxidantes en favor de las primeras (Winston y Di Giulio
1991, Matés 2000, Nordberg y Arnér 2001).
Los antioxidantes se dividen principalmente en dos grandes grupos, los
enzimáticos y los no enzimáticos. Entre los antioxidantes enzimáticos destacan la
superóxido dismutasa (SOD), la catalasa (CAT), y la glutatión peroxidasa (GPX).
Entre los antioxidantes no enzimáticos, se incluyen por un lado moléculas
reductoras de pequeño tamaño y naturaleza hidrosoluble como el glutatión reducido
(GSH) y el ascorbato (vitamina C) y por otro, algunas vitaminas de naturaleza
liposoluble como la vitamina E (α-tocoferol), el antioxidante más importante frente
a la peroxidación lipídica y los carotenoides. Aunque sobre estos últimos existe
cierta controversia en cuanto a su función antioxidante (Hartley y Kennedy 2004,
Constantini y Møller 2008). Dada la estrecha relación entre la contaminación por
hidrocarburos y el estrés oxidativo, algunos de los compuestos mencionados
anteriormente han sido usados como biomarcadores de contaminación.
11
Introducción
Biomarcadores de contaminación
Se puede definir un biomarcador como la medida de fluidos corporales, células o
tejidos que indican en términos bioquímicos o celulares la presencia de
contaminantes (Livingstone 1993, Cajaraville et al. 2000, Montserrat et al. 2007).
Los biomarcadores serían el punto de unión entre niveles de contaminación y
estado de salud ambiental, entre causas y efectos de la contaminación.
Se distinguen dos grandes grupos:
•
los de exposición, que están más relacionados con los niveles de
contaminantes, entre los que destaca el sistema monooxigenasa de función
mixta (MFO, sistema enzimático de fase I). Así son usados como
biomarcadores la detección de niveles elevados de actividad enzimática del
citocromo P450 y EROD (7-etoxiresorufin-O-desetilasa), así como niveles
elevados de las transaminasas en plasma sanguíneo (como la LDH o
lactato deshidrogenasa y la AST o aspartato aminotransferasa) que serían
indicadores de daño celular (Golet et al. 2002, Jewett et al. 2002, Box et al.
2007).
•
los de efecto, que son indicativos de las consecuencias de dichos
contaminantes sobre los seres vivos. Destacan la peroxidación lipídica y la
genotoxicidad (formación de aductos de ADN) (Amat et al. 2006,
Eriyamremu et al. 2008)
Por último, es importante mencionar los criterios que se han de seguir para la
elección de un biomarcador. En primer lugar, el cambio biológico ha de deberse
únicamente a la presencia de contaminantes, de ahí que sea necesario tener en
cuenta la variabilidad debida a factores tales como la época del año, el sexo o la
temperatura a la hora de obtener una medida fiable. En segundo lugar, este cambio
biológico debe estar relacionado con un efecto adverso sobre algún aspecto de la
bioquímica o fisiología del animal, teniendo un tiempo de respuesta corto y
abarcando un rango que incluya tanto las condiciones óptimas como las letales
(Moore 1985, Widdows 1985).
Aunque existe una amplia variedad de estudios que analizan biomarcadores de
contaminación, prácticamente no existen trabajos que usen las señales sexuales
12
Introducción
dependientes de carotenoides como indicadoras de contaminación (ver Bortolotti et
al. 2003, Arellano-Aguilar y Garcia 2008)
Las señales sexuales dependientes de carotenoides
Características de los carotenoides
Con el nombre de carotenoides se denominan a más de 600 moléculas terpenoides
formadas por 40 átomos de carbono ordenados formando cadenas poliénicas
conjugadas, en ocasiones terminadas en anillos de carbono que pueden ser
sustituidos por diferentes grupos funcionales. Los carotenoides que están formados
solamente por moléculas de carbono e hidrógeno son conocidos como carotenos
(ej: β-carotenos); esta ausencia de grupos funcionales les confieren características de
liposolubilidad y poca polaridad. Por el contrario, las xantofilas (ej: zeaxantina y
cantaxantina) son carotenoides que poseen moléculas de oxígeno y presentan mayor
polaridad.
Los carotenoides son los pigmentos responsables de la mayoría de los
colores amarillos, anaranjados y rojos de frutos y verduras. El color es debido a la
presencia en su molécula de un cromóforo, consistente total o principalmente en
una cadena de dobles enlaces conjugados. Así, por ejemplo, los colores naranja de la
zanahoria y rojo del tomate, se deben a la presencia de β-caroteno y licopeno,
respectivamente. Los carotenoides, están presentes en todos los tejidos
fotosintéticos junto con las clorofilas, así como en tejidos vegetales no
fotosintéticos formando parte de los cromoplastos (Hill y McGraw 2006). Estos
compuestos son sintetizados exclusivamente por algas, bacterias, hongos y plantas
(Goodwin 1984, Latscha 1990, Surai 2002), por lo que los animales los incorporan
en su organismo a través de la dieta (Figura 2). No obstante, lo que si pueden hacer
los animales es transformarlos en otros carotenoides a través de diferentes rutas
metabólicas (Brush 1990, Møller et al. 2000). Se ha sugerido además que los
carotenoides responsables de las coloraciones rojas, en los animales, son más
costosos de producir que los implicados en las coloraciones amarillas (Hill 1996,
Andersson et al. 2007), aunque no esta claro el porqué.
13
Introducción
Figura 2. Las coloraciones rojas, naranjas o amarillas exhibidas por muchos animales son
dependientes de carotenoides.
Función de los carotenoides
Ya que los animales adquieren los carotenoides por la dieta, las coloraciones
producidas por estos compuestos pueden indicar, al receptor de la señal, la
capacidad individual para la búsqueda y consumo de estos pigmentos (Hill 1991).
Además de ser pigmentos, los carotenoides también tienen importantes funciones
fisiológicas. Así por un lado pueden estimular el sistema inmune (Lozano 1994,
Chew y Park 2004) y por otro pueden actuar como antioxidantes inactivando
especies reactivas de oxígeno (EROs), protegiendo de esta forma a los tejidos del
daño oxidativo (Krinsky y Yeum 2003, Young y Lowe 2001; Rao y Rao 2007). En
este contexto, se ha sugerido que existe un compromiso en usar los carotenoides
como pigmentos en la señal o usarlos en funciones fisiológicas (Lozano 1994, von
Schantz et al. 1999). Apoyando esta idea varios estudios experimentales han
mostrado que la activación del sistema inmune afecta a la expresión de las señales
sexuales mediadas por carotenoides (Faivre et al. 2003, Alonso-Alvarez et al. 2004,
Velando et al. 2006, ver Figura 3). Esta hipótesis se vio reforzada con los resultados
de varios experimentos (llevados a cabo en aves y peces), en los que a animales a los
que se les daba un suplemento de carotenoides en el alimento mostraban un
aumento tanto en la respuesta inmune como en la expresión de la señal (Blount et
al. 2003, Grether et al. 2004, McGraw y Ardia 2003), indicando que ambas
funciones estaban limitadas por la disponibilidad de estas moléculas.
Por otra parte, como subproducto de la activación del sistema inmune el
organismo genera sustancias prooxidantes (Finkel y Holbrook 2000); así, el estrés
oxidativo podría estar mediando en la relación anteriormente descrita entre la
expresión de las señales dependientes de carotenoides y la activación del sistema
14
Introducción
inmune. De esta forma, se han propuesto dos hipótesis que sugieren que la
intensidad de las coloraciones dependientes de carotenoides indicarían el estado
antioxidante del individuo (von Schantz et al. 1999, Hartley y Kennedy 2004). Sin
embargo estas hipótesis difieren en el mecanismo que subyace a la expresión de
estas señales. Por un lado, se sugiere que solamente los individuos con buenas
defensas antioxidantes (o con bajos niveles de EROs) podrían permitirse desviar los
carotenoides de su función antioxidante para usarlos como pigmentos (von Schantz
et al. 1999). Como alternativa, se ha propuesto que la intensidad de la coloración
dependiente de carotenoides sería un índice de los antioxidantes no pigmentarios
del individuo (Hartley y Kennedy 2004). Esta hipótesis se basa en que la coloración
de los carotenoides es alterada y destruida por procesos oxidativos (Woodall et al.
1997, Siems et al. 1999), de tal forma que solamente los individuos con altos niveles
de antioxidantes podrían prevenir la perdida del color de sus carotenoides
protegiéndolos contra la oxidación. Así, los resultados de dos recientes estudios
muestran que un aumento en la disponibilidad de antioxidantes (incoloros) provoca
un aumento en la intensidad de las señales sexuales mediadas por coloraciones
dependientes de carotenoides (Bertrand et al. 2006, Pike et al. 2007a). Aunque en
ninguno de estos trabajos se decantan con seguridad por ninguna de las dos
alternativas expuestas anteriormente, dejando el campo abierto para futuras
investigaciones.
En el contexto de la presente tesis y debido a las características funcionales
que presentan las coloraciones dependientes de carotenoides, los animales que
exhiben este tipo de coloraciones son un buen modelo para poner a prueba el papel
de estas señales como indicadoras de contaminación ambiental y también para
analizar los mecanismos que subyacen a la expresión de estas señales.
15
Introducción
Figura 3. En este ejemplo se muestra el efecto de la activación inmune
sobre la expresión de una señal sexual mediada por carotenoides. A) Se
muestra el cromatograma del análisis de los carotenoides, presentes en el
pico de los machos de mirlo común, mediante cromatografía líquida de alta
resolución. B y C) Se observan los cambios en el color del pico del mirlo
tras una inyección intraperitoneal de células rojas de cordero (SRBC), que
provocaron la activación del sistema inmune. D) Se observa gráficamente las
diferencias en el color del pico de ejemplares inyectados con SRBC y
ejemplares control (media ± error estándar). Datos Faivre et al. (2003)
Importancia de la expresión de las coloraciones dependientes de
carotenoides
La expresión de las señales dependientes de carotenoides es importante, dentro del
contexto de la selección sexual, ya que puede ser usada como señal de dominancia,
por ejemplo para la defensa de los territorios de cría, o para el acceso a la pareja. De
este modo, en distintas especies se ha demostrado experimentalmente que los
ejemplares más coloreados son más dominantes y tienen mayor capacidad de lucha
que los menos coloreados a la hora de defender un territorio de cría (Pryke et al.
16
Introducción
2002, Figura 4) o que las hembras prefieren a los machos más coloreados a la hora
de reproducirse (Pike et al. 2007b, Figura 5). Debido a que las coloraciones
dependientes de carotenoides han evolucionado por razones sociales dentro de la
población. Las alteraciones que se pudiesen producir en estas señales tras un
episodio de contaminación podrían tener consecuencias importantes en la toma de
decisiones, por parte de los ejemplares, durante la época de cría, lo que podría tener
efecto en el éxito reproductivo a nivel poblacional (Doutrelant et al.2008).
Figura 4. En un experimento llevado a cabo
en esta ave (Euplectes ardens) se manipuló el
tamaño y el color del collar rojo de la
garganta (una señal sexual dependiente de
carotenoides) antes y después de que se
establecieran los territorios de cría. Los
resultados mostraron que la coloración de
los machos afectaba al establecimiento y
defensa de los territorios. Así los machos
más rojos tenían una mayor ventaja que los
menos rojos o no coloreados. (Pryke et al.
2002).
Figura 5. En un trabajo experimental
usando como modelo esta especie de pez
(Gasterosteus aculeatus), en la que los machos
presentan una señal sexual dependiente de
carotenoides. Los resultados revelaron que
los ejemplares macho suplementados con
más carotenoides en la dieta aumentaban la
intensidad de la expresión de la señal
respecto de los que recibieron menos
carotenoides. Además, las hembras
preferían a los ejemplares con mayor
coloración (Pike et al. 2007b).
Mareas negras en la costa gallega
Desde el año 1970 hasta la actualidad se han producido en la costa gallega cinco
accidentes de grandes petroleros: Polycommander 1970, Urquiola 1976, Andros
Patria 1978, Aegean Sea 1992 y Prestige 2002. Entre todos ellos liberaron al medio
17
Introducción
unas 300.000 toneladas de hidrocarburos, con consecuencias nefastas para los
ecosistemas marinos costeros de Galicia (http://otvm.uvigo.es/). A estos derrames
hay que añadir los hidrocarburos procedentes de los “sentinazos”, palabra con la
que se conoce a la limpieza ilegal de los depósitos de los buques en alta mar, aunque
de estos últimos no hay datos oficiales disponibles. El accidente y posterior
hundimiento del buque petrolero Prestige, a día de hoy, fue el último y el más
mediático de las mareas negras ocurridas en Galicia.
El Prestige
El 13 de Noviembre del año 2002, el buque petrolero Prestige con bandera de
Bahamas y con 77.000 toneladas de fuel pesado en sus tanques sufrió un fallo
estructural en su costado derecho a unas 30 millas de la costa de Fisterra. Como
consecuencia de la brecha, el barco comenzó a derramar parte de su carga. En los 6
días posteriores el barco siguió un rumbo errático, como consecuencia de las
decisiones tomadas por las autoridades competentes, y finalmente el día 19 el barco
se partió en dos y se hundió a 3.800 m de profundidad (42º 10.8 N 012º 03.6 W), a
unas 165 millas al oeste de las islas Cíes (Figura 6). Hasta ese momento se llevaban
liberadas aproximadamente unas 20.000 toneladas de fuel. Tras el hundimiento, el
barco siguió liberando fuel de sus tanques, aproximadamente unas 40.000 toneladas,
hasta que en el verano del 2003 se llevaron a cabo las operaciones necesarias para
recuperar el fuel que quedaba en sus bodegas (más información en
http://webs.uvigo.es/c04/webc04/prestige/prestige.htm).
Figura 6. Rumbo que siguió el buque petrolero Prestige, desde el aviso de socorro el 1311-2002 hasta el hundimiento el 19-11-2002. Se observa en color más oscuro el fuel
derramado durante esos días, unas 20.000 Tm (Albaigés et al. 2007). En la derecha se
observa una de las últimas imágenes tomadas del petrolero antes de su hundimiento.
18
Introducción
El Prestige y las aves marinas
Las 63.000 toneladas de fuel liberadas por el Prestige llegaron en varias mareas negras
a las costas gallegas y a las cantábricas (en la Península Ibérica) y a parte de la costa
oeste francesa entre los años 2002 y 2003, afectando a la flora y fauna de estas
zonas. Entre los integrantes de la fauna costera afectados por los vertidos de
petróleo se encuentran las aves marinas. Estas aves son de los animales con mayor
riesgo de sufrir los impactos negativos de los derrames de hidrocarburos (Piatt y
Ford 1996; Lance et al. 2001; Peterson et al. 2003) ya que pasan parte de su vida en
contacto con la superficie del mar y debido a que las zonas costeras, donde las aves
se congregan para criar, pueden recibir los impactos de las mareas negras (Piatt et al.
1990, Irons et al. 2000).
La marea negra del Prestige produjo una mortalidad masiva de miles de aves
marinas durante el derrame (García et al. 2004), debido a los efectos letales directos
de la contaminación. Así, se vieron afectadas las principales especies de estas aves
que crían en Galicia, con consecuencias negativas para las poblaciones de estas
especies (Velando et al. 2005a). A modo de ejemplo, el derrame mató a un grupo
numeroso de cormoranes moñudos (especialmente a hembras), una especie de ave
marina catalogada en España como en peligro de extinción (Velando y Alvarez
2004), con consecuencias negativas para la poblaciones gallegas (Velando et al.
2005a; Martínez-Abraín et al. 2006, Figura 7).
Las aves que no murieron permanecieron en contacto con la
contaminación persistente en el medio (Freire y Labarta 2003) con consecuencias
negativas para su salud (Alonso-Alvarez et al. 2007). Por otro lado, se ha señalado
que el derrame de petróleo del Prestige afectó de forma indirecta, a través de la
disminución en la disponibilidad de presas, a las poblaciones de cormorán moñudo
(Phalacrocorax aristotelis) en las islas Cíes, una de las principales colonias de cría de
esta
especie
(Velando
et
al.
2005b,
Figura
8;
ver
http://webs.uvigo.es/avelando/prestige/)
19
Introducción
Figura 7. Proporción de sexos de los cormoranes moñudos adultos y subadultos
encontrados muertos después del derrame de petróleo del Prestige a lo largo de la costa
gallega. Se observa que el efecto de las marea negra en esta especie fue mayor en las
hembras que en los machos (Martínez-Abraín et al. 2006).
Figura 8. En la figura de la izquierda se muestra la representación gráfica de la evolución
de la población reproductora del cormorán moñudo situada en una colonia de las Islas
Cíes, entre el año 1992 y el 2003. Los puntos blancos representan el tamaño estimado
procedente del conteo de los nidos. El punto negro representa la estación reproductiva
siguiente al derrame del Prestige. Se observa una disminución en el número de parejas
reproductoras en la estación siguiente al derrame. En el gráfico de la derecha se observa la
captura de bolo (peces de la familia Ammodytidae, consumidos mayoritariamente por los
cormoranes en la dieta Velando y Freire 1999), por unidad de esfuerzo en la Ría de Vigo
(media ± error estándar). Se observa como el bolo disminuyó en la estación siguiente al
derrame de petróleo. Parece que la población de cormorán moñudo se vio afectada de
forma indirecta por el derrame de petróleo, ya que este afectó a su principal fuente de
alimento (Velando et al. 2005b).
20
Introducción
Las zonas afectadas por el derrame del Prestige albergan colonias de todas
las aves marinas que crían en Galicia (gaviota patiamarilla, cormorán moñudo,
gaviota sombría, gaviota tridáctila, arao común, paiño común y recientemente la
pardela cenicienta y el gavión. Figura 9), y en algunas de las cuales se han
documentado importantes efectos de la contaminación (Velando et al. 2005a,b,
Martínez-Abraín et al. 2006, Alonso-Alvarez et al. 2007). La gaviota patiamarilla
(Larus cachinnans) es el ave más numerosa de esta comunidad, reproduciéndose en
Galicia aproximadamente la mitad de los efectivos presentes en España (Munilla
1997a, Martí y Del Moral 2003).
Figura 9. Zonas costeras afectadas por las mareas negras del Prestige. Se señalan las
colonias de cría de la gaviota patiamarilla que fueron afectadas (“oiled”) y poco
afectadas (“unoiled”) por la marea negra del Prestige, muestreadas en el presente
estudio (Pérez et al. 2008)
La gaviota patiamarilla
La gaviota patiamarilla es un ave marina con reproducción iterópara (se reproducen
más de una vez a lo largo de su ciclo vital) y con un ciclo vital largo. Posee además
una alta supervivencia adulta, una elevada mortalidad juvenil, y un éxito reproductor
anual bajo. Es un ave dimórfica en tamaño (Figura 10), los machos son un 15-20%
más grandes que las hembras (Alonso-Alvarez y Velando 2003). Ambos sexos
21
Introducción
presentan coloraciones dependientes de carotenoides en patas, pico, anillos oculares
y comisuras del pico (Cramp y Simmons 1983, Figura 10).
En cuanto a la alimentación, es una especie oportunista cuya dieta en esta
población se compone fundamentalmente de pescado, basura y alguna especie de
crustáceo como el cangrejo Polybius henslowii, el cual es consumido especialmente a
partir de la fecha de incubación (Munilla 1997a,b). En la época de reproducción, se
alimentan a una distancia de unos 40 km próximos a la colonia de cría (Oro et al.
1995).
Figura 10. Pareja de gaviota patiamarilla (Larus michahellis) durante la reproducción. El
ejemplar que aparece en primer plano es el macho. En esta especie existe dimorfismo
sexual en tamaño, siendo el macho de un 15-20% mayor que la hembra.
En Galicia cría mayoritariamente en las islas siendo la colonia de las islas
Cíes, donde cría en los acantilados de su parte occidental, la que presenta mayor
número de efectivos, unas 18.400 parejas (Arcea 2001). A partir de marzo, las
gaviotas se concentran en las colonias (especialmente los machos) donde desarrollan
conductas agresivas y combates con el fin de adquirir los territorios para la
reproducción (Tinbergen 1953). El tamaño del territorio en torno al nido es muy
importante para los pollos, ya que favorecen los movimientos de los pollos antes del
vuelo, y su supervivencia (Tinbergen 1956, Alonso-Alvarez y Velando 2001). Los
22
Introducción
machos son mayoritariamente los que conquistan y defienden los territorios
(Southern 1981), y el tamaño del territorio que consiguen depende de la condición
del ejemplar (Alonso-Alvarez y Velando 2001). En un mismo territorio, los machos
pueden construir varios nidos de los cuales la pareja sólo ocupará uno (Pérez 2005).
Las gaviotas construyen el nido en el suelo, tiene forma de cuenco y los
materiales que usan para su construcción suelen ser vegetales del territorio o
proximidades. Durante el cortejo, los machos ceban a las hembras, estas son
restrictivas a la hora de copular y cambian copulas por cebas (Velando 2004). La
puesta comienza a finales de abril (Alonso-Alvarez 1998), el tamaño de puesta varía
entre uno y tres huevos siendo este último el tamaño modal (Alonso-Alvarez y
Velando 2003). El intervalo de tiempo entre la puesta del primer y segundo huevo
suele ser pequeño extendiéndose más cara a la puesta del tercero. El volumen de los
huevos va a depender de la condición nutricional de la hembra, así hembras con
mejor condición ponen huevos más grandes (Pérez 2005). El comienzo de la
incubación es asincrónica, comenzando antes de que finalice la puesta de todos los
huevos y suele durar de 27-31 días (Cramp y Simons 1983), en la incubación
participan los dos miembros de la pareja. La condición nutricional de las madres
influye en la cantidad de recursos que reparten a los huevos, lo que afecta a la
supervivencia de los embriones, especialmente a los embriones macho, durante la
eclosión (Pérez et al. 2006). Los pollos son alimentados por los padres durante 3540 días, a partir de los cuales abandonan los territorios de cría (Cramp y Simons
1983).
Las colonias de cría de la gaviota patiamarilla se vieron afectadas por la
marea negra del Prestige. Los resultados de un estudio llevado a cabo en las colonias
afectadas y no afectadas por el derrame de crudo, indican que las gaviotas adultas
que criaron en las colonias afectadas por el vertido presentaban daños subletales,
especialmente daños hepáticos, diecisiete meses después del accidente (AlonsoAlvarez et al. 2007, Tabla 1).
23
Introducción
Tabla 1. Niveles de varios compuestos plasmáticos, hematocrito y condición corporal de las
gaviotas adultas que se reprodujeron en las colonias afectadas y no afectadas por la marea
negra del Prestige en el año 2004. Las abreviaturas hacen referencia a: AST, aspartato
aminotransferasa; GGT, gamma glutamiltransferasa; Ca, calcio; iP, fósforo inorgánico.
(Alonso-Alvarez et al. 2007).
La gaviota patimarilla es un buen modelo para estudiar el uso de las
señales sexuales como medida de calidad ambiental. Debido a que presentan señales
sexuales dependientes de carotenoides y a que recientemente estuvieron expuestas a
un evento de contaminación por petróleo, la marea negra generada por el accidente
y posterior hundimiento del Prestige.
24
Objetivos
OBJETIVOS
El objetivo principal de esta tesis es estudiar si las señales sexuales de un ave marina
pueden reflejar la exposición a una contaminación por petróleo. La hipótesis de
partida es que las gaviotas después de un episodio de contaminación, como el
ocurrido tras el accidente del Prestige, quedan expuestas a los hidrocarburos
policíclicos aromáticos del petróleo (HPAs), los cuales una vez ingeridos son
transportados a través de la sangre, hasta el hígado donde una serie de sistemas
enzimáticos, como el citocromo P450, los transforman en otros compuestos para
facilitar su eliminación. Esta transformación genera sustancias prooxidantes,
especies reactivas del oxígeno (EROs), que el organismo combate con la
movilización de antioxidantes lo que genera posibles efectos en la coloración. Con
lo que la demanda de antioxidantes ante un evento de contaminación puede afectar
negativamente a la expresión de este tipo de señales (ver Figura 11).
En este contexto, von Schantz y colaboradores (1999) sugirieron que los
organismos se enfrentan a un compromiso entre usar los carotenoides como
antioxidantes o usarlos como pigmentos. Por otro lado, otro mecanismo posible es
el que propusieron Hartley y Kennedy (2004) que sugiere que el color de los
carotenoides puede ser alterado por procesos oxidativos, especialmente cuando hay
escasez de antioxidantes. Así, según ambas hipótesis la demanda de antioxidantes
ante un evento de contaminación puede afectar a la coloración dependiente de los
carotenoides (ver Figura 11). Además, los carotenoides juegan un papel importante
en la función inmune (que puede ser dependiente, también, del estrés oxidativo),
aunque no se aborda en la presente tesis.
25
Objetivos
Figura 11. Vías mediante las que la contaminación por petróleo podría afectar a la
coloración dependiente de carotenoides en la gaviota patiamarilla. Después de un evento de
contaminación, como el generado por la marea negra del Prestige. Por un lado, los
hidrocarburos policíclicos aromáticos (HPAs) pueden provocar la inmunosupresión del
sistema inmune que a su vez se verá afectado por los radicales libres generados durante el
proceso de transformación de los HPAs. Así, los carotenoides pueden ser usados para
estimular el sistema inmune en detrimento de su función pigmentaria y esto puede afectar,
por lo tanto a la coloración. Esta posible vía no será tratada en la presente tesis (en la figura
esta vía aparece señalada con el número 1). Pero además, la contaminación generada por una
marea negra, como la del Prestige, puede desencadenar una demanda en el organismo de
antioxidantes para intentar contrarrestar las EROs generadas durante el proceso de
transformación de los HPAs. Así, en este contexto, los carotenoides podrían ser usados
como antioxidantes en detrimento de su uso como pigmentos lo que se reflejará en la
expresión de las señales sexuales mediadas por estas moléculas (hipótesis sugerida por von
Schantz et al. 1999, en la figura vía número 2). Otra posibilidad es que el organismo al
demandar antioxidantes para combatir las EROs, generadas durante el proceso de
transformación de los HPAs, podría disminuir la disponibilidad de antioxidantes que evitan
la oxidación de los carotenoides con consecuencias en la pigmentación (hipótesis de Hartley
y Kennedy 2004, vía señalada con el número 3 en la figura).
Siguiendo las recomendaciones de Hill (1995) (ver el apartado anterior sobre
señales sexuales), primero se han abordado una serie de objetivos particulares que
en conjunto permiten contestar el objetivo principal. Así por una parte, en el anexo
1 y anexo 2, se aborda si las coloraciones dependientes de carotenoides se ven
afectadas por la disponibilidad de antioxidantes y si estas coloraciones se ven
afectadas por una exposición al petróleo. De esta forma:
26
Objetivos
•
Se contesta una de las premisas clave de la hipótesis de trabajo, si la
coloración producida por los carotenoides depende de la disponibilidad de
antioxidantes, anexo 1.
•
Se analiza experimentalmente si la coloración dependiente de carotenoides
puede indicar una exposición a hidrocarburos policíclicos aromáticos,
anexo 2.
En los tres anexos siguientes se aborda el tema de la tesis desde un punto de
vista aplicado. Se analiza el efecto que un caso real de contaminación por petróleo
(la marea negra generada por el accidente y posterior hundimiento del buque
petrolero Prestige), produjo en la expresión de las señales dependientes de
carotenoides de las gaviotas patiamarillas. De tal forma que:
•
Primero, se estudia si las gaviotas patiamarillas estuvieron expuestas a la
contaminación de la marea negra generada por el derrame del buque
Prestige y para ello se estudia si los hidrocarburos policíclicos aromáticos
analizados en la sangre son biomarcadores de este tipo de eventos de
contaminación, anexo 3.
•
Una vez conocido el grado de exposición de las gaviotas se analizan
experimentalmente los efectos tóxicos que se producen tras la exposición a
los hidrocarburos policíclicos aromáticos, anexo 4.
•
En último lugar, se estudia si las señales dependientes de carotenoides son
útiles para el seguimiento de los efectos tóxicos como los que se
produjeron tras la marea negra del Prestige, anexo 5.
27
Métodos
MÉTODOS
La metodología empleada en cada estudio se detalla en cada anexo, así en este
apartado únicamente se hará referencia a la zona de estudio, al trabajo de campo, a
las principales metodologías puestas a punto y a los análisis más relevantes de la
presente tesis.
Zona de estudio
El trabajo de campo se llevó a cabo durante dos épocas reproductivas consecutivas.
Durante la época reproductiva del año 2004 se eligieron varias colonias de cría de la
gaviota patiamarilla (situadas a lo largo de la costa gallega y una en la costa asturiana)
afectadas y no afectadas por la marea negra generada por el Prestige (ver Figura 9).
Durante la época reproductiva siguiente, el año 2005, se llevaron a cabo los trabajos
experimentales en las colonias de cría situadas en la isla de Monteagudo (islas Cíes).
Las islas Cíes fueron elegidas para llevar a cabo los trabajos experimentales
de esta tesis, por albergar una de las colonias más importantes que la gaviota
patiamarilla posee en Galicia, unas 18.400 parejas (Arcea 2001). Estas islas se sitúan
en la entrada de la ría de Vigo (Figura 12), y forman parte del Parque Nacional
Marítimo-Terrestre de las Islas Atlánticas de Galicia. Las islas están
permanentemente ocupadas por personal de Parques Nacionales y reciben la visita
de turistas entre junio y septiembre, aunque ocasionalmente también son visitadas
algún fin de semana antes de la temporada turística. En estas islas, las aves marinas
crían en su parte occidental, siendo estas zonas de difícil acceso, las más escarpadas
orográficamente, y con acceso restringido por ser zonas de protección especial.
29
Métodos
Figura 12. En el esquema superior izquierdo se observa la situación del archipiélago de las
islas Cíes en la entrada de la ría de Vigo. En la foto superior derecha se observan las tres islas
que componen este archipiélago protegido dentro Parque Nacional Marítimo-Terrestre de las
Islas Atlánticas de Galicia. En la foto central se observa la orografía abrupta de la parte
occidental de la Isla de Monteagudo en primer plano y la isla Do Faro al fondo (islas Cíes),
donde se sitúan las áreas reproductivas de la gaviota patiamarilla usadas para desarrollar los
trabajos experimentales de esta tesis.
30
Métodos
Figura 13. En la foto de la izquierda se observa A Valgada y en la foto de la derecha A
Percha, áreas de cría de la gaviota patiamarilla situadas en la isla de Monteagudo (islas
Cíes).
Trabajo de campo
En el año 2004, se capturaron las gaviotas en las colonias de las islas: Cíes, Ons,
Vionta, Lobeiras, Coelleira y Ansarón, colonias repartidas a lo largo del litoral
gallego y en una colonia situada en el litoral asturiano en la isla de Las Pantorgas
(ver Figura 9). Una vez que las gaviotas eran capturadas, se marcaba el nido (con
una estaca de madera numerada), se les tomaban varias medidas morfométricas
(tamaño del ala, tarso, pico y cabeza) y el peso. Además, se tomaba una fotografía
del lado derecho del pico (con una caja diseñada especialmente para tal función). En
el año 2004, pusimos a punto esta técnica durante el trabajo de campo, por lo que
sólo pudieron ser usadas las fotografías tomadas en las últimas colonias
muestreadas. Por último, a las gaviotas capturadas, se les extraía una muestra de
sangre de la vena ulnar situada en el ala. La sangre se almacenaba a 4ºC y al final del
día se centrifugaba para separar la fracción plasmática de la celular, que eran
posteriormente almacenadas en nitrógeno líquido. En el laboratorio las muestras se
almacenaron a -80ºC hasta que fueron analizadas.
En el año 2005 se llevaron a cabo los trabajos experimentales en las áreas
adyacentes de cría de A Valgada y A Percha situadas en la isla de Monteagudo
(Figura 13), desde el 25 de abril hasta el 2 de junio. Los nidos se marcaron con
estacas numeradas, que fueron previamente asignadas, aleatoriamente, a los tres
tratamientos alimenticios. Así las gaviotas del grupo experimental denominado
“vitamina E”, recibieron un suplemento de vitamina E diluida en aceite vegetal en
una rebanada de pan, las denominadas “control” recibieron aceite vegetal y pan y
por último las denominadas “petróleo” recibieron trazas de petróleo procedente del
31
Métodos
Prestige diluido en aceite vegetal, durante siete días consecutivos después la
alimentación era similar a la de las gaviotas del grupo control. Cada día se realizaba
un recorrido por cada nido para suministrarle el alimento que le correspondiese
(Figura 14), y se hacía un seguimiento de la puesta. Una vez que se completaba la
puesta (cinco días después de que se pusiese el primer huevo), se paraba la
alimentación y se empezaban a capturar a los adultos. La captura se realizaba
cuando los adultos estaban incubando, con trampas-nasa situadas encima del nido
(Figura 14). A las gaviotas capturadas se les realizaba el mismo protocolo de
muestreo que en el año 2004, medidas, peso, foto del pico y muestra de sangre.
Además a tres gaviotas, que habían muerto recientemente por causas naturales en la
colonia, se les quitó el pico para determinar los carotenoides responsables de su
coloración.
Figura 14. A la izquierda se observa una imagen de como era suministrado el alimento a las
gaviotas (aceite vegetal, vitamina E diluida en aceite vegetal o petróleo diluido en aceite
vegetal). Este alimento era esparcido en una rebanada de pan que era troceada y escondida en
la vegetación próxima al nido correspondiente. En la imagen de la izquierda se observa una
gaviota incubando dentro de una trampa-nasa situada encima del nido.
Trabajo de laboratorio
En la presente tesis se han puesto a punto varias técnicas para lograr los objetivos
propuestos. En primer lugar, se determinaron los carotenoides presentes tanto en el
plasma sanguíneo como en el pico de las gaviotas patiamarillas, para ello estos
compuestos se extrajeron usando etanol como solvente (anexo 1, Figura 16). Una
vez extraídos se determinaron y cuantificaron mediante cromatografía líquida de alta
resolución (HPLC, protocolo en el anexo 1, Figura 16). Esta es la primera vez que
los carotenoides responsables del color amarillo y rojo del pico de las gaviotas
patiamarillas son analizados. Por otro lado, se ha desarrollado el protocolo de
32
Métodos
HPLC para determinar y cuantificar la vitamina E plasmática (ver anexo 1).
Asimismo, se cuantificaron 15 HPAs presentes en las células sanguíneas usando
HPLC, la primera vez que se han determinado HPAs en este tipo de tejido en
animales silvestres. Por último, se analizaron las fotografías de los picos de las
gaviotas mediante un programa informático de análisis de imágenes para cuantificar
el tamaño de la mancha roja.
Análisis de las muestras
Para alcanzar los objetivos propuestos para el anexo 1 se extrajeron y analizaron los
carotenoides del pico de las gaviotas patiamarillas, de esta forma sabríamos que
carotenoides eran los responsables de la coloración roja y de la amarilla que
presentan en el pico (Figura 15). Además, se analizaron en las muestras sanguíneas
de los machos de las gaviotas del grupo vitamina E y grupo control (experimento
llevado a cabo en la colonia de las Islas Cíes en el año 2005), los niveles plasmáticos
de la vitamina E, carotenoides totales y los carotenoides responsables de la
coloración amarilla y roja del pico para saber si hubo un efecto del suplemento con
la vitamina E sobre los compuestos plasmáticos mencionados. Asimismo, se estudió
el efecto del suplemento de la vitamina sobre la señal sexual mediante el análisis de
las fotografías realizadas a los picos de las gaviotas de los dos grupos
experimentales.
33
Métodos
Figura 15. En la imagen de la izquierda se observa el color que presentan las extracciones
de los carotenoides de la zona amarilla del pico (vial superior) y de la mancha roja (vial
inferior). En la imagen de la derecha se observan los cromatogramas de la extracción de la
zona amarilla, (cromatograma superior) y de la mancha roja (cromatograma inferior). En
el cuadro amarillo se engloban los carotenoides que aparecen en ambas extracciones y en
el cuadro rojo los carotenoides que aparecen solamente en la extracción de la mancha
roja. Se señalan los carotenoides identificados y no identificados.
Para estudiar los objetivos propuestos para el anexo 2, se analizaron las
muestras sanguíneas procedentes de los machos y las hembras del grupo de petróleo
y del grupo control (experimento llevado a cabo en la colonia de las Islas Cíes en el
año 2005). Se determinan los niveles sanguíneos de quince hidrocarburos
policíclicos aromáticos (HPAs) y los niveles plasmáticos de vitamina E,
carotenoides totales y los carotenoides responsables de las coloraciones del pico,
evaluando el resultado de la exposición al petróleo sobre los niveles de estos
compuestos. Asimismo, se determinó el efecto del petróleo sobre la señal
dependiente de carotenoides mediante el análisis de las fotografías de los picos.
Previamente a la presente tesis, se había sugerido que medir directamente
hidrocarburos en los tejidos de las aves probablemente no era útil porque no
reflejaría adecuadamente la exposición al petróleo (Trust et al. 2000), ya que las aves,
al igual que todos los vertebrados, poseen sistemas enzimáticos que pueden
metabolizar los HPAs originales en otros compuestos para facilitar su eliminación
del organismo. Esta idea se apoyaba en que estos compuestos, hasta ahora, fueron
detectados en los tejidos de los vertebrados en concentraciones bajas (Ariese et al.
1993, Di Giulio et al. 1995). De esta forma el objetivo del anexo 3 consiste en
34
Métodos
comprobar si la exposición al petróleo se refleja en el análisis de los hidrocarburos
en sangre. Así, por primera vez en la sangre de un ave marina, se analizan los
hidrocarburos policíclicos aromáticos (HPAs). De esta forma, se examinaron las
muestras sanguíneas de las gaviotas muetreadas en el año 2004 en las colonias
afectadas por la marea negra del Prestige y en las no afectadas. Además, se utilizaron
las muestras sanguíneas de las gaviotas del experimento de exposición al petróleo,
mencionado anteriormente, para evaluar si una exposición directa a los HPAs se ve
reflejada en los niveles sanguíneos de HPAs. Por último, en este capítulo se
comparan los niveles sanguíneos de HPAs de las gaviotas de las islas Cíes durante
dos años consecutivos para estudiar la evolución temporal de la carga contaminante
del petróleo en las gaviotas.
Para alcanzar los objetivos del anexo 4, se estudiaron los efectos tóxicos
que el petróleo provoca en las gaviotas patiamatrillas tras su ingestión. Para esto, se
analizaron los niveles plasmáticos de dos enzimas transaminasas (la aspartato
aminotransferasa AST y la gamma glutamiltransferasa GGT) y los de glucosa y
fósforo inorgánico en las muestras de sangre de las gaviotas procedentes del
experimento de petróleo llevado a cabo en la colonia de las islas Cíes en el año
2005. Estos compuestos son indicadores de daños hepáticos y renales de los efectos
subletales del petróleo del Prestige (Alonso-Alvarez et al. 2007).
Por último en el anexo 5, se estudia la relación entre los indicadores de
efectos tóxicos del petróleo y la expresión de la mancha roja, una señal sexual, en las
gaviotas. Para esto, en las gaviotas muestreadas en las colonias afectadas por la
marea negra del Prestige, en el año 2004, se analizó la relación entre la condición
corporal, los niveles plasmáticos de glucosa, fósforo inorgánico, o la de las dos
transaminasas (AST y GGT), y el tamaño de la mancha roja del pico en las
fotografías tomadas a las gaviotas muestreadas.
35
Resultados
RESULTADOS
En este apartado se hará referencia únicamente a los resultados más relevantes de la
presente tesis. Para ver de forma detallada los resultados encontrados en cada
estudio hay que remitirse al capítulo o anexo correspondiente.
Coloración por carotenoides y estrés oxidativo
La disponibilidad de antioxidantes incoloros afecta a la mancha roja que
poseen las gaviotas patiamarillas en el pico (Anexo 1)
En este estudio se analizó experimentalmente si la disponibilidad de un antioxidante
plasmático no coloreado afecta a la expresión de la mancha roja del pico de las
gaviotas patiamarillas. Así, en primer lugar, se determinó por primera vez que
carotenoides estaban presentes en la mancha roja del pico. Los resultados de los
análisis cromatográficos muestran que los carotenoides responsables de la
coloración del pico son diez, de los cuales seis pudieron ser identificados. De estos
diez carotenoides cinco estaban presentes, tanto en la extracción de la zona amarilla
del pico, como en la extracción de la mancha roja (cantaxantina, β-criptoxantina, βcaroteno y dos carotenoides no identificados, que serán denominados en la presente
memoria como “otros carotenoides”, Figura 15). Además, se encontró que otros
cinco carotenoides estaban presentes exclusivamente en la extracción de la mancha
roja (astaxantina, luteina, zeaxantina y dos carotenoides no identificados, que serán
denominados como “carotenoides de la mancha roja”, Figura 15).
El suplemento de vitamina E (un antioxidante incoloro) durante el cortejo
provocó un aumento de su concentración en el plasma de los machos (P < 0,001;
Figura 16). Además, los machos vitaminados presentaron un aumento de un 22%
en la disponibilidad de carotenoides plasmáticos respecto de los machos control (P
= 0,01; Figura 16), lo que indica que la disponibilidad de antioxidantes afecta a la
movilización de carotenoides plasmáticos. Este aumento fue debido especialmente
al aumento de los “carotenoides de la mancha roja”, (P = 0,02; Figura 17), más que
de los “otros carotenoides”, cuyos niveles plasmáticos no difirieron entre los dos
grupos experimentales (P > 0,05; Figura 17).
37
Resultados
Finalmente, el suplemento de antioxidantes también afectó a la mancha
roja del pico, de tal forma que la mancha de los machos suplementados fue un 9%
más grande que la de los machos control (P = 0,02; Figura 16).
Figura 16. Efecto del suplemento de vitamina E sobre: (a) los niveles plasmáticos de
vitamina E; (b) los niveles plasmáticos de los carotenoides totales y (c) el tamaño de la
mancha roja del pico. Valores medios (± error estándar).
38
Resultados
Figura 17. Efecto del suplemento de la vitamina E en la alimentación sobre los niveles
plasmáticos de los carotenoides que se encuentran sólo en la mancha roja del pico
“carotenoides de la mancha roja” y sobre los niveles de los “otros carotenoides”. Valores
medios (± error estándar).
El petróleo afecta a la mancha roja del pico de la gaviota patiamarilla (Anexo
2)
En este anexo se analizó experimentalmente el efecto de la exposición al petróleo
en las señales dependientes de carotenoides. En el estudio experimental realizado en
el campo, las gaviotas expuestas al petróleo tuvieron mayores niveles sanguíneos de
HPAs totales (HPATs, suma de los 15 HPAs) que las gaviotas control (P = 0,036;
Figura 22 B). Asimismo, las gaviotas expuestas al petróleo presentaron una
concentración plasmática de vitamina E y carotenoides mayor, de un 31% (P =
0,04; Figura 18), y un 27% (P = 0,01; Figura 18) respectivamente, que las gaviotas
control; lo que sugiere una movilización de antioxidantes después de un evento de
estrés oxidativo, como el que se produce tras la exposición al petróleo. Por otro
lado, independientemente del tratamiento, las hembras presentaron mayores niveles
de carotenoides plasmáticos que los machos (P < 0,01).
39
Resultados
Los carotenoides plasmáticos correlacionaron negativamente con los
niveles sanguíneos de los hidrocarburos policíclicos aromáticos (Figura 19).
Además, en las hembras, los niveles plasmáticos de los “carotenoides de la mancha
roja” correlacionaron negativamente con los niveles sanguíneos de los HPAs
totales, relación que no se observó en los machos (sexo * HPATs, P = 0,022; Figura
20). En último término, el tamaño de la mancha roja de las gaviotas
experimentalmente expuestas al crudo del Prestige fue un 16% menor que la mancha
de las gaviotas controles (P = 0,02; Figura 21).
Figura 18. Efecto del suplemento de petróleo en los niveles plasmáticos de vitamina E (a)
y carotenoides totales (b). Valores medios (± error estándar).
40
Resultados
Figura 19. Relación entre los niveles sanguíneos de los hidrocarburos policíclicos
aromáticos y los niveles plasmáticos de los carotenoides, en las gaviotas del grupo control
(puntos blancos y línea discontinua, R2 = 0,07) y en las gaviotas del grupo expuesto
experimentalmente al petróleo del Prestige (puntos negros y línea continua, R2 = 0,24).
41
Resultados
Figura 20. Relación entre los niveles sanguíneos de los hidrocarburos policíclicos
aromáticos totales y los niveles plasmáticos de los carotenoides exclusivos de la mancha
roja del pico, en los machos de gaviota patiamarilla (puntos negros y línea continua, R2
= 0,01) y en las hembras (puntos blancos, y línea discontinua, R2 = 0,51).
42
Resultados
Figura 21. Efecto del alimento suplementado con petróleo sobre el tamaño de la
mancha roja del pico de las gaviotas patiamarillas. Valores medios (± error estándar).
La toxicidad del petróleo y la coloración por carotenoides
Evaluación de la exposición de las gaviotas al vertido de petróleo del
Prestige mediante el análisis sanguíneo de los hidrocarburos policíclicos
aromáticos (Anexo 3)
En este estudio se analizó el nivel de exposición de las gaviotas patiamarillas a la
contaminación del petróleo, 17 meses después de la marea negra del Prestige. Para
ello se analizaron los niveles de HPAs en la sangre de gaviotas nidificantes,
muestreadas en siete colonias. En este estudio, se encontró que las gaviotas que
criaban en las colonias afectadas por la marea negra presentaban el doble de
concentración de hidrocarburos policíclicos aromáticos (la suma de la
concentración sanguínea de los 15 HPAs analizados), con respecto a la sangre de
aquellas gaviotas que criaron en las colonias no afectadas (P = 0,011; Figura 22 A).
Además, se observó que la concentración total de HPAs en sangre de las gaviotas
de las islas Cíes (colonia afectada), disminuyó aproximadamente un tercio en dos
estaciones reproductivas consecutivas, 2004 y 2005 (P = 0,03; Figura 22 A); lo que
43
Resultados
sugiere que la concentración de estos compuestos en el medio disminuyó con el
tiempo.
Por otro lado, los resultados del estudio experimental corroboraron que la
ingestión de petróleo en la dieta se ve reflejada en la concentración total de HPAs
en la sangre de las gaviotas (P = 0,036; Figura 22 B). No obstante, los resultados del
experimento también indicaron que la degradación y el metabolismo son específicos
de cada compuesto. Así, la relación entre los niveles sanguíneos de los HPAs y el
tiempo transcurrido entre el fin de la alimentación y la captura de los adultos fue no
lineal en seis compuestos (Figura 23). Cuatro de ellos (fluoreno, fluoranteno,
dibenzo(a,h)antraceno y benzo(a)pireno) mostraron un patrón de respuesta similar,
con una mayor concentración en las gaviotas capturadas más tarde en relación con
el fin de la ingesta de petróleo. Por otro lado, los niveles sanguíneos de
indeno(1,2,3-cd)pireno disminuyeron con el tiempo transcurrido y los de
benzo(b+j)fluoranteno disminuyeron a partir de los 15 días después de la ingesta
(Figura 23). Los otros compuestos analizados no presentaron ningún tipo de
relación significativa con el tiempo transcurrido desde el fin de la alimentación (P >
0,05).
44
Resultados
Figura 22. Valores medios (± error estándar) de los hidrocarburos policíclicos aromáticos en
las células sanguíneas de las gaviotas patiamarillas. En A) se muestra la concentración en la
sangre de las gaviotas procedentes de colonias afectadas y no afectadas (barras negras y
blancas, respectivamente) por la marea negra del Prestige en el año 2004 y del año 2005 en las
Islas Cíes (barra gris). En B) se muestra la concentración sanguínea de las gaviotas
suplementadas con aceite vegetal (grupo control, barra blanca) y la de las gaviotas
suplementadas con aceite vegetal más petróleo del Prestige (grupo petróleo, barra negra). Las
abreviaturas de las colonias son: PA=Pantorgas, AN=Ansarón, CO=Coelleira, LO=Lobeiras,
VI=Vionta, ON=Ons, CI=Cíes 2004 and CI05=Cíes 2005). * P <0.05
45
Resultados
Figura 23. Relación de los niveles sanguíneos de seis hidrocarburos policíclicos aromáticos
analizados y el tiempo transcurrido entre el fin de la alimentación y la captura de las gaviotas
suplementadas con petróleo del Prestige.
Efectos subletales tras la exposición al petróleo en la gaviota patiamarilla
(Anexo 4)
En este capítulo se estudia experimentalmente el efecto tóxico, a corto plazo, de la
exposición al petróleo en la gaviota patiamarilla. Los resultados se compararon con
los de un trabajo previo en el que se analizaron los daños subletales en las gaviotas
afectadas por la marea negra del Prestige (Alonso-Alvarez et al. 2007), diecisiete
meses después del accidente. Así, en el trabajo experimental, al igual que en el
trabajo mencionado anteriormente, las gaviotas expuestas al petróleo presentaron
una menor concentración plasmática de glucosa y fósforo inorgánico (P = 0,01 y P
= 0,009, respectivamente; Figura 24) con respecto a las de las gaviotas no expuestas.
Además, la exposición al petróleo afectó a los niveles de aspartato aminotransferasa
(AST) de los machos (P = 0,043; Figura 24), en contraste con los resultados previos
que indicaban que tanto machos como hembras de las colonias afectadas por el
Prestige presentaban altos niveles de actividad plasmática de AST (Alonso-Alvarez et
al. 2007).
46
Resultados
En cuanto al efecto de la exposición al petróleo sobre la actividad
plasmática de la gamma glutamiltransferasa (GGT) se encontró una interacción
entre el sexo y el tratamiento. Así, la actividad plasmática de la GGT fue mayor en
las hembras del grupo control que en las hembras suplementadas con petróleo,
mientras que en los machos no se observó ningún efecto (sexo * tratamiento, P =
0,033; Figura 24). Los resultados encontrados en las hembras fueron opuestos a los
del trabajo previo; las hembras procedentes de las colonias afectadas por el Prestige
presentaban mayores niveles plasmáticos de GGT que las procedentes de colonias
no afectadas por la marea negra (sexo * área, P < 0,01).
Figura 24. Niveles plasmáticos de varios parámetros bioquímicos de las gaviotas
patiamarillas: (a) glucosa, (b) fósforo inorgánico, (c) aspartato aminotransferasa (AST) y (d)
gamma glutamiltransferasa (GGT). Las barras blancas hacen referencia a las gaviotas
suplementadas con aceite vegetal y las barras negras se corresponden con las gaviotas
suplementadas con aceite vegetal más trazas de petróleo procedente del Prestige. Valores
medios (± error estándar).
47
Resultados
Efectos subletales y coloración tras la exposición al petróleo en la gaviota
patiamarilla (Anexo 5)
En este capítulo se analiza si la expresión de las señales dependientes de
carotenoides puede ser útil para detectar los efectos subletales a largo plazo
producidos por la contaminación por petróleo. Para ello, diecisiete meses después
del naufragio, muestreamos veintisiete gaviotas en las colonias afectadas por el
derrame de petróleo del Prestige. En estas gaviotas, el tamaño de la mancha roja del
pico correlacionó positivamente con la condición corporal (P = 0,011; r =0.48), y
negativamente con los niveles plasmáticos de la aspartato aminotransferasa (P =
0,017; r = -0.46; Figura 25), lo que indica que la señal dependiente de carotenoides
refleja el estado nutricional y los daños hepáticos en la gaviota patiamarilla.
Figura 25. En las fotografías se muestra el tamaño de la mancha roja del pico en dos
gaviotas, una con bajos y otra con altos niveles de aspartato aminotransferasa (AST),
fotografías a y b, respectivamente. En c) se muestra la relación entre el tamaño de la mancha
roja del pico y los niveles plasmáticos de la aspartato aminotransferasa.
48
Discusión
DISCUSIÓN
Coloración por carotenoides y estrés oxidativo
En los anexos 1 y 2 de esta tesis se muestra por primera vez, con dos trabajos
experimentales, que el estrés oxidativo afecta a la coloración de un ave marina
producida por carotenoides. En primer lugar, se muestra que el aumento de un
antioxidante incoloro (vitamina E, una situación de bajo estrés oxidativo), provoca
un aumento en los niveles de carotenoides plasmáticos y esto afecta positivamente a
la expresión de la señal sexual (anexo 1); y, en segundo lugar, se muestra que en una
situación de alto estrés oxidativo (como la provocada por el aumento de los
radicales libres que se producen tras la exposición de los hidrocarburos policíclicos
aromáticos) provocó una movilización plasmática de vitamina E y carotenoides, lo
que afectó negativamente a la expresión de la señal sexual (anexo 2).
Los resultados del anexo 1 son consistentes con dos estudios
experimentales recientes, llevados a cabo en vertebrados, que muestran que un
suplemento en la dieta de antioxidantes incoloros aumenta la intensidad de las
coloraciones producidas por carotenoides (Bertrand et al. 2006, Pike et al. 2007a).
Los resultados de estos trabajos y los de los dos primeros anexos de la presente tesis
afianzan la hipótesis que sostiene que la expresión de la coloración producida por
los carotenoides es una señal honesta de la disponibilidad de los antioxidantes del
individuo (von Schantz et al. 1999, Hartley y Kennedy 2004).
Como se mencionó anteriormente, el mecanismo por el cúal estas señales
indican la disponibilidad de antioxidantes es controvertido, sugiriéndose dos vías: el
compromiso de la función pigmentaria con el uso de los carotenoides como
antioxidantes (von Schantz et al. 1999) y, alternativamente, la protección de la
función pigmentaria por el resto de antioxidantes (Hartley y Kennedy 2004; ver
introducción). Esta última idea se basa en que los antioxidantes incoloros pueden
ser usados para proteger a los carotenoides de su oxidación (Woodall et al. 1997,
Siems et al. 1999), esperable sólo en los ejemplares con altos niveles de
antioxidantes.
En el anexo 1 se muestra que los carotenoides plasmáticos responsables
de la mancha roja aumentaron tras la ingestión de un antioxidante incoloro, pero en
cambio este aumento no se produjo en el resto de carotenoides. Este resultado
49
Discusión
podría apoyar la hipótesis de la protección si los carotenoides de la mancha roja
fueran más susceptibles a la oxidación que los “otros carotenoides”. Sin embargo,
esto no parece que sea el caso; así, la ingestión de vitamina E afectó de forma muy
diferente a dos carotenoides con la misma tasa de oxidación (Woodall et al.1997), la
astaxantina presente en la mancha roja y la cantaxantina que no esta presente (ver
anexo 1). De esta forma, los resultados del anexo 1 sugieren que un incremento en
la disponibilidad de antioxidantes (vitamina E), promueve un mecanismo activo que
incrementa la cantidad de carotenoides en la señal, más que una protección pasiva
de estos compuestos (Hartley y Kennedy 2004). Por otro lado, los resultados del
anexo 2, son congruentes con la hipótesis del compromiso entre el uso de los
carotenoides para las funciones vitales o el uso de estos compuestos como
pigmentos (von Schantz et al. 1999). Así, los resultados muestran que la exposición
al petróleo (situación de alto estrés oxidativo) provoca un aumento en la
movilización de carotenoides plasmáticos acompañado de una disminución en la
expresión de la señal dependiente de carotenoides, resultado que va en contra de la
hipótesis de Hartley y Kennedy (2004).
Los resultados del anexo 2 también muestran una relación negativa entre
los niveles plasmáticos de los carotenoides y los niveles sanguíneos totales de los
hidrocarburos policíclicos aromáticos, lo que sugiere que los carotenoides
plasmáticos pueden estar implicados directa o indirectamente en la degradación de
los HPAs. Los HPAs una vez ingeridos son transportados al hígado donde una serie
de sistemas enzimáticos, como el citocromo P450, los transforman estructuralmente
para facilitar su degradación (Meador et al. 1995, Ramos y García 2007). En esta
transformación se generan radicales libres (Lewis 2002) que el organismo inactiva
con enzimas y sustancias antioxidantes (Matés 2000, Nordberg y Arnér 2001). Los
HPAs también inducen otros efectos tóxicos en el organismo, como daños
hepáticos (anexo 4), que en muchos casos pueden generar más radicales libres
(Stephensen et al. 2003, Sturve et al. 2006). En nuestro experimento, la demanda de
antioxidantes tras la ingestión de petróleo es la explicación más plausible al aumento
de antioxidantes plasmáticos, como la vitamina E, en las gaviotas expuestas al
petróleo y también del aumento de los carotenoides. Aunque recientemente se ha
cuestionado el papel antioxidante de los carotenoides en las aves (Costantini y
Møller 2008). En las gaviotas, los carotenoides afectan a la susceptibilidad a la
50
Discusión
peroxidación de lípidos de los embriones y a la capacidad antioxidante (Blount et al.
2002a,b), lo que sugiere un papel importante de los carotenoides como
antioxidantes sistémicos en estas especies. Además, ya que los carotenoides
modulan la respuesta inmune (Blount et al. 2003, McGraw y Ardia 2003, Velando et
al. 2006), el incremento de los niveles plasmáticos de los carotenoides en las
gaviotas expuestas al petróleo podría ser un mecanismo para contrarrestar los
efectos inmunosupresivos de los HPAs (White et al. 1994). En cualquier caso, los
resultados del anexo 2 sugieren que las gaviotas movilizaron carotenoides para
hacer frente a los efectos dañinos de la ingestión de los HPAs; indicando que los
carotenoides pueden jugar un papel importante en los procesos de detoxificación
tras la contaminación por petróleo.
En resumen, los resultados de los anexos 1 y 2 sugieren que el mecanismo
que subyace a la expresión de las coloraciones producidas por carotenoides es el
compromiso entre las funciones fisiológicas, relacionadas con el estrés oxidativo, y
pigmentarias de los carotenoides (von Schantz et al. 1999). Cuando un organismo
tiene un exceso de antioxidantes puede usar los carotenoides como pigmentos
(anexo 1) y cuando aumenta la demanda de antioxidantes para combatir los
radicales libres generados, por ejemplo, tras un episodio de contaminación, usa los
carotenoides para combatir estos radicales libres (anexo 2), o a una posible
inmunodepresión. Además, los resultados del anexo 2 sugieren que altos niveles
plasmáticos de carotenoides no deben ser interpretados como situaciones de bajo
estrés oxidativo, ya que altos niveles también pueden deberse a un aumento en la
demanda de estos compuestos para combatir el estrés oxidativo.
La toxicidad del petróleo y la coloración por carotenoides
Como vimos en el anexo 2, las señales dependientes de carotenoides pueden
reflejar una exposición a hidrocarburos. Siguiendo el esquema propuesto por Hill
(1995; ver introducción), para validar el uso aplicado de las señales, primero
debemos demostrar que las gaviotas estuvieron expuestas a un contaminante,
segundo que este contaminate provoca efectos tóxicos en el organismo y tercero
que la toxicidad se ve reflejada en la expresión de la señal sexual de los ejemplares
expuestos. Siguiendo este esquema, y tras demostar que la exposición al petróleo
afecta a las señales (anexo 2), en los tres últimos anexos de la tesis se muestra, que
51
Discusión
las gaviotas estuvieron expuestas al fuel del Prestige (anexo 3), que la exposición al
fuel del Prestige provoca daños tóxicos (especialmente hepáticos) en las gaviotas
(anexo 4), y que la mancha roja reflejó los daños hepáticos en gaviotas afectadas
por la marea negra del Prestige (anexo 5). Estos resultados sugieren que este tipo de
señales son útiles para rastrear los efectos tóxicos producidos tras una marea negra.
Lamentablemente no disponemos de datos de los niveles sanguíneos de los
hidrocarburos policíclicos aromáticos en las gaviotas antes de la marea negra del
Prestige. Así, no se pudo llevar a cabo la comparación de los niveles de los
hidrocarburos antes y después del accidente (before-after-control-impact, BACI).
Sin embargo, la comparación zonas afectadas frente a zonas no afectadas (ejemplos
previos: Dean et al. 2002, Esler et al. 2002, Golet et al. 2002) y el patrón temporal
posterior al derrame (anexo 3) han servido para analizar el patrón de exposición al
petróleo en las gaviotas patimarillas producido tras el derrame del fuel del Prestige.
Además los resultados encontrados se ven reforzados con los resultados de los
estudios experimentales.
Los resultados del anexo 3 muestran, por primera vez, que los
hidrocarburos policíclicos aromáticos se detectan en la sangre de las aves y que este
análisis refleja muy bien la exposición al petróleo, en contra de lo que se pensaba
anteriormente (Trust et al. 2000). Así, por un lado, se muestra que la concentración
en sangre de estos compuestos separa los ejemplares procedentes de las colonias
que estuvieron afectadas de los procedentes de colonias poco o nada afectadas por
la contaminación de la marea negra del Prestige. Asimismo, estos resultados son
corroborados experimentalmente, las gaviotas que fueron expuestas al petróleo
procedente del Prestige presentaron mayores concentraciones sanguíneas de HPAs
que los ejemplares control.
Por otro lado, en concordancia con lo descrito en otros organismos
marinos (Soriano et al. 2006), encontramos que la contaminación en las gaviotas
disminuye a medida que aumenta el tiempo transcurrido desde el derrame de
petróleo, como es esperable tras un evento catastófico de contaminación. Así, se
encontró que la disminución de la carga total de HPAs en la sangre de las gaviotas
muestreadas en la misma colonia disminuyó aproximadamente un tercio en dos
años consecutivos. Este resultado sugiere que en los primeros momentos, tras el
vertido del Prestige, la concentración de HPAs en las gaviotas debió haber sido
52
Discusión
mayor que la que encontramos en el año 2004, diecisiete meses después del
accidente.
Por último, en este estudio, se encontró que los HPAs tienen distintos
patrones de permanencia en el organismo. Para interpretar este resultado, hay que
tener en cuenta que los eritrocitos sanguíneos tienen una vida media en la
circulación sanguínea de unos 30 días (Clark 1988) y que, además, en los
vertebrados una parte de los HPAs ingeridos, después de ser transformados en el
hígado, son expulsados del organismo, pero otros HPAs permanecen en la
circulación enterohepática aumentando su permanencia en el organismo (Ramesh et
al. 2004). De este modo, los diferentes patrones temporales encontrados en los
distintos HPAs probablemente indican diferentes tasas de metabolización y
permanencia en el hígado de estos compuestos. Estos resultados sugieren que las
proporciones de HPAs en los tejidos, y especialmente en la sangre de vertebrados
no tienen porque reflejar la proporción en el petróleo original (como en este caso,
en el fuel del Prestige).
En relación a los efectos del petróleo, de forma experimental encontamos
que una exposición menor a la encontrada en las poblaciones naturales (anexo 3),
tiene efectos tóxicos en la gaviotas patiamarillas (anexo 4). Se corroboran, así, los
daños subletales encontrados, en esta especie, en las colonias afectadas por el
derrame del Prestige (Alonso-Alvarez et al. 2007). Los resultados de ambos trabajos
coinciden en que la exposición al petróleo provoca una disminución de glucosa y
fósforo inorgánico, probablemente debido a alteraciones en el funcionamiento del
hígado como consecuencia de la contaminación por petróleo. Estos daños
hepáticos fueron corroborados con los altos niveles de las transaminasas, la
aspartato aminotransferasa (AST) y gamma glutamiltransferasa (GGT), encontrados
en las gaviotas expuestas al fuel en ambos trabajos (Alonso-Alvarez et al. 2007 y
anexo 4), y que son indicadores de daños renales y hepáticos (Lewandowski et al.
1986, Brugère-Picoux et al. 1987, Hochleithner 1994, Harr 2002). Aunque hay que
resaltar que los efectos del petróleo sobre los niveles plasmáticos de la enzima AST
son distintos tras una exposición a corto plazo que tras una exposición a largo
plazo. Así mientras a corto plazo los niveles de esta enzima son significativos en los
machos, no así en las hembras, esta diferenciación por sexos no ocurre en las
gaviotas que probablemente estuvieron expuestas al petróleo durante un largo
53
Discusión
periodo de tiempo. En cuanto a la GGT, los resultados de los dos trabajos (AlonsoAlvarez et al. 2007 y anexo 4), coinciden en que el petróleo afectó a los niveles
plasmáticos de la GGT en las hembras pero no en los machos. Así los resultados
del anexo 4, indican una respuesta en los niveles de la AST y la GGT dependiente
del sexo de la gaviota tras una exposición reciente al petróleo.
Los resultados de los niveles en las transaminasas plasmáticas encontrados
en los machos y en las hembras de las gaviotas patiamarillas sugieren una diferente
respuesta adaptativa dependiente del sexo en estas aves tras una exposición al
petróleo y que varía con el tiempo. Las diferencias entre sexos en la respuesta al
petróleo coinciden con la diferente movilización de carotenoides encontrada entre
machos y hembras en el anexo 2. Las hembras movilizaron más carotenoides que
los machos y además, en las hembras, la movilización de los carotenoides de la
mancha roja se relacionó negativamente con los niveles sanguíneos de los HPAs.
En su conjunto, estos resultados sugieren que las hembras se enfrentan a la
toxicidad de los HPAs de forma diferente que los machos; lo que podría estar
relacionado con algún mecanismo de protección de la descendencia. Las hembras,
por ejemplo, podrían estar usando los carotenoides para combatir los HPAs
evitando así que la toxicidad de estos compuestos llegue a la descendencia a través
del huevo. Son necesarios más estudios sobre las diferencias entre sexos en los
procesos de detoxificación y su valor adaptativo.
En último lugar, encontramos que la mancha roja del pico de las gaviotas,
reflejó los daños hepáticos de las gaviotas que criaban en zonas afectadas por el
derrame del Prestige (anexo 5). Así, la coloración roja del pico correlacionó
negativamente con los niveles plasmáticos de la transaminasa aspartato
aminotransferasa, asociada con daños hepáticos (Alonso-Alvarez et al. 2007 y
anexo 4). Las gaviotas muestreadas en este estudio estuvieron expuestas a grandes
cantidades de petróleo del Prestige como se muestra en el anexo 3, lo que provocó
un gran cantidad de daños tóxicos en estas aves (ver anexo 4 y Alonso-Alvarez et
al. 2007).
En el arao columbino (Cepphus columba) en el que tras el vertido del Exxon
Valdez, los niveles de la AST correlacionaban con la activación del citocromo
hepático, P4501A, (Golet et al. 2002) el cual es específicamente sensible a la los
hidrocarburos policíclicos aromáticos (Collier y Varanasi 1991, Trust et al. 2000).
54
Discusión
Así, la AST parece ser un buen indicador de daños tóxicos producidos por la
exposición al petróleo (ver anexo 4 y Alonso-Alvarez et al. 2007).
Además de la exposición al petróleo, los altos niveles de AST encontrados
podrían ser debidos a otros contaminantes. Las gaviotas, además de organismos
marinos, consumen desperdicios (Munilla 1996a), que podría ser una vía de entrada
de contaminantes. En el momento en que las gaviotas fueron capturadas, tan solo
un 15% de las egagrópilas (restos de alimentos no digeridos que son regurgitados),
analizadas en estas colonias contenía desperdicios, por lo que su efecto en los
niveles de AST, aunque no descartable, probablemente sea menor. A falta de
muestras fotográficas de ejemplares procedentes de colonias control, no podemos
afirmar que el vertido del Prestige afectase a la coloración de las gaviotas, pero si que
esta refleja los daños hepáticos producidos al menos en parte, por la marea negra
del Prestige que han sido previamente documentados (Alonso-Alvarez et al. 2007).
La mancha roja del pico además de estar relacionada con daños hepáticos
también indica la condición corporal de las gaviotas. Este resultado es coherente
con lo mostrado en un estudio previo, en el que se encontró que la mancha roja que
posee en el pico el gavión (Larus marinus, una especie emparentada con la gaviota
patiamarilla), correlacionaba con la condición corporal (Kristiansen et al. 2006). En
aves, se ha demostrado experimentalmente un efecto de la ingestión de alimento
sobre colores producidos por los carotenoides (piquero de patas azules, Sula
nebouxii, Velando et al. 2006). El transporte y la absorción de los carotenoides
pueden verse afectados por la disponibilidad de lípidos y lipoproteínas (Salomón y
Bulux 1993) cuyos niveles, en la gaviota patiamarilla, disminuyen en situaciones de
baja condición nutricional (Alonso-Alvarez y Ferrer 2001). En las colonias de
estudio, no hay evidencias de que el petróleo del Prestige afectase a la condición
nutricional de las gaviotas patiamarillas (ver anexo 4 y Alonso-Alvarez et al. 2007).
Así, los resultados sugieren que el tamaño de la mancha roja de las gaviotas es
sensible a dos presiones ambientales distintas, una la condición nutricional y otra los
efectos generados como respuesta a agentes tóxicos (indicada por los niveles de
AST). En futuros estudios en los que se analice el efecto de un contaminante sobre
una señal sexual mediada por carotenoides se debería controlar la condición
corporal de los ejemplares analizados para evitar que los efectos del contaminante
sean enmascarados.
55
Discusión
En resumen, los resultados mostrados en la presente tesis apuntan a que el
posible mecanismo que subyace a la expresión de las señales sexuales mediadas por
carotenoides es el estrés oxidativo. Además, estos resultados validan el uso de las
señales sexuales como indicadoras de contaminación por petróleo. Ya que las
gaviotas patiamarillas pertenecen a un complejo de especies ampliamente
distribuidas a lo largo del hemisferio norte, los resultados podrían ser usados como
una herramienta en las futuras evaluaciones de los efectos subletales de los derrames
de petróleo en las aves marinas.
56
Conclusiones
CONCLUSIONES
•
La mancha roja en el pico de las gaviotas es una señal sexual dependiente
de carotenoides y refleja el estatus antioxidante del individuo.
•
La coloración amarilla y roja del pico de las gaviotas es debida a la
deposición de distintos carotenoides en estas dos coloraciones.
•
La movilización de antioxidantes plasmáticos afecta a la colación roja del
pico.
•
La exposición al petroleo afecta negativamente a la coloración de la gaviota
patiamarilla.
•
Existe un compromiso entre el uso de los carotenoides para las funciones
fisiológicas y el uso de estos compuestos como pigmentos.
•
Los compromisos funcionales de los carotenoides son diferentes en
machos y hembras de la gaviota patiamarilla.
•
Los carotenoides plasmáticos están relacionados con la degradación del
petróleo.
•
La concentración total de hidrocarburos policíclicos aromáticos en sangre
en la gaviota patiamarilla refleja la exposición al petróleo.
•
Los hidrocarburos policíclicos aromáticos presentan distinto metabolismo,
lo que ha de tenerse en cuenta en los estudios en los que se analicen estos
compuestos.
•
Existe una diferente respuesta, a los efectos tóxicos del petróleo, por parte
de las gaviotas tras una exposición al petróleo tanto a largo como a corto
plazo.
•
La glucosa, el fósforo inorgánico, la aspartato aminotransferasa (AST) y la
gamma glutamiltransferasa (GGT) son buenos indicadores de exposición
al petróleo. Hay que tener en cuenta el sexo de las gaviotas patiamarillas en
el uso de las transaminasas AST y GGT como indicadoras de exposición al
petróleo.
•
El tamaño de la mancha roja del pico, una coloración dependiente de
carotenoides, de las gaviotas patiamarillas indica el grado de los daños
hepáticos tras la exposición a una contaminación por petróleo.
57
Conclusiones
•
El tamaño de la mancha roja del pico se ve afectada por la condición del
ejemplar por lo que esta medida debe incluirse en los estudios de las
señales dependientes de carotenoides tras una exposición a agentes
contaminantes.
58
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70
Parte 2
Anexo 1
La disponibilidad de antioxidantes
incoloros afecta a la mancha roja que
poseen las gaviotas patiamarillas en el
pico
Pérez, C., Lores, M., Velando, A. 2008. Availability of nonpigmentary
antioxidant affects red coloration in gulls. Behavioral Ecology, 19: 967-973.
Anexo 1
La disponibilidad de antioxidantes incoloros afecta a la mancha roja que
poseen las gaviotas patiamarillas en el pico
Cristóbal Pérez, Marta Lores, Alberto Velando
Las coloraciones rojas, naranjas y amarillas exhibidas por peces y aves pueden
funcionar como señales sexuales honestas de la calidad del portador de la señal. Sin
embargo, los mecanismos que subyacen a la expresión de estos rasgos y la
información que expresan, es controvertida. Ya que los carotenoides son
antioxidantes y que la pigmentación de los carotenoides es decolorada como
consecuencia de procesos oxidativos, se ha sugerido que este tipo de
pigmentaciones pueden señalar el estado antioxidante del individuo. Nosotros
contrastamos esta hipótesis en la gaviota patiamarilla (Larus michahellis), un ave
marina que muestra una mancha roja, dependiente de carotenoides, en la mandíbula
inferior. En las gaviotas, por medio de un experimento de alimentación
suplementaria, modificamos la disponibilidad de un antioxidante incoloro (vitamina
E) antes de la puesta de los huevos. Durante el periodo de incubación, las parejas
experimentales fueron capturadas para medirles la intensidad del color y el tamaño
de la mancha roja del pico. También se midieron los niveles plasmáticos de
peroxidación de lípidos, la capacidad antioxidante total y los carotenoides.
Encontramos que los machos que recibieron el suplemento de vitamina E
presentaban manchas rojas más grandes que los machos control, sin embargo la
intensidad de la coloración roja de la mancha no se vio afectada por el experimento.
Asimismo, encontramos que solamente los carotenoides plasmáticos involucrados
en la coloración roja del pico estuvieron afectados por el suplemento de
antioxidantes. En general, nuestros resultados muestran evidencias experimentales
que apoyan la hipótesis que sostiene que la coloración dependiente de carotenoides
refleja el estado antioxidante del portador de estas señales.
75 Anexo 1
Availability of non-pigmentary antioxidant affects red coloration in gulls
Cristóbal Pérez1, Marta Lores2 & Alberto Velando1
Departamento de Ecoloxía e Bioloxía Animal. Facultade de Bioloxía. Universidade de Vigo, As LagoasMarconsende s/n 36310 Vigo. Spain. 2Departamento de Química Analítica, Nutrición e Bromatoloxía.
Facultade de Química. Instituto de Investigacións e Análises Alimentarios. Universidade de Santiago de
Compostela, Avda. das Ciencias s/n 15782 Santiago de Compostela. Spain.
1
Abstract
Red, orange and yellow carotenoid-based colorations displayed by fishes and birds
may function as honest sexual signals of the bearer’s quality. However, the
mechanisms underlying the expression of these traits and the information they
convey are still controversial. Since carotenoids are antioxidants and carotenoidbased pigmentation is bleached as a consequence of oxidative processes, it has been
suggested that the pigmentation may signal antioxidant status. We tested this
hypothesis in the yellow-legged gull (Larus michahellis), a seabird that exhibits a
carotenoid-based red spot on the lower mandible. The availability of a nonpigmentary antioxidant (i.e. vitamin E) to the gulls was modified before egg laying
by means of a supplementary feeding experiment. During the incubation period,
breeding pairs were captured to assess the intensity of the color and the size of the
red bill spots. We measured the plasma level of lipid peroxidation, total antioxidant
capacity and carotenoids. We found that males that received vitamin E supplements
had larger red spot than control birds, but that color intensity was not affected by
the supplements. Moreover, we found that only those plasma carotenoids involved
in the red coloration were affected by the antioxidant supplementation, suggesting
an active mechanism to increase red coloration. Overall, our results provide
experimental evidence for the hypothesis that carotenoid-based coloration reflects
the bearer’s antioxidant status in male gulls.
Introduction
Many animals exhibit elaborate ornamental traits that have evolved as signals of the
bearer’s quality and can be evaluated by prospective mates or opponents (Anderson,
1994). One of the major goals of animal communication studies has been to identify
the information content of these signals and the entailed costs that may prevent
77
cheating (Zahavi, 1975; Grafen, 1990; Espmark et al., 2000). However, the currency
of these costs is still under debate and may depend on the nature of the signal
involved.
The red, orange and yellow carotenoid-based colorations displayed by
fishes and birds can be considered as good examples of honest sexual signals (e.g.
Olson & Owens, 1998; Badyaev & Hill, 2000; Pike et al., 2007). However, the
information conveyed by these traits and the mechanisms underlying their
expression are still controversial and have been subject of intensive research in
recent decades. Vertebrates can transform carotenoids through different metabolic
routes (Brush, 1990; Møller et al., 2000) but cannot synthesize them de novo and,
therefore, must acquire them from food. Several experimental studies involving
carotenoid supplementation have shown that an increase in dietary carotenoids
leads to enhanced carotenoid-based ornament expression (e.g. McGraw & Ardia,
2003; Bertrand et al., 2006). The honesty of carotenoid-based signals may also be
reinforced by the important physiological functions of carotenoids (Lozano, 1994;
von Schantz et al., 1999; Møller et al., 2000). One of the functions attributed to
carotenoids is their antioxidant activity (Krinsky, 2001; Young & Lowe, 2001; Rao
& Rao, 2007). Accordingly, it has been suggested that oxidative stress is the
proximate cause of the genuine information revealed to prospective females
through male carotenoid-dependent traits (von Schantz et al., 1999).
Organisms produce reactive oxygen species (ROS) as by-products of
physiological functions, which provoke oxidative damage to DNA, proteins and
lipids (Finkel & Holbrook, 2000). To mitigate oxidative injury, organisms use
endogenous enzymes, such as superoxide dismutase, catalase and glutathione
peroxidase, as well as extracellular antioxidants such as uric acid, vitamin E, vitamin
C and carotenoids (Godin & Garnett, 1992; Fang et al., 2002; Rao & Rao, 2007). In
this context, it has been suggested that there is a trade-off between allocation of
carotenoids to the sexual signal or to functions of antioxidant defense (“antioxidant
trade-off” hypothesis; von Schantz et al., 1999). Thus, oxidative stress may also
underlie the relationship between carotenoid-dependent expression and current
immunological status (Faivre et al., 2003; Alonso-Alvarez et al., 2004 ; Velando et
al., 2007).The activation of the immune system produces reactive oxygen species
that must be counteracted by the mobilization of bodily antioxidants, including
Anexo 1
carotenoids, to balance oxidative stress at the expense of the expression of sexual
coloration (Blount et al., 2003; Faivre et al., 2003; Alonso-Alvarez et al., 2004;
Grether et al., 2004).
Although commonly assumed, the importance of carotenoids in the tradeoff between coloration and free-radical scavenging remains controversial (Hõrak et
al., 2006; Costantini et al., 2007; Isaksson et al., 2007). Thus, it has been suggested
that antioxidant activity is not the main biological role of carotenoids (Hartley &
Kennedy, 2004), but that as they are bleached as a consequence of oxidative
processes (Woodall et al., 1997), they may reflect the healthy functioning of systems
that prevent their oxidation (“protection” hypotheses; Hartley & Kennedy, 2004).
The common prediction implicit in both hypotheses (the antioxidant
trade-off and the protection hypotheses) is that increasing the availability of other
(non-pigmentary) antioxidants should favor the expression of carotenoid-based
signals (Bertrand et al., 2006; Pike et al., 2007). Thus, individuals with greater access
to other antioxidants may use carotenoids for coloration rather than for antioxidant
defense purposes (von Schantz et al., 1999; Blount et al., 2000) or, alternatively,
high levels of antioxidant defenses may diminish the oxidation of carotenoids
(Hartely & Kennedy, 2004).
In this study, we tested the prediction that antioxidant availability
modulates carotenoid-based coloration in a wild population of the yellow-legged
gull (Larus michahellis), a seabird in which both sexes show intense integumentary
carotenoid-based coloration in legs, eye rings, gape flanges, gape and bill spots
(Cramp & Simmons, 1983). In related gull species, red coloration reflects body
condition (Kristiansen et al., 2006) and is related to carotenoid intake (Blount et al.,
2001). We focused on the red spot area on the lower mandible because expression
of this trait is very variable throughout the reproductive period, and is enhanced
during courtship (Cramp & Simmons, 1983). Moreover, in a recent experimental
study, we found that the red bill spot is used by individuals after pairing to modify
the reproductive decisions of their mate (J. Morales et al., unpublished data). During
courtship period in established pairs, we modified the availability of non-pigmentary
antioxidant by means of a supplemental vitamin E feeding study. We predicted that
carotenoid supply may enhance the expression of the red bill spot. We also
79
measured the effect of treatment on the levels of lipid peroxidation, total
antioxidant capacity and total carotenoids in plasma. Moreover, we differentiated
the carotenoids present exclusively in the red bill spot from all other carotenoids.
We expected increases in all plasma carotenoids and such increases related to their
oxidation susceptibility (Woodall et al., 1997) if carotenoids are passively protected
(Hartely & Kennedy, 2004). In contrast, we expected an effect especially on redspot carotenoids if the antioxidant availability promotes an active allocation to the
signal.
Materials and methods
Experimental procedure
This study was carried out in 2005, in the Illas Cíes (Ría de Vigo, Galicia, NW of
Iberian Peninsula). In mid-April, during the courtship period of the yellow-legged
gull (Larus michahellis), forty nest sites in the central part of the colony were
randomly allocated either to a feeding treatment (n = 20) or to a control group (n =
20). Food supplementation was begun 10.17 ± 4.62 days (range 3-19 days) before
egg laying. The experimental pairs were fed daily with 198 mg of vitamin E (αtocopherol acetate. Sigma-Aldrich); an individual dose of 112 mg/kg of body
weight, similar to dosages used in poultry (i.e. Sahin et al., 2002; Grobas et al.,
2002). The daily amount of vitamin E supplied was within the estimated natural
range of intake (2-757 mg of vitamin E per day), estimation based on daily food
consumption of gulls (280.6 g of food; Munilla, 1997; Hunt et al., 2005) and the
amount of vitamin E in the main prey (0.2 mg/g in marine invertebrates and 2.7
mg/g in marine fish; Sikorski 1990). Indeed, the increase of Vitamin E in
experimental gulls (see results) was within the natural range reported in seabirds (i.e.
Murvoll et al., 2007). Vitamin E was mixed with vegetable oil and placed on a slice
of bread, which was hidden under vegetation and close to the nest (50 cm
approximately). This was done to prevent non-target birds eating the bread. In a
previous feeding experiment performed in the same way, the behavior of gulls
around the territory was observed and it was reported that no food item was stolen
by other birds (Pérez et al., 2006). The control group was provided with the bread
and vegetable oil, but without vitamin E. The period of supplementation prior to
Anexo 1
laying did not differ significantly between treatment groups (t16 = 0.13, P = 0.89).
Supplemental feeding was halted five days after the first egg was laid.
Between five and twenty six days after egg laying was completed (i.e. the
third egg was laid) 10 control and 8 supplemented males were trapped. Head, bill
and tarsus length were measured (to the nearest 1 mm). Body mass was also
determined (to the nearest 10 g). The tarsus length allowed confirmation of the sex
of the birds by means of a discriminant function (Bosch, 1996). The bill was
photographed against a white standard, together with a standard red color and a
millimetric scale, inside a black box, with a digital camera (Nikon Coolpix 5200).
The distance from the lens to the bill (15 cm) was held constant. The red spot area
was measured by the same person (CP) by use of image analysis software (analySIS
FIVE) blindly with respect to treatment. Repeatability of the method performed 3
times on 6 randomly selected photographs was very high (r = 0.98, F5,12 = 161.01, P
< 0.001). The intensity of color (redness) of the red spot was measured using three
pixel values in RGB color-space on the central part of the spot. A single redness
intensity value was calculated according to Pike et al. (2007). Redness is expressed as
the proportion of the R-value to the red standard color.
A blood sample (about 1.5 ml) was taken from the brachial vein, with a
25G heparinized needle. The blood was immediately transferred to plastic tubes and
maintained on ice in cool boxes (4ºC), then centrifuged in the laboratory at the end
of the day. Plasma and blood cells (pellet) were frozen separately at -80 ºC until
analysis. Eggs were measured (to the nearest 0.01 mm) to calculate egg volume
(Volume = length x width2 x 0.52, Hoyt, 1979)
Biochemical assays
The carotenoids responsible for the red spot and orange bill pigmentation were
identified by analysis of the bills of three gulls that were found dead, by highperformance liquid chromatography (HPLC). The red and orange pigmented layers
were separated from the bill keratin with a scalpel and cut into small pieces,
avoiding exposure of the samples to direct light and high temperatures. The tissue
was placed in a tube and covered with absolute ethanol. The solution was mixed on
a vortex mixer, sonicated for 15 minutes in an ultrasonic bath (BRANSON, model
81
5510) and subsequently centrifuged at 10000 rpm for 10 minutes. The supernatant
was collected in a new tube, dried under a nitrogen atmosphere and diluted again in
200 µl of methanol. The carotenoids and vitamin-E contained in the plasma
samples were also measured by HPLC. Plasma samples (50 µl) were diluted in 250
µl of absolute ethanol in tubes, avoiding exposure of the plasma to high
temperatures and direct light (Alonso-Alvarez et al., 2004). The solution was
processed in the same way as described above.
Samples (20 µl) were injected into a HPLC system (JASCO Comparison
Proven, model 1500) fitted with a SecurityGuard column and a C18 reverse-phase
analytical column (15 cm x 4.6 mm x 3 µm) (SphereClone type ODS(2),
Phenomenex). The mobile phase was methanol-milliQ water (90:10 v/v) in gradient
elution (gradient: 0-21 min 90:10 v/v, 21-25 min 100:0 v/v, 25-35 min 90:10 v/v)
and the flow rate, 1.5 ml/min. Carotenoids were determined at 445 nm with a UV
detector (JASCO Comparison Proven, model UV-1570) and quantified by use of
external standards (canthaxanthin, astaxanthin and β-carotene, Dr. Ehrenstorfer
GmbH; Lutein, Sigma-Aldrich; zeaxanthin, echinenone and β-cryptoxanthin, LGC
Promochem S.L.). The calibration curves for the carotenoids present in the samples
showed high correlation coefficients (in all cases R2 > 0.99). The concentration of
the unknown carotenoids was calculated in relation to the lutein standard. VitaminE (α-tocopherol) was simultaneously determined from the same extract with the
same column, mobile phase, gradient and flow rate but with a fluorescence detector
(JASCO Comparison Proven, model FP-1520). The excitation and emission
wavelengths used were 295 nm and 330 nm respectively. Concentrations were
calculated in relation to the vitamin-E standard (α-tocopherol, Sigma-Aldrich;
calibration curve, R2 = 0.99). Concentrations of carotenoids and vitamin-E are
expressed in µg/ml.
Lipid peroxidation was quantified with a lipid peroxidation assay kit
(Calbiochem, cat nº 437634). This method measures malondialdehyde (MDA) and
4-hydroxyalkenals (HAE), which are end products derived from peroxidation of
polyunsaturated fatty acids and related esters.
The total antioxidant capacity was measured by the method described by
Erel (2004). Basically, it consists in the use of the molecule 2,2´-azinobis-(3-
Anexo 1
ethylbenzothiazoline-6-sulfonate) (ABTS*+), which is decolorized by antioxidants
according to their concentration and antioxidant capacity. The change in color is
measured as the change in absorbance at 415 nm (BIO-RAD, microplate reader
550). The antioxidant capacity is expressed as mmol Trolox equivalent/l
Data analyses
Student’s t-tests were used for comparison between groups when data met the
assumptions of homocedasticity and normality; otherwise, Mann-Whitney U-test
was used. The effects of the vitamin E-supplementation on red spot size, spot
redness and plasma concentrations of vitamin-E and carotenoids were analyzed
using General Linear Models (GLMs). In the models the experimental treatment
was included as factor and laying date, number of days elapsed from laying to
capture, tarsus length and bill size (in the red bill spot model) as covariates. The full
models are reported, as recommended by Whittinghan et al. (2006). When it was
necessary
(plasma
carotenoids),
data
were
transformed
with
Box-Cox
transformation in order to met model requirements (normality and homocedasticity
variance structure). Data are expressed as means ± SE.
Results
Carotenoids present in bill and plasma
The HPLC analysis revealed that ten different carotenoids were present in
the orange and red bill coloration (Table 1.1), and that five of these were exclusively
present in the red bill spot (hereafter called “red spot carotenoids”; Table 1.1). Red
spot carotenoids were present in plasma of male gulls, with lutein predominating
(Table 1.1). Another two carotenoids were also found in plasma (Table 1.1,
hereafter referred to together as “other carotenoids”).
83
Table 1.1. Carotenoids present in samples of bill and plasma from yellow-legged gulls (Larus
michahellis), as determined by HPLC analysis. Carotenoids exclusively present in the red bill spot
are shown in bold type. The retention time is expressed in minutes. Plasma concentration
(mean ± standard errors) is expressed in µg ml−1.
Retention
Carotenoid
Bill1
Plasma concentration
time
Control
Vitamin E
1
Astaxanthin
6.58
R
0.25 ± 0.0
0.72 ± 0.85
Unknown 1
9.21
R
3.09 ± 0.05
3.26 ± 0.26
Unknown 2
9.60
R
3.45 ± 0.20
3.58 ± 0.35
Lutein
10.07
R
4.31 ± 0.62
6.28 ± 3.98
Zeaxanthin
10.51
R
0.75 ± 0.31
1.20 ± 0.45
Cantaxanthin
14.59
O, R
0.31 ± 0.09
0.30 ± 0.08
Unknown 3
17.30
-
3.51 ± 1.40
3.75 ± 1.75
β-Cryptoxanthin
19.70
O, R
-
-
Unknown 4
21.33
O, R
-
-
Unknown 5
22.45
O, R
-
-
β-carotene
25.14
O, R
-
-
Presence of carotenoids in the orange area (O) and red spot area (R) of the bill
Vitamin E supplementation experiment
The body size did not differ between experimental groups (tarsus length,
t16 = 1.42, P = 0.18; bill size, t16 = 0.02, P = 0.98), nor the time elapsed from laying
to capture (Z = 0.45, P = 0.65). Moreover, vitamin E supplementation did not
affect body mass (t16 = 0.65, P = 0.53), clutch size (Z = 0.41, P = 0.68) or egg
volume (t16 = 0.25, P = 0.81).
Anexo 1
Table 1.2. General Linear Models showing treatment effects with all covariates (full models)
and the model that retained only variables that caused a significant increase in deviance
(minimal adequate models)
Dependent
variable
Bill spot
size
P
F
P
treatment
23.501
6.30
0.03
7.36
0.02
laying date
0.143
0.02
0.88
0.035
0.00
0.96
DF*
1,12
18.234
8.37
0.01
4.82
0.04
tarsus
-6.421
3.96
0.07
9.91
0.01
treatment
13.602
23.45
< 0.01
20.49
< 0.001
0.116
0.15
0.70
0.447
4.12
0.06
-1.535
2.54
0.13
0.015
6.47
0.02
8.53
0.01
0.000
0.23
0.64
0.001
1.37
0.26
0.000
0.06
0.81
6.33
0.02
laying date
days until
capture
tarsus
1,13
treatment
Total
carotenoids
Red spot
carotenoids
Minimal model
F
days until
capture
bill width
Vitamin E
Full model
Parameter
estimate
Variables
laying date
days until
capture
tarsus
1,13
treatment
0.022
4.13
0.04
laying date
0.000
0.04
0.84
0.000
0.33
0.57
-0.002
0.24
0.63
days until
capture
tarsus
1,13
Significant values are represented in bold type
*DF: Degrees of freedom
Interestingly, vitamin E supplementation affected the size of the red bill
spot (Table 1.2), so that the red spot of supplemented male gulls was 9% larger than
that of controls (Figure 1.1a). The size of the red bill spot was related to the bill
width (Table 1.2). In contrast, the redness of the bill spot did not differ in the
different groups (F1,16 = 0.30, P = 0.59).
85
Figure 1.1. Effect of vitamin E supplementation on a) size of the red bill spot; b) plasma
levels of vitamin E; c) levels of total carotenoids in plasma and d) levels of total antioxidant
capacity in plasma. Values are expressed as means ± standard errors.
As expected, plasma vitamin E concentration was significantly higher in
male gulls that received the vitamin E diet than in control gulls (Table 1.2; Figure
1.1b). Vitamin E supplemented males also showed a significantly higher
concentration (22%) of plasma carotenoids than control gulls (Table 1.2; Figure
1.1c). These differences were due to red spot carotenoids, present at higher levels in
plasma from supplemented male gulls than in plasma from controls (Table 1.2;
Figure 1.2). Nevertheless, the concentrations of other carotenoids did not differ
between groups (F1,16 = 0.66, P = 0.43; Figure 1.2). The antioxidant capacity of gulls
fed the vitamin-E diet was twice that of control gulls, although the difference was
Anexo 1
not significant (F1,16 = 3.09, P = 0.10; Figure 1.1d). Plasma levels of lipid
peroxidation markers did not differ between treatments (F1,16 = 0.02, P = 0.88).
Figure 1.2. Effect of vitamin E supplementation on plasma carotenoids exclusively
associated with the red bill coloration (red spot carotenoids) and on other carotenoids.
Values are expressed as means ± standard errors.
Discussion
We found that male gulls supplied with a non-pigmentary antioxidant (vitamin E)
had higher levels of vitamin-E and total carotenoids in plasma than control males,
specifically those carotenoids responsible for the red spot coloration. Moreover, we
found that vitamin E supplemented males had larger red bill spot than control
males. Since the saturation of red spot did not differ, the results suggest that a
greater proportion of carotenoids were used to enlarge red spot area in antioxidant
supplemented males. The results provide the first evidence under field experimental
conditions supporting the hypothesis that antioxidant availability modulates
carotenoid-based coloration.
The role of oxidative stress as a mechanism mediating the expression of
sexual signals has been the focus of several studies in recent years (e.g. Moreno &
Osorno, 2003; Alonso-Alvarez et al., 2004; Kurtz et al., 2006; Torres & Velando,
87
2007). Our findings are consistent with the results of two recent experimental
studies with captive vertebrates, which showed that supplementation with nonpigmentary antioxidants enhances expression of carotenoid-based sexual colorations
in the stickleback, Gasterosteus aculeatus, (Pike et al., 2007) and in the zebra finch,
Taeniopygia guttata, (Bertrand et al., 2006). Overall, the results of these and the
present study provide experimental evidence supporting the hypothesis that
carotenoid-based coloration is an honest signal of the availability of antioxidants in
an individual (von Schantz et al., 1999; Hartley & Kennedy, 2004).
Two principal mechanisms have been proposed as underlying the honesty
of carotenoid-based coloration as a signal of the individual’s antioxidant status. The
antioxidant trade-off hypothesis suggests that only individuals with good
antioxidant defenses (or with low levels of ROS) can afford to divert carotenoids
away from the detoxification system instead of allocating them to sexual signaling
(von Schantz et al., 1999; Blount et al., 2000). Alternatively, it has been proposed
that carotenoid-based coloration is an index of non-pigmentary antioxidants
(“protection hypothesis” Hartley & Kennedy, 2004). This hypothesis is based on
the idea that carotenoid coloration is altered and destroyed by oxidation (Woodall et
al., 1997; Siems et al., 1999), and that therefore only individuals with high levels of
antioxidants can prevent carotenoid-based coloration from diminishing.
The enlargement of the red spot area caused by vitamin E
supplementation may be consistent with both hypotheses. According to the
protection hypothesis, an increase in the antioxidant defenses should protect
carotenoids from bleaching. Therefore, the protective effect of vitamin E may be
higher in those carotenoids that are more susceptible to oxidation than in other
carotenoids. We found that vitamin E supplementation increased the concentration
of plasma carotenoids responsible for the red spot coloration, but not the others.
This result would be consistent with the protection hypothesis if red spot
carotenoids were more vulnerable to oxidation than other carotenoids. However,
this is not the case, as for example, the experiment affected to a red spot carotenoid,
astaxanthin (Z=2.05, P=0.04), but not to cantaxanthin (Z=0.47, P=0.64) a
carotenoid that was not exclusive to the red spot, even though both carotenoids
present similar bleaching rates when are exposed to a free radical attack (Woodall et
al., 1997). Thus, the results of the experiment suggest that rather than passive
Anexo 1
protection of carotenoids (Hartley & Kennedy, 2004), an increase in vitamin E may
promote an active mechanism to increase the amount of carotenoids responsible
for the red spot coloration. Further experimental studies should confirm this idea.
Our findings may be consistent with the antioxidant trade-off hypothesis,
as vitamin E supplementation may have allowed the male gulls to divert specific
carotenoids from the detoxification system in order to allocate them to red
coloration (von Schantz et al., 1999; Blount et al., 2000). Indeed, in other bird
species, experimental activation of the immune system resulted in an increase of
oxidative damage, which in turn caused a parallel decrease in carotenoid-based
coloration (Alonso-Alvarez et al., 2004; Velando & Torres, 2007). Overall, these
results suggest that oxidative stress is one of the principal mechanisms underlying
the trade-off between carotenoid coloration and self-maintenance (von Schantz et
al., 1999).
It is also possible that specific carotenoid coloration is produced through
metabolic pathways that modify dietary carotenoids (Fox & Hopkins, 1966; Stradi et
al., 2001; Hill & McGraw, 2006; Hudon et al., 2007) thereby producing a release in
reactive oxygen species, such as those catalyzed by cytocrome P450 (Paolini et al.,
1999; Lewis, 2002). The cytochrome P450 reaction cycle produces different active
oxygen species, specifically superoxide and peroxide (Lewis, 2002), which can cause
cellular damage leading to oxidative stress and its toxic consequences (De Groot &
Sies, 1989; Goeptar et al., 1995). Under this scenario, only individuals with a high
antioxidant status may be able to activate oxidative pathways for carotenoid
transformation, explaining why carotenoids responsible for red colorations are more
costly to produce (Hill, 1996; Andersson et al., 2007).
In our experiment, vitamin E supplementation did not affect the levels of
lipid peroxidation products, which suggests that extra antioxidants were not used to
combat oxidation injury. This supports the idea that vitamin E supplemented birds
transformed carotenoids into those needed for red coloration, and were thus able to
afford the costs associated with carotenoid transformation. Consequently, vitamin
E supply may have been used to inactivate the ROS produced in this
transformation and to some extent explain the lack of significant differences in the
antioxidant capacity of total plasma. The oxidative cost of carotenoid
89
transformation may also fit with the observed results in previous experiments with
captive animals (Bertrand et al., 2006; Pike et al., 2007). Thus, in the experimental
study on sticklebacks, fishes received the same amount of dietary (yellow and red)
carotenoids, but red coloration increased in males fed on a high-antioxidant diet
(Pike et al., 2007). This may indicate that sticklebacks with high antioxidant
availability were able to transform dietary (from yellow to red) carotenoids. In other
study, Bertrand et al. 2006 manipulated the availability of carotenoids and of
melatonin, a colorless antioxidant, in zebra finches. They found that the
concentration of plasma carotenoids did not change in the birds that received
melatonin supplements but that these birds presented redder bills than controls
(Bertrand et al., 2006). However, antioxidant supply in zebra finches may have
affected specific red carotenoids (which were not measured) in the bill due to the
transformation of dietary carotenoids.
Although our experimental design does not allow us to unravel the
mechanism underlying the honesty of carotenoid-based coloration, the results of
our study appear to be consistent with the idea that carotenoid allocation to sexual
signals is costly and only individuals with high antioxidant status are able to afford
these costs. Further studies are needed to tease out apart the two hypotheses on the
cost of carotenoid allocation into sexual signals (the antioxidant trade-off or the
transformation hypothesis).
Acknowledgments
We are grateful to Judith Morales, Will Cresswell and two anonymous referees for
constructive comments on the manuscript. We wish to express our gratitude to the staff at
the Parque Nacional de las Islas Atlánticas de Galicia and Naviera Mar de Ons, for logistic
support, and Carmen Díez and Julio Eiroa for help in field work. A.V. was supported by a
Ramon y Cajal Fellowship (Ministerio de Educación y Ciencia, Spain). The present study was
founded by the program Plan Nacional I+D+I 2004-2007 (Ministerio de Educación y
Ciencia, Spain).
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El petróleo afecta a la mancha roja
del pico de la gaviota patiamarilla
Este anexo está basado en: Pérez, C., Lores, M., Velando, A. Carotenoids are
mobilized after exposure to heavy fuel oil, with negative consequences on bird
coloration. En revisión
Anexo 2
El petróleo afecta a la mancha roja del pico de la gaviota patiamarilla
Cristóbal Pérez, Marta Lores, Alberto Velando
Se ha sugerido que las señales dependientes de condición pueden ser una medida
útil para evaluar la calidad ambiental. En este contexto, la coloración dependiente de
carotenoides de las aves marinas puede ser especialmente valiosa para monitorear y
detectar los efectos subletales de la contaminación, como la que se produce tras los
derrames de petróleo. Sin embargo, el efecto de la ingestión de petróleo en los
ornamentos sexuales todavía no ha sido explorado. En este estudio, contrastamos la
hipótesis de que la contaminación por petróleo aumenta el estrés oxidativo, lo que
perjudica a la señal dependiente de carotenoides, en una población natural de
gaviota patiamarilla (Larus michahellis), un ave marina que posee intensas
coloraciones dependientes de carotenoides. Medimos el efecto que una exposición
al petróleo provoca en los niveles plasmáticos de varios antioxidantes no
enzimáticos. Así, analizamos los niveles plasmáticos de carotenoides, vitamina E y la
actividad antioxidante del plasma. Además, como una medida de daño oxidativo
celular analizamos la peroxidación de los lípidos plasmática. Predecimos que la
ingestión de petróleo se verá reflejada en la coloración dependiente de carotenoides
de las gaviotas, como consecuencia del estrés oxidativo. Se encontró un efecto
significativo del petróleo en la concentración plasmática de vitamina E y
carotenoides. Además como consecuencia de la ingestión de petróleo, las gaviotas
expuestas a la contaminación por hidrocarburos presentaron un menor tamaño de
la mancha roja del pico respecto de las gaviotas control. Este estudio llevado a cabo
en un ave marina en libertad, muestra por primera vez evidencias de que la señal
dependiente de carotenoides está asociada con procesos de detoxificación de
compuestos contaminantes y por lo tanto es indicativa de la calidad ambiental. Así,
la coloración dependiente de carotenoides puede ser útil para monitorear y detectar
los efectos subletales de los contaminantes a nivel de la población. Asimismo,
debido a que las gaviotas patiamarillas pertenecen a un complejo de especies
ampliamente distribuidas a lo largo del hemisferio norte, los resultados podrían ser
usados como una herramienta en las futuras evaluaciones de los efectos subletales
de los derrames de petróleo en las aves marinas.
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Anexo 2
Carotenoids are mobilized after exposure to heavy fuel oil, with negative
consequences on bird coloration
Cristóbal Pérez1, Marta Lores2 & Alberto Velando1
Departamento de Ecoloxía e Bioloxía Animal. Facultade de Bioloxía. Universidade de Vigo, As LagoasMarconsende s/n 36310 Vigo. Spain. 2Departamento de Química Analítica, Nutrición e Bromatoloxía.
Facultade de Química. Instituto de Investigacións e Análises Alimentarios. Universidade de Santiago de
Compostela, Avda. das Ciencias s/n 15782 Santiago de Compostela. Spain.
1
Abstract
It has been suggested that condition-dependent signals may be a useful measure of
environmental quality. In this context carotenoid-based coloration in seabirds may
be especially valuable for monitoring and detecting the sublethal effects of oil
pollution in the environment, as occurs after large oil-spills. However, the effect of
the ingestion of oil on sexual ornaments has not previously been explored. In this
study, we tested the hypothesis that oil pollution enhances oxidative stress and
impairs the expression of a carotenoid-based signal in a wild population of the
yellow-legged gull (Larus michahellis), a seabird with intense carotenoid-based
coloration. We measured the effect of pollution on the plasma levels of different
non-enzymatic antioxidants by analyzing carotenoids, vitamin E and plasma
antioxidant activity. In addition, we also analyzed plasma lipid peroxidation
products as a measure of oxidative cellular damage. We predict that oil ingestion
will be reflected by the carotenoid-based coloration of gulls, as a consequence of
oxidative stress. We showed a significant effect of oil on concentration of plasma
vitamin E and carotenoids. Moreover, as a consequence of oil ingestion gulls
exposured to oil pollution showed a smaller size of the red bill spot respect to
control ones. For the first time in a free-living seabird, this study provides evidence
that a carotenoid-based signal is associated with a detoxification process due to
pollution exposure and is hence indicative of environmental quality. Carotenoidbased coloration may be useful in monitoring and detecting the sublethal effects of
pollutants at the population level. Since the yellow-legged gull belongs to a complex
of species widely distributed throughout the Northern hemisphere, the results might
provide a tool for future evaluations of sublethal effects of oil spills in seabirds.
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Introduction
Many animals exhibit elaborate ornamental traits that have evolved as signals of the
bearer’s quality and can be evaluated by prospective mates or opponents (Anderson,
1994). The honesty of these traits is often based on the fact that their maintenance
entails an associated cost that only can be afforded by animals of higher quality (see
Zahavi & Zahavi, 1997). The expression of such traits thus often signals reliable
information about the physiological condition of the bearer (Hamilton & Zuk,
1982; Grafen, 1990). Sexual signals display high phenotypic plasticity, and their
expression, relative to other traits, is particularly sensitive to the cascade of
physiological mechanisms produced by stressful events (Hill, 1995; Buchanan,
2000). Accordingly, it has been suggested that condition-dependent signals may be a
useful measure of environmental quality as they represent the sum of environmental
pressures on the animal (Hill, 1995). In this context, carotenoid-based coloration
may be especially valuable for monitoring and detecting the sublethal effects of
toxic chemicals in the environment, because in many cases the mechanisms
underlying both coloration and pollutant damage are interconnected (Dauwe &
Eens, 2008).
The carotenoid-based colorations displayed by fishes and birds are considered as
good examples of honest sexual signals (e.g. Olson & Owens, 1998; Badyaev & Hill,
2000; Pike et al., 2007). Carotenoids are lipid-soluble pigments and can only be
synthesized by algae, bacteria, fungi and plants (Goodwin, 1984), and thus
vertebrates must obtain them by food. As well as being involved in the expression
of colour signals, carotenoids also have important physiological functions, and act
as immunoenhancers in the immune function and as oxygen radical scavengers in
antioxidant activity (Lozano, 1994; von Schantz et al., 1999).
Recent studies showed that the availability of colourless antioxidants affects the
expression of carotenoid-based signals (Bertrand et al., 2006; Pike et al., 2007; Pérez
et al., 2008a), indicating that oxidative stress plays a key role regulating these
colourations. Since many pollutants induce oxidative stress (Kappus, 1987), it is
expected that these pollutants will increase antioxidant demand and lead to reduced
expression of carotenoid-based coloration, which should therefore be useful in
ecotoxicological monitoring (Camplani et al., 1999; Dauwe & Eens, 2008). Indeed,
other biomarkers of oxidative stress are commonly used in environmental pollution
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monitoring (McCarthy & Shugart, 1990). Two recent experimental studies in captive
birds and fishes showed that carotenoid coloration was impaired when the animals
were exposed to pollutants (Bortolotti et al., 2003; Arellano-Aguilar & Garcia,
2008). However, carotenoid coloration is rarely used as a biomarker of pollutantmediated oxidative stress.
Here, we experimentally evaluated the effect of natural-occurring oil pollution (from
the Prestige oil spill) on oxidative stress and coloration in a free-living seabird. Large
quantities of petroleum products are released into the marine environment as a
result of tanker wrecks. Such catastrophic events have a dramatic impact on marine
ecosystems, and affect a broad range of organisms, including seabirds (e.g. Peterson,
2003). The life history characteristics of seabirds make them particularly vulnerable
to oil pollution (Peterson et al., 2003; Velando et al., 2005) because they spend
much of their lives on the ocean’s surface and because their populations
concentrate in habitats prone to high levels of exposure to oil (Clark, 1984).
Moreover, since seabirds occupy high trophic positions, important toxic effects
(due to persistent exposure to oil) are expected in these organisms (Alonso-Alvarez
et al., 2007a,b). One of the most recent examples of a large marine oil spill occurred
in November 2002 when the supertanker Prestige sank off the Galician coast (NW
Spain). The tanker spilled between 40,000 and 63,000 tonnes of heavy fuel oil into
the Atlantic Ocean, polluting coastal areas as far apart as Portugal and France. The
Prestige oil spill was the biggest catastrophe of its type in Europe and thousands of
seabirds died in the following months (Camphuysen et al., 2002; Martínez-Abraín et
al., 2006). Moreover, polycyclic aromatic hydrocarbons (PAHs), the most toxic
components present in crude oils, are being detected in the marine food chain (e.g.
Laffon et al., 2006; Ordas et al., 2007; Pérez et al., 2008b).
The acute toxicity of PAHs is mainly attributed to oxidative stress and cellular
damage associated with the metabolic response, such as cytocrome P450 catalytic
activity, by which they are eliminated from tissues (Gonzalez, 2005; Shimada, 2006;
Ramos & García, 2007). Enhanced production of Radical Oxygen Species (ROS)
due to PAH ingestion may lead to oxidative stress, possibly resulting in damage
such as mutagenesis, carcinogenesis, protein oxidation and degradation,
carbohydrate damage, and lipid peroxidation (Livingstone et al., 1990; Winston &
Di Giulio, 1991; Sole et al., 1995). Damaging effects of exposure to petroleum
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products in seabirds after oil spills are well documented (e.g. Seiser et al., 2000;
Balseiro et al., 2005; Alonso-Alvarez et al., 2007a,b). However, as far as we know,
the effect of the ingestion of oil on sexual ornaments has not previously been
explored.
In this study, we tested the hypothesis that oil pollution enhances oxidative stress
and impairs the expression of a carotenoid-based signal in a wild population of the
yellow-legged gull (Larus michahellis), a seabird species in which both sexes show
intense integumentary carotenoid-based coloration of legs, eye rings, gape and bill
spots (Cramp & Simmons, 1983). We focused on the red spot area on the lower
mandible because expression of this trait is very variable throughout the
reproductive period. In a recent experimental study, we found that brood food
provisioning by both males and females depend on the size of red bill spot of their
partner (Morales, J., Alonso-Alvarez, C., Pérez, C., Torres, R., Serafino, E. &
Velando, A. unpublished data). Furthermore, we found that the size of the red spot
depends on antioxidant availability (Pérez et al., 2008a), indicating that oxidative
stress modulates ornament expression. Thus, we predict that the oxidative stress
produced by oil ingestion may divert the carotenoids from their pigmentary
function to the antioxidant function. To test this idea, we measured the effect of
pollution on the plasma levels of different non-enzymatic antioxidants by analyzing
carotenoids, vitamin E and plasma antioxidant activity. In addition, we also analyzed
plasma lipid peroxidation products as a measure of oxidative cellular damage.
Finally, we predict that oil ingestion will be reflected by the carotenoid-based
coloration of gulls, as a consequence of oxidative stress.
Material and methods
The fieldwork was carried out on a breeding colony of yellow-legged gulls (Larus
michahellis) on the Illas Cíes (Ría de Vigo, Galicia, NW Iberian Peninsula). At the end
of April 2005, during the gulls’ courtship period, we randomly allocated 36 breeding
pairs to the experiment: 16 were fed oil (oil-supplemented group) and 20 were
treated as controls (control group). The experiment was designed to avoid
unnecessary harm to animals while still eliciting a measurable response. Thus, the
number of experimental subjects was kept as low as possible (Dawkins & Gosling,
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1996) and the amount of oil used was well below the dosage used in previous
experiments (e.g. Butler & Lukasiewicz, 1979; Leighton, 1991). The oilsupplemented group was restricted to 16 pairs, which were fed daily (from April
26), with 0.04 mL of Prestige oil (kindly provided by the Instituto Español de
Oceanografía) mixed with vegetable oil and place on a slice of bread, during seven
consecutive days (0.3 mL in total per pair; individual daily PAHs dose: 59.15 µg),
and from May 3 until the end of egg-laying, gulls were fed with bread and vegetable
oil. Pairs from the control group were fed in a similar manner but without fuel oil
(for more details about food supplementation see Pérez et al., 2005; Pérez et al.,
2008b). Food supplementation was begun 10.41 ± 1.06 days (range 2-22 days)
before egg laying. The period of supplementation prior to laying did not differ
significantly between treatment groups (t27 = 0.08, P = 0.94). Supplemental feeding
was halted five days after the first egg was laid and between one and twenty one
days after egg laying was complete (i.e. after the third egg was laid). Twenty control
and twelve oil-supplemented gulls were trapped in the nest (one gull per pair).
Three birds sampled during harsh weather conditions were excluded in the present
study because no spot size was estimated and the small blood samples were only
used to estimate PAH concentration (Pérez et al., 2008a). Thus, in the present
study, nineteen control (8 females and 11 males) and ten oil-supplemented (5
females and 5 males) gulls were included in the analyses. The time between the end
of supplemental feeding and the capture of gulls did not differ between
experimental groups (t27 = 0.05, P = 0.96). Head, bill width (measured in the
broader area of the lower mandible) and tarsus length were measured (to the nearest
1 mm). Body mass was also determined (to the nearest 10 g). The tarsus length
allowed confirmation of the sex of the birds by means of a discriminant function
(Bosch, 1996). The bill was photographed against a white standard, together with a
red standard and a millimetric scale, inside a black box, with a digital camera (Nikon
Coolpix 5200). The distance from the lens to the bill (15 cm) was maintained
constant. The red spot area was measured by the same person (CP) by use of image
analysis software (analySIS FIVE) and blindly with respect to treatment. The
repeatability of the method performed 3 times on 6 randomly selected photographs
was very high (r = 0.98, F5,12 = 161.01, P < 0.001). The intensity of colour (redness)
of the red spot was measured as three pixel values in RGB colour space in the
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Anexo 2
central part of the spot. A single redness intensity value was calculated according to
Pike et al., (2007).
A blood sample (about 1.5 mL) was taken from the brachial vein, with a
25G heparinized needle. The blood was immediately transferred to plastic tubes and
maintained on ice in cool boxes (4ºC), then centrifuged in the laboratory at the end
of the day. Plasma and blood cells (pellet) were frozen separately at -80 ºC until
analysis.
Biochemical assays
Blood cells were analyzed to determine and quantify haematological levels of PAHs
from oil spilled by the Prestige. PAH levels were determined by high performance
liquid chromatography (HPLC) coupled to a wavelength programmable
fluorescence detector. Samples (100 µL) were injected into a HPLC system fitted
with a Waters PAH analytical column (250 mm x 4.6 mm x 5µm). The mobile phase
was acetonitrile:water in gradient elution and at a flow rate of 1.2 mL/min (see
Pérez et al., 2008b). The PAHs analysed were acenaphthene, anthracene,
benz(a)anthracene, benzo(a)pyrene, benzo(b+j)fluoranthene, benzo(g,h,i)perylene,
benzo(k)fluoranthene, chrysene, dibenz(a,h)anthracene, fluorene, fluoranthene,
indeno(1,2,3-c-d)pyrene, naphthalene, phenanthrene and pyrene. To estimate the
individual degree of oil contamination, the sum of concentrations from all these
hydrocarbons was used as a variable (thereafter PAH levels).
The carotenoids and vitamin E contained in the plasma samples were also
measured by high-performance liquid chromatography (HPLC). Plasma samples (50
µL) were diluted in 250 µL of absolute ethanol (Alonso-Alvarez et al., 2004) in
tubes protected from high temperatures and direct light. The solution was mixed on
a vortex mixer and subsequently centrifuged at 10000 rpm for 10 minutes. The
supernatant was collected in a new tube, dried under a nitrogen atmosphere and
diluted again in 200 µL of methanol. Samples (20 µL) were injected into a HPLC
system (JASCO Comparison Proven, model 1500), fitted with a SecurityGuard
column and a C18 reverse-phase analytical column (15 cm x 4.6 mm x 3 µm)
(SphereClone type ODS (2), Phenomenex). The mobile phase was methanol-milliQ
water (90:10 v/v) in gradient elution (gradient: 0-21 min 90:10 v/v, 21-25 min 100:0
v/v, 25-35 min 90:10 v/v) and the flow rate, 1.5 mL/min. Carotenoids were
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Anexo 2
determined at 445 nm with a UV detector (JASCO Comparison Proven, model UV1570) and quantified by use of external standards (canthaxanthin, astaxanthin and βcarotene, Dr. Ehrenstorfer GmbH; Lutein, Sigma-Aldrich; zeaxanthin, echinenone
and β-cryptoxanthin, LGC Promochem S.L.). The calibration curves for the
carotenoids present in the samples revealed high correlation coefficients (in all cases
R2 > 0.99). The concentration of the unknown carotenoids was calculated in
relation to a lutein standard. Vitamin E (α-tocopherol) was simultaneously
determined from the same extract with the same column, mobile phase, gradient
and flow rate but with a fluorescence detector (JASCO Comparison Proven, model
FP-1520). The excitation and emission wavelengths used were 295 nm and 330 nm
respectively. Concentrations were calculated in relation to the vitamin E standard
(α-tocopherol, Sigma-Aldrich; calibration curve, R2 = 0.99). Concentrations of
carotenoids and vitamin-E were expressed in µg/mL. As we have previously
identified the carotenoids present exclusively in the red bill spot (astaxanthin, lutein,
zeaxanthin and two unidentified carotenoids, see Pérez et al., 2008a), we calculated
their concentrations in plasma (thereafter called “plasma red carotenoids”).
The plasma antioxidant activity was measured by the method described by
Erel (2004). Basically, it consists of the use of the molecule 2,2´-azinobis-(3ethylbenzothiazoline-6-sulphonate) (ABTS*+), which is decolorized by antioxidants
according to their concentration and antioxidant capacity. The change in colour is
measured as the change in absorbance at 415 nm (BIO-RAD, microplate reader
550). We used “plasma antioxidant activity” instead of the common term “total
antioxidant capacity” because, rather than total antioxidants, this method quantifies
only the reaction of antioxidants present in the aqueous phase of the plasma (Miller
et al., 1995; Young, 2001; Prior et al., 2003). Lipid peroxidation was quantified with
a lipid peroxidation assay kit (Calbiochem, cat nº 437634). This method measures
malondialdehyde (MDA) and 4-hydroxyalkenals (HAE), which are end products
derived from peroxidation of polyunsaturated fatty acids and related esters.
Data analyses
The effects of the oil-supplementation on red spot size, intensity of red spot, blood
levels of PAHs, and plasma concentrations of vitamin E, carotenoids, plasma
antioxidant activity and end products derived from the lipid peroxidation were
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Anexo 2
analyzed with General Linear Models (GLMs). In the models the experimental
treatment and sex were included as factors and body mass, bill width (in the red bill
spot model), blood levels of PAHs, luminosity and R-value of red standard (in the
bill spot redness model), as covariates. Non significant terms (P value > 0.05), were
backward dropped by a stepwise elimination procedure. Moreover, the full models
are reported, as recommended (Whittinghan et al., 2006). Additionally, we explored
the effect of carotenoids on the models by including them as covariates. Data are
expressed as means ± standard errors. Sample sizes varied somewhat among
statistical analysis as plasma volumes were not always sufficient for all biochemical
analysis.
Results
Oil supplementation did not affect body mass (F1,26 = 0.06, P = 0.81; Sex F1,26 =
28.29, P < 0.001), nor did it affect laying date (t27 = 0.08, P = 0.94), clutch size (Z =
0.38, P = 0.70) or egg volume (t27 = 0.97, P = 0.34). The oil supplemented gulls
showed a raise in the bloods levels of PAHs (17%), respect to control gulls but this
effect was not significant in the present data set (F1,25 = 1.51, P = 0.23; note that the
effect of oil ingestion was significant when the whole data set was analysed: P
=0.036; Pérez et al., 2008a).
Antioxidants, carotenoids, and oxidative damage
The concentration of plasma vitamin E was significantly higher (31%) in gulls that
received the oil diet than in control gulls (Table 2.1, Fig. 2.1a). Plasma antioxidant
activity was higher (43%, Fig. 2.1c) in gulls fed with Prestige oil than in control gulls,
but the difference was not significant (minimal model, F1,21 =0.78, P = 0.39).
Nevertheless, there were sex-related differences in the relationship between plasma
antioxidant activity and blood levels of PAHs (Table 2.1). Thus, in females,
antioxidant activity was negatively related to blood levels of PAHs, but in males
such a relationship was positive (Table 2.1, Fig. 2.2). Moreover, plasma levels of
vitamin E were negatively correlated to carotenoids levels (parameter estimate -0.44,
F1,25 = 4.95, P = 0.03), but plasma antioxidant activity was not associated to
carotenoids (F1,21 = 2.64, P = 0.12).
Total plasma carotenoids were affected by the treatment; thus oilsupplemented gulls showed higher levels (27%) than controls (Table 2.1, Fig. 2.1b):
108
Anexo 2
Moreover, plasma from female gulls showed higher concentrations of carotenoids
than plasma from males (Table 2.1). In addition, plasma carotenoids were negatively
related to the blood levels of PAHs (Fig. 2.3). Similar results were achieved when
the analysis was repeated by separating the carotenoids into those involved in the
red signal (see Pérez et al., 2008b) and the others (Table 2.1). However, the
interaction between sex and blood levels of PAH had a significant effect on plasma
levels of red spot carotenoids (Table 2.1). Thus, the negative relationship between
red carotenoids and PAH blood levels was only observed in females (Fig. 2.4).
Interestingly, the significant effect of the treatment on carotenoids disappeared
when PHA levels were removed from the model (P = 0.09).
109
Anexo 2
Table 2.1. General Linear Models showing treatment effects with all covariates (full models)
and the model that retained only variables that caused a significant increase in deviance
(minimal adequate models).
Dependent
variable
Vitamin E
Antioxidant
activity
Total
carotenoids
Red
carotenoids
Other
carotenoids
Lipid
peroxidation
Bill spot
size
Bill spot
redness
1Degree
110
Variables
Intercept
treatment (control)
sex (female)
weight
TPAHs
sex*treatment
TPAHs *treatment
TPAHs *sex
Intercept
treatment (control)
sex (female)
weight
TPAHs
sex*treatment
TPAHs*treatment
TPAHs *sex
Intercept
treatment (control)
sex (female)
weight
TPAHs
sex*treatment
TPAHs *treatment
TPAHs *sex
Intercept
treatment (control)
sex (female)
weight
TPAHs
sex*treatment
TPAHs *treatment
TPAHs *sex
Intercept
treatment (control)
sex (female)
weight
TPAHs
sex*treatment
TPAHs *treatment
TPAHs *sex
Intercept
treatment (control)
sex (female)
weight
TPAHs
sex*treatment
TPAHs *treatment
TPAHs *sex
Intercept
treatment (control)
sex (female)
bill depth
TPAHs
sex*treatment
TPAHs *treatment
TPAHs *sex
Intercept
treatment (control)
sex (female)
bill depth
R-red standard
Luminosity
TPAHs
sex*treatment
TPAHs *treatment
TPAHs *sex
of freedom. 2Parameter estimate
Full model
1Df
1,18
1,18
1,18
1,18
1,18
1,18
1,19
1,17
2Pe
24.13
-1.29
-8.82
-0.01
-0.02
1.18
-0.05
0.07
-0.71
-0.74
2.25
0.01
0.01
-0.30
0.01
-0.03
15.50
-5.53
10.05
-0.01
-0.03
-0.05
0.04
-0.09
11.58
-2.53
7.92
0.01
-0.01
0.74
0.01
-0.07
2.99
1.41
1.69
0.01
0.02
0.94
0.02
0.02
42.20
58.69
-4.89
0.15
0.29
16.00
-1.40
0.31
-253.89
34.34
68.82
19.54
0.64
5.39
-0.32
-0.72
1.13
0.01
-0.03
-0.01
0.36
-0.01
-0.01
-0.04
0.01
0.01
F
P
0.02
1.95
0.26
0.03
0.11
0.43
0.86
0.89
0.18
0.61
0.87
0.75
0.52
0.37
1.10
4.84
0.45
0.29
0.26
0.66
7.26
0.31
0.04
0.51
0.59
0.61
0.43
0.01
3.12
8.08
0.08
6.93
0.01
0.76
3.73
0.94
0.01
0.78
0.02
0.98
0.40
0.07
3.29
38.22
0.04
21.77
0.81
0.05
16.57
0.37
<0.01
0.92
0.03
0.65
0.91
0.05
6.28
1.32
0.24
4.00
0.72
3.32
0.81
0.02
0.27
0.63
0.06
0.41
0.08
0.38
1.48
0.01
2.60
0.42
0.18
3.11
0.16
0.24
0.96
0.12
0.53
0.68
0.09
0.69
3.00
7.22
7.61
0.54
0.11
1.03
5.35
0.10
0.15
0.13
0.47
0.74
0.32
0.03
0.03
0.50
0.22
9.04
5.41
0.39
0.72
0.02
0.29
0.85
0.49
0.64
0.01
0.03
0.54
0.41
0.90
0.59
Minimal model
F
P
4.82
0.04
1.65
4.73
0.04
0.02
0.27
0.61
-0.03
15.70
-3.00
4.79
6.94
0.01
7.29
20.38
0.01
<0.01
-0.06
8.28
<0.01
10.70
-1.92
8.69
6.48
18.33
0.02
<0.01
-0.01
6.80
0.02
-0.07
2.89
-1.11
6.07
0.02
5.02
0.03
-0.02
4.88
0.04
-18.84
22.08
6.23
0.02
9.24
7.26
0.01
0.48
-0.01
37.37
12.76
<0.01
<0.01
2Pe
16.85
-3.72
0.01
Anexo 2
Figure 2.1. Effect of oil supplementation on levels of plasma: a) vitamin E; b)
total carotenoids; c) antioxidant activity; d) levels of lipid peroxidation products.
Values are expressed as means ± standard errors.
Figure 2.2. Relationship between plasma antioxidant activity and blood levels of
total PAH (sum of 15 compounds; see methods) in male gulls (filled dots and solid
line; R2 = 0.29) and female gulls (open dots and dashed line; R2 = 0.18).
111
Anexo 2
Lastly, the plasma levels of lipid peroxidation markers were higher in gulls
fed with Prestige oil than in control gulls (13%; Figure 2.1d), although the
difference was not significant (minimal model, F1,26 = 1.75, P = 0.20). Lipid
peroxidation markers were also not affected by the sex of gulls or blood levels of
PAHs (minimal model, P > 0.2).
Figure 2.3. Relationship between the blood levels of total PAHs (sum of 15
compounds; see methods) and the plasma levels of carotenoids for control gulls (open
dots and dashed line; R2 = 0.07) and oil-supplemented gulls (filled dot and solid line;
R2 = 0.24).
112
Anexo 2
Figure 2.4. Relationship between the blood levels of total PAHs (sum of 15 compounds;
see methods) and the plasma levels of red spot carotenoids for male (filled dots and solid
line; R2 = 0.01) and female gulls (open dots and dashed line; R2 = 0.51).
Carotenoid-based signal
As expected, oil supplementation affected the size of the red bill spot (Table 2.1):
the red spot of oil supplemented gulls was 16% smaller than that of controls gulls
(Fig. 2.5). The blood levels of PAHs did not affect the size of the bill spot (minimal
model, Table 2.1). Nevertheless, in the full model, there was a significant interaction
between PAH levels and spot size (Table 2.1). Thus, females with higher blood
levels of PAHs showed smaller red spot (R2 = 0.24), but this effect was not found
in males (R2 = 0.09). The redness of the bill spot did not differ between groups
(minimal model, F1,24 = 0.23, P = 0.64). Moreover, males and females did not show
differences either in the size of bill spot or in the redness of the spot (P > 0.2 in all
cases). Interestingly, the blood levels of PAHs did not affect the size and the
redness of the bill spot (P > 0.5 in both cases). Redness or size of the bill spot did
not correlate with plasma levels of carotenoids (P > 0.2 in both cases).
113
Anexo 2
Figure 2.5. Effect of oil fed supplementation on size of red bill spot in yellow-legged
gulls. Values are expressed as means ± standard errors
Discussion
In this study, we showed that the plasma levels of vitamin E and carotenoids were
higher in gulls experimentally exposed to oil pollution (ingestion of Prestige oil) than
in control gulls. In addition, as expected, the mobilization of the plasma carotenoids
provoked a reduction in the expression of a carotenoid-based signal. For the first
time in a free-living seabird, this study provides evidence that a carotenoid-based
signal is associated with a detoxification process due to pollution exposure and is
hence indicative of environmental quality.
The toxicity of PAHs, the most toxic compounds in crude oils, is
associated with the adaptive response through which they are eliminated from
tissues. Thus, it is widely held that much of the acute toxicity of PAHs is due to
oxidative stress and cellular damage arising from cytochrome P450 catalytic activity.
Thus, PAHs are absorbed through the intestine and then transported via the blood
to the liver, where they are transformed into polar compounds by the microsomal
mixed function oxidase system (MFO), especially the cytochrome P450
monooxygenase, in order to be easily excreted (Meador et al., 1995; Ramos &
García, 2007). The cytochrome P450 reaction cycle produces different reactive
114
Anexo 2
oxygen species (ROS), specifically superoxide and hydrogen peroxide (Lewis, 2002),
which the organism will counteract by activating the antioxidant systems (Matés,
2000; Nordberg & Arnér, 2001). Antioxidant systems act to prevent oxidative
damage by eliminating ROS and they may be induced as an adaptive response after
exposure to PAHs, allowing an organism to partially or totally overcome oxidative
stress in a polluted environment (Di Giulio et al., 1989; Winston & Di Giulio,
1991). For this reason, induction of antioxidant defence components have been
proposed as biomarkers that provide an early warning of oil exposure (Cossu et al.,
1997; Cheung et al., 2001).
In the present study, the gulls that were exposed to crude oil mobilized
antioxidants, probably to counteract petroleum-hydrocarbon-induced free radical
toxicity. Thus, we found that oiled gulls showed higher levels of vitamin E and
carotenoids than control gulls. Lipid peroxidation products did not differ between
experimental groups, suggesting that the mobilization of antioxidants allowed gulls
to overcome -to some extent- oxidative stress promoted by oil ingestion. Vitamin E
is a major lipid-soluble antioxidant (Halliwell & Gutteridge, 1999; Surai, 2002)
involved in detoxification processes (see Murvoll et al., 2005) and is the most
important antioxidant agent protecting polyunsaturated fatty acids against lipid
peroxidation (e.g. İnal et al., 1999; Mateo et al., 2003). The increase in enzymatic
antioxidant defences after acute exposure to oil pollution has been well documented
in animals, and interpreted as an adaptive response to overcome oxidative stress
(e.g. Achuba & Osakwe, 2003; Reid & MacFarlane, 2003). Our study highlights for
first time that this mobilization also occurs on non enzymatic antioxidants.
Interestingly, although plasma antioxidant activity also increased in oiled gulls, the
experimental effect was not significant. This assay measures the overall capability of
the hydrosoluble fraction of the plasma to absorb different species of ROS (Miller
et al., 1995; Young, 2001; Prior et al., 2003). Therefore, the results suggest that
ingestion of oil in gulls increased the transportation of lipid soluble antioxidants
(Vitamin E and carotenoids) to damaged tissues, rather than enhancing antioxidant
protection of the hydrophilic fraction of plasma. Nevertheless, male and female
gulls showed a different plasma antioxidant activity in relation to the blood levels of
PAHs, suggestive of a sex-specific strategy to combat the PAHs (see below).
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Anexo 2
In birds, carotenoids are mobilized under stressful conditions (Eraud et al.,
2007). Accordingly, we found that plasma carotenoids were enhanced by oil
ingestion, which suggests an adaptive response to overcome the oxidative stress
promoted by acute exposure to heavy fuel oil. Indeed, in oil-supplemented gulls,
those birds with higher levels of plasma carotenoids showed reduced blood levels of
PAHs, suggesting that carotenoids improved PAH degradation. The ROS produced
by PAH degradation may be counteracted by the mobilization of carotenoids to
balance oxidative stress (see Krinsky et al., 2003; Rao & Rao, 2007). For instance,
oxidative damage to DNA is reduced when the total concentration of carotenoids
in plasma is high (e.g. Zhao et al., 2006; Thomson et al., 2008). Nevertheless, in
birds, the role of carotenoids as free radical scavengers remains controversial
(Hartley & Kennedy 2004; Costantini & Møller 2008), and it has been suggested
that antioxidant activity is not the main biological role for carotenoids in birds
(Hartley & Kennedy, 2004). However, in gulls, experimental carotenoid
supplementation reduces the susceptibility of eggs to lipid peroxidation and
increases the antioxidant capacity of adult birds (Blount et al., 2002a,b).
Alternatively (or additionally), the carotenoids are modulators of the immune
response (see Blount et al., 2003; McGraw & Ardia, 2003; Velando et al., 2006).
Thus, the increase in plasma levels of carotenoids in oiled gulls may also be a
mechanism to counteract the well documented immunodepressive effects of PAHs
(White et al., 1994). In any case, the present results suggest that the organism
mobilized carotenoids to overcome the harmful effects of PAHs ingestion,
highlighting the fact that carotenoids may play an important role in the
detoxification process of the oil pollution. Although, oil ingestion increased both
vitamin E and carotenoids, there was a negative relationship between plasma levels
of both compounds, indicating that birds mobilized differentially both compounds.
In oiled gulls, mobilization of the plasma carotenoids had negative
consequences on the size of the red spot. These findings suggest that carotenoids
are probably a limiting resource under oil exposure and that they are prioritized for
oil detoxification at the expense of carotenoid-based coloration. Plasma levels of
carotenoids did not correlate with red spot size, indicating that carotenoids were not
exclusively used to its pigmentary function. In a previous experiment, we found that
red coloration is affected by the availability of colourless antioxidant (Pérez et al.,
116
Anexo 2
2008a). It has been suggested that carotenoid-based coloration indicates the
antioxidant capacity because colourless antioxidants mitigate the oxidative
decolouration of carotenoids, making them available for sexual signalling (Hartley &
Kennedy, 2004). Nevertheless, yellow-legged gulls supplemented with vitamin E
mobilized only those plasma carotenoids involved in the red coloration, irrespective
of their bleaching rates (Pérez et al., 2008a), this suggest that an increase in
antioxidants may promote an active mechanism to increase the amount of specific
carotenoids rather than a passive protection of carotenoids (Hartley & Kennedy,
2004). These findings and the enhancement of plasma carotenoids followed by a
reduction of gull coloration in an oxidative stress scenario (oil ingestion) observed
in the present study are consistent with the hypothesis that there is a trade-off
between allocation of carotenoids to the sexual signal and to physiological functions
(von Schantz et al., 1999). Importantly, plasma levels of carotenoids increased under
low (Pérez et al., 2008a) and high (present study) oxidative stress scenarios. Thus,
high levels of carotenoids (or antioxidants), in plasma should be not interpreted as
indicative of low levels of oxidative stress.
Oil ingestion had similar effects on the red spot size of males and females.
Nevertheless, we found sex-related differences in the relationship between PHA
levels and antioxidant activity or carotenoids. In females these relationships were
negative, suggesting that females use the antioxidant activity and carotenoids
(specifically the red spot carotenoids), to combat the PAHs ingestion. This could
also explain the negative relationship between PHA levels and red-spot size found
in females. This effect was not found in males (although the interaction was only
significant in the full model). These results may be explained by sex-related tradeoffs between antioxidants and their function (i.e. egg yolk transfer or coloration), to
the different role that sex-specific hormones (androgens) play in the regulation of
circulating carotenoids (see Blas et al., 2006), to sex-related differences in feeding
habits described in gulls (Pons, 1994), or to sex-specific strategies to combat PAHs.
Females were captured just after laying, thus the availability of antioxidant defences
and carotenoids could be reduced after laying effort (Blount et al., 2002b; Morales
et al., 2009). Indeed, it has been suggested that females coloration is limited by
carotenoid allocation to fecundity (Morales et al., 2009). In the yellow legged gulls
after the Prestige oil spill, breeding females, but no males, showed high enzymatic
117
Anexo 2
activity of gamma-glutamyl transferase (i. e. related to oxidative damage) in oiled
colonies, effect that was attributed to female laying effort (Alonso-Alvarez et al.,
2007a). Overall, our results suggest that females are facing a higher trade-off
between colouration and PHA degradation. Thus, female coloration could be more
‘informative’ of oil pollution. Here, we did not detect this effect, but further studies
could confirm this idea.
The feeding ecology of yellow-legged gulls make them susceptible to
continued exposure to remnant oil (Alonso-Alvarez et al., 2007a), because they
frequently occur and feed on coastal and near-shore environments, such as the areas
that received much of the oil spilled from the Prestige (Pérez et al., 2008b). We
previously found that yellow-legged gulls breeding in colonies affected by the oil
spill were exposed to residual Prestige oil (Pérez et al., 2008b), with important longterm sub-lethal effects (Alonso-Alvarez et al., 2007a,b). Here we experimentally
demonstrate that yellow-legged gulls exposed to the Prestige heavy fuel oil (in feed)
showed reduced coloration. Because carotenoid-based traits have evolved for social
reasons, the disruption by alteration after exposure to oil pollution of this signal
may have significant consequences in the reproductive output. In yellow-legged
gulls, the red spot size is a mutually selected sexual signal; thus, in a previous study,
we found that food provisioning by both parents depend on the size of red bill spot
of their partner (Morales, J., Pérez, C., Alonso-Alvarez, C., Torres, R., Serafino, E.
& Velando, A. unpublished data). Moreover, since red spot at the bill of gulls elicit
chick begging behaviour (Tinbergen & Perdeck, 1950), a reduced spot could also
influence chick growth. Thus, impaired coloration may have important reproductive
consequences. There is a risk of underestimating the impact of contamination by
overlooking the behavioural consequences of chronic exposure. Therefore,
integrating studies of biochemical, physiological, ecological and behavioural
approaches are imperative when quantifying the real impact of pollution on wildlife
(Zala & Penn, 2004).
The present study provides experimental evidence regarding the plasticity
of carotenoid-based colour integuments in response to toxic chemicals in the
environment, a long-standing hypothesis (Hill, 1995). Our results are consistent
with those of previous studies in captive vertebrates in which pollutants were
mirrored in carotenoid-based signals (Bortolotti et al., 2003, Arellano-Aguilar &
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Anexo 2
Garcia, 2008). Since mechanisms underlying both coloration and pollutant damage
are probably interconnected (Dauwe & Eens, 2008), carotenoid-based coloration
may be useful in monitoring and detecting the sublethal effects of pollutants at the
population level.
Our study gives support to the use of seabirds as biomonitors of oil toxic
effects in a non destructive manner. The inclusion of seabird coloration of breeding
sabirds in routine monitoring programs is therefore promising. Carotenoid
coloration in breeding seabirds will be indicative of the health of the population
monitored, since it probably represents the sum of all environmental pressures on
the animal (Hill, 1995). The existence of baseline data on seabird coloration, blood
PHA levels (Pérez et al., 2008a) and seabird diet will allow the use of temporal and
spatial replicated design (known as before-after-control-impact BACI) to
disentangle complex indirect effects (see Velando et al., 2005). Thus, for example,
by comparing carotenoid-coloration before and after a spillage event it is possible to
distinguish between pollution effects and natural and spatial variation. Since the
yellow-legged gull belongs to a complex of species widely distributed throughout
the Northern hemisphere (e.g. Liebers et al., 2001, 2004), the results provide a tool
for future evaluations of short- and long-term effects of oil pollution, with little
disturbance, in seabirds.
Acknowledgments
We are grateful to the staff at the Parque Nacional de las Islas Atlánticas de Galicia and
Naviera Mar de Ons, for logistic support, and Carmen Díez and Julio Eiroa for help in the
field work. A.V. was supported by a Ramon y Cajal Fellowship (Ministerio de Educación y
Ciencia, Spain). The present study was funded by the program Plan Nacional I+D+I 20042007 (Ministerio de Educación y Ciencia, Spain).
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Evaluación de la exposición de las
gaviotas al vertido de petróleo del Prestige
mediante el análisis sanguíneo de los
hidrocarburos policíclicos aromáticos
Pérez, C., Velando, A., Munilla, I., López-Alonso, M., Oro, D. 2008. Monitoring
Polycyclic Aromatic Hydrocarbon pollution in the marine environment after
the Prestige oil spill by means of seabird blood analysis. Environmental
Science & Technology, 42, 707-713.
Anexo 3
Evaluación de la exposición de las gaviotas al vertido de petróleo del
Prestige mediante el análisis sanguíneo de los hidrocarburos policíclicos
aromáticos
Cristóbal Pérez, Alberto Velando, Ignacio Munilla, Marta López-Alonso, Daniel Oro
En este estudio contrastamos el uso de la sangre de un ave marina como
bioindicador de contaminación por hidrocarburos policíclicos aromáticos (HPAs)
en el medio marino. Los análisis sanguíneos realizados durante la época
reproductiva en las gaviotas patiamarillas (Larus michahellis) señalaron cambios
espaciales y temporales de contaminación coherentes con el pulso masivo de
contaminación de petróleo tras la marea negra procedente del Prestige. Así en el año
2004, la concentración sanguínea de hidrocarburos en gaviotas procedentes de
colonias afectadas por el derrame de petróleo fue el doble de las concentraciones
encontradas en las gaviotas de colonias no afectadas. Además, los niveles de los
HPAs totales en sangre de las gaviotas disminuyeron cerca de un tercio durante dos
estaciones reproductivas consecutivas (2004 y 2005) en una colonia afectada.
Asimismo, mediante un experimento de campo en el cual a las gaviotas se les
administró petróleo en la dieta, se encontró que la concentración total de los HPAs
procedentes de las gaviotas suplementadas con petróleo fue un 30% mayor que en
las gaviotas control. Esto sugiere que la concentración de los HPAs totales en la
sangre es sensible a la ingestión de pequeñas cantidades de petróleo. En resumen,
nuestro estudio muestra tanto evidencias sobre los patrones espaciales y temporales
de la contaminación por petróleo en los ecosistemas marinos después del vertido de
petróleo del Prestige, como evidencias del uso de una forma no destructiva de las
aves marinas como bioindicadores de contaminación por petróleo en el medio
marino.
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Monitoring PAH pollution in the marine environment after the Prestige oilspill by means of seabird blood analysis
Cristóbal Pérez1, Alberto Velando1, Ignacio Munilla1, Marta López-Alonso2, Daniel Oro3
Departamento de Ecoloxía e Bioloxía Animal, Facultade de Ciencias, Campus Lagoas-Marconsende,
Universidade de Vigo, 36310 Vigo, Spain. 2Departamento de Patoloxía Animal. Facultade de Veterinaria.
Universidade de Santiago de Compostela. 27002 Lugo, Spain. 3IMEDEA (CSIC-UIB), C/Miquel
Marqués 21, 07190, Esporles, Majorca, Spain.
1
Abstract
In this study we tested the use of seabird blood as a bioindicator of polycyclic
aromatic hydrocarbon (PAH) pollution in the marine environment. Blood cells of
breeding yellow-legged gulls (Larus michahellis) were able to track spatial and
temporal changes consistent with the massive oil pollution pulse that resulted from
the Prestige oil spill. Thus, in 2004, blood samples from yellow-legged gulls breeding
in colonies that were in the trajectory of the spill doubled in their total PAH
concentrations when compared to samples from unoiled colonies. Furthermore,
PAH levels in gulls from an oiled colony decreased by nearly a third in two
consecutive breeding seasons (2004 and 2005). Experimental evidence was gathered
by means of an oil-ingestion field experiment. The total concentration of PAHs in
the blood of gulls given oil supplements was 30% higher compared to controls.
This strongly suggested that measures of PAHs in the blood of gulls are sensitive to
the ingestion of small quantities of oil. Our study provide evidence that seabirds
were exposed to residual Prestige oil 17 months after the spill commenced and gives
support to the non destructive use of seabirds as biomonitors of oil pollution in
marine environments.
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are globally distributed environmental
contaminants which attract considerable concern because of their known toxic and
bioaccumulative effects in animals (Moore et al., 1989; Meador et al., 1995). In
humans, health risks associated to PAH exposure include cancer (Baars, 2002) and
DNA damage (Laffon et al., 2006). The major sources affecting the presence and
distribution of PAHs in the environment are anthropogenic (van Metre et al., 2002).
In the marine environment, these include large oil spills from tankers, oil discharges
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by all kinds of ships and activities associated with offshore oil and gas exploration
and production (Wells, 2001).
Immediate negative impacts are expected from oil pollution in coastal and
offshore environments through acute mortality of marine organisms directly
exposed to oil (Paine et al., 1996; daSilva et al., 1997). For example, lethal shortterm effects of large oil spills often involve substantial seabird losses (Votier et al.,
2005). Nonetheless, marine organisms can also become affected to the long-term
exposure of the persistent and bioaccumulative components of oil via several
indirect processes mediated through the ecosystem (Broman et al., 1990; Meador et
al., 1995). Direct effects immediately following an oil spill typically attract the
greatest public and scientific concern (Salomone, 2002; Paine et al., 1996). In
contrast, sublethal effects due to chronic oil exposure have rarely been explored
(some exceptions: Esler et al. 2002; Alonso-Alvarez et al., 2007). Such research is
more costly to conduct because it involves longer time frames and requires
evaluation of multiple mechanisms of potential impact to biological systems
(Peterson et al., 2003).
Petroleum products are toxic to seabirds (Leighton, 1991). Life history
characteristics of seabirds make them particularly vulnerable to oil pollution
(Peterson et al., 2003) because they spend much of their lives on the ocean’s
surface, and because their populations concentrate in habitats prone to high oil
exposure (Clark, 1984). Moreover, because seabirds are placed in high trophic
positions, they are likely to be good candidates to monitor the marine ecosystem
(Clark, 1984). In fact, seabirds also been used to follow polluting agents as heavy
metals and organochlorines (Arcos et al., 2002; Braune, 2007). Nevertheless, very
few studies have monitored PAH concentrations in bird tissues; in these studies the
approaches mainly used are based upon the examination of birds either found dead
or sacrificed (Broman et al., 1990; Kayall & Connell, 1995; Custer et al., 2000; Troisi
et al., 2006) though eggs have also been used to follow the Sea Empress oil spill
(Shore et al., 1999). Scarcity of data about PAHs in seabird tissues probably reflects
the view that vertebrates are not good models to assess oil contamination because
of their high ability in metabolizing PAHs (Hall & Coon, 1988; Varanasi et al.,
1989). In common with all vertebrates, birds have well-developed mixed function
oxygenase (MFO) systems that can rapidly metabolise parent PAHs into hydrophilic
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products that are more easily excreted, thereby, making it difficult to determine the
chemical structure of the original compound. For example, PAHs were metabolized
by chicken embryo within two weeks after injection into eggs (Naf et al., 1992).
Consequently, only minor concentrations of parent compounds are usually
detectable in vertebrate tissues (Ariese et al., 1993; Di Giulio et al., 1995) and it has
been postulated that directly measuring oil constituents in bird tissues does not
accurately reflect exposure to xenobiotic parent compounds (Trust et al., 2000).
Alternative techniques as PAH metabolite bile burden have been developed or the
induction of cytochrome P450 (Trust et al., 2000, Esler et al., 2002; Troisi et al.,
2006). However, these measures normally require freshly killed animals.
Here, we present the analysis of PAHs in seabird blood as a convenient
and relatively rapid method with little disturbance to birds for monitoring PAH
contamination in the marine environment. Since blood cells are continuously being
produced and have a lifespan of several weeks (Clark, 1988), the presence of PAHs
in blood cells probably indicates a recent incorporation during erythropoiesis. As far
as we know, no previous studies have investigated the presence of PAHs in the
blood of birds exposed to oil (but see 30 for an example in mammals). We
evaluated the adequacy of yellow-legged gulls (Larus michahellis formerly Larus
cachinnans) as indicators of PAH pollution derived from the Prestige oil spill by
measuring the concentration of 15 Prestige oil PAHs in their blood.
The Prestige wreck, off Galicia (NW Spain) in November 2002, was one of
the most recent examples of a large marine oil spill. It resulted in the released to the
marine environment of approximately 60,000 tonnes of oil products in the eights
months following the wreck, spreading pollution from Northern Portugal to France
(Figure S1 in the Supporting Information (SI)). The Prestige oil spill is considered
the biggest large-scale catastrophe of its type in Europe. Since incorporation of oil
from the Prestige is currently being detected in the marine food chain (Fernandez et
al., 2006; Laffon et al., 2006; Morales-Caselles et al., 2006), chronic exposure of
seabirds would be expected, as they are long lived and upper trophic level
consumers. In the present study, two complementary approaches were used; firstly,
we compared PAHs levels in the blood of adult yellow-legged gulls captured in
unoiled and oiled breeding colonies, seventeen months after the event. Secondly, we
performed an oil-ingestion experiment by supplementing a sample of gulls with oil
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(Butler & Lukasiewicz, 1979). This experiment allowed us to evaluate whether
seabird blood reflected direct exposure to PAHs and to study the dynamics of
PAHs incorporation in blood (Leighton et al., 1985). In addition, since it is expected
that oil incorporation in the food web from the spill will lessen with time, we
compared PAH values from gulls sampled at the oiled colony of Illas Cíes in two
consecutive years.
Materials and methods
Spatial study
Bird sampling was performed in seven insular yellow-legged gull breeding colonies
distributed along the coast of North-western Spain (Figure 1). Since yellow-legged
gulls feed mainly on marine organisms (Munilla, 1997; >80% in 2004) at an average
distance of less than 40 km away from the breeding colony (Oro et al., 1995), PAHs
in blood probably indicates contamination at local scale. Three of the colonies were
located in an area that was free from the impact of the Prestige oil spill (unoiled area:
Coelleira, Ansarón and Pantorgas), whereas the other four were in the pathway of
the spill (oiled area: Cíes, Ons, Vionta and Lobeiras). In total, 61 adults (32 females
and 29 males) were nest-trapped in 2004 while incubating (May 19 to June 5), 17
months after the Prestige wreck.
Oil-ingestion experiment
In order to evaluate the effect of oil ingestion on the presence of PAHs in the
blood of gulls, we performed a field experiment at the Illas Cíes breeding colony
(Figure 1). At the end of April 2005, during the courtship period of gulls, we
randomly allocated 36 breeding pairs to the experiment of which 16 were fed oil
(oil-supplemented group) and 20 were treated as controls (control group). Between
one and thirty days after egg laying was complete (i.e.: the third egg was laid) 18
control (10 females and 8 males) and 12 (8 females and 4 males) oil-supplemented
gulls were trapped at the nest (one gull per pair) and a blood sample was taken (see
further details in supporting information). The comparison between the
concentration of PAHs in control adults with respects to adults sampled in 2004
were used to estimate temporal changes in the PAH contamination after the Prestige
oil spill.
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Blood sampling and PAH analysis
Blood cells were analyzed to determine and quantify haematological levels of PAHs.
A blood sample (1-2 ml, depending on body mass) was taken from the ulnar vein
with a heparinized 25G needle. Blood was immediately transferred to plastic tubes
that were kept cool in ice boxes (4ºC), and centrifuged at the end of the day. Blood
cells were transferred into cryovials which were kept frozen at -80ºC until analysis.
The PAHs that were selected for analysis were the 15 PAHs (Table 3.1) constituents
of the oil spilled by the Prestige (CSIC, 2003) according to PAH priority pollutants
listed by the United States Environmental Protection Agency (US EPA) (Keith &
Telliard, 1979). PAH levels were determined by high performance liquid
chromatography (HPLC) coupled to a wavelength programmable fluorescence
detector (see further details in supporting information).
Statistical analysis
Spatial comparisons of PAH values were tested by means of a generalized mixed
model (PROC MIXED in SAS software; SAS Institute, 2001) including the area
(oiled vs. unoiled) as fixed factor and the identity of each colony as a random factor.
In order to avoid type II errors due to small sample size (see ethical considerations
above), the effect of oil ingestion was analyzed using one-tailed tests and
significance levels set at 0.05, as recommended in studies which involve
manipulations that are potentially detrimental to animals (Still, 1982). For each
PAH, regression curves were fitted to data from the oil-supplemented group as a
means to examine significant non-linear relationships between the blood levels at
the time of capture and time since ingestion. Furthermore, data were subject to a
Principal Component Analysis (PCA), in order to analyze the underlying effect of
the Prestige oil spill on the individual concentrations of the PAHs found in the blood
of gulls. This analysis included the adults sampled in the temporal study and the
experimental birds as well. Data are expressed as mean ± SE.analyzed
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Anexo 3
Results
Spatial and temporal distribution of PHA pollution
In 2004, 17 months after the Prestige disaster, the concentration of ΣPAHs in the
blood cells of gulls from oiled colonies was, on average, 120% higher than
concentrations found in gulls from unoiled colonies (F1,59 = 5.44, P = 0.011; Figure
3.1A). Gulls from Lobeiras, the colony most heavily affected by the spill, showed
the highest ΣPAHs values (Figure 1A). Differences between oiled and unoiled
colonies were significant for four compounds (naphthalene, fluorene, anthracene
and pyrene; Table 3.1) and in the oiled colonies, PAH profiles in gull blood were
clearly dominated by naphthalene (Table 3.1).
The temporal comparison between gulls sampled in 2004 and 2005 (control group
in the experimental study) at Illas Cíes showed an overall decrease in ΣPAHs levels
with time (Table 3.1, the Σ PAHs in blood decreased by 170%). Accordingly, the
majority of oil compounds showed reduced concentrations in blood in 2005 (Table
3.1).
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Anexo 3
Figure 3.1. Mean (±SE) PAH levels in the blood cells of yellow-legged gulls from A)
unoiled and oiled colonies (open and black bars, respectively) and illas Cíes in 2005, and B)
from gulls fed vegetable oil (control group, open bar) and vegetable oil plus Prestige oil (oilsupplemented group, black bar). (Colony abbreviations are: PA=Pantorgas, AN=Ansarón,
CO=Coelleira, LO=Lobeiras, VI=Vionta, ON=Ons, CI=Cíes 2004 and CI05=Cíes 2005).
* P <0.05
Oil ingestion experiment
The oil-supplemented group showed higher ΣPAHs concentrations in blood than
control gulls (Figure 3.1B; t28 = 1.87, P = 0.036). Overall, specific PAH
concentrations in oil-supplemented gulls were significantly higher for five
compounds (anthracene, fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene,
dibenz(a,h)anthracene; Figure S2). The relative abundances of individual
hydrocarbons in the blood samples of oil-supplemented gulls was not in accordance
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Anexo 3
with their proportions in the oil supplements (r = -0.14, P = 0.61). Moreover, their
relative abundances in blood correlated inversely with molecular weight (r = -0.71, P
= 0.003) and the number of rings (r = -0.749, P = 0.001).
When the effect of time after ingestion was analyzed, a specific pattern for
each compound was found. Thus, six compounds showed significant non-linear
responses (Figure 3.2). Of these, fluorene, fluorantene, benzo(a)pyrene and
dibenzo(a,h)anthracene) showed similar response patterns: oil-supplemented gulls
trapped at the end of the experiment consistently showed higher blood
concentrations than birds trapped in the few days after ingestion (Figure 3.2). In
contrast, the concentration of indeno(1,2,3-cd) pyrene decreased according with the
time of capture and, benzo(b+j)fluoranthene concentration started to decrease in
birds captured 15 days after the oil ingestion. The other compounds did not show a
significant relationship with the time from oil ingestion (P > 0.05).
Figure 3.2. Significant relationship of PAHs of blood cells levels from gulls fed with
Prestige heavy fuel oil and elapsed time between the end of oil feeding and the capture of
gulls.
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Principal Component Analysis
The factorial analysis revealed the presence of three main factors accounting for
61% of the total variance observed. The first component (PC1) explaining 28.3 of
the total variance; probably represents total oil pollution, thus it is highly correlated
with PAHs (r = 0.92, P =0.003). The second and third components explained 18.4
and 13.9 of the variance, respectively. These two components clearly separated oiled
from unoiled colonies (Figure 3.3): oiled colonies showed positive values in PC2
and PC3, whereas unoiled colonies showed negative values in PC2. Thus, PC2
ordered the colonies according to their degree of exposure to the Prestige oil. In the
experimental birds, the supplementation of Prestige oil increased the PC2 but not the
PC3 values, further validating the PC2 component as indicator of Prestige pollution.
Accordingly, the PC3 component (highly correlated with benz(a)anthracene and
pyrene) probably indicates oil pollution from others sources. Interestingly, the gulls
sampled at Illas Cíes in 2005 (CI05; Figure 3.3) displayed lower values in the PC2
and PC3 components when compared to the 2004 samples (CI; Figure 3.3)
suggesting a reduced exposure to oil contamination for gulls in 2005.
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Anexo 3
Figure 3.3. Principal Component Analysis (PCA) diagram of 15 Prestige oil PAHs, PAH
levels in oiled colonies (closed circles) and unoiled colonies (open circles) and PAH levels
in gulls subject to the oil ingestion experiment. The long broken line shows the
comparison between gulls sampled at Cíes in the 2004 and 2005 (CI05, control group in
the experiment) breeding periods. The closed circle at the end of the shorter arrow shows
PAH levels from oil-supplemented gulls in the experiment (Colony abbreviations are:
PA=Pantorgas, AN=Ansarón, CO=Coelleira, LO=Lobeiras, VI=Vionta, ON=Ons,
CI=Cíes 2004 and CI05=Cíes 2005).
Discussion
To our knowledge, this is the first field study in which levels of PAHs were
measured non-destructively in a vertebrate with the purpose to monitor oil
pollution in the marine environment after a large oil spill. Overall, our study
provides reliable support to the potential use of seabird blood as a monitoring tool
for oil exposure. This view is based upon observational and experimental evidences.
First, the technique was able to track spatial and temporal changes consistent with
the massive oil pollution pulse that resulted from the Prestige wreck in 2002 (Bosch,
2003). Thus, yellow-legged gulls sampled in oiled colonies doubled total PAH
concentrations when compared to gulls from unoiled colonies. Furthermore, PAH
143
Anexo 3
levels in gulls from a colony in the trajectory of the spill (Illas Cíes) decreased by
nearly a third in one year. On the other hand, our field experiment strongly
suggested that the profile of PAHs in the blood of gulls is likely to be influenced by
the composition of recently ingested oil and that measures of PAHs in the blood of
gulls are sensitive to the ingestion of small quantities of oil.
Polycyclic aromatic hydrocarbons are constituents of oil that, upon
ingestion, are rapidly metabolized, thereby, making it difficult to determine the
chemical structure of the original compound. For this reason, it has been postulated
that low concentrations of parent PAHs should be expected in vertebrate tissues
(Naf et al., 1992; Ariese et al., 1993; Di Giulio et al., 1995). Nonetheless, we found
higher concentrations of parent PAHs in the blood cells of yellow-legged gulls that
were exposed to the Prestige oil (either experimentally or at the moment of the spill)
respect to unexposed gulls. The mean concentration of parent PAH compounds (n
= 15), analyzed in blood cells of yellow-legged gulls, were 139.53 ± 21.42 ng/g dry
weight (range 6.48 - 860.78 ng/g; equivalent to 86.12 ± 13.22 ng/g wet weight) in
the range of values reported for other seabird tissues. Thus, for example, in muscle
tissues of silver gulls (Larus novaehollandiae) and australian pelicans (Pelecanus
conspicillatus) the mean concentration values were 85 and 75 ng/g ww respectively
(Σ12PAHs; Kayall & Connell, 1995); in herring gull (Larus argentatus) muscle the
mean values were 37.8 ± 12.5 ng/g ww (Σ 8PAHs; Wan et al., 2007), whereas in the
liver of oil exposed guillemots (Uria aalge) the mean values were 250 ± 90 ng/g
(range 40 - 970 ng/g, ww; Σ10PAHs; Troisi et al., 2006). Inter-specific comparisons
of PAHs levels should be treated with caution due to high intra-specific variability
as shown by our results and because PAHs concentrations probably differ broadly
among tissues. Thus, for example, in eider ducks (Somateria mollissima), the mean
value was 7.8 ng/g dw in liver, 46 ng/g in gallbladder and 9.7 ng/g in adipose tissue
(Σ7PAHs; Broman et al., 1990), suggesting important within organism variability.
The spatial comparison of PAH levels in the blood of yellow-legged gulls
breeding in oiled versus unoiled colonies, strongly suggests that yellow-legged gulls
were exposed to residual Prestige oil 17 months after the spill commenced. Acute
toxicity is expected when seabirds exposed to the spill ingest oil by preening (Briggs
et al., 1996). However, contaminated prey are also a potential source of ingestion
144
Anexo 3
and continued incorporation of oil products through trophic processes has been
documented for seabird species after a large oil spill (Esler et al., 2002). The life
history characteristics of yellow-legged gulls make them susceptible to continued
exposure to remnant oil (Alonso-Alvarez et al., 2007) because they frequently occur
and feed in coastal and nearshore environments, which are the same areas that
received much of the oil spilled from the Prestige. Adult yellow-legged gulls in
North-western Spain are sedentary and feed extensively on benthic and intertidal
marine organisms (Munilla, 1997). Sublethal effects derived from continued oil
exposure have been recently documented for yellow-legged gulls in North-western
Spain (Alonso-Alvarez et al., 2007).
In the oiled colonies, most of the PAH profiles in gull blood were
dominated by naphthalene (22-38%), indicating a petrogenic (i.e.: derived from
petroleum) source (Page et al., 1999). Although after the wreck, the composition of
the Prestige oil was probably altered by weathering (Fernández-Varela et al., 2006),
naphthalene was also the dominant parent compound found in subsurface waters
(Gónzalez et al., 2006) and intertidal sediments (46) from oiled areas immediately
after spill. In contrast, gulls from unoiled colonies showed low naphthalene
percentages (6-12%), and profiles were dominated by PAHs with a large number of
benzene rings (≥4 rings), especially in Pantorgas and Ansarón colonies, indicative of
a rather pyrogenic source of contamination. In other studies, naphatalene and
tricyclic PAHs also dominated samples from seabird species, including gulls,
affected by petrogenic contamination (Kayall & Connell, 1995; Troisi et al., 2006).
The differences on PHA profiles between the gull blood and the Prestige crude oil
can be due to oil alterations by weathering, changes in PAH composition in the prey
tissues, or specific metabolization of PHA compounds by gulls (see below).
There is no information about PAH levels in the blood of yellow-legged
gulls before the Prestige wreck to complete the classic before-after-control-impact
(BACI) approach (Osenberg et al., 1994). Nevertheless, the comparison of gulls
sampled at Illas Cíes in 2004 and 2005 is consistent with the expected reduction in
PAH levels with time after acute oil incorporation during the spill. Thus, ΣPAHs
concentrations in the blood of gulls decreased threefold in just one year, down to
the 2004 values from unoiled colonies. Interestingly, the reduction in PAH levels
with time also suggest that PAH concentrations right after the wreck may even have
145
Anexo 3
been higher than those found in 2004 samples (17 moths later). Except for five
compounds, the majority of hydrocarbons decreased their concentrations abruptly.
This reduction was not related to molecular weight or the number of aromatic rings,
suggesting an overall reduction in oil exposure by yellow-legged gulls in coastal
North-western Spain in 2005. Although the reduction in PAH levels should be
treated with caution because it was estimated in a single colony, our results are in
agreement with studies on other marine organisms (mussels, Mytilus galloprovincialis),
that found that ΣPAHs also decreased substantially with time after the Prestige event
(Soriano et al., 2006).
In our experiment, gulls fed with oil increased their blood concentration of
PAHs by 30% with respect to controls, hence revealing that PAHs levels in the
blood of yellow-legged gulls were in some extent directly related with oil ingestion.
A rough extrapolation from the experiment indicates that the ingestion of 3.25 µg
of ΣPAHs resulted in an increase of 1 ng/g of PAHs in blood. However, the
relative abundances of PAHs in blood were not in accordance with the composition
of the oil ingested. Interestingly, heavier compounds showed lower concentrations
in blood, suggesting that gulls mobilized and metabolized PAH compounds
differentially depending on their number of rings or molecular weight. Note that
vertebrate erythrocytes have a finite programmed lifespan in blood circulation (30
days in birds; Clark, 1988), thus PAHs found in blood cells were mobilized recently.
However, the incorporation of ingested PAHs into the blood cells during
erythropoiesis is complex and specific of each compound, while differences in
metabolization should also be expected (Lee et al., 1985, Naf et al., 1992).
Differences in the mobilization and metabolization of PAHs by gulls were also
evident in the study of the temporal pattern of PAHs in blood since oil ingestion.
Although our experiment was not designed to entirely cover the metabolism of
these compounds in seabird blood, six of the PAHs analyzed presented significant
short-term patterns of change. In four compounds, the highest concentrations in
blood were measured towards the end of the experiment. In vertebrates, ingested
PAHs are transported to the liver and some fraction is transformed in excretable
compounds, but some PAHs remain in the enterohepatic circulation extending the
residence time of PAHs in the body (Ramesh et al., 2004). The increase of some
146
Anexo 3
PAHs in oil-fed gulls at the end of the experiment may be due to the incorporation
during the erythropoiesis of enterohepatic circulating PAHs. Interestingly, different
temporal patterns of PAH compounds in experimental gulls probably indicates
different rates of metabolization and residence in the liver. The experimental study
suggests that using gull blood as a monitoring tool may underestimate the exposure
to heavier PAHs and that acute exposure to some PAH may not be adequately
reflected if samples are taken too shortly after an oil pollution event.
Lastly, the factorial analysis revealed that the variance in the blood
concentration of PAHs could be grouped in three main factors. While the first
factor (PC1) represented total oil pollution in blood, the other two components
(PC2 and PC3) clearly segregated oiled and unoiled colonies. In addition, PC2
probably indicated exposure to the Prestige oil. Two main lines of evidence further
support the use of this component as proxy of Prestige pollution. First, the PC2 was
highly correlated with the amount of ΣPAHs in the sediments close to the colonies
shortly after the Prestige spill (r = 0.96, P = 0.01; data from Gonzalez 2006).
Moreover, experimental gulls fed with Prestige oil, increased their PC2 but not their
PC3 scores. The PC3 scores probably indicated oil contamination from other
sources (i.e.: chronic). Interestingly, the PC3 score of Illas Cíes was lower in 2005
than in 2004, suggesting that lower levels of (chronic) oil pollution were operating.
Enforcement of controls of illegal oil discharges from passing ships after such a
large and visible oiling incident as the Prestige spill could explain this pattern (6).
In summary, our study not only provides evidence on the temporal and
spatial patterns of oil contamination in the marine ecosystems of North-western
Spain after the Prestige oil spill but also gives support to the use of seabirds as
biomonitors of oil pollution in a non destructive manner. Monitoring programs
based upon the analysis of PAHs in seabird blood are therefore promising,
providing that harm and disturbance to seabird individuals and populations is kept
to a minimum.
147
Anexo 3
Acknowledgments
We want to express our gratitude to Parque Nacional de las Islas Atlánticas de Galicia,
Naviera Mar de Ons, Confraría de Celeiro and the Punta Roncadoira crew (Delegación da
Consellería de Pesca en Celeiro) for logistic support and the IEO for kindly providing a
sample of Prestige oil. Carmen Díez, Julio Eiroa, David Álvarez and Manolo Pajuelo assisted
in field work. A.V. was supported by a Ramon y Cajal Fellowship (Ministerio de Educación y
Ciencia, Spain). The present study was founded by the program Plan Nacional I+D+I 20042007 (Ministerio de Educación y Ciencia, Spain).
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SUPPORTING INFORMATION
Oil-ingestion experiment
Ethical considerations were taken into account in the design to avoid unnecessary
harm to animals while still eliciting a measurable response. Thus, the number of
experimental subjects was kept as low as possible (Still, 1982) and we opted for an
amount of oil that was well below the dosage used in previous experiments (Butler
& Lukasiewicz, 1979, Leighton, 1991). The oil-supplemented group was restricted
to 16 pairs that were fed daily with 0.04 ml of Prestige oil (kindly provided by
Instituto Español de Oceanografía under the control of the Spanish Technical
Bureau of Marine Spills; otvm.uvigo.es) during seven consecutive days (0.3 ml in
total per pair; individual daily PAHs dose: 59.15 µg; Table S1). Oil was dissolved in
6 ml of vegetable oil and spread over a slice of white bread. To minimize the risk of
theft by non-target birds, the oiled bread was placed in the territory hidden in
vegetation as close to the nest as possible (Pérez et al., 2006). Pairs from the control
group were fed in a similar manner with bread and vegetable oil.
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Tabla S1. Relative composition (%) of 15 PAHs in the oil used in the ingestion experiment
and the individual daily dose ingested by experimental yellow-legged gulls.
%
Total1 (µg)
Dose2(ng/g)
Naphthalene
14.54
8.60
9.82
Acenaphthene
5.61
3.32
3.78
Fluorene
1.01
0.60
0.68
Phenanthrene
3.79
2.24
2.55
Anthracene
26.32
15.57
17.76
Fluoranthene
5.78
3.42
3.90
Pyrene
4.55
2.69
3.06
Benz[a]anthracene
17.50
10.35
11.80
Chrysene
6.48
3.83
4.37
Benzo[b+j]fluoranthene
9.04
5.35
6.10
Benzo[k]fluoranthene
1.35
0.79
0.91
Benzo[a]pyrene
2.16
1.28
1.46
Dibenz[a,h] anthracene
0.27
153
0.18
Benzo[g,h,i]perylene
0.12
0.07
0.08
Indeno[1,2,3-cd]pyrene
1.13
0.67
0.77
ΣPAH
100
59.15
67.47
Polycyclic Aromatic Hydrocarbon
1
2
Total amount of PAHs present in on the crude oil daily ingested by individual gulls
PAH dose in relation to adult body mass (876.7±41.4 g)
Analysis of PAHS
After microwave extraction with a 1:1 mixture of acetone and hexane, the extract
was cleaned-up using a deactivated alumina column with hexane as eluant. PAH
levels were determined by high performance liquid chromatography (HPLC)
coupled to a wavelength programmable fluorescence detector (Viñas-Diéguez,
2002). Samples (100 µl) were injected into a HPLC system fitted with a Waters
PAH analytical column (250 mm x 4.6 mm x 5µm). The mobile phase was
acetonitrile:water in gradient elution and at a flow rate of 1.2 ml/min. The column
oven temperature was maintained at 27 °C. For every group of 10 blood samples, a
blank sample was included and processed through extraction and cleanup
154
Anexo 3
procedures to check for any external sources of contamination. From the analysis of
serial dilution of standards (SRM 2977), the limit of detection was calculated (Table
S2). Recovery of PAHs was analyzed by adding a mixture of PAHs (200 ng/g of
each compound) to a pool of blood cells and compared with the original values
(Table S2).
Table S2. Limits of detection and percentage recoveries (±SE) of 15 PAHs analyzed.
Polycyclic Aromatic
Hydrocarbons
Naphthalene
Detection limit
(ng/g)
0.02
Recovery
(% ± SE)
77.44 ± 1.53
Acenaphthene
0.01
84.50 ± 1.78
Fluorene
0.01
102.11 ± 2.04
Phenanthrene
0.01
94.68 ± 2.70
Anthracene
0.02
90.92 ± 1.71
Fluoranthene
0.01
94.44 ± 1.36
Pyrene
0.01
97.12 ± 1.13
Benz[a]anthracene
0.04
81.71 ± 1.79
Chrysene
0.03
95.11 ± 0.78
Benzo[b+j]fluoranthene
0.05
93.13 ± 0.94
Benzo[k]fluoranthene
0.05
93.55 ± 1.09
Benzo[a]pyrene
0.05
96.47 ± 4.01
Dibenz[a,h] anthracene
0.02
97.53 ± 1.66
Benzo[g,h,i]perylene
0.01
94.58 ± 0.34
Indeno[1,2,3-cd]pyrene
0.05
93.75 ± 0.29
The accuracy and precision of the analytical procedure were tested using
two different methods. Since no certified quality controls on blood samples are
available, we used a mussel tissue, a rich lipid matrix. First, we compared PAH
levels analyzed by HLPC and GC-MS in the same mussel samples. The PAH
concentrations determined by HPLC did not differ than those concentrations by
GC-MS (P > 0.1), except for fluorene concentration that was 8% lower in HPLC
compared with GC-MS. In addition, certified quality control from the National
Institute of Standard and Technology (Gaithersburg, USA; NIST SRM 2977) was
used to compare with our analytical procedure. All PAH compounds were within
the certified range except for benz[a]anthracene, benzo[g,h,i]perylene levels that
were 6 and 11%, respectively, lower and fluorine, benzo[b+j]fluoranthene that were
6 and 5%, respectively, higher than certified values. Moreover, our analytical
155
Anexo 3
procedure was involved in the “Prestige 2004 Intercalibration Trial” held by the
Instituto Español de Oceanografía (unpublished data). From the basis of IUPAC
classification (Thompson & Wood, 1993) the method proficiency was judged as
satisfactory (|z|<2).
Figure S2. Levels of 15 PAHs in the blood cells of yellow-legged gulls subject to an oil
ingestion experiment: control group (open bars) and oil-supplemented group (black bars).
n.s. p >0.05, * p < 0.05, **p < 0.01.
References
Butler, R.G. & Lukasiewicz, P.A 1979. Field study of the effect of crude oil on Herring gull
(Larus argentatus) chick growth. Auk, 96, 809-812
Leighton, F.A. 1991. The toxicity of petroleum oils to birds:an overview. In: The effects of
oil in wildlife: research, rehabilitation and general concerns. Ed. by White, J. and Frink,
L. Sheridan Press, Hanover, PA
Pérez, C., Velando, A., Dominguez, J. 2006. Parental food conditions affect sex-specific
embryo mortality in the yellow-legged gull (Larus michahellis). Journal of Ornithology,
147, 513-519.
Still, A.W. 1982.On the numbers of subjects used in animal behaviour experiments. Animal
Behavior, 30, 873-880.
Thompson, M. & Wood, R. 1993. The International harmonized protocol for the proficiency
testing of (chemical) analytical laboratories. Pure and Applied Chemistry, 65, 2123-2144.
156
Anexo 3
Viñas-Diéguez, L. 2002. Evaluación de Hidrocarburos Aromáticos Policíclicos (HAPs) por
Cromatografía Líquida de Alta Eficacia (CLAE) en el Entorno Marino Gallego. PhD
dissertation. Universidade de Vigo. Spain.
157
Anexo 4
Efectos subletales tras la exposición al
petróleo en la gaviota patiamarilla
Alonso-Alvarez, C., Pérez, C., Velando, A. 2007. Effects of acute exposure to
heavy fuel oil from the Prestige oil spill on yellow legged gulls (Larus
michahellis). Aquatic Toxicology, 84, 103-110.
Anexo 4
Efectos subletales tras la exposición al petróleo en la gaviota patiamarilla
Carlos Alonso-Álvarez, Cristóbal Pérez, Alberto Velando
Grandes cantidades de petróleo y derivados son liberados al medio marino como
resultado del naufragio de petroleros. Estos eventos catastróficos provocan severos
impactos en los ecosistemas marinos, afectando a un amplio rango de especies entre
las que se encuentran las aves marinas. Estas aves están situadas en los niveles
superiores de la cadena alimentaria marina, por lo que son esperables efectos
tóxicos importantes en estos organismos. El reciente vertido de petróleo procedente
del Prestige nos da la oportunidad de contrastar dichos efectos. Un estudio previo
señaló que las gaviotas patiamarillas (Larus michahellis) en las áreas afectadas por el
petróleo (17 meses después del derrame), mostraban diferencias tanto en la
bioquímica del plasma como en los niveles sanguíneos de los hidrocarburos
policíclicos aromáticos totales (HPAsT) comparadas con las gaviotas de áreas no
contaminadas. En este estudio se crearon dos grupos experimentales de gaviotas en
el campo, uno de ellos fue alimentado con petróleo procedente del Prestige (Pgaviotas) diluido en aceite vegetal mientras el otro grupo de gaviotas, denominado
control
(C-gaviotas)
fueron
alimentadas
únicamente
con
aceite
vegetal.
Coherentemente con los resultados del trabajo anteriormente citado, las gaviotas
alimentadas con petróleo mostraron una reducción en los niveles plasmáticos de
glucosa y fósforo inorgánico, así como, una tendencia en la reducción de los niveles
de creatinina. Además, se encontró una relación negativa entre la concentración de
glucosa y los niveles de los HPAsT. Los machos alimentados con petróleo
mostraron una mayor actividad plasmática de aspartato aminotransferasa (AST)
cuando se compararon con los controles, resultado que no se encontró en las
hembras. Con respecto a la actividad plasmática de la gamma-glutamiltransferasa
(GGT), los resultados fueron contrarios a los del estudio previo. La actividad de la
GGT aumentó en las C-hembras, probablemente relacionado con el incremento del
metabolismo del hígado durante la puesta de los huevos, pero este efecto no se
encontró en las P-hembras. Las diferencias encontradas respecto al estudio previo
posiblemente reflejen diferentes repuestas adaptativas de estas enzimas ante una
exposición aguda a corto plazo al petróleo. Las gaviotas patiamarillas pertenecen a
161
Anexo 4
un complejo de especies ampliamente distribuidas a través del hemisferio norte, por
lo que los resultados mostrados podrían proporcionar una herramienta para futuras
evaluaciones de efectos a corto y a largo plazo de los derrames de petróleo. La
disminución de glucosa y fósforo inorgánico plasmáticos son esperables en
exposiciones a corto y a largo plazo al petróleo, mientras que las respuestas
enzimáticas de la AST y la GGT dependerán del sexo del los individuos y del patrón
temporal de exposición al petróleo
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Anexo 4
Effects of acute exposure to heavy fuel oil from the Prestige spill on a seabird
Carlos Alonso-Álvareza, Cristóbal Pérezb, Alberto Velandob
a Unidad de Ecología, Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCM, JCCM),
Ronda de Toledo s/n, 13005. Ciudad Real, Spain. 1Departamento de Ecoloxía e Bioloxía Animal,
Facultade de Ciencias, Campus Lagoas-Marconsende, Universidade de Vigo, 36310 Vigo, Spain
Abstract
Large quantities of petroleum products are released into the marine
environment as result of tanker wrecks. Such catastrophic events have a dramatic
impact on marine ecosystems, affecting a broad range of species. Seabirds are
placed at the uppermost trophic level of the marine food chain. Therefore,
important toxic effects are expected in these organisms. The recent Prestige oil spill
gave the opportunity to test this. A previous study reported that yellow-legged gulls
(Larus michahellis) breeding in the oiled area (17 months after the spill) showed
differences both in plasma biochemistry and in the total circulating levels of
polycyclic aromatic hydrocarbons (TPAHs) in blood regard to gulls sampled in
clean areas. In the present study, wild yellow-legged gulls were fed with heavy fuel
oil from the Prestige oil spill (P-gulls) and compared with control gulls (C-gulls) fed
only with the vehicle (vegetable oil). Consistent with the cited previous findings,
gulls fed with fuel oil showed reduced glucose and inorganic phosphorus levels in
plasma, as well as a trend to significantly reduced creatinine values. In addition,
glucose concentration was negatively related to TPAH levels. Males but not females
fed with fuel oil showed higher plasma activity of aspartate aminotransferase (AST)
than controls. With regard to plasma activity of gamma-glutamyl transferase (GGT),
the results were opposite to the previous study. The GGT activity increased in Cfemales, apparently to meet with increased liver metabolism due to egg laying
demands, but not in P-females. Differences to the previous study possibly reflect
different adaptive responses of these enzymes to an acute short-term exposure to
heavy fuel oil. Since the yellow-legged gull belongs to a complex of species widely
distributed throughout the Northern hemisphere, the results as a whole might
provide a tool for future evaluations of short- and long-term effects of oil spills on
seabirds. Decreased glucose and inorganic phosphorus levels in plasma are expected
in both short- and long-lasting exposures to fuel oil, whereas responses of AST and
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Anexo 4
GGT enzymes would depend on both the sex of individuals and the temporal
pattern of exposure.
Introduction
Different compounds present in petroleum products (such as polycyclic
aromatic hydrocarbons [PAHs]; i.e. list in Keith & Telliard, 1979) are able to induce
carcinogenic and immunotoxic effects as well as endocrine disruption in vertebrates
(e.g. Gelboin & Ts’o, 1981; Nicolas, 1999; Reynaud & Deschaux, 2006). Large
quantities of petroleum products are released into the marine environment as result
of tanker wrecks. Such catastrophic events have a dramatic impact on marine
ecosystems, affecting a broad range of organisms, including seabirds (e.g. Peterson,
2001; Velando et al., 2005a,b). One of the last examples of a large marine oil spill
took place in November 2002 when the supertanker Prestige sank in the Galician
coast (NW Spain). The tanker spilled between 40,000 and 63,000 tonnes of heavy
fuel oil into the Atlantic Ocean (e.g. Marcos et al., 2004; Dieguez et al., 2007),
causing pollution from Portugal to France. The Prestige oil spill was the biggest
catastrophe of its type in Europe and thousands of seabirds died in the following
months (Camphuysen et al., 2002; Martinez-Abraín et al., 2006). Moreover, toxic
compounds present in the Prestige oil spill, such as PAHs, are currently being
detected in the marine food chain (e.g. Fernandez et al., 2006a, 2006b; Laffon et al.,
2006; Morales-Caselles et al., 2006; Ordas et al., 2007).
Seabirds are placed at the uppermost trophic level of the marine food chain.
Important toxic effects of the petroleum products would be therefore expected in
these organisms. In the past, many studies have reported damaging effects of
exposure to petroleum products in seabirds after oil spills (e.g. Newman et al., 1999;
Seiser et al., 2000; Golet et al., 2002; Balseiro et al., 2005). However, as far as we
know, only one study on seabirds has determined the presence of PAHs in the
organism, exploring the relationship between the total blood circulating levels of
PAHs (TPAH) and the health status of individuals (Alonso-Alvarez et al., 2007).
That study was carried out on the most common seabird of Galician coasts, the
yellow-legged gull (Larus michahellis). The species was used to monitor the impact of
the Prestige oil spill on the marine ecosystem. The previous knowledge on the plasma
biochemistry of this particular seabird (e.g. Alonso-Alvarez & Ferrer, 2001; Alonso-
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Anexo 4
Alvarez & Velando, 2003; Alonso-Alvarez, 2005) allowed the interpretation of the
results, which suggested different sub-lethal effects associated to a long-term
exposure to fuel oil. Thus, birds sampled in oiled areas showed reduced levels of
glucose, inorganic phosphorus (iP), total protein and creatinine as well as higher
levels of two aminotransferases, namely gamma-glutamyl transferase (GGT) and
aspartate aminotransferase (AST) (Alonso-Alvarez et al., 2007).
In birds, high GGT levels in blood are commonly used as an index of liver
disease, as well as damages in biliary ducts and renal epithelium (reviewed by
Lewandowski et al., 1986, Hochleithner, 1994 & Harr, 2002). Meanwhile, high AST
concentration in blood would be indicative of hepatocellular disease in birds
(Brugere-Picoux et al., 1987; Harr, 2002). Both GGT and AST enzymes are
involved in the transamination of glucogenic amino acids (i.e. glutamate and
aspartate, respectively; Stevens, 1996) in order to produce glucose (e.g. Stevens,
1996). Therefore, changes on these parameters would not only support the idea of
liver damages, but also explain the decrease in glucose levels in the earlier study (i.e.
Alonso-Alvarez et al., 2007). Moreover, the study reported positive correlations
between the total blood concentrations of PAHs (TPAH) and a number of the
analysed substances. Although such correlations do not imply causation, the results
are suggestive of an effect of these particular compounds on seagull physiology. In
summary, the results as a whole suggested fuel-oil induced damages on vital organs
(i.e. liver and kidney). However, an experimental demonstration of these effects was
still necessary.
Here, we have carried out such an experiment. Wild yellow-legged gulls were
experimentally fed with heavy fuel oil from the Prestige oil spill and compared with
control gulls fed only with vehicle (i.e. vegetable oil). The study provided the
opportunity to compare the sub-lethal effects of a long-term exposure to toxic
compounds in the oil spill (i.e. Alonso-Alvarez et al., 2007) with the effects induced
by a short-term acute exposure in a vertebrate species. The same biochemical
parameters were analysed as in the earlier correlational study (Alonso-Alvarez et al.,
2007). In addition, the total concentration of fifteen PAHs found in the Prestige
heavy fuel oil (Bosch, 2003; CSIC, 2003) was determined in the red cell fraction of
blood.
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Materials and methods
Experimental Procedure
Thirty one yellow-legged gull couples were randomly chosen at their breeding
colony (Illas Cíes, Galicia, NW Spain). This population suffered the impact of the
Prestige oil spill in 2002 (Alonso-Alvarez et al., 2007). Twelve breeding pairs chosen
at random were daily fed with 0.04 ml of unweathered heavy fuel oil from the
Prestige oil spill (petrol-fed birds, “P-birds”), kindly provided by Instituto Español de
Oceanografía under the control of the Spanish Technical Bureau of Marine Spills
(otvm.uvigo.es). In the heavy fuel oil provided to birds, the concentration of the
fifteen PAHs (described below) was 17.75 μg/mL.
Ethical considerations were taken into account in the experiment design to
minimize the damage caused by fuel oil while still eliciting a measurable response.
To avoid provoking unnecessary damage to the gulls and to keep the number of
subjects needed as low as possible (Still, 1982), we chose an amount of fuel oil (0.3
ml in 7 days) well below the dosage used in previous experiments (e.g. Butler &
Lukasiewicz, 1979; Leighton et al., 1985).
Fuel oil was mixed with vegetable oil and placed on a slice of bread, which was
hidden under vegetation and close to the nest (50 cm approximately) to avoid the
risk of being eaten by non-target birds. Yellow-legged gulls are territorial, defending
aggressively several meters around their nest site (Alonso-Alvarez, 2001; AlonsoAlvarez & Velando, 2001). Moreover, a previous experience providing food in the
same way and registering behavior of gulls around the territory reported that no
food item was stolen by other birds (Perez et al., 2006). The other 19 pairs (control
birds, “C-birds”) were fed in the same way, but the bread did not contain fuel oil,
but only vegetable oil. There were no differences in body size (i.e. tarsus length or
head length) between gulls assigned to each group (p-values > 0.20). All gulls were
fed from April 26 (just before the egg-laying period in the population) to the time
when the clutch was completed (mean, range: 2.8, 1-3 eggs). Fuel oil diet was,
however, restricted to seven consecutive days (until May 3). From the end of the
seven-day period to the end of the egg-laying period all birds received bread with
only vegetable oil (mean ± SE: 9.2 ± 0.98 days, range 1-21 days). There was no
difference between treatments in the number of days elapsed from the start of
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Anexo 4
feeding to the end of egg-laying (complete feeding period) (F1,29 = 0.29, p = 0.597;
means ± SE: 9.84 ± 1.1 days and 8.75 ± 1.88 days for C- and P-gulls, respectively).
One of the members of the couple was randomly captured at their nest site
during incubation (10 C-females, eight P-females, nine C-males and four P-males).
A blood sample (about 1.5 mL) was immediately taken from the ulnar vein with a
heparinized 25G needle. Blood was then transferred to plastic tubes and
microcapillaries filled from them. Both tubes and filled microcapillaries were
maintained cooled in ice boxes (4ºC), and centrifuged at the end of the day. Plasma
and blood cells (pellet) were separately frozen in liquid nitrogen (-196ºC).
Hematocrit values were daily determined from centrifuged microcapillaries.
Several morphometric measures including the head and tarsus length (to 1mm)
were measured on each bird. Body mass was also determined (to 1g). The tarsus
length allowed sexing birds by means of a discriminant function (Bosch, 1996). The
number of days elapsed from the end of the laying to the capture date did not differ
between groups (F1,29 = 1.02, P = 0.321; means ± SE: 8.68 ± 1.1 days and 10.8 ± 2.1
days for C- and P-gulls, respectively).
TPAH levels
Fifteen of the most toxic polycyclic aromatic hydrocarbons (PAHs), according
to U.S. Environmental Protection Agency (EPA) data (Keith & Telliard, 1979) and
present in the Prestige heavy fuel oil (CSIC, 2003), were measured in the red blood
cell fraction (pellet). The PAHs analysed were acenaphthene, anthracene,
benz(a)anthracene, benzo(a)pyrene, benzo(b+j)fluoranthene, benzo(g, h, i)perylene,
benzo(k)fluoranthene, chrysene, dibenz(a,h)anthracene, fluorene, fluoranthene,
indeno(1,2,3-c-d)pyrene, naphthalene, phenanthrene and pyrene. To estimate the
individual degree of oil contamination, the sum of concentrations from all these
hydrocarbons was used as a variable (TPAH level).
The concentrations of PAHs were determined by high performance liquid
chromatography (HPLC). After microwave extraction with a mixture of
acetone:hexane 1:1, the extract was cleaned-up using a deactivated alumina column
with hexane as eluant. The PAHs were determined by HPLC coupled to a
wavelength programmable fluorescence detector (Viñas-Diéguez 2002). The
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method used certified quality controls from the National Institute of Standards and
Technology (Gaithersburg, USA; references NIST SRM 1647d and 2977).
Moreover, the method was involved in the “Prestige 2004 Intercalibration Trial”
organized by the Instituto Español de Oceanografía (www.ieo.es).
Biochemical measurements from plasma
The measurements were carried out using a spectrophotometer (A-25,
Biosystems SA, Barcelona), and commercial kits and certified controls from
Biosystems (Biosystems SA, Barcelona). The analyzed parameters were (method in
parenthesis): AST activity (NADH-method), calcium concentration (arsenaze III),
cholesterol concentration (cholesterol oxidase), creatinine concentration (alkaline
picrate), GGT activity (carboxy substrate), glucose concentration (glucose oxidase),
inorganic phosphorus concentration (phospho-molybdate reaction), total protein
concentration (biuret reaction) and uric acid concentration (uricase method). The
within-assay coefficient of variation (CV) for all these parameters ranged between
0.4% - 2.9%. The analyses were carried out in the same assay session. The
experimental groups were alternated in the reader plate. There was not enough
sample volume for all tests in all the samples, and thus degrees of freedom showed
in results (Table 1) could vary.
Breeding output
Reproduction was monitored by regular visits to the colony. Egg width and
length were measured with a caliper to the nearest 0.01 mm. We calculated egg
volume using the function 0.51 x length x (width)2 , proposed by Hoyt (1979).
Statistical analyses
We tested differences between P- and C-birds by running several ANCOVA
models including the experimental treatment and the sex as fixed factors, testing its
interaction. Additionally, other potentially confounding factors and covariates were
introduced in the models in order to control for individual variability. We always
started from the saturated model with all factors and covariates, removing nonsignificant terms by a backward stepwise procedure. Covariates were: body mass
and body size of gulls, total egg volume of the clutch and the number of days
elapsed from the beginning of the treatment to the end of egg-laying (complete
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Anexo 4
feeding period). In addition, the influence of clutch size was tested as a fixed factor
with three levels (1-3 eggs).
The tarsus and head length of adults were also added as covariates when
analysing size-corrected body mass (e.g. Garcia-Berthou, 2001; Stevenson &
Woods, 2006). Since adult body mass follows a bimodal distribution (i.e. yellowlegged gulls are sexually dimorphic in size; Bosch, 1996), the analyses on this
variable were performed separately for each sex.
The interaction between circulating levels of TPAH and the experimental
treatment was also tested in order to know if the PAHs acquired from the
experimental diet or from the environment correlated with different responses.
In order to avoid type II errors due to the reduced sample size (see ethical
considerations above), we used one-tailed P-values in those analyses where an a
priori prediction was clear, as recommended in studies which involve manipulations
that are potentially detrimental to the animals (Still, 1982). Thus, we predict that Pbirds would show a higher TPAH level in blood than controls (above). Moreover,
we predicted some differences between treatments following results in the previous
study on the impact of the Prestige oil spill on yellow-legged gulls (Alonso-Alvarez
et al., 2007). In this way, we predicted lower glucose, total protein, creatinine and
phosphorus levels and higher AST activities in P-birds. Similarly, we predicted
higher GGT activities in P-females (i.e. Alonso-Alvarez et al., 2007). Calcium
concentrations and AST activities were log-transformed whereas creatinine
concentrations were square root transformed to meet the normality requirements
for parametric analyses. SAS statistical software was used (version 8.2; SAS
Institute, 2001). DMS tests were used for pairwise post hoc comparisons. Results are
given as means ± SE.
Results
TPAH in blood
As expected, TPAH values of P-gulls were higher than TPAH values of C-gulls
(75.79 ± 9.13 and 56.42 ± 5.38 ng/g, respectively; F1,29 = 3.80, P = 0.031). Neither
the sex of the bird nor the treatment x sex interaction showed significant effects
(both P’s > 0.75).
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Anexo 4
Breeding output, body mass and hematocrit variability
The date of laying of the first egg did not differ between treatments (F1,29 =
0.14, P = 0.716; Julian calendar: 127.8 ± 1.1 and 127 ± 1.9 days, for C- and P-gulls
respectively). Neither clutch nor the total volume of eggs (F1,29 = 0.23, P = 0.634;
212 ± 8.5 and 205 ± 15.8 cm3, for C- and P-gulls respectively) differed between
experimental groups. Neither the size-corrected body mass (both sexes) nor the
haematocrit value showed differences between the treatments (Table 4.1).
Table 4.1. Circulating levels of several biochemical parameters, the haematocrit value and the
size-corrected body mass from adult yellow-legged gulls fed with heavy fuel oil from the
Prestige oil spill or fed with vehicle only (controls)
Parameter
Control
Petrol-fed
Treatment effect
Mean
SE
Mean
SE
F
df
p
Glucose (mg/dL)
471.8
10.48
441.7
20.96
6.02
1,25
0.010a
iP (mg/dL)
2.99
0.22
2.45
0.36
6.51
1,25
0.009a
Creatinine (mg/dL)
0.79
0.02
0.56
0.05
2.49
1,28
0.063a
Ca (mg/dL)
13.22
0.39
12.46
0.59
3.88
1,26
0.274
Uric Acid (mg/dL)
6.52
1.05
4.67
0.47
1.87
1,28
0.183
GGT (U/L)
15.30
4.40
5.87
3.92
1.80
1,24
0.192
AST (U/L)
289
19.8
310.8
27.3
1.74
1,24
0.200
Cholesterol (mg/dL)
410.9
19.3
420.8
16.99
0.13
1,28
0.722
Total protein (g/dL)
52.76
3.03
51.5
2.01
0.37
1,28
0.547
Hematocrit (%)
44.11
0.80
43.67
1.24
0.10
1,29
0.751
Male size-corrected
body mass (residuals)
-0.15
0.33
0.18
0.14
0.70
1,9
0.426
0.01
0.38
-0.01
0.66
0.48
1,14
0.499
b
Female sizecorrected
body mass (residuals)
b
The effect of the treatment from ANCOVA models is also shown. Other significant factors and covariates
are described in results. a One-tailed p-values established from a priori predictions (see methods). bSizecorrected body masses were standardized residuals from the model including tarsus and head length as
covariates (always p < 0.05)
Effects on plasma parameters
Gulls experimentally fed with heavy fuel oil showed significantly lower plasma
concentrations of glucose and iP than controls (Table 1 and Fig. 1). Creatinine
concentrations also tended to be lower in fuel oil supplemented gulls, but the
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Anexo 4
difference was not significant (Table 4.1 and Fig. 4.1; sex x treatment interaction
was not significant: P > 0.20). In the case of plasma glucose levels, the interaction
between experimental group and the concentration of circulating PAHs was
significant (F1,25 = 6.64, P = 0.016). Thus, whereas TPAH did not correlate with
glucose levels in controls (r = +0.16, P = 0.531), a clear negative relationship was
present in P-gulls (r = -0.66, P = 0.019; Fig. 4.2). Meanwhile, when the clutch size
was retained in the model testing variability in iP levels (F1,25= 8.39, P = 0.002),
phosphorus values decreased with increasing clutch size (5.78 ± 0.01, 2.69 ± 0.42
and 2.65 ± 0.19 mg/dL, for 1-, 2- and 3-egg clutches).
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Anexo 4
Figure 4.1. Plasma levels of several biochemical parameters from yellow-legged gulls fed
with vehicle (vegetable oil) or fed with vehicle plus heavy fuel oil from the Prestige oil spill
(white and dark bars, respectively). Creatinine and AST values were transformed to meet
with the normality assumption (methods). Data are separately presented for male and
female individuals. The main effect of the treatment is nonetheless described in Table 1.
Significance of within-sex post hoc comparisons are only represented for models showing
a significant treatment x sex interaction (see Results; * P < 0.05; ** P < 0.010; n.s.: non
significant). Bars are means ± SE.
In the model on plasma calcium levels, the treatment x sex interaction was
close to significance (F1,24= 3.67, P =0.068; Fig. 1). Thus the effect of fuel-oil
supplementation was evident in females but not in males. P-females showed lower
calcium levels than C-females (11.8 ± 0.73 and 13.6 ± 0.45 mg/dL, respectively; P =
0.034), whereas males did not show any effect (13.6 ± 0.81 and 12.8 ± 0.56 mg/dL
for P- and C-males respectively; P = 0.490).
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With regard to the enzymes, the effect of the experiment on AST activities was
evident in males but not in females (sex x treatment, F1,24= 4.58, P =0.043; Fig. 4.1).
Thus, males fed with petrol showed higher AST activities than controls (P = 0.041).
However, P-male activities did not differ from those found in females (P > 0.37).
The number of days from the beginning of the experimental feeding to the end of
the egg laying (complete feeding period) correlated positively with AST activities (r
= +0.38, P = 0.044) and was retained in the model on AST variability (F1,24= 7.16, P
=0.013). Meanwhile, GGT activity was significantly affected by the interaction
between treatment and sex (F1,24= 5.12, P =0.033), but in the opposite direction
from that previously predicted (Fig. 4.1). Thus, C-females clearly showed higher
GGT activities than P-females (P = 0.009), whereas males from both groups did
not differ (P = 0.558).
Figure 4.2. The relationship between plasma glucose and total polycyclic aromatic
hydrocarbon (TPAH) levels in the blood of yellow-legged gulls fed with heavy fuel oil
from the Prestige oil spill.
Discussion
Here we experimentally demonstrate that yellow-legged gulls exposed to the
Prestige heavy fuel oil (in feed) showed reduced levels of glucose and inorganic
173
Anexo 4
phosphorus. Thus, the present findings agree with previous correlational results
from yellow-legged gulls captured several months after the Prestige oil spill. In that
time, gulls captured in oiled areas also showed lower glucose and inorganic
phosphorus levels than gulls from unoiled areas (Alonso-Alvarez et al., 2007).
Interestingly, the results also revealed sex-specific differences in sensitivity to heavy
fuel oil in other parameters (i.e. aminotransferase activities and calcium levels).
Moreover, the findings highlight the differences of acute and long-term exposure to
fuel oil on aminotransferase enzymes.
Low glucose levels as a result of ingestion of petroleum products have been
also reported in American coots (Fulica americana) exposed to plumage oiling and
crude oil intake after the Unocal-Metrolink spill (Newman et al., 2000). Thus, our
results support the conclusion that petroleum products spilled on the marine
environment are able to induce a decrease in circulating glucose levels in seabirds,
not only after long-term exposures (i.e. 17 months; Alonso-Alvarez et al., 2007), but
also after short episodes of ingestion. Nonetheless, Golet et al., (2002) reported
higher glucose levels in blood from pigeon guillemots (Cepphus columba) sampled in
areas affected by the Exxon Valdez oil spill. These authors suggested that the effect
was due to an increased adrenal response because corticosterone, the hormone
responsible of the physiological stress response, also showed high levels in these
birds. However, contrary to this conclusion, other authors reported decreased
adrenocortical response in oiled seabirds (i.e. Gorsline & Holmes, 1982).
Our results also suggest that PAHs could be the specific compounds
responsible for such a decrease in glucose levels. Thus, TPAH values were
negatively correlated to glucose concentration in P-gulls. Those birds supporting
higher blood levels of PAHs also showed lower levels of glucose in plasma.
Nonetheless, we must also consider that other toxic compounds present in the
Prestige fuel oil could have contributed to the observed effects. This seems to be
particularly probable when the oil is weathered (see e.g. Barron et al., 1999; Booth et
al., 2007).
From a mechanistic perspective, the lower glucose levels in P-birds may
suggest, at a first glance, a decrease in food intake. Circulating glucose concentration
is tightly regulated because it is used as energy source for most tissues and cells (e.g.
the central nervous system; Castellini & Rea, 1992). Here, the glucose values of gulls
174
Anexo 4
from both experimental groups were higher than those reported in captive yellowlegged gulls fed ad libitum (Alonso-Alvarez & Ferrer, 2001) and than levels present
in wild gulls sampled in the previous year (Alonso-Alvarez et al., 2007). Moreover,
P-birds did not show lower body masses, which would have indicated reduced food
intake. In the same way, they did not show lower haematocrit values, and hence,
anaemia (e.g. Newman et al., 1999; Balseiro et al., 2005). The results agree with our
previous study (Alonso-Alvarez et al., 2007) where gulls sampled in oiled areas
showed reduced levels of glucose, but no effect on nutritional condition was
detected. Thus, overall the results strongly suggest that glucose differences are not
caused by reduced feeding. Instead, reduced glucose levels are probably due to the
impact of TPAH or other toxic compounds present in fuel oil on glycogenic tissues.
The liver is the main detoxification organ, but has also a pivotal role in glycogenesis
(e.g. Stevens, 1996; Whittow, 2000). Hence, its malfunction could have led to
reduced glucose synthesis and then reduced circulating glucose.
Liver malfunction could also explain differences in inorganic phosphorus
concentrations. Avian embryos exposed to fluoranthene and benz(k)fluoranthene,
that is, two of the PAHs detected in our birds, showed liver necrosis and a decrease
of alkaline phosphatase (ALP) activity (Kertesz & Hlubick, 2002). ALP is involved
in phosphorus metabolism (Whittow, 2000). Since ALP is produced by liver, kidney,
intestines and bones (Lewandowski et al., 1986; Hochleithner, 1994), any toxin
acting on any of these tissues could potentially affect its activity, and consequently,
iP levels. Studies analysing both acute and chronic exposures of seabirds to crude or
fuel oil reported decreased iP concentrations. Thus, reduced iP levels have been
reported in female mallards (Anas platyrhynchos) experimentally fed with crude oil
(Stubblefield et al., 1995) and on pigeon guillemots sampled in the Exxon Valdez
oiled sites seven years after the spill (Golet et al., 2002). Meanwhile, the apparent
impact of fuel oil on female calcium levels could be associated to the effort of
eggshell production. Accordingly, Stubblefield et al., (1995) detected decreased
levels of plasma calcium in female but not male mallards fed with crude oil, as well
as reduced eggshell thickness.
Moreover, gulls fed with fuel oil also showed a trend to significantly lower
creatinine levels. A significant difference in the same direction was observed
between gulls sampled in oiled and unoiled sites (Alonso-Alvarez et al., 2007).
175
Anexo 4
Creatinine is produced in phosphocreatine catabolism, which is induced by creatine
kinase to produce energy for the muscle activity (Wyss & Kaddurah-Daouk, 2000).
Captive yellow-legged gulls maintained in a reduced space had lower values than
those observed in the present study (Alonso-Alvarez & Ferrer, 2001). Thus, low
creatinine levels could indicate decreased muscular metabolism. Otherwise, reduced
creatinine levels could be associated with a reduced rate of glomerular filtration in
kidneys, as deducted from bibliography in humans (e.g. Herget-Rosenthal et al.,
2007). However, the absence of differences in uric acid values would not support
this idea (i.e. high uric acid levels are often indicative of kidney failure in birds;
Hochtleiner, 1994).
Males fed on fuel oil showed higher AST activities than control males. Several
studies on seabirds suffering from long-term exposures to oil spills show an
increase of AST activities. Thus, pigeon guillemots captured in areas affected by the
Exxon-Valdez oil spill seven years earlier showed higher AST activities than birds
from non-affected areas (Seiser et al., 2000; Golet et al., 2002). Similarly, yellowlegged gulls from oiled areas showed higher AST activities 17 months after the spill,
AST being also positively correlated to TPAH levels in blood (Alonso-Alvarez et al.,
2007). However, avian studies on the effects of an acute exposure to oil spills on
AST activities are not so consistent. Thus, whereas pigeon guillemots fed with crude
oil did not show significant changes on AST activities (Prichard et al., 1997), in
American coots AST activities decreased after the birds had suffered plumage oiling
and ingested large amount of crude oil (Newman et al., 2000). Therefore, acute and
chronic long-term exposures could result in different responses. Interestingly,
females did not show significant differences in AST levels. Both groups showed
high AST activities similar to those of P-males, suggesting that control females
display higher hepatic damages than males from chronic oil exposure. In that
scenario, the experimental increase of toxic compounds present in fuel oil over that
threshold would have not been able to raise the AST production in females. On the
other hand, females, but not males, showed a positive correlation between AST
activities and the days feeding (females: r = +0.52, p = 0.040; males: r = 0.06, p =
0.85). Since fuel oil was only restricted to the first seven days, this result can be
associated to a stronger sensitivity of females than males to feeding on vegetable oil.
176
Anexo 4
We have previously reported higher GGT activities in female, but not male,
yellow-legged gulls from oiled areas after the Prestige oil spill (Alonso-Alvarez et al.,
2007). Here, females were again the affected sex. Interestingly, the pattern was now
reversed. Control females showed higher GGT activities than P-females. Although
a decrease in GGT activity after fuel oil exposure seems contraintuitive, decreased
GGT levels after acute exposure to some toxic compounds (i.e. PCBs) has been
reported in mice (Hori et al., 1997). Moreover, a decrease in GGT activity has been
also described in mussels (Mytilus edulis) experimentally exposed to PAHs
(Krishnakumar et al., 1997). An alternative interpretation would be that P-females
would have not been able to increase GGT activity in response to some
physiological requirement such as C-females did. In female mallards, serum GGT
activity increased 20-fold during the egg laying period (Fairbrother et al., 1990).
Interestingly, the time elapsed from the date of clutch end to the blood sampling
was inversely related to GGT values in females (r = -0.55, p = 0.029), but not in
males (r = 0.37, p = 0.244). This suggests that healthy females were able to increase
GGT activity as a response to the physiological requirements of egg-laying (e.g.
Christians & Williams, 1999; Barboza & Jorde, 2002). Overall, the results indicate a
sex-related sensitivity of GGT and AST enzymes to acute oil exposures.
Sex-specific effects of oil contamination on seabirds have been rarely explored,
and their demographic consequences are commonly ignored. A recent study
indicated that when female-skewed mortality occurs, a large decrease in breeding
numbers is expected, because unmated males can defer breeding (Martinez-Abraín
et al., 2006), and this may in part explain the considerable decline observed in oiled
colonies after oil spills (Velando et al., 2005b). Sex-related sensitivity to fuel oil,
such as reported in this study, could be affecting the population dynamics and
constrains the recovery of gulls in oil-affected colonies. Unfortunately, the lack of
an appropriate monitoring of yellow-legged gull populations (especially before the
Prestige event) has prevented to obtain definitive conclusions at least to the present
time. Gulls are long-lived birds (e.g. Annett & Pierotti, 1999) and hence delayed
effects such as reduced lifespan or reduced offspring fecundity could arise in future
years. Nonetheless, the study of another seabird sharing the same colonies as gulls
(the European shag; Phalacrocorax aristotelis) has reported a clear negative impact in
terms of population growth rate and breeding success (Velando et al., 2005a). Thus,
177
Anexo 4
further investigation is still necessary to disentangle the long-term effects of the oil
spill on the gull population.
In summary, several findings of the present experiment corroborate the
correlational results of a previous study, which suggested sub-lethal effects derived
from long-term exposures to fuel oil in the same seabird species (Alonso-Alvarez et
al.,
2007).
Meanwhile,
the
study
also
revealed
different
patterns
in
aminotransferases, probably an adaptive response to endure the acute short-term
exposure to fuel oil. Since the yellow-legged gull belongs to a complex of species
widely distributed throughout the Northern hemisphere (e.g. Liebers et al., 2001 &
2004), the results as a whole might provide a tool for future evaluations of shortand long-term effects of oil spills in seabirds. Decreased glucose and inorganic
phosphorus levels in plasma are expected in both short- and long-lasting exposures
to fuel oil, whereas different responses of AST and GGT are expected depending
on both the sex of the individuals and the temporal pattern of exposure.
Acknowledgments
We acknowledge Xunta de Galicia staff (Spain) and Naviera Mar de Ons for providing us
with logistic support during the sampling campaigns. We are grateful to Rafael Mateo and
Lorenzo Perez-Rodriguez for their support in spectrophotometry. We also acknowledge
Marta Lopez-Alonso for her logistic support in the HPLC analyses and Carmen Díez and
Julio Eiroa for field assistance. C. A.-A. and A.V. were supported by a Ramon y Cajal
Fellowship (Ministerio de Educación y Ciencia, Spain). The present study was founded by the
program Plan Nacional I+D+I 2004-2007 (Ministerio de Educación y Ciencia, Spain).
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184
Anexo 5
Efectos subletales y coloración tras la
exposición al petróleo en la gaviota
patiamarilla
Este anexo está basado en: Pérez, C., Munilla, I., López-Alonso, M., Velando, A.
Sublethal effects on seabirds after the Prestige oil-spill are mirrored in
carotenoid-based coloration.
Anexo 5
Efectos subletales y coloración tras la exposición al petróleo en la gaviota
patiamarilla
Cristobal Pérez1, Ignacio Munilla2, Marta López-Alonso3, Alberto Velando1
Se ha sugerido que las señales sexuales pueden ser usadas para evaluar la calidad
ambiental, ya que es probable que la intensidad de estas señales represente la suma
de las presiones ambientales a las que se enfrentan los animales. Un buen ejemplo
de las señales sexuales son aquellas dependientes de carotenoides exhibidas por
muchos peces y aves. De acuerdo a esta idea, se ha propuesto que las coloraciones
dependientes de carotenoides pueden ser especialmente valiosas para monitorear y
detectar los efectos subletales de los contaminantes tóxicos en el medio ambiente.
Nosotros evaluamos si la coloración dependiente de carotenoides del pico de las
gaviotas patiamarillas adultas refleja los efectos subletales inducidos por el petróleo
en las colonias afectadas por la marea negra Prestige. En el año 2004, tomamos una
muestra sanguínea a veintisiete gaviotas adultas que fueron capturadas en el nido,
mientras estaban incubando, en cuatro colonias de cría insulares afectadas por la
marea negra del Prestige. Analizamos los parámetros bioquímicos plasmáticos
indicadores de efectos subletales después de una contaminación por petróleo. Así
fueron analizados los niveles plasmáticos de glucosa, proteina total, creatinina,
fósforo inorgánico, aspartato aminotransferasa (AST) y la gamma-glutamil
transferasa (GGT) y además, medimos el área de la mancha roja del pico. El tamaño
de la mancha roja del pico correlacionó positivamente con la condición corporal y
negativamente con los niveles plasmáticos de la aspartato aminotranferasa (AST),
una enzima comúnmente usada como indicadora de daños hepáticos en aves, y que
aumentó en las colonias afectadas por la marea negra. Este trabajo muestra
evidencias de que el color de los tejidos con coloraciones dependientes de
carotenoides puede ser usado como medida de calidad ambiental. Además,
subrayamos la importancia que tienen la evaluación de las señales sexuales y el
comportamiento de los animales, cuando se cuantifica el impacto real de una
contaminación por petróleo sobre la fauna.
187
Anexo 5
Sublethal effects on seabirds after the Prestige oil-spill are mirrored in
carotenoid-based coloration
Cristobal Pérez1, Ignacio Munilla2, Marta López-Alonso3, Alberto Velando1
1Departamento de Ecoloxía e Bioloxía Animal. Facultade de Bioloxía. Universidade de Vigo. Campus
Lagoas-Marconsende. 36310 Vigo, Spain.2Departamento de Bioloxía Celular e Ecoloxía. Facultade de
Bioloxía. Universidade de Santiago de Compostela. 15782 Santiago de Compostela, Spain.3Departamento
de Patoloxía Animal. Facultade de Veterinária. Universidade de Santiago de Compostela. 27002 Lugo,
Spain.
Abstract
It has been suggested that sexual signals may be a useful measure of environmental
quality as they represent the sum of environmental pressures on the animal. A good
example of sexual signals are those mediated by carotenoid-based colorations as
exhibited by fishes and birds. Accordingly, it has been proposed that carotenoidbased coloration may be especially valuable in monitoring and detecting the
sublethal effects of toxic pollutants in the environment. Here we evaluate whether
the carotenoid-based coloration in the bill of adult yellow-legged gulls reflects oilinduced sublethal effects in breeding colonies affected by the Prestige oil spill. In
2004, we took a blood sample from twenty seven adult birds that were nest-trapped
while incubating, in four insular breeding colonies located in the pathway of the
Prestige oil spill. We analyzed plasma biochemical parameters indicative of sublethal
effects of oil contamination in gulls, including glucose, total protein, creatinine,
inorganic phosphorus, aspartate aminotransferase (AST) and gamma-glutamyl
transferase (GGT). Moreover, we measured the size of the red bill spot area, in
order to evaluate the effect of oil pollution on this carotenoid-based sexual signal.
We showed that in yellow-legged gulls breeding in oiled colonies, 17 months after
the Prestige wreck, the size of their red bill spot area was positively related to body
condition while negatively related with aspartate aminotransferase (AST) plasma
levels, an enzyme that is commonly used as an indication of hepatic damage in
birds. Hence, the present study provides support to the idea that carotenoid-based
colour integuments may be useful measures of environmental quality. Moreover, we
highlight the importance of the evaluation of sexual signals and the behavior of the
animals when assessing the real impact of oil pollution on wildlife.
189
Introduction
The expression of the ornamental traits often signals reliable information about the
physical condition of the bearer (Hamilton & Zuk 1982; Grafen 1990). Moreover,
due to their high phenotypic plasticity, the expression of sexual signals, relative to
other traits, is particular sensitive to the cascade of physiological mechanisms
produced by stressful events (Hill 1995; Buchanan 2000). Accordingly, it has been
suggested that sexual signals may be a useful measure of environmental quality as
they represent the sum of environmental pressures on the animal (Hill 1995).
Carotenoid-based colorations exhibited by fishes and birds can be
considered as good examples of reliable condition-dependent signals (e.g. Olson &
Owens 1998; Badyaev & Hill 2000; Pike et al. 2007b). Thus, it has been proposed
that carotenoid-based coloration may be especially valuable in monitoring and
detecting the sublethal effects of toxic pollutants in the environment, because
mechanisms underlying both coloration and pollutant damage are interconnected
(Dauwe & Eens 2008). Indeed, we have proved experimentally that oil exposure
promotes carotenoid mobilization with negative consequences on pigmentation in
the yellow-legged gull (Larus michahellis), a seabird that shows intense integumentary
carotenoid-based coloration in both sexes (Anexo 1). These results suggest that
carotenoid-based coloration may be useful for monitoring sublethal effects on
seabirds following catastrophic oil pollution pulses at sea. Here, this hypothesis is
evaluated for the first time in the aftermath of a real oil pollution event, the Prestige
oil spill.
Large quantities of petroleum products are released into the marine
environment as a result of tanker wrecks, thus affecting a broad range of organisms,
including seabirds (e.g. Peterson 2001; Velando et al. 2005). The Prestige oil spill was
the biggest catastrophe of its type in Europe and thousands of seabirds died in the
following months (García et al. 2003, Martínez-Abraín et al. 2006). Although acute
mortality resulting in large numbers of seabird casualties draw much public
attention, long-term sub-lethal exposures to petroleum products have commonly
been ignored (but see Esler et al. 2002; Golet et al. 2002). One of the few exceptions
to date is the yellow-legged gull in northwestern Spain after the Prestige oil spill. In
this species, recent research has shown that the Prestige oil spill was responsible for a
delayed impact of a sublethal nature, operating 17 months after the catastrophe
190
Anexo 5
(Alonso-Alvarez et al. 2007a,b) with birds from colonies that were in the path of the
oil spill showing consistently higher oil contamination levels compared to birds
from unoiled colonies (Pérez et al. 2008b).
In this study, we present seabird coloration as a convenient method for
monitoring bird condition after an acute oil pollution event, with little harm and
disturbance to individuals and populations. As it was previously found that the
Prestige oil spill induced damages on vital organs, such as liver and kidney (as shown
by higher levels of two aminotransferases: aspartate aminotransferase, AST and
gamma-glutamyl transferase, GGT) in yellow-legged gulls breeding at oiled colonies
(Alonso et al. 2007a,b). We evaluate whether variation in carotenoid-based
coloration reflecte these oil-induced sublethal effects in yellow-legged gulls from
colonies affected by the Prestige oil spill.
Material and methods
This study was carried out in four insular yellow-legged gull breeding colonies of
Northwestern Spain (Cíes, Ons, Vionta, and Lobeiras), located in the pathway of
the Prestige oil spill (see Pérez et al. 2008b). In total 27 adult birds (19 females and 8
males) were nest-trapped in 2004 while incubating (May 23 to June 5), 17 months
after the Prestige wreck. Head, bill, and tarsus length were measured (to the nearest 1
mm) and body mass was recorded (to the nearest 10 g). Tarsus length allowed
determination of sex by means of a discriminant function (Bosch 1996). A body
condition index was estimated using the residuals of the regression of body mass
against wing length and sex.
The bill was photographed with a digital camera (Nikon Coolpix 5200)
against a white standard, together with a standard red colour and a millimetric scale,
inside a black box. The distance from lens to bill (15 cm) was held constant. The
size of the red spot area was measured by the same person (C.P.) with the aid of an
image analysis software (analySIS FIVE) blindly with respect to treatment. A test on
the repeatability of the measuring method was performed 3 times on 6 randomly
selected photographs and was very high (r = 0.98, F5,12 = 161.01, P < 0.001). We
focused on the red carotenoid-based spot of the lower mandible because of
previous evidence that gulls that were exposed to the Prestige oil mobilized
191
antioxidants (vitamin E and carotenoids) probably to counteract the toxic effects
provoked by oil, and that this mobilization resulted in a reduction of the size of the
red bill spot area compared to control subjects (Pérez et al. 2008a).
A blood sample (1–2 mL, depending on body mass) was taken from the
ulnar vein with a heparinized 25 G needle. Blood was immediately transferred to
plastic tubes, maintained cooled in ice boxes (4 °C), and centrifuged at the end of
the day. Plasma was removed from tubes and both plasma and blood cell fractions
(pellets) were kept frozen with liquid nitrogen (−196 °C).
Biochemical assays
Blood cells were analyzed to determine and quantify haematological levels of PAHs
that were present in the oil spilled by the Prestige (see Pérez et al. 2008b). Plasma
chemicals were measured in a Lambda PerkinElmer spectrophotometer (Wellesley,
USA) using commercial kits and certified controls from Spinreact labs (Girona,
Spain; http://www.spinreact.com). We concentrated on biochemical parameters
(method in parenthesis) indicative of sublethal effects triggered by the Prestige oil
spill (Alonso-Alvarez et al. 2007a,b): glucose (glucose oxidase), total protein (biuret
reaction), creatinine (kinetic Jaffee reaction), inorganic phosphorus (“iP”;
molybdenum blue reaction), aspartate aminotransferase (“AST”; NADH-method)
and gamma-glutamyl transferase (“GGT”; carboxy substrate). The coefficient of
variation (CV) for all these parameters ranged between 3.79–9.39%.
Data analyses
We tested the effect of the blood levels of PAHs and plasma biochemicals on the
size of the red bill spot area using a General Linear Model (GLM). In the model,
colony and sex were included as factors while date of capture, body condition
(estimated as the residuals between body weight, body size and sex), AST, GGT,
creatinine, inorganic phosphorus, glucose, total protein and PAHs were included as
covariates. Non-significant terms were backward dropped using a stepwise
elimination procedure and afterwards each non-significant variable was added to the
minimal model to estimate the F statistic. Data are expressed as means ± standard
errors.
192
Anexo 5
Results
In the sampled yellow-legged gulls the size of the red bill spot area was not related
to date of capture, colony or sex (p > 0.15 in all cases). Consequently, the final
model on the size of this red ornament included only two variables, body condition
(F1,25 = 7.50, P= 0.011) and plasma levels of aspartate aminotranferase (F1,25 = 6.54,
P = 0.017). Thus, the size of the red bill spot area was positively correlated with
individual condition (Figure 5.1, r = 0.48) and negatively with plasma levels of
aspartate aminotranferase (Figure 5.2, r = -0.46), indicative of liver damage in birds
(Brugère-Picoux et al. 1987, Harr 2002). Blood levels of PAHs and the rest of
biochemical parameters did not correlate with the size of the red bill spot area (P >
0.1, in all cases).
Figure 5.1. Relationship between the size of the red bill spot area and body condition
(estimated as the residuals between body weight, body size and sex) in breeding yellowlegged gulls from oiled colonies (r = 0.48)
193
Figure 5.2. Pictures showing the red bill spot area in two gulls with low (a) and high (b)
plasma levels of AST. The figure (c) shows the relationship between the size of red bill
spot area and plasma levels of AST.
Discussion
We found that, in yellow-legged gulls that were breeding in colonies affected by the
Prestige oil spill, the size of the red bill spot area reflected body condition and hepatic
damage. In earlier studies, we had shown that the Prestige oil spill induced sub-lethal
damages on breeding yellow-legged gulls, as birds from oiled colonies, showed
higher plasma levels of several blood parameters indicative of physiological
disorders including liver and kidney damage (Alvarez et al. 2007a,b). In addition, we
have previously presented experimental evidence showing that, in yellow-legged
gulls, the size of the red spot area of the bill is indicative of the antioxidant status of
individuals (Pérez et al. 2008a) and that exposure to oil pollution promotes
antioxidant mobilization with negative consequences on red spot size (Anexo 1).
194
Anexo 5
Thus, in addition to this experimental evidence, the present study provides support
to the long-standing hypothesis (Hill 1995), that carotenoid-based colour
integuments may be useful measure of environmental quality in the wild, as they
represent the sum of environmental pressures on the animal (including the response
to toxic chemicals).
In this study on oiled gull colonies, we found that individuals with high
plasma levels of aspartate aminotransferase (AST) showed reduced red bill spots.
The AST enzyme is commonly used as an index of hepatocellular disease in birds
(Brugère-Picoux et al. 1987; Harr 2002). AST plasma levels increase after oil
pollution in relation to the activation of the hepatic cytochrome P450 (see Seizer et
al. 2000; Golet et al. 2002), in order to convert hydrocarbons into more polar
compounds that can be more easily excreted from the organism (Meador et al. 1995;
Ramos & García 2007). The cytochrome P450 reaction cycle produces different
reactive oxygen species (ROS), specifically superoxides and peroxides (Lewis 2002)
which the organism will counteract by activating the antioxidant system (Matés
2000; Nordberg & Arnér 2001). After oil exposure, carotenoids are mobilized to
overcome the harmful effects of PAH ingestion (Anexo 1) thus combating the
prooxidant substances generated by the degradation of oil hydrocarbons (Yilmaz et
al. 2007) and counteracting the immunodepressive effects of oil exposure (White et
al. 1994). Hence, birds with high levels of hepatic damage, as shown by high levels
of AST, should be diverting carotenoids away from sexual signals to use them into
the oil detoxification process (Anexo 1).
AST plasma levels in yellow-legged gulls from colonies affected by the
Prestige oil spill, probably reflect hepatic damages derived from Prestige oil pollution
(Alonso-Alvarez et al. 2007a,b), thus suggesting that the levels of this enzyme are
elevated as a consequence of oil exposure (Alonso-Alvarez et al. 2007b). Other
(unknown) toxic compounds could have also contributed to elevated plasma levels
of AST. Gulls feeding on polluted environments as refuse landfills habits are prone
to a higher exposure to toxic compounds (e.g.: Fossi et al. 1995) and yellow-legged
gulls in Galicia are known to feed on refuse. Nonetheless, only 11% of the pellets
collected in the colonies at the time of the study (N = 211) contained remains of
refuse. Further support for the hypothesis that plasma AST levels were mainly
induced by exposure to Prestige oil comes from our previous finding that breeding
195
yellow-legged gulls from these colonies were, at the time, exposed to residual Prestige
oil (Pérez et al. 2008b), with important consequences on AST levels (Alonso-Alvarez
et al. 2007a,b).
Interestingly, in the sampled gulls, the size of the red bill spot area was not
related to blood levels of total polycyclic aromatic hydrocarbons. Since vertebrate
erythrocytes have a finite programmed lifespan in blood circulation (about 30 days
in birds; Clark 1988), blood levels of PAHs are indicative of recent ingestion and
mobilization (Pérez et al. 2008b), rather than the long-term exposure typically
involved in sublethal effects. In fact, an earlier experiment showed that the size of
the red bill spot area was negatively affected by heavy fuel oil exposure, although it
did not correlate with blood levels of PAHs (Anexo 1).
In addition to hepatic damages, we have also found that the size of red bill
spot area is a good indicator of body condition in yellow-legged gulls, in accordance
with a recent study on wild black-backed gulls (Larus marinus), a closely related
species (Kristiansen et al. 2006). A previous experimental study on another seabird,
the blue-footed booby (Sula nebouxii), showed a strong effect of food ingestion
upon carotenoid integument coloration (Velando et al. 2006). Some mechanisms of
carotenoid utilization may explain the reduced red spot signal shown by gulls in low
condition. The absorption and transport of carotenoids can be sensitive to lipids
and lipoproteins (Solomon & Bulux 1993) that are reduced during poor nutritional
conditions (Alonso-Alvarez & Ferrer 2001). However, there was no evidence that
the Prestige oil spill was affecting the nutritional condition of yellow-legged gulls
(Alvarez et al. 2007a,b). Thus, our results suggest that, in gulls, the size of the red bill
spot area is sensitive to several independent environmental pressures, such as
nutritional conditions and exposure to toxic chemicals, as evidenced by AST plasma
levels.
As a conclusion, our study gives support to the hypothesis proposed by
Hill (1995), that carotenoid-based colour integuments may be useful measures of
environmental quality. In a previous experimental study with yellow-legged gulls, we
found that signal expression was reduced after oil ingestion and here we showed
that in colonies affected by an oil spill, the carotenoid based coloration reflected
hepatic damages as those produced by oil ingestion. Moreover, our results suggest
that the Prestige oil spill was responsible for a delayed impact on seabird coloration,
196
Anexo 5
operating 17 months after the wreck. Since carotenoid-based traits have evolved for
social reasons, their disruption as a result of exposure to oil pollution may have
significant consequences in decision-making by gulls during the breeding period,
thus affecting reproductive output. Thus, there is a risk of underestimating the
impact of oil pollution on seabirds by overlooking the behavioural and population
consequences of long-term sublethal effects, such as those derived from impaired
sexual signals. Therefore, an evaluation of sexual signals in the animals affected will
add further knowledge to the assessment of the real impact of oil pollution on
wildlife.
Acknowledgments
We wish to express our gratitude to the staff at the Parque Nacional de las Islas Atlánticas de
Galicia and Naviera Mar de Ons, for logistic support, and Carmen Díez for help in field
work. A.V. was supported by a Ramon y Cajal Fellowship (Ministerio de Educación y
Ciencia, Spain). The present study was founded by the program Plan Nacional I+D+I 20042007 (Ministerio de Educación y Ciencia, Spain).
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Resumen
Resumen
Introducción
Las coloraciones basadas en los carotenoides exhibidas por muchos animales son un
buen ejemplo de señales sexuales honestas dependientes de condición. Esto se cree
que se debe a que el coste que acarrea su mantenimiento solo se lo pueden permitir
los animales que posean una mayor calidad. Sin embargo, la interpretación de la
información que expresan estas señales, así como los mecanismos que determinan
su expresión, han generado una gran controversia en el mundo científico a lo largo
de las últimas décadas. En particular, se sabe que la expresión de estas señales lleva
asociada una plasticidad fenotípica que depende mucho de las condiciones
ambientales, siendo particularmente sensible a la cascada de mecanismos
fisiológicos que se desencadenan en muchos animales durante los episodios de
estrés. En este contexto, se ha sugerido que estas señales podrían ser útiles para
medir la calidad ambiental, ya que es probable que la intensidad de estas señales
represente la suma de las presiones ambientales a las que se enfrentan los animales.
Los carotenoides, además de actuar como pigmentos, también tienen
funciones fisiológicas importantes para los animales. Así, por un lado pueden
estimular el sistema inmune y por otro pueden actuar como antioxidantes,
inactivando especies reactivas de oxígeno (EROs) y protegiendo de esta forma a los
tejidos del daño oxidativo. Dentro de esta última función, se han propuesto dos
hipótesis que sugieren que la intensidad de las coloraciones dependientes de
carotenoides estaría indicando el estado antioxidante del individuo. Una primera
hipótesis sugiere que solamente los individuos con buenas defensas antioxidantes (o
con bajos niveles de EROs) podrían permitirse desviar los carotenoides de su
función antioxidante para usarlos como pigmentos. Como alternativa, la segunda
hipótesis propone que la intensidad de la coloración dependiente de carotenoides
sería un índice de los antioxidantes no pigmentarios del individuo. Esta última
hipótesis se basa en que la coloración de los carotenoides es alterada y destruida por
procesos oxidativos, de tal forma que solamente los individuos con altos niveles de
antioxidantes podrían prevenir la perdida del color de sus carotenoides ya que los
protegerían contra la oxidación. Por lo tanto estas dos hipótesis difieren en el
mecanismo que subyace a la expresión de estas señales
201
Resumen
Como consecuencia de las características funcionales que presentan las
coloraciones dependientes de carotenoides, los animales que exhiben este tipo de
coloraciones son un buen modelo para poner a prueba el papel de estas señales
como indicadoras de contaminación ambiental, además de servir para analizar los
mecanismos que subyacen a la expresión de estas señales.
El objetivo principal de esta tesis es estudiar si las señales sexuales
dependientes de carotenoides que exhibe un ave marina (la gaviota patiamarilla,
Larus cachinnans) pueden reflejar la exposición a la contaminación por petróleo.
Hasta ahora, ningún estudio había contestado a esta pregunta desde un punto de
vista integrador. Así, en esta tesis se analiza el modo en que un contaminante afecta
a un ave marina y como éste provoca efectos en sus señales sexuales dependientes
de carotenoides. Además, se propone el uso aplicado de estas señales como
instrumento para monitorear el nivel de contaminación a la que están expuestas.
Para contrastar esta hipótesis, se usa como modelo de estudio a la gaviota
patiamarilla, un ave marina con un ciclo de vida largo y que exhibe coloraciones
dependientes de carotenoides en el pico. Además, varias colonias de esta especie
estuvieron expuestas a la contaminación marina por petróleo que generó el
accidente y posterior hundimiento del buque petrolero Prestige en las costas gallegas
en el año 2002.
Métodos
El trabajo de campo se desarrolló durante dos periodos reproductivos
consecutivos de la gaviota patiamarilla, durante los años 2004 y 2005. En el año
2004 se estudió el posible uso de la sangre de la gaviota para detectar una
exposición a largo plazo al petróleo, así como el efecto que esta exposición podría
provocar en las señales dependientes de carotenoides. Para realizar este estudio se
eligieron seis colonias de cría de esta especie de gaviota situadas a lo largo de la
costa gallega y una colonia en la costa asturiana. Entre estas colonias, cuatro se
vieron afectadas por la marea negra generada por el Prestige y otras tres colonias no
se vieron afectadas. A las gaviotas muestreadas se les tomó una serie de medidas
morfométricas (tamaño del ala, longitud del tarso y ancho del pico), se pesaron, se
les extrajo una muestra de sangre (para analizar los distintos compuestos
sanguíneos, ver más abajo) y se les quitó una fotografía del lado derecho del pico
(para analizar el tamaño de la mancha roja que poseen estas gaviotas en el pico).
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Resumen
Durante la época reproductiva siguiente, en el año 2005, y con el objetivo
de estudiar si la disponibilidad de antioxidantes y la exposición al petróleo afectan a
la coloración dependiente de carotenoides, se llevaron a cabo dos trabajos
experimentales en la isla de Monteagudo (islas Cíes). En primer lugar se marcaron
una serie de nidos que fueron asignados aleatoriamente a los tres tratamientos
alimenticios: suplemento con vitamina E - antioxidante extracelular incoloro-,
suplemento con petróleo y control –animales sin suplementar-. Una vez que se
completaba la puesta, se detuvo la manipulación de la alimentación y se empezaron
a capturar a los adultos, siguiendo el mismo protocolo de muestreo que en el año
2004. Además a tres gaviotas que habían muerto recientemente por causas naturales
en la colonia, se les quitó el pico para determinar los carotenoides responsables de
su coloración.
Los carotenoides presentes en el plasma sanguíneo de las gaviotas
patiamarillas se determinaron y cuantificaron mediante cromatografía líquida de alta
resolución (HPLC). Así, en esta tesis se muestra el primer trabajo en el que se
determinaron los carotenoides responsables de la coloración del pico de esta
especie. Además de la importancia y novedad de los resultados obtenidos en esta
tesis, se ha desarrollado un protocolo de HPLC que ha permitido determinar y
cuantificar la vitamina E plasmática. Asimismo, usando técnicas de HPLC, se han
podido cuantificar por primera vez 15 hidrocarburos policíclicos aromáticos en las
células sanguíneas de animales silvestres. Además, también se han analizado los
niveles plasmáticos de glucosa, de fósforo inorgánico y de las enzimas aspartato
aminotranferasa (AST) y gamma-glutamiltransferasa (GGT).
Las fotografías de los picos de las gaviotas se analizaron mediante un
programa informático de análisis de imágenes para cuantificar el tamaño de la
mancha roja.
Resultados
Los carotenoides responsables de la coloración del pico son diez, de los cuales seis
pudieron ser identificados mediante la técnica de HPLC empleada. De estos diez
carotenoides cinco estaban presentes tanto en la zona amarilla del pico como en la
mancha roja y fueron denominados como “otros carotenoides”. Además, se
encontró que otros cinco carotenoides estaban presentes exclusivamente en la
mancha roja, denominándose como “carotenoides de la mancha roja”.
203
Resumen
Se encontró que en los machos y durante el cortejo, el suplemento
experimental con vitamina E provocó un aumento en la concentración plasmática
tanto de la vitamina E como de los carotenoides, especialmente de aquellos
carotenoides que eran los responsables de la coloración de mancha roja del pico
(“carotenoides de la mancha roja”). El suplemento con este antioxidante también
afectó a la mancha roja del pico, de tal forma que la mancha de los machos
suplementados fue más grande que la de los machos control.
El estudio experimental sobre el efecto de una exposición al petróleo sobre
las señales sexuales dependientes de carotenoides mostró que las gaviotas expuestas
al petróleo tuvieron mayores niveles plasmáticos de vitamina E y de carotenoides
que las gaviotas control. Estos resultados sugieren una movilización de
antioxidantes después de un evento de estrés oxidativo, como el que se produce tras
la exposición al petróleo. Las hembras presentaron mayores niveles de carotenoides
plasmáticos que los machos, independientemente del tratamiento. Además los
carotenoides plasmáticos se correlacionaron negativamente con los niveles
sanguíneos de los hidrocarburos policíclicos aromáticos. En las hembras, los niveles
plasmáticos de los “carotenoides de la mancha roja” se correlacionaron
negativamente con los niveles sanguíneos de los hidrocarburos policíclicos
aromáticos (HPAs) totales, relación que no se observó en los machos. Por último,
tal y como ocurría con el suplemento con vitamina E, el suplemento con petróleo
también afectó a la mancha roja del pico. En este caso, el tamaño de la mancha roja
de las gaviotas expuestas al crudo del Prestige fue menor que el de las gaviotas no
expuestas.
Se estudió el posible uso de la composición de la sangre de las gaviotas
como un bioindicador de contaminación marina por hidrocarburos policíclicos
aromáticos. Se encontró que las gaviotas que criaban en las colonias afectadas por la
marea negra del Prestige presentaban el doble de concentración de HPAs en la sangre
que las gaviotas que criaban en las colonias no afectadas. Además, se observó que la
concentración total de los HPAs analizados en la sangre de las gaviotas de la colonia
situada en las islas Cíes disminuyó aproximadamente un tercio en dos estaciones
reproductivas consecutivas 2004 y 2005. Por otro lado, se demostró
experimentalmente que la ingestión de petróleo por la dieta se ve reflejada en la
concentración total de los HPAs en sangre de las gaviotas. No obstante, los
204
Resumen
resultados sugieren que su degradación y el metabolismo sanguíneo son específicos
de cada HPA. Así, cuando se estudiaron los niveles sanguíneos de los HPAs en
relación al tiempo que había transcurrido entre el fin de la alimentación y la captura
de los adultos, se observaron respuestas significativas y no lineales en seis
compuestos.
Los resultados del efecto tóxico que puede provocar una exposición a
corto plazo al petróleo en las gaviotas patiamarillas se compararon con los de un
trabajo previo en el que se habían analizado los daños subletales que mostraban las
gaviotas afectadas por la marea negra del Prestige (Alonso-Alvarez et al. 2007)1. Se
encontró que en el trabajo experimental desarrollado en esta tesis, al igual que en el
trabajo previo, las gaviotas expuestas al petróleo presentaron una menor
concentración plasmática de glucosa y fósforo inorgánico que las gaviotas no
expuestas. Sin embargo, el trabajo experimental llevado a cabo en esta tesis
demostró que la exposición al petróleo afectó a los niveles de aspartato
aminotransferasa (AST) de los machos, al contrario de lo que se había observado en
el trabajo de Alonso-Alvarez et al. (2007). En cuanto al efecto de la exposición al
petróleo sobre la actividad plasmática de la gamma glutamiltransferasa (GGT), ésta
fue mayor en las hembras del grupo control que en las hembras suplementadas con
petróleo, al contrario de lo que se había descrito en el trabajo previo. Sin embargo,
al igual que en este trabajo, no se observó ningún efecto en la concentración
plasmática de GGT en los machos
Por último se estudió la utilidad de la expresión de las señales sexuales
dependientes de carotenoides como instrumento
para detectar los efectos
subletales producidos por la contaminación por petróleo. Se encontró que el
tamaño de la mancha roja del pico se correlacionó positivamente con la condición
corporal y negativamente con los niveles plasmáticos de la AST. Esto indica que la
señal sexual dependiente de carotenoides puede reflejar el estado nutricional y los
daños hepáticos sufridos por las gaviotas patiamarillas que criaron en las colonias
afectadas por el petróleo procedente del Prestige (diecisiete meses después de la
marea negra) y por lo tanto puede considerarse como un buen indicador de la
calidad ambiental.
1
Alonso-Alvarez, C., Munilla, I., López-Alonso, M., Velando, A. 2007. Sublethal toxicity of the
Prestige oil spill on yellow-legged gulls. Environment International, 54, 773-781.
205
Resumen
Discusión
Por medio de dos trabajos experimentales, en esta tesis se demuestra por primera
vez, que el estrés oxidativo afecta a la coloración producida por carotenoides de un
ave marina:
-
El aumento de un antioxidante incoloro (situación de bajo estrés
oxidativo), provoca un aumento en los niveles de carotenoides plasmáticos
y esto afecta positivamente a la expresión de la señal dependiente de
carotenoides.
-
Bajo una situación de alto estrés oxidativo (e.g., la exposición a los HPAs),
se produce una movilización plasmática de vitamina E y carotenoides, lo
que afecta negativamente a la expresión de la señal dependiente de
carotenoides.
Los resultados obtenidos afianzan la hipótesis que sostiene que la expresión de la
coloración reproducida por los carotenoides es una señal honesta dependiente de la
disponibilidad de los antioxidantes del individuo. Además, estos resultados sugieren
que un aumento en la disponibilidad de antioxidantes (vitamina E), promueve un
mecanismo activo que incrementa la cantidad de carotenoides, más que producir
una protección pasiva de estos compuestos.
Los resultados derivados de este trabajo son también congruentes con la hipótesis
del compromiso entre el uso de los carotenoides para las funciones vitales y el uso
de estos compuestos como pigmentos. Así, la exposición al petróleo (situación de
alto estrés oxidativo), provoca un aumento en la movilización de carotenoides
plasmáticos acompañado de una disminución en la expresión de la señal
dependiente de carotenoides. Sin embargo, este resultado va en contra de lo que se
esperaría en función de la hipótesis de la protección de los carotenoides por
sustancias antioxidantes. En este trabajo se observa una relación negativa entre los
niveles plasmáticos de los carotenoides y los niveles sanguíneos de los
hidrocarburos policíclicos aromáticos. Esto sugiere que los carotenoides
plasmáticos pueden estar implicados directa o indirectamente en la degradación de
los HPAs. Por lo tanto, el mecanismo que subyace a la expresión de las coloraciones
producidas por carotenoides es el estrés oxidativo, mediando en el compromiso
entre las funciones fisiológicas y pigmentarias de estos compuestos. Así, cuando un
organismo tiene sus funciones antioxidantes cubiertas puede usar los carotenoides
206
Resumen
como pigmentos. Sin embargo, cuando aumenta la demanda de antioxidantes para
combatir los radicales libres generados tras un episodio de estrés ambiental (e.g.,
contaminación), los organismos usan los carotenoides para combatirlos.
En esta tesis también se valida el uso de las señales dependientes de
carotenoides de una forma aplicada. Para ello, se demuestra que las gaviotas
patiamarillas estuvieron expuestas a un contaminante, que este provocó efectos
tóxicos en el organismo y que esta toxicidad se vió reflejada en la expresión de la
señal sexual de los ejemplares expuestos al contaminante. En este sentido, en esta
tesis se muestra, por primera vez, que los hidrocarburos policíclicos aromáticos se
pueden detectar en la sangre de un ave marina y que este análisis refleja muy bien la
exposición a una contaminación por petróleo. Se muestra además que la
concentración en sangre de estos compuestos separa los ejemplares procedentes de
las colonias que estuvieron afectadas por la contaminación de la marea negra del
Prestige de los procedentes de colonias poco o nada afectadas por esta marea.
Asimismo estos resultados observacionales son corroborados experimentalmente,
así las gaviotas expuestas al petróleo procedente del Prestige presentaron mayores
concentraciones sanguíneas de HPAs que los ejemplares no expuestos. Además, la
contaminación en las gaviotas disminuye a medida que aumenta el tiempo
transcurrido desde la exposición al petróleo. Esta reducción sugiere que la
concentración de HPAs en los primeros momentos tras la exposición a la marea
negra del Prestige pudo incluso haber sido mayor que la encontrada en las gaviotas
muestreadas en el año 2004 (diecisiete meses después del accidente). Se señala
también que los HPAs tienen distintos patrones de permanencia en el organismo, lo
que indica probablemente diferentes tasas de metabolización y permanencia en el
hígado de estos compuestos.
Se encuentra además que una exposición al petróleo tiene efectos tóxicos
en las gaviotas patiamarillas, corroborándose de esta forma los efectos subletales
encontrados en esta especie en las colonias afectadas por el derrame del petróleo.
Así, la exposición al petróleo provoca una disminución plasmática de glucosa y
fósforo inorgánico, probablemente debido a alteraciones en el funcionamiento del
hígado. Estos resultados son congruentes con los altos niveles de las transaminasas,
AST y GGT, encontrados en las gaviotas expuestas al fuel y que son indicadores de
daños renales y hepáticos. Además, se muestra una diferente respuesta en machos y
207
Resumen
hembras, de gaviota patiamarilla, en relación a los niveles de AST y GGT tras una
exposición al petróleo a largo y a corto plazo lo que sugiere una diferente respuesta
adaptativa dependiente del sexo en estas aves. Así, este resultado puede estar
relacionado con la diferente movilización de carotenoides encontrada entre machos
y hembras tras una exposición a hidrocarburos. De este modo, las hembras
movilizan más carotenoides plasmáticos que los machos y además en las hembras, la
movilización de los carotenoides plasmáticos responsables de la mancha roja se
corresponde negativamente con los niveles sanguíneos de los HPAs. Este resultado
sugiere que en las hembras podría estar operando algún mecanismo de protección
de la descendencia. Así las hembras podrían usar los carotenoides para combatir los
HPAs evitando así que la toxicidad de estos compuestos llegue a la descendencia a
través del huevo. Aunque son necesarios más trabajos para confirmar esta
suposición.
Por último, la mancha roja del pico de las gaviotas, una señal sexual
producida por los carotenoides, refleja la toxicidad del petróleo en las gaviotas que
criaron en las colonias afectadas por el derrame del Prestige. Aunque no se puede
concluir que la marea negra del Prestige haya afectado a la coloración de las gaviotas
si podemos afirmar que esta refleja los daños hepáticos de los individuos afectados
por esta marea. La mancha roja del pico también indica la condición corporal de las
gaviotas. Sin embargo no hay evidencias de que el petróleo del Prestige afectase a la
condición nutricional de las gaviotas patiamarillas Así, los resultados sugieren que el
tamaño de la mancha roja de las gaviotas es sensible a dos presiones ambientales
distintas, una es la condición nutricional y otra son los efectos generados como
respuesta a agentes tóxicos (como los efectos producidos en los niveles de AST tras
la exposición al petróleo).
En general los resultados mostrados en la presente tesis apuntan a que el
posible mecanismo que subyace a la expresión de las señales sexuales mediadas por
carotenoides es el estrés oxidativo. Además, estos resultados validan el uso de las
señales sexuales como indicadoras de contaminación por petróleo.
208
Abstract
Abstract
Introduction
The carotenoid-based colorations displayed by many animals are considered as good
examples of honest sexual condition-dependent signals. This can be explained due
to the fact that the maintenance of these signals entails an associated cost that only
can be afforded by animals of higher quality. However, there has been controversy
regarding the information conveyed by these traits and the mechanisms underlying
their expression, being subject of intensive research over the last decades. Sexual
signals display high phenotypic plasticity in relation to environmental change, and
their expression is particularly sensitive to the cascade of physiological mechanisms
produced by stressful events. Accordingly, it has been suggested that these
condition-dependent sexual signals may be a useful as bioindicators of
environmental quality, as their expression intensity may represent the sum of
environmental pressures affecting the animal.
Besides being commonly used as colorants, carotenoids also fulfill essential
physiological functions in animals. They either act as inmunoenhancers, or as
antioxidants, thus acting as oxygen radical scavengers (ROS), thus protecting the
tissues of oxidative damage. Accordingly, there have been proposed two hypotheses
that suggest that the intensity of carotenoid-based colorations would indicate the
individual antioxidant status. The first hypothesis suggests that only those
individuals with good antioxidant status (or with low levels of reactive oxygen
species, ROS), could divert the carotenoids of their antioxidant function to be used
as pigments. The second hypothesis proposes that the intensity of the carotenoidbased coloration would be a good indicator of the amount of individual colorless
antioxidants. In this case, and as a result of the oxidative process occurring in the
organism, the carotenoids may be bleached out and only those individuals with high
levels of antioxidants may be able to prevent the carotenoid bleaching. Therefore,
both hypotheses differ in the mechanisms underlying the expression of these
signals.
Due to the functional characteristic of the carotenoid-based signals,
animals displaying these traits can be considered as good models to test hypothesis
regarding the role of these signals as indicators of environmental health. Moreover,
209
Abstract
these traits could also be used to evaluate the mechanisms underlie the expression
of these signals.
The main goal of this thesis is to study whether the carotenoid-based
sexual signals displayed by a seabird, the yellow-legged gull (Larus cachinnans) can
reflect the effects of exposure to oil pollution. This question is analyzed, for first
from an integrative point of view. Thus, in this thesis is demonstrated not only that
the pollutant affect the seagull under study but also the effects that this pollutant
provoke in the sexual signals of this species. Moreover, this work proposes the use
of these traits as a biomonitoring tool for environment pollution. The yellow-legged
gulls was used as a model in this study because of its long life cycle and because this
species shows intense carotenoid-based colorations in the bill. Moreover, several
colonies of this gull have been recently exposed to the marine oil spill released when
the supertanker Prestige sank along the Galicia coast in November 2002.
Methods
The field work conducted to fulfill the studies reported in this thesis was performer
in two consecutives breeding periods of the yellow-legged gulls, during 2004 and
2005.
The use of blood to detect the long-term oil exposure and the effect that
this exposure provokes in the carotenoid-based sexual signals was studied in 2004.
With this purpose, bird sampling was performed in seven insular colonies, six
located in Galicia and one in Asturias (northwest of Iberian Peninsula). Among
these colonies, four were affected by the Prestige oil pollution and others three were
unaffected. Several measurements were taken to the gulls studied, including weight
an morphometric measures (wing size, tarsus length and bill width), blood samples
(to analyze the different blood compounds, see below) and a picture of the right
side of the bill (to analyze the bill red spot). In 2005, two experimental studies were
carried out in the Cíes Island to study whether the antioxidant availability and the
oil exposure affect to the carotenoid-based coloration in the gulls, as well as to test
whether their bloodcould be used as a biomarker to evaluate the toxic effects of oil..
Durin the breeding period of gulls the nests were assigned randomly to one of the
three
experimental
feeding
treatments:
vitamin
E
supplementation,
oil
supplementation and control diet (no supplementation). Once the egg laying was
complete, the adult experimental gulls were trapped in the nest, following the same
210
Abstract
sampling protocol used in the 2004 breeding period (see above). Moreover, the bill
of three gulls that had died recently by natural causes in the colony was extracted in
order to determine the carotenoids responsible of the bill coloration.
The plasma carotenoid concentrations and, for first time, those
carotenoids responsible for the bill pigmentation were analyzed by high
performance liquid chromatography (HPLC). Moreover, a HPLC protocol has been
developed to measure the levels of vitamin E in plasma. Another novelty of this
study is that, for first time, fifteen polycyclic aromatic hydrocarbons (PAHs) were
identified, using same chromatographic technique, in the blood cells of wild birds.
Furthermore, levels of glucose, inorganic phosphorus and the enzymatic activity of
aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) were also
estimated in plasma. Finally, the bill red spot size was analyzed using image analysis
software in the bill photographs.
Results
The HPLC analysis revealed that ten different carotenoids were responsible of the
bill coloration of the yellow-legged gull. Five of these carotenoids were present in
the red and orange bill coloration (hereafter called “other carotenoids”) and the
other five were exclusively present in the red spot (hereafter called “red spot
carotenoids”).
It was experimentally analyzed if the plasma colorless antioxidant
availability (vitamin E) modulates carotenoid-based coloration in a wild population
of the yellow-legged gull. The results showed that the plasma vitamin E and
carotenoids concentration were significantly higher in male gulls that received the
supplementation of vitamin E than in the gulls under the control diet. The
differences in the concentration in plasma carotenoids were associated to the red
spot carotenoids, which were present at higher levels in plasma from male gulls
supplemented with vitamin E than in plasma from controls. Interestingly, vitamin E
supplementation also affected the size of the red bill spot, being larger in the male
gulls fed with vitamin E supplementation.
The experimental analysis of the effects of the oil pollution on the
carotenoid-based sexual signal showed that the gulls exposed to oil presented higher
levels of plasma vitamin E and carotenoids than the control gulls, with the females
showing higher plasma carotenoids levels than males. These results suggest that the
211
Abstract
plasma antioxidants were mobilized after an exposure to oil (i.e., oxidative stress
event). Moreover, the concentration of plasma carotenoids were negatively related
to the blood levels of PAHs. In the females, the levels of “red spot carotenoids”
were negatively related to blood levels of PAHs, but this relationship was not
observed in males. Finally, and as occurred with the supplementation of vitamin E,
oil supplementation also affected the size of the red spot, which was smaller in oil
supplemented gulls than in controls gulls.
Regarding the possible use of seabird blood as a bioindicator of PAHs
pollution in the marine environment, this study demonstrates that gulls from
colonies affected by the Prestige oil spill showed twofold of the HPAs blood levels
than gulls breeding in the colonies unaffected by the spill. Moreover the PAHs
blood levels from gulls of the Cíes island colony decreased 3-fold in two
consecutives breeding periods, suggesting that the concentration of these
compounds in the environment decrease with time. Moreover, the ingestion of oil
in the diet was reflected in the blood levels of PAHs in gulls. However, the results
suggest that the rates of their degradation and blood metabolism are specific of each
PAH. Thus, when the effect of time after ingestion was analyzed, a specific pattern
for each compound was found. Accordingly, six compounds showed significant
non-linear responses.
The results of the short-term toxic effects of the oil ingestion in yellowlegged gulls were compared with the results found in a previous work described by
Alonso-Alvarez et al. 200712 in which the authors analyzed the sub-lethal effects
associated to a long-term exposure to the same oil. In accordance with the results
reported in this paper, in the experimental study conducted in this thesis, gulls fed
with heavy fuel oil showed significantly lower plasma concentrations of glucose and
inorganic phosphorus than controls. Moreover, the effect of oil exposition on AST
activities was evident in males but not in females, which differ from the results
obtained by Alvarez et al. (20071) in which both males and females from colonies
affected to Prestige oil showed higher levels of plasma AST activity. In relation to
GGT activity and in opposition to the results of Alonso-Alvarez et al (2007), control
1
Alonso-Alvarez, C., Munilla, I., López-Alonso, M., Velando, A. 2007. Sublethal toxicity of the
Prestige oil spill on yellow-legged gulls. Environment International, 54, 773-781.
212
Abstract
females clearly showed higher GGT activities than oil females, whereas there were
no differences between males from both groups.
Finally, this thesis analyzed if the expression of the carotenoid-based
sexual signals could be used as tool to detect the long-term sub-lethal effects of
Prestige oil pollution. It was found that the size of the bill spot was related positively
with body condition and negatively with the plasma levels of AST. These results
show that the carotenoid-based sexual signal can reflect the nutritional condition
and the hepatic damages suffered by the yellow-legged gulls breeding in “oiled
colonies” 17 months after the Prestige wreck, and hence can be considered as a good
indicator of environment quality.
Discussion
By mean of two experimental studies, the present thesis showed, for first time, that
the oxidative stress affects the carotenoid-based coloration of a seabird.
1.
The increase of a non pigmentary antioxidant (vitamin E, a situation of
low levels of oxidative stress), cause an increase in the concentration of
plasma carotenoids, which affects positively to the expression of the
carotenoid-based signal.
2.
In a situation of high levels of oxidative stress ((e.g., after a exposure to
PAHs), the plasma vitamin E and carotenoids are mobilized, affecting
negatively to the expression of the carotenoid-based signal.
The results obtained in this study support the hypotheses that the expression of the
carotenoid-based coloration is an honest signal which depends on the individual
availability of antioxidants. Moreover, an increase in vitamin E may promote an
active mechanism that increases the amount of carotenoids rather than a passive
protection of these compounds. The results reported in this thesis are also
consistent with the hypothesis that there is a trade-off between the use of
carotenoids in either physiological or as pigments functions. Accordingly, the oil
exposure (situation of high levels of oxidative stress), causes an increase in the
mobilization of plasma carotenoids accompanied by a decrease in the expression of
the sexual signal. However, this result doesn´t agree with the hypothesis that the
antioxidants protect the carotenoids of their bleaching. A negative relation between
the levels of plasma carotenoids and the blood levels of the PAHs was observed,
suggesting that the carotenoids can be implied directly or indirectly in the PAH
213
Abstract
degradation. Therefore, these results suggest that the mechanism underlying the
expression of the carotenoid-based coloration is the oxidative stress, which
mediates the trade-off between the physiological and the pigment functions of the
carotenoids. Consequently, when the antioxidant functions of an organism are
covered, it can use the carotenoids as pigments. However, when the demand of
antioxidants increase, for example to combat the free radicals generated after a
pollution event, the organism uses the carotenoids to combat the free radicals.
As explained above, the present thesis also validates the applied use of the
carotenoid-based signals. The results show, for first time, that the PAHs can be
detected in the blood of a seabird and that their levels reflect the exposure to oil
pollution. In addition, this study showed that the blood concentration of this
compounds separate the gulls from colonies that are affected by the Prestige oil spill
from those unaffected.. This study also shows that the effect of the exposure to
pollution in the seagulls decrease with the time elapsed since the oil release.
Accordingly, this reduction suggests that the concentration of the PAHs short after
the exposure to the Prestige oil spill could be even higher than the concentration
found in the gulls sampled in 2004 breeding period (seventeen months after the
Prestige wreck). It also point out that the different temporal patterns of PAHs found
in gulls exposed to oil may indicate different rates of metabolization and residence
of these compounds in the liver of the animals.
Another result reported in this thesis is that an acute oil exposure had
toxic effects in seagulls, supporting the sub-lethal effects found in this species in the
colonies affected by the oil spill. Accordingly, the exposure to oil caused a decrease
of glucosa and inorganic phosphorus in the plasma levels, probably due to the liver
malfunction caused by the oil pollution. These results are congruent with the higher
plasma levels of the AST and GGT found in the gulls exposed to oil, which are also
indicators of kidney and liver damage. Moreover it showed a different response in
males and females in relation to the plasma levels of AST and GGT after short- and
long-lasting exposures to fuel oil. This probably suggests a different sex-dependent
adaptative response in this species of seabird. Thus, this result can be related to the
different mobilization of carotenoids found in males and females after an exposure
to oil pollution. Accordingly, females mobilized more plasma carotenoids than
males, and in females the plasma carotenoids responsible of the bill red spot
214
Abstract
coloration was negatively related to the blood levels of PAHs. This could be related
to some mechanism of protection of the offspring, so females could use the
carotenoids to combat the PAHs preventing that the toxicity of this compounds
pass to the offspring through the egg. However, more studies are necessary to
confirm this assumption.
Finally, this study demonstrates that the bill red spot of the gulls (a
carotenoid-based signal), reflects the oil toxicity affecting the breeding gulls from
colonies exposed to the Prestige oil spill. Although it can not be concluded that the
Prestige oil spill affected directly to the coloration of the gulls it caused toxic damage
in the liver of the animals, which in turns cause changes in their coloration. Bill red
spot also reflects toxic damages also indicate the body condition of gulls. However
there are not evidences that the oil from Prestige affected to the nutritional condition
of the yellow-legged gulls. Thus, the results suggest that the size of red spot of the
yellow-legged gulls is sensitive to two different environmental pressures, one caused
by changes in the the nutritional conditions and the other produced as a response to
toxic chemicals, as evidenced by AST plasma levels.
In general the results of this thesis suggest that the possible mechanism underlying
the expression of the carotenoid-based sexual signals is the oxidative stress and
validates the use of the sexual signals as indicators of oil pollution.
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