Social behaviour in sib mating fungus farmers

Social behaviour in sib mating
fungus farmers
Intra- and interspecific cooperation in ambrosia
beetles
Masterarbeit
der Philosophisch-naturwissenschaftlichen Fakultät
der Universität Bern
vorgelegt von
Peter H. W. Biedermann
2007
Leiter der Arbeit:
Prof. Dr. Michael Taborsky
Zoologisches Institut
Abteilung für Verhaltensökologie
Universität Bern
Von ganzem Herzen meinen
Eltern Johanna und Hubert Biedermann gewidmet
Wer nichts weiß, liebt nichts.
Wer nichts tun kann, versteht nichts.
Wer nichts versteht, ist nichts wert.
Aber wer versteht,
der liebt, bemerkt und sieht auch...
Je mehr Erkenntnis einem Dinge innewohnt,
desto größer ist die Liebe...
Wer meint, alle Früchte
würden gleichzeitig mit den Erdbeeren reif,
versteht nichts von den Trauben.
Paracelsus
Jeder kann zaubern, jeder kann seine Ziele erreichen,
wenn er warten kann, wenn er fasten kann.
Hermann Hesse
3
Summary / Zusammenfassung........................................................... 6
Chapter I: General introduction...................................................... 10
What is cooperation? ............................................................................................................ 10
Intraspecific relationships – From alloparental helping to eusociality ................................ 11
Interspecific relationships – From parasitism to mutualism ................................................ 12
The evolution of inbreeding ................................................................................................. 13
The evolution of haplodiploidy ............................................................................................ 14
The evolution and maintenance of agriculture in ambrosia beetles –
A comparison with other insect farmers .............................................................................. 15
Literature .............................................................................................................................. 17
Chapter II: Towards eusociality in Xyleborinus
saxeseni ......................................................................................................................... 20
Introduction ......................................................................................................................... 20
Material & Methods ........................................................................................................... 22
Results................................................................................................................................. 28
Discussion........................................................................................................................... 35
Literature ............................................................................................................................. 38
Appendix 1.......................................................................................................................... 42
Appendix 2: Methodological details ................................................................................ 43
Chapter III: A detailed study of behavioural
observations ............................................................................................................. 46
Results for the different developmental stages and sexes .................................................... 46
Results for behavioural correlations with certain age stages of the gallery ......................... 52
Results on the phenology of galleries .................................................................................. 56
Literature .............................................................................................................................. 60
Chapter IV: Optimal allocation of resources: The
value of males ....................................................................................................... 61
Introduction .......................................................................................................................... 61
Material & Methods ............................................................................................................. 63
Results .................................................................................................................................. 64
4
Discussion ............................................................................................................................ 67
Literature .............................................................................................................................. 68
Chapter V: Chemical analysis of beetle secretions
and actinomycete bacteria ..................................................................... 70
Introduction .......................................................................................................................... 70
Material & Methods ............................................................................................................. 72
Results & Discussion ........................................................................................................... 74
Acknowledgements .............................................................................................................. 76
Literature .............................................................................................................................. 76
Schlussbemerkungen.................................................................................... 80
Curriculum Vitae .............................................................................................. 83
5
Summary
“The fruits of cooperativeness depend upon the particular conditions of the environment and
are available to only a minority of animal species during the course of their evolution.”
(E.O.Wilson in The Insect Societies 1971)
The evolution of cooperation represents a puzzle for evolutionary biology. Since Darwin we
know that, natural selection cannot possibly produce any modification in any given species
exclusively for the good of another species (Darwin, 1859). In the modern, selfish-gene view
it is difficult to explain how cooperation could evolve in the same species, because it is also
vulnerable to the invasion of non-cooperators.
The ambrosia beetles in the tribe Xyleborini are a fascinating model system because they
allow, insights into the factors promoting intraspecific cooperation. Although poorly studied,
species in this tribe are expected to show different levels of sociality, depending on certain
life history traits. Ambrosia beetles are also one of the few groups that are able to grow their
own food. They live in close relationships with mutualistic ambrosia fungi they obligatorily
depend on. Stabilising mechanisms and conflict resolutions in this mutualism are fairly
unknown.
I studied the level of cooperation in Xyleborinus saxeseni and its promoting factors on the
intraspecific level and tried to give insights into interspecific cooperation between this beetle
and the fungi it grows on. The Xyleborini are a species-rich tribe of bark-beetles (Scolytinae)
with haplodiploid sex determination and regular sib mating. This mating habit led to strongly
female biased sex-ratios and relatedness coefficients that approach one within galleries. Males
are flightless and short-lived, while females disperse and excavate breeding galleries in the
hardwood of freshly dead trees. They have mycetangia (spore carrying organs) to vertically
transfer their ambrosia fungi from the natal gallery to a new gallery where they cultivate them
on the surface of the gallery walls. Depending on the species, larvae exhibit
xylomycethophagy or mycethophagy, while adults feed exclusively on the fungus. In all
Xyleborini females show intensive maternal care, including gallery excavation, maintenance
of the fungus gardens, removal of debris from the gallery, and blocking of the entrance tunnel
for protection.
The habit of predispersal mating inside the family and monogynous gallery initiation leads to
high levels of relatedness within a brood chamber. Haplodiploid sex determination causes
relatedness asymmetries inside the gallery: females are more closely related to their sisters
(75%), than they would be to their own female offspring (50%). Immense relatedness and
haplodiploidy, together with high costs on independent breeding and the potential for
participation in brood care within the natal gallery, would predict selection for higher levels of
cooperation within the Xyleborini. Fungus gardening in the strict sense (e.g. tending, cultivar
selection,…) cannot be performed by single individuals, as the entirely eusocial attine ants
and macrotermite termites show. In ambrosia beetles the mutualistic interrelationship with the
fungi is hardly studied, but one eusocial ambrosia beetle is known, and for others less
advanced levels of sociality are proposed.
In Chapter 1 I demonstrate that in Xyleborinus saxeseni adult female offspring remain in their
natal gallery for some time. There is a significant positive relationship between the delay of
dispersal in adult females and opportunities to help the mother (i.e. the number of eggs, larvae
and pupae, that are dependent on brood-care). I was able to produce at least two overlapping
generations in the lab. All individuals spent a large part of their time with mutually beneficial
behaviours and some time with altruistic tasks. Nevertheless, some females reproduce
6
together with the mother in the second generation. This should be expected under favourable
conditions, in a system without much potential for a conflict over reproduction (because of
immense relatedness). However, it is not unlikely that some females refrain totally from
reproduction, for the purpose of helping their relatives. Anyway, to our current knowledge
Xyleborinus saxeseni is a subsocial species, but with the potential for primitive eusociality,
what has both not been described for the tribe Xyleborini before.
These results show that adult daughters are flexible to decide between three strategies: (1) to
disperse under favourable outbreeding conditions, or when the productivity of the natal
gallery decreases, (2) to stay in the natal gallery and help the relatives to grow more offspring,
or (3) to reproduce in the natal gallery. The third strategy is likely for a few daughters if
fungal production is high, because female egg-production is limited to around three eggs per
day. However, female production is only possible when the daughters are fertilized already.
This is one condition for predispersal sib-mating to evolve, and more conditions are discussed
in Chapter 2. Males are produced together with the first females to assure complete
fertilization of sisters. The production of males is limited to a number necessary for fertilizing
all sisters (sex-ratios around 1 male : 20 females), although small galleries stay male-less.
This biased sex-ratio is (1) due to reduction of male-male competition by the mother,
allocating resources in the females instead, (2) potentially influenced by vertically transmitted
bacteria in the female ovaries, that are solely transmitted by the females, and/or (3) might be
caused by the fungus cultivar that is also vertically transmitted by the females.
Mechanisms of fungal agriculture are relatively well studied in attine ants, less in termites and
little studied in ambrosia beetles. The main differences of the ambrosia beetles compared to
the other two groups is that they do not have to provide their cultivars with substrate, but
rather build their galleries into it. Therefore, substrate provisioning as the main task for
workers in eusocial attine ant and termite societies, is not necessary in the ambrosia beetles.
Further tasks of remaining daughters in ambrosia beetles like gallery and garden maintenance
and protection have not been shown clearly to increase productivity of the gallery, although
there are strong hints that they have beneficial influences on the fungi. Beetle excretes have
been shown to get utilized by the fungi again and fungal grazing actually promotes further
sporulation. In Chapter 1 we saw that a decrease in beetles and larvae led at some point to an
invasion of detrimental fungi, that destroyed the gardens and killed the brood.
During the course of their rapid speciation, bark beetles have evolved different levels of
interdependence with their fungi – from parasitic relationships, to commensalistic
relationships, up to mutualistic relationships. Ambrosia beetles obligatorily depend on their
fungi for feeding. It is likely that this dependence is exploited by some fungal partners in
certain species. Nevertheless, fungus cultivation appeared to promote intraspecific
cooperation in ambrosia beetles.
7
Zusammenfassung
„Die Früchte von Kooperation hängen von speziellen Umweltbedingungen ab und sind nur
für eine Minderheit von Tierarten im Verlauf ihrer Evolution erreichbar.“ (E.O.Wilson in The
Insect Societies 1971)
Die Entstehung von Kooperation ist eines der größten Rätsel der Evolutionsbiologie. Seit
Darwin ist klar, dass in durch die natürliche Selektion keine Veränderung innerhalb einer Art
Bestand haben die ausschließlich dem Vorteil einer anderen Art dienen (Darwin, 1859). In der
modernen Ansicht, in der auch Gene egoistisch handeln, ist es sogar schwierig zu erklären
wieso Kooperation innerhalb einer Art entstehen kann. Diese wäre nämlich wiederum durch
Ausbeutung von nicht-kooperativen Elementen anfällig.
Die Ambrosiakäfer des Tribus Xyleborini sind faszinierende Modellorganismen weil sie
Einblicke in Faktoren erlauben, die zu innerartlicher Kooperation führen. Obwohl kaum
bekannt, scheinen Arten dieser Gruppe abhängig ihrer Lebensweise unterschiedliche Stadien
von Sozialverhalten entwickelt zu haben. Ambrosiakäfer sind auch eine der seltenen Gruppen
die fähig sind ihre eigene Nahrung zu züchten. Sie leben in engen Lebensgemeinschaften mit
mutualistischen Ambrosiapilzen, die essentiell für ihr Überleben sind. Den Mutualismus
stabilisierende Faktoren und Konfliktlösungen sind ungeklärt.
Ich untersuchte den Grad der Kooperation und die dafür selektionierenden Faktoren im
Kleinen Holzbohrer (Xyleborinus saxeseni). Ich versuchte außerdem Einsichten in die
Beziehung zwischen ihm und seinen gezüchteten Pilzen zu geben. Xyleborini sind eine
artenreiche
Gruppe
von
Borkenkäfern
(Scolytinae),
mit
haplodiploider
Geschlechtsbestimmung und regulären Geschwisterbruten. Dieses Paarungssystem führte zu
stark weibchenlastigen Geschlechterverhältnissen und Verwandtschaftskoeffizienten von
beinahe 1 innerhalb einzelner Galerien. Männchen sind flugunfähig und kurzlebig, während
Weibchen ausfliegen und Brutgalerien im Kernholz von frischem Totholz anlegen. Sie
besitzen Mycetangien (Organe die Pilzsporen übertragen) um Ambrosiapilze vertikal
zwischen der Geburtsgalerie und einer neuen Galerie zu übertragen. Dort züchten sie dann die
Pilze an den Gangwänden des Galeriesystems. Artabhängig sind Larven mycetho- (nur
Pilznahrung) oder xylomycethophag (Holz und Pilz als Nahrung), während ausgewachsene
Käfer reine Pilzfresser sind. Alle Weibchen der Xyleborini zeigen intensive Brutpflege.
Außerdem erweitern sie das Galeriesystem, Pflegen die Pilzgärten, Entfernen Abfall und Kot
aus den Gängen und blockieren den Eingangstunnel gegen Eindringlinge.
Ihre Lebensweise sich innerhalb der eigenen Familie zu verpaaren und das Gründen von
Galerien durch einzelne Weibchen führt zu hohen Verwandtschaftsgraden innerhalb einer
Brutkammer. Asymmetrische Verwandtschaftsverhältnisse werden durch haplodiploide
Geschlechtsbestimmung verursacht: Weibchen sind enger mit ihren Schwestern (75%)
verwandt als sie es zu ihrem eigenen weiblichen Nachwuchs sein würden (50%). Immense
Verwandtschaft und Haplodiploidität, Schwierigkeiten eigenständig zu Brüten und die
Möglichkeiten bei der Brutpflege in der Geburtsgalerie mitzuhelfen würden alle Selektion in
Richtung von höherem Sozialverhaltens bei Xyleborini erwarten lassen. Pilzzucht in strengem
Sinn (d.h. mit Pilzpflege, mit Selektion von bestwachsenden Pilzen,...) kann nicht von
einzelnen Individuen bewältigt werden, wie auch die ausschließlich eusozialen
(staatenbildenden) pilzzüchtenden Ameisen und Termiten zeigen. Bei Ambrosiakäfern ist die
Beziehung Pilz-Käfer kaum untersucht, aber eine eusoziale Art ist bereits bekannt und für
andere werden weniger hoch entwickelte Sozialsysteme erwartet.
Im ersten Kapitel zeige ich dass erwachsene Töchter des kleinen Holzbohrers eine Zeit in der
Geburtsgalerie verbleiben obwohl sie bereits auswandern könnten. Dabei gibt es einen
8
signifikanten Zusammenhang zur Anzahl vorhandener Eier, Larven und Puppen, die gepflegt
werden müssen. Es gelang mir im Labor zwei überlappende Generationen in einer Galerie zu
züchten. Alle Individuen verbrachten den Grossteil ihrer Zeit mit kooperativen und etwas Zeit
mit selbstlosen (altruistischen) Verhaltensweisen. Einige der Töchter begannen jedoch
gemeinsam mit der Mutter Eier zu legen. Unter optimal Bedingungen, wenn kaum Konflikte
um die Fortpflanzung herrschen (aufgrund der hohen Verwandtschaft) ist das aber zu
erwarten. Allerdings ist nicht unwahrscheinlich, dass manche Weibchen ganz auf die eigene
Reproduktion verzichten um ihren Verwandten stattdessen zu helfen. Nach der gegenwärtigen
Datenlage gehen wir jedoch aus, dass der Kleine Holzbohrer „nur“ eine subsoziale Art ist, mit
dem Potential für primitive Eusozialität. Beides wurde für den Tribus Xyleborini noch nicht
beschrieben.
Diese Ergebnisse zeigen dass erwachsene Töchter flexibel sind zwischen 3 Strategien zu
wählen: (1) Unter optimalen Bedingungen oder wenn die Produktivität der Galerie nachlässt
abzuwandern. (2) In der Geburtsgalerie zu bleiben und Verwandten bei der Brutpflege zu
helfen, oder (3) sich selbst in Geburtsgalerie fortzupflanzen. Die dritte Strategie ist für einen
Teil der Töchter wahrscheinlich, wenn die Pilzproduktion hoch ist. Weibchen können nur
etwa drei Eier pro Tag legen. Möglich ist diese Strategie allerdings nur wenn die Weibchen
bereits befruchtet wurden. Das geht nur wenn sie sich bereits mit Geschwistern verpaart
haben. Andere Bedingungen für die Entstehung von Geschwisterbruten werden im zweiten
Kapitel diskutiert. Männchen werden zusammen mit den ersten Weibchen produziert, um
sicher zu gehen, dass alle Schwestern von Anfang an befruchtet sind. Die
Männchenproduktion ist auf ein Mindestmass reduziert, gerade so viele um alle Schwestern
zu befruchten (Verhältnis 1 M : 20 W). Kleine Galerien haben oft gar kein Männchen. Dieses
verschobene Geschlechterverhältnis könnte verursacht werden durch: (1) Mütter, die BruderBruder Konflikte zu vermeiden versuchen und Ressourcen besser in Weibchen anlegen. (2)
Potentiell, vertikal von Weibchen übertragene Bakterien in ihren Ovarien, die Produktion von
Männchen erschweren. (3) Den Pilze der auch ausschließlich von Weibchen vertikal
übertragen wird.
Pilzzucht ist sehr gut in pilzzüchtenden Ameisen, weniger in Termiten und kaum in
Ambrosiakäfern untersucht. Der Hauptunterschied zu den beiden anderen pilzzüchtenden
Tiergruppen ist, dass die Käfer ihre Pilzgärten im Substrat (Holz) anlegen, anstatt das Substrat
anzutransportieren. Daher entfällt die Beschaffung des Substrats, die bei Ameisen und
Termiten die Hauptaufgabe der Arbeiter (-innen) ist. Weitere potentielle Aufgaben der
Töchter bei Ambrosiakäfern, wie die Aufrechterhaltung und der Schutz der Galerie und
Pilzgärten konnten noch nicht klar als produktionssteigernd identifiziert werden, obwohl es
Hinweise für ihre positive Wirkung auf die Pilze gibt. Exkrete der Käfer werden von den
Pilzen wiederverwertet und das Abgrasen des Pilzes regt diesen zu weiterer Sporulation an.
Im ersten Kapitel wird gezeigt, dass eine Abnahme von Käfern und Larven in der
Brutkammer zu einem Eindringen von Fremdpilzen führt, die ab einem bestimmten Zeitpunkt
die Gärten und Brut zerstören.
Während ihrer schnellen Artbildung haben Borkenkäfer unterschiedliche Grade von
Verhältnissen mit Pilzen entwickelt – von parasitischen und kommensalistischen bis zu
mutualistischen Beziehungen. Für Ambrosiakäfer sind ihre Pilze sogar überlebenswichtig.
Allerdings wird diese Abhängigkeit der Käfer wahrscheinlich von verschiedenen Pilzen
ausgenutzt. Nichtsdestotrotz scheint Pilzzucht bei Ambrosiakäfern innerartliche Kooperation
stark zu fördern.
9
Chapter I: General introduction
Behavioural or physiological mechanisms that operate between an individual and its own
offspring are normally friendly and cooperative. That is probably one reason why lots of
people still believe that cooperative interactions evolve for the good of the species. For
biologists this view has changed since Hamilton (Hamilton, 1964) and Williams (Williams,
1966) showed that natural selection is inherently selfish and cooperative acts can only evolve
under special conditions. All targets of natural selection are thought to maximize their own
fitness, but nevertheless cooperative acts are ubiquitous. Cooperation can be found across taxa
and it pervades all levels of biological organizations from genes to cells to organisms to
societies. That is why mechanisms for the evolution of cooperation are especially interesting
for applied research in genetics (e.g. Burt & Trivers, 2006), microbiology and medicine (e.g.
Crespi, 2001; West, Griffin, Gardner & Diggle, 2006) or economics (e.g. Noe, Van Hoof &
Hammerstein, 2001). Probably the most famous model systems to study these mechanisms
intraspecifically are the order hymenoptera, comprising species with various levels of
sociality. Another promising group to study both intra- and interspecific cooperation is the
bark-beetle tribe Xyleborini, living in close associations with bacteria, fungi and mites. (e.g.
Peleg & Norris, 1972; Norris, Baker & Chu, 1969).
What is cooperation?
Cooperative behaviours by definition provide a benefit to another individual (recipient),
which is selected for because of its beneficial effect on the actor (West, Griffin & Gardner,
2007). This term includes mutually beneficial and altruistic behaviours. Sachs et al. (Sachs,
Mueller, Wilcox & Bull, 2004) proposed direct reciprocity (cooperation with individuals that
return benefits), shared genes (e.g. kin selection) and byproduct benefits (cooperation with
others as a byproduct of selfish actions) as the three general models to explain the existence
and maintenance of cooperation.
In direct reciprocity, an individual benefits a specific partner by accepting own costs. In turn
the partner reciprocates or compensates that benefit back to the donator. The cooperative traits
have to be costly (in contrast to byproduct benefits) and directed reciprocation can operate
between species and non-relatives (in contrast to shared genes). The iterated prisoners
dilemma is the most famous model of direct reciprocity (Trivers, 1971). Cooperation may be
selected if (1) the association lasts long enough that a feedback on the fitness can operate (the
fitness between two individuals are coupled through repeated interactions), or (2) through
choice of cooperative partners. For the first assumption repeated interactions of partners are
needed, while under the second some kind of partner recognition is necessary.
If the partners are members of the same species shared genes can select for the evolution of
cooperation. The cooperative individual does not need to benefit from its act, if it shares a
proportion of its genes (depending on the average degree of relatedness) with the recipient
(Hamilton, 1964). To prevent cheaters exploiting the behaviour an individual has to recognize
kin or shared genes (e.g. “green beard selection”). This partner choice can be dispensable if
benefits are given to relatives based on context-dependent spatial association, as in offspring
sharing a nest. However, competition between close relatives can overwhelm selection for
cooperation in special cases (West, Murray, Machado, Griffin & Herre, 2001; West, Pen &
Griffin, 2002).
10
One-way byproduct cooperative traits benefit the actor and only incidentally benefit others.
That is why it is not seen as cooperation in the original sense. Nevertheless two-way
byproducts are original forms of cooperation, termed byproduct mutualism (West-Eberhard,
1975). Synergistic behaviours that are more profitable when performed in groups, like
flocking, selfish herds and Müllerian mimicry are examples of byproduct mutualism. It
evolves via individual selection whenever the benefits increase disproportionately with group
size. A special example of byproduct mutualism is byproduct reciprocity (or
pseudoreciprocity: Connor, 1986). An individual may be selected to be more cooperative to
an actor from which it receives byproduct benefits.
Fig. 1.1 Picture of an opened brood chamber of Xyleborinus saxeseni, with the entrance
tunnel at the bottom. All larval stages and adult female beetles (that could already reproduce
independently) are visible. The yellowish layers on the chamber walls are the fungal beds. In
the direction of the strains one can see that the wood has stained black, which is caused by the
penetrating fungal hyphens. (Picture by P. Biedermann)
Intraspecific relationships – From alloparental helping to eusociality
Sometimes it is difficult to reproduce individually due to environmental constraints. In such
cases it may be advantageous to increase the transmission of copies of ones own genes by
helping or raising relatives. The higher the relatedness between individuals (potentially due to
11
inbreeding), the higher the probability that such (reproductive) altruism can evolve. In the
absence of inbreeding, unusual levels of relatedness occur in haplodiploid organisms
(relatedness asymmetries). Males are haploid and pass all their genes to their daughters. Full
sisters share more genes with each other (r = 0.75), than they would share with their own
offspring (r = 0.50). Therefore, females should prefer to raise sisters compared to selfish
reproduction (Hamilton, 1964). However, in all of these cases opportunities to help the
relatives are necessary.
If reproductive altruism occurs, individuals within a population show different levels of
lifetime reproductive success (LRS). This intragroup skew in LRS can be displayed for each
cooperative species, on a common scale – the eusociality continuum (Sherman, Lacey, Reeve
& Keller, 1995). This units alloparental helping, common in cooperative vertebrates, and
reproductive division of labour up to eusociality, mainly in arthropods, under one single term.
Haplodiploidy in combination with inbreeding genetically predisposes Xyleborini for helping
each other. Additionally independent gallery foundation appears to be difficult and costly.
However, opportunities for help are relatively small and the breeding substrate (usually in
dead trees) is suitable for only a few generations. The only eusocial ambrosia beetle settles in
living trees and the worker caste has not more than 6 females. The social system that may be
found in some Xyleborini is thus probably more similar to a “helper at the nest”-system found
in some cooperative vertebrate species, where selfish reproduction is delayed for some time.
Interspecific relationships – From parasitism to mutualism
The absence of indirect fitness gains (kin selection) in interspecific interactions led to the
currently accepted view that mutualisms are reciprocal exploitations that nonetheless provide
net benefits to each partner (e.g. Maynard-Smith & Szathmáry, 1995; Doebeli & Knowlton,
1998; Herre, Knowlton, Mueller & Rehner, 1999a). The interests of interacting partners are
hardly ever the same, creating potential for conflicts of interest, that shape or destabilize
associations. Conflicts depend on the extent to which the survival and reproductive interests
of both partners are aligned. There is no general theory of mutualism that approaches the
explanatory power that “Hamilton’s rule” appears to hold for the understanding of withinspecies interactions (Herre et al., 1999a), however there has been tries towards such a
unification (e.g. Foster & Wenseleers, 2006). Factors that align both partners interests have
been identified the passage of the partner from parent to offspring (vertical transmission), the
genotypic uniformity of symbionts within individual hosts, repeated interactions between two
species (spatial population structure), and restricted options outside the relationship for both
partners, all may select for higher levels of cooperation (Herre et al., 1999a). Vertical
transmission is known to lower virulence in parasitic relationships, because both symbionts
and hosts benefit from successful host reproduction that could lead to mutualistic
relationships (e.g. Ferdy & Godelle, 2005; Stewart, Logsdon & Kelley, 2005; Yamamura,
1993). Vertical transmission will tend to reduce genetic diversity by providing a potential
bottleneck each generation (Douglas, 1996). The resulting genetic homogeneity of symbionts
within a host reduces selection for traits on symbionts competition, that would be detrimental
to the host. However, strict vertical transmission is rare, and sporadic cases of horizontal
transmission appear to be the rule (e.g. Herre, Knowlton, Mueller & Rehner, 1999b;
Maynard-Smith et al., 1995; Frank, 1994).
In Xyleborini the fungal cultivars are expected to be clonally propagated (vertical
transmission) within nests and beetle generations, through the mycetangia (fig.1.2) in the
founding females (e.g. Rollins, Jones, Krokene, Solheim & Blackwell, 2001, Francke12
Grosmann, 1967). Fungal hyphens may occasionally penetrate neighbouring galleries, causing
horizontal, inter- or intraspecific exchanges of the cultivar (e.g. Batra, 1963b, Gebhardt,
Begerow & Oberwinkler, 2004). Thus, as in the attine ants (Sachs et al., 2004; Mueller,
2002), the association between beetle and fungal lineages should persist through a partner
fidelity feedback (both interests are aligned), that is occasionally disturbed by horizontal
cultivar transfer. Lab data shows that X. saxeseni tries to build galleries more distantly than
expected from a random distribution, probably to impede horizontal cultivar transfer (own
unpublished data). However, the lateral transmission is potentially most likely from large
productive galleries (galleries with nonexploiting cultivars). The question whether If the
beetle-fungus mutualism is solely held up by partner fidelity feedbacks or additionally by
mechanisms of partner choice (to pick between productive and unproductive cultivars),
requires testing in the future.
Fig. 1.2. Longitudinal section of the mycetangium (mycangium) of Xyleborinus saxeseni.
I Pronotum. – II Mesonotum. – III Metanotum. – Is Intersegmental membran. – d Gland cells.
– dlm Different muscles. – pd Fungal depot. – e Elytra. (drawing from Francke-Grosmann
1956)
The evolution of inbreeding
Genetic distance between mating partners can strongly influence offspring fitness (e.g.
Mitton, 1993). The less related parents are, the higher is the genetic variability of their
offspring. Those offspring are expected to cope best with changing environmental conditions
and parasitic pressures. The same pressures are thought to be responsible for the evolution of
sex. Deleterious mutations are more likely to be expressed in homozygous individuals,
seeming that extreme inbreeding can eliminate the advantages of sex. Under certain
environmental conditions, if it is hardly possible to find a mating partner outside the natal
nest, sibmating might be favoured, especially in populations that already purged their
deleterious mutations and are capable to go through inbreeding periods. Temporal stability of
the environment and low parasitic pressures are further preconditions for a sibmating system
to evolve (see above). Inbreeding bark beetles are especially common on tropical islands.
They are expected to be much better colonizers than outbreeding species, because before a
population can be established they have to go through periods of severe inbreeding (Jordal,
Beaver & Kirkendall, 2001).
13
Xyleborini excavate their galleries in a sterile environment - the heartwood of freshly dead
trees. Pressures from parasites are expected to be quite low (e.g. Kenis, Wermelinger &
Gregoire, 2004; Eichhorn & Graf, 1974) and the environment appears to be relatively stable.
Furthermore, suitable host trees are sparsely distributed in forests, like island in a sea of
unsuitable habitat. I suppose that predispersal mating causing inbreeding is selected because
of these strong environmental pressures. New mutations in each inbreeding group may
relatively quickly lend to speciation. Indeed outbreeding has been found to be less beneficial
compared to inbreeding in Xylosandrus germanus, even if it occurs between neighbouring
galleries (Peer & Taborsky, 2005). All Xyleborini are sibmating, which promoted the rapid
diversification of the tribe: 1200 species of the 3400 usually outbreeding ambrosia beetle
species (including 10 tribes) are found in the tribe Xyleborini (Farrell, Sequeira, O'Meara,
Normark, Chung & Jordal, 2001; Jordal, Normark & Farrell, 2000).
The evolution of haplodiploidy
Inbreeding appears to be associated with haplodiploidy (e.g.Hamilton, 1967, Smith, 2000;
Normark, 2004). The occurrence and probable origin of four of the six lineages of male
haploidy among insects (Hymenoptera, Thysanoptera, Micromalthus, certain Scolytidae) is
under the bark of dead trees (Hamilton, 1978). One of these lineages – the tribe Xyleborini in
the Scolytidae invariably exemplifies this original life-style. The selective force behind the
transition to haplodiploidy and its maintenance is still controversial. There are four main
hypotheses (e.g. Normark, 2004, Smith, 2000, Hamilton, 1967, Hamilton, 1993): (1) Females
that can produce viable fatherless sons have a deterministic transmission-genetic advantage
over females that cannot, (2) purging of deleterious mutations is more efficient when selection
occurs in the haploid phase, (3) haplodiploidy allows maternal control of sex ratio, which may
be advantageous under inbreeding, and maternally transmitted endosymbionts in
heterogametic males destroy or disable the paternal chromosome set, which is then eliminated
during spermatogenesis. Only the X-chromosome is transmitted to the offspring, which are
therefore daughters. In insects, haplodiploidy tends to arise in lineages that rely on maternally
transmitted bacteria for nutrition (e.g. Buchner, 1965) and that have gregarious broods, often
with local male competition. Under brother-brother competition there is strong selection on
less investment in males, and thus selecting on maternally transmitted elements to kill males
(Hamilton, 1993, Normark, 2004). Recent models show that inbreeding and competition
under sibmating are conditions that align interests of the hosts and maternally transmitted
bacteria, by eliminating paternal genes (e.g. Normark, 2004; Normark, 2006; Engelstadter,
Charlat, Pomiankowski & Hurst, 2006; Engelstadter & Hurst, 2006).
In the Xyleborini, specific environmental conditions under the bark of dead trees (e.g.
difficulty to find a mating partner outside the nest, low parasitic pressures, or a relatively
stable habitat) favour predispersal sibmating. Inbreeding, biased sex-ratios and haplodiploidy,
potentially in combination with maternally transmitted bacteria (e.g. Peleg & Norris, 1972;
Peleg & Norris, 1973) appear to have evolved in parallel under these environmental
conditions. The overall life-style in Xyleborini, e.g. the use of inbreeding and male-numbers
that are optimised on outbreeding possibilities and numbers of sisters (Peer & Taborsky,
2004, Chapter II), which optimally conserves resources for perpetuating its genes is
remarkable (Norris, 1993). However, there needs much research to be done in the future.
Questions which should be addressed on: How common are maternally transmitted bacteria in
this tribe? What is the role of the vertically transmitted mutualistic ambrosia fungi in the
evolution of biased sex-ratios and haplodiploidy? Some of these questions will be further
discussed in Chapter III.
14
The evolution and maintenance of agriculture in ambrosia beetles –
A comparison with other insect farmers
The following paragraph reviews briefly fungal agriculture and its potential evolution in
ambrosia beetles, in close backing with a recently published paper on agriculture in insects
(Mueller, et al. 2005). This should provide first ideas to unravel the beetle–fungus symbiosis
in the future.
Agriculture has evolved once in ants (45-65 Mya), once in termites (24-34 Mya) and seven
times in ambrosia beetles (20-60 Mya, once in Xyleborini 30-40 Mya (Farrell et al. 2001)).
The transition to agriculture appears to be irreversible because there is no known case of
reversal from agricultural back to non-agricultural life in any of this nine lineages.
There are two main models for the transition to agriculture in insects (Mueller et al. 2001): (a)
in the “transmission-first” model – which is the likely model for the beetles – the insects first
serve as vectors for the fungi then begin to derive nutrition from them and later become the
fungus cultivators; (b) in the “consumption-first” model (termites) the insects have the fungi
in their generalistic diet, then specialize on the fungi and later adapt to cultivate them. There
is now a third hypothesis where one insect lineage (the attine ants) first exploit an preexisting
insect-fungus association (the fungi ancestral with ambrosia beetles) and later starts to
cultivate them (Sanchez-Pena 2005). This hypothesis is neither supported by the phylogenetic
relationship of the fungi nor by the estimated dates of origin (Mueller, et al. 2005).
Regarding the transition to agriculture in ambrosia beetles some of the seven agricultural
origins appeared to follow the transmission-first route, while others followed the consumption
first-route. Some of the more primitive non-gardening scolytines act as fungal vectors without
dependence on their fungal associates. Others carry fungi in mycangia and feed as larvae on
ungardened mycelium that colonizes host plants and feed as adults on spore layers lining
pupal chambers (e.g. Ayres et al. 2000; see fig.1.3. for different gallery types). The transitions
to agriculture is usually associated with a shift to angiosperm hosts (Farrell et al. 2001).
The origin of 1300 xyleborine species in 20 million years is more than twice the rate for the
Platypodinae (1500 species in 60 million years) and may reflect the combination of polyphagy
enabled by ambrosia feeding together with inbreeding and haplodiploidy in the Xyleborini
(Jordal et al. 2000).
These beetles have adapted to their mutualists by evolving mycangia, modifications of
mandibles and guts, and glandular or physiological modifications. The ambrosia fungi
modified their growth specifically to efficient consumption and digestion by beetle larvae. It
consists of tightly packed conidiophores with copious spores and is only formed in the
presence of the beetles (French and Roeper 1972). Multiple lineages of ambrosia fungi
(Ambrosiella, Rafaelea) have the same ambrosial morphology (Blackwell and Jones 1997),
suggesting that convergent evolution allows for easy grazing by beetles. Additionally
ambrosia fungi are adapted to survive storage in the mycangia of the beetles.
15
Fig. 1.3. Different types of galleries in Scolytides. Black are parts that get tunneled by the
foundress; the rest is excavated by the growing larvae. - a-f.) different bark breeders; - g-k.)
ambrosia beetles: Xyloterus lineatus (g); Xyleborinus saxeseni (h); most common type e.g.
Xyleborus monographus (i); Xyleborus dispar (k); the last three systems belong to the tribe
Xyleborini. (drawings from Lengerken 1939)
All insect fungus gardeners propagate their primary fungi as clonal monocultures within their
nests and mostly across generations too (trophophoresy from parent to offspring in the ants
and the beetles). Sexual reproduction may be disadvantageous by continually breaking up
16
successful gene combinations (Wulff 1985, Six 2003). In the Xyleborini, the primary fungus
is strictly asexual (Rollins et al. 2001), but the auxiliary fungi are often sexual (FranckeGrosmann 1967). Solely asexual fungi might be problematic because of disease control, but
behaviours against that involve (1) to sequester the gardens from the environment (i.e. ants);
(2) to monitor gardens intensively; (3) to access occasionally population reservoirs of
genetically variable cultivars (known for the ants); (4) to manage an array of “auxiliary”
microbes providing disease suppression and other services. Secondary mutualistic microbes
can potentially evolve at the same rate as the coevolving garden pests, enabling quick
responses to the emergence of new disease genotypes (Mueller and Gerardo 2002). The small
size of the garden allows for intensive monitoring by a single or by a small group of females.
Beneficial microbes can rapidly evolve into detrimental ones (Morrissey et al. 2002). The
solutions that the farming insects have to deal with this are (mostly shown in the ants): (1)
selection on spatially limited microbial consortia; (2) propagation of crops with fast
generational turnovers, thus minimizing the time for the evolution of any deleterious traits in
the microbes; and (3) partial or complete sterilization of the substrate before planting.
In ambrosia beetles, as in ants, only one primary cultivar is associated with a particular beetle
species within a particular geographic region. Some beetles are associated with different
primary cultivars in different geographic regions, as a result of cultivar exchange between and
within beetle species (Batra 1963, Gebhardt et al. 2004). This is possible when different
female beetles colonize the same tree and the fungi cross-contaminate neighbouring galleries.
Xyleborinus saxeseni harbours different primary cultivars in North America and in Europe
(Francke-Grosmann 1975).
If the gardening insects are removed or if they abandon their nests, the garden is quickly
overrun by “weedy” fungi. These fungi normally coexist at low levels along with the crop
(Leach et al. 1940, Batra 1979, Norris 1979). By sequestering the gardens in galleries in
wood, they are buffered against fungivores, wind-borne pathogens and arthropods (e.g. mites,
collembolans). Temperature, humidity and level of facilitating microbes can also be regulated.
Sociality may have facilitated the evolution of agriculture because the benefits of agriculture
increase with the division of labour. Termites, attine ants and one ambrosia beetle are
eusocial, while the other known ambrosia beetles are subsocial (a single female cares for her
brood) or communal (several reproductive females cooperate in gardening and brood care)
(Mueller et al. 2005).
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19
Chapter II: Towards eusociality in
Xyleborinus saxeseni
Biedermann, Peter H.W.1; Taborsky, Michael.1
1
Department of Behavioural Ecology, Institute of Zoology, University of Berne,
Wohlenstrasse 50a, CH-3032 Hinterkappelen, Switzerland
Correspondence: [email protected]
Advanced sociality should be expected in the haplodipoloid Xyleborini, a tribe of
ambrosia beetles (bark beetles; Coleoptera: Curculionidae: Scolytinae) cultivating
fungi. All other known fungus cultivating insects (the attine ants and macrotermitine
termites) are eusocial, and eusociality has been described in one species of
ambrosia beetles. Xyleborinus saxeseni lives in large colonies and shows delayed
dispersal of adult daughters, which makes it particularly likely to show advanced
levels of sociality. We established a breeding population of this species in the lab and
conducted the first extensive behavioural study performed on ambrosia beetles. Our
results show that all developmental stages help in cooperative fungus garden
maintenance and mutual grooming. Additionally, larvae and females take part in
altruistic cleaning and protection of their home galleries. The retention period of
females in the natal gallery after reaching adulthood depends upon the number of
offspring present. Some daughters do reproduce in their natal gallery. Conflict over
reproduction is probably low or absent, however, because of high relatedness due to
regular sibling mating and haplodiploidy. We discuss the excellent suitability of
ambrosia beetles for studies of evolutionary mechanisms underlying cooperative
breeding and eusociality.
Introduction
The focus on social evolution has diverged between studies of different taxa (Helms
Cahan et al. 2002). While vertebrate studies have focused traditionally on ecological
factors promoting sociality (e.g. Alexander et al. 1991, Koenig et al. 1992, Cockburn
1998), research on social insects has mainly addressed the genetic structure within
the group (e.g. Hamilton 1964, Bourke and Franks 1995, Queller and Strassmann
1998). The highest level of cooperation among animals is characterized by a
combination of overlapping generations, cooperative brood care and reproductive
division of labour, and has been termed “eusociality” (Batra 1966a, Michener 1969,
Wilson 1971). It is known from some groups of insects, particularly the termites and
many hymenoptera, and a few other taxa (Costa 2006). To unravel evolutionary
mechanisms underlying advanced sociality, taxa with variable levels of sociality
should be studied where the importance of genetic, ecological and demographic
factors can be tested separately. Hamilton (1967, 1978) proposed that the ancestral
habitat and conditions in which a genetic predisposition (haplodiploidy) for higher
20
sociality has evolved most often is behind the bark of dead trees and under high
levels of inbreeding (see also Normark 2004). At least two of the most highly
advanced forms of social life have originated in this type of habitat (termites and ants
(Hamilton 1978)). There is one more group exemplifying Hamilton’s “biofacies of
extreme inbreeding and arrhenothoky”, which also uses this habitat: the beetle family
Curculionidae, comprising among others the ambrosia beetles (subfamilies
Platypodinae and Scolytinae; (Kirkendall 1993, Normark et al. 1999). Despite being
of exceptional interest in the context of evolutionary mechanisms of sociality (e.g.
Hamilton 1978, Kirkendall et al. 1997, Costa 2006), the social behaviour of these
beetles has been hardly studied. There is one species known that is probably
eusocial (Kent and Simpson 1992), and others are apparently subsocial (a single
female cares for the brood) or communal (several reproductive females cooperate in
brood care (Mueller et al. 2005). For studies of social evolution the great advantage
of these beetles over other taxa is that they comprise species with different mating
systems (exhibiting both inbreeding and outbreeding), variation in their genetic makeup (diploidy / haplodiploidy) and life histories adapted to different environmental
conditions. Cooperation and advanced sociality should be expected particularly in
species that live in mutualistic association with fungi and which have lost their ability
to digest wood (e.g. Francke-Grosmann 1956, Batra 1963, Haack and Slansky 1987):
the ambrosia beetles. In one tribe of this group, the Xyleborini (Scolytinae), which is
entirely haplodiploid and shows a very high degree of inbreeding, the demands of
fungus agriculture may render cooperation highly profitable (Mueller et al. 2005).
This largest tribe among the ambrosia beetles includes more than 1200 species
(Normark et al. 1999) and is the only other haplodiploid lineage in holometabolic
insects apart from the hymenoptera (Mable and Otto 1998). Both, rapid diversification
and haplodiploidy are likely to be a result of strong inbreeding and living in closed
social groups under the bark, which favours local mate competition (Jordal et al.
2000, Normark 2004). Mating takes place almost exclusively between siblings within
the natal brood chamber, and haplodiploid sex determination allows females to
allocate resources optimally to offspring by biasing their sex-ratio (Peer and Taborsky
2004). In all Xyleborini sex-ratios are highly female biased and usually one to three
brothers fertilize all their sisters, which may sometimes exceed 100 in number
(Fischer 1954, Hosking 1972). Males develop from unfertilised eggs and are smaller
than females, they are unable to fly and never fully sclerotize. The regular inbreeding
among full siblings leads to relatedness levels approaching one within colonies. This
means that females are equally related to their sisters, own offspring and all other
offspring produced by colony members.
Xyleborini in temperate regions settle in freshly dead trees, soon after bacterial
fermentation has started. Favourable hosts are rare and usually outspaced, and flight
mortality due to predation and adverse environmental conditions like strong winds is
potentially high (Dahlsten 1982). After finding a suitable host the foundress
excavates a gallery and inseminates it with spores of the ambrosia fungus she
carries in special mycetangia (Francke-Grosmann 1956, Batra 1963). The start of
fungus cultivation is difficult because the substrate is often unsuitable for growing
fungi or even detrimental to them, which leads to failure rates of up to 80% in
Xyleborinus saxeseni in the field (Fischer 1954, Peer and Taborsky 2007). The
alternative to this risky dispersal with high failure probability is to help family
members in an already established, productive (natal) gallery. Dispersal decisions
should depend on the expected future productivity of the natal gallery and ecological
factors influencing the success probability to found an own gallery. If females decide
21
to stay, they can either help to raise siblings or reproduce by themselves. Due to the
obligatory inbreeding, conflict over reproduction is probably very low. In Xylosandrus
germanus the proportion of sibmating s = 0,96 (inbreeding coefficient F = 0,85; K.
Peer pers. comm.). Behavioural observations of the closely related species X.
saxeseni suggest that male dispersal and outbreeding is even less frequent (own
obs.). In this species only a very small proportion of nests is successful, contributing
to the next generation by producing large galleries with up to several hundred
individuals (Hosking 1972; own obs.).
X. saxeseni is predisposed to a high level of sociality because (1) it colonizes on host
trees very early in their degradation process, which provides the opportunity for
rearing successive generations in the same gallery system, and (2) it excavates big
larval chambers, which causes individuals of different generations to be in close
contact with one another and favours cooperative behaviours among group
members. In accordance with this expectation a previous field study found that
daughters delay dispersal from their natal galleries when there are high numbers of
dependent offspring present (Peer and Taborsky 2007).
In this study we established groups of X. saxeseni in the lab to answer the following
questions: (1) Do females behave cooperatively before dispersing from their natal
galleries and to what effect for other group members? (2) Do group members perform
altruistic behaviours? (3) Which factors determine the retention period of adult
daughters in their natal gallery? (4) Do daughters reproduce in their natal galleries?
Material & Methods
Study system
After dispersal from their natal gallery, solitary females of X. saxeseni excavate a
gallery (see fig. 2.1.) in a freshly dead tree and cultivate a primary ambrosia fungus
(Ambrosiella sulfurea) together with some auxiliary fungi on the surface of the gallery
walls. After this fungus has started to grow, the female oviposits 5 -10 eggs in a sidebranch of the main gallery (Batra 1963), own lab data). If the ambrosia fungus does
not grow or other fungi invade the tunnels, foundresses sometimes keep up
tunnelling for 12 –14 weeks or disperse again before (Hosking 1972; own lab data).
~
In the lab they usually lay the first egg after 5 to 50 days (N = 93, X = 18d). Eggs
hatch about four days later, and 1st instar larvae moult to 2nd instars within three
days. 2nd and 3rd instar larvae are not distinguishable by sight. The developmental
period of these two instars becomes more plastic, depending on the amount of
fungus accessible to the growing offspring. On average both instar periods together
take six days (N = 12, Range = 4 –17d). Pupae hatch within another six days. The
time to full sclerotization depends on the amount of food accessible to the teneral
beetles and takes on average 9 days (N = 11, Range = 6 –15d; own lab data).
In the lab brood sizes are usually small with up to 30 emerging females in total,
although some galleries may produce up to 90 females (own obs.). Under favourable
conditions in the field brood sizes of several hundreds coexisting in one or more
brood chambers of a gallery system have been reported (Hosking 1972, Peer and
Taborsky 2007). In contrast to most other Xyleborini, developing larvae do not
develop in separate branches of the gallery, but enlarge a central brood chamber
together by xylomycetophagous feeding (Roeper 1995). Males are nearly blind,
smaller than females and never fully sclerotize. Due to local mate competition, sex
22
~
~
ratios are highly female biased ( X = 0.07 in the field, Peer and Taborsky 2007; X =
0,04 in the lab, own data). Despite extreme cases of up to 80 females to one male,
only 3,2% of founding females are not fertilized and produce solely male offspring.
~
Founder females terminate egg production on average within 35 days ( X , Range =
15 – 65d, N = 93), but there is potential for a second period of egg production.
Habitat desiccation is the main reason of gallery abandonment by the beetles in the
lab (28,1 %), followed by “invasion of green mould” (26 %), “invasion of mites” (15,6
%), and “invasion of other fungi” (13,5 %; own data). In the field tree trunks can hold
humidity for a longer time and may provide a suitable habitat for at least two or three
overlapping generations. In good hosts, two years of gallery maintenance could be
possible (e.g. Lengerken 1939, Hosking 1972).
Fig. 2.1. a-b.) Brood chambers of Xyleborinus saxeseni in hard wood (vertical section;
natural size); - c.) two affiliated brood chambers (natural size); - d.) two associated brood
chambers (slightly reduced size). (drawings from Lengerken 1939)
Observations
We bred X. saxeseni in the lab in glass tubes filled with artificial medium mainly
containing sawdust (for details see Peer and Taborsky 2004). We used one founder
female per tube, which had been stored in the fridge for at least one night. These
females had been collected in the field or originated from a lab brood. Methods of
23
gallery collection in the field and lab rearing are reported in more detail elsewhere
(Biedermann 2007). In the breeding tubes, large parts of the galleries and brood
chambers were excavated along the tube wall, which facilitated observations of
activity and development. When a gallery was established successfully the tube was
wrapped in paper to keep galleries dark, but light could enter through the entrance.
For observations the tubes were put under a binocular (6,4x – 16x magnification) with
an artificial light source (max. 6W). In a pilot study we found that larval and adult
beetle behaviour was disturbed neither by the light source nor when the gallery was
turned changing the axis of gravity (unpublished data). After the observations tubes
were immediately wrapped in the paper again and stored in a climate chamber in the
same position as before.
Fig. 2.2. Various stages of Xyleborinus saxeseni. 1.) Egg. – 2.) 1st instar larvae. – 3.) 3rd
instar larve. – 4.) Female pupae. – 5.) Female imago. – 6.) Male imago. – 7.) Elytral declivity.
– 8.) Mouth parts of larvae. – 14.) Male genitalia. (drawings from Hopkins 1898)
24
Behavioural scans of all visible individuals were made at different stages of the
gallery, encompassing its development from initiation to abandonment. Larvae in the
1st instar stage were distinguished by size from larvae in the 2nd and 3rd instar stages.
Pupae and adult beetles were sexed by morphology and size. “Teneral” adults are
recently-hatched beetles with weak sclerotization and brownish elytra. Fully
sclerotized females are dark-brown to black (see fig. 2.2. for the different
developmental stages and sexes). Age was not determined in adult males, because
they never fully sclerotize. We differentiated between 15 behaviours as described in
table 1. For further analysis these behaviours were combined into four behavioural
classes: altruistic behaviours, cooperative or mutually beneficial “grazing” behaviours,
selfish “feeding” behaviours, and other selfish behaviours. Behaviours that are costly
to the actor and beneficial to the rest of the group, now or in the future, were termed
altruistic. We identified “blocking”, “shuffling” and “digging” as altruistic behaviours in
adult beetles and “balling” and “shuffling” as altruistic behaviours in larvae.
“Blocking”, “digging” and “shuffling” were described in Xyleborini before (e.g.
Kirkendall 1983, 1993), but to our knowledge the altruistic behaviours in the larvae
have not been observed in any other bark beetle species. The cooperative or
mutually beneficial behaviour “grazing” (from wall / from gallery members) is
beneficial for both, the actor and other group members. All other behaviours were
either termed as selfish “feeding”, other selfish behaviours or not classified, because
they were performed only by males (mating behaviours) or were shown outside of the
gallery (dispersal behaviour) (for semantics see West et al. 2007).
We performed scan observations which produced similar results as focal individual
observations as found in a pilot study (Biedermann 2007), but which were less time
consuming. In five galleries we were able to distinguish between the founder females
and their adult daughters, because the latter remained lighter than their mothers for
some days. In these galleries we made focal observations (10 min) to test for
differences between the time budgets of founder females and their adult female
daughters. Additionally, we compared all behaviours of larvae, females and males
between six successive gallery stages: (1) founder female with or without eggs, but
without hatched offspring (N = 4), (2) founder female with eggs, larvae and pupae (N
= 5), (3) founder female with larvae and pupae, but without eggs (N = 4), (4) all
stages (adult / teneral beetles, larvae and pupae) and with eggs (N = 14), (5) all
stages but without eggs (N = 23), and (6) post larval period (no eggs, larvae and
pupae) (N = 16).
25
Behaviour
Shown by
Balling
L
Shuffling
L, F
Blocking
Digging
F
M, F
Definition
to bend the body ventrally and form balls
of frass and faeces
to move frass and faeces with the body (L)
or the legs and elytra (F)
to be inactive in the entrance tunnel and
close it
to excavate new tunnels
to groom an egg, larva, pupa, or adult
beetle with the mouth (L) or tasters
(maxilla, labium)
to groom or feed on the fungal layer on the
gallery walls (M, F), or fungal hyphens
containing medium (L), with the tasters
and/or mandibles
to feed on the fungal layer covering the
glass of the tube with tasters and mandibles
to eat a larva, a pupa, or an adult beetle
with the mandibles
Class
A
A
A
A
C
Grazing from gallery
members (grooming)
L, F, M
Grazing fungus from
wall
L, F, M
Grazing glass
L, F, M
Cannibalising
L, F, M
Inactive
L, M, F
to be inactive without moving
SO
SO
C
SE
SE
Defecating
Locomotion
Self grooming
L, M, F
to defecate
L, M, F
F
Mating attempt
M
to creep (L) or walk (M, F)
SO
to groom oneself with the legs
SO
to mount something, a teneral or adult
female and extending the copulatory organ
Mating
M
to copulate with a female
Dispersing
F, M
to walk on the outer surface (M, F) and
attempt to fly away (F)
Table 2.1. Ethogram of the observed behaviours of larvae (L), females (F) and males (M).
The last column combines the behaviours in four groups,that were used for analysis: A –
altruistic behaviours, C – cooperative / mutually beneficial behaviours, SE – selfish feeding
behaviours and SO – other selfish behaviours.
Dissection of the ovaries
Female ovaries were dissected after storage in 95% Ethanol. Dissection was done
from dorsal, under a binocular with 6,4x – 40x magnification with very fine tweezers
(Dumont medical 5; Dumont biology 5). In X. saxeseni fully developed ovaries consist
of two pairs of ovarioles (see fig. 2.3.). Ovaries were separated in three distinct
classes: (1) not or not fully developed ovaries (not all ovarioles are developed), (2)
mature ovaries (all four ovarioles are developed, but no oocytes are visible), and (3)
egg carrying ovaries (ovarioles contain one or more oocytes). For this study we only
distinguished between ovaries with (class 3) or without eggs (classes 1 and 2).
26
We dissected teneral and adult females from 16 field galleries collected at the end of
summer 2001 in the Spilwald. All females from galleries containing eggs and a
minimum of 15 females from galleries without eggs that contained more than 20
females were dissected. A few females could not be dissected, because they had
~
been destroyed during gallery collection (dissected females: X = 93,5%, Range = 19
–100, N = 16). Thus, we always consider the number of egg-layers found in a gallery
as a minimum estimate. However, the distinction between monogynous and
polygynous (multiple egg-layers) galleries is important: With one exception all
monogynous galleries were fully dissected; in this one gallery all non-dissectible
females (59,3%, N = 27) were very light tenerals; i.e. they could not have produced
eggs. For all galleries of which females were dissected data were available on the
number of eggs, larvae, pupae, tenerals and adults, in additionally to data on
dispersal activity. Dispersal was recorded during the period from the foundation of the
~
gallery until it was dissected ( X = 72 days, Range = 19 –309d, N = 9). In seven
galleries the dispersal activity was not known: However, a previous study showed
that 90% of galleries that had not yet started dispersal contain 18 females or less
(Peer unpublished data). Therefore, we classified four of the seven galleries
(containing 26, 31, 33 and 36 females) as “dispersal started”.
A
B
Fig. 2.3. Fully developed female reproductive organ of Xyleborinus saxeseni. A: Terminal
filaments (Terminalfilamente); Oocytes (Eiröhren); Oviductus (Eileiter); Uterus (Uterus);
Spermatheca (Receptaculum seminis); attached gland (Anhangdrüse); Spermiduct
(Samenkanal) (drawing from Fischer 1954); B: Picture of egg-carrying ovaries (by P.
Biedermann).
Foundress removal experiment
In this experiment we checked for the influence of the presence of a foundress on the
behaviour and dispersal activity of daughters. We removed the blocking foundress
together with some medium from half of the galleries and measured behaviour in the
remaining daughters on four days before the intervention and four days after it and
27
compared it to the behaviour of a control group recorded for the same period. We
recorded all behaviours listed in table 1 and additionally the number of dispersing
daughters during these periods. The foundress was removed by skimming the first
centimetre of medium with the blocking individual, which was dissected to check its
reproductive status. If this female had ovaries with eggs it was regarded to be the
foundress, and if this was not the case this gallery was excluded from the
experiment. In the control group we skimmed the first centimetre of the medium at
the time when no female was blocking.
Statistical analyses
For measuring time budgets the proportion of time that all individuals belonging to a
certain developmental stage (e.g. 1st instar larvae, teneral females, etc.) spent with a
behaviour in one gallery was combined (i.e. each gallery contributes one value). The
distribution of behavioural data was usually non-normal. Therefore, we used mainly
non-parametric statistics. Friedman’s ANOVA, with post hoc comparisons (see Siegel
and Castellan (1988), pp. 180-181), was used for analysing the proportion of time
different developmental stages and sexes from the same gallery spent with selfish or
cooperative behaviours. For comparisons between single behaviours from the same
gallery we took the Wilcoxon matched pairs signed-ranks test. Two independent
groups were compared by the Mann-Whitney U-test. If data were not significantly
different from normal distributions (p>0.1) we used the t-test to compare two groups.
For correlation analysis we used either Kendall’s tau (Bonferroni corrected) or
Spearman rank correlation coefficients. We used Kendall’s tau instead of the
Spearman’s coefficient because it should preferably be used in small data sets with a
large number of tied ranks (Field 2005), which was often the case. Analyses were
performed with SPSS (Release 14.0, © SPSS Inc., 1989-2005).
Results
1. Cooperative behaviours and their mutual benefits
All larvae and females showed behaviours that are potentially of mutual benefit,
about as often as purely selfish behaviours (fig. 2.5., for behaviours see fig. 2.4.).
Grazing was the most common behaviour in larvae and females, occurring in more
than 75% of the galleries. All developmental stages and sexes differed in the
proportion of time spent with selfish feeding (Friedman: χ ² (4) = 30.34, p < 0.001, N =
24) and cooperative grazing (Friedman: χ ² (4) = 12.24, p = 0.016, N = 24). Both
behavioural classes were more common in teneral females than males. The relative
proportion of grooming, as a component of grazing increased when females reached
adulthood: pure feeding decreased significantly (p < 0.05; difference between rank
sums = 1.68, critical value = 1.07), while grazing did not change significantly (p >
0.05; difference between rank sums = 0.5, critical value = 0.64). In the period without
larvae teneral females tended to graze less than before (stage 7 vs. 5+6; t-test, t =
1.975, p = 0.057, N = 36 + 8). Adult daughters and the founder female showed no
significant difference in all recorded behaviours under focal animal sampling (U-tests:
p > 0.05, N = 5 galleries).
The body surface of all single beetles (primarily founding females) was overgrown by
a thin fungal layer within a few weeks. This fungal growth apparently was responsible
28
for the death of the single founder female in at least 7 out of 29 documented cases.
We made similar observations with single beetles close to the abandonment of a
gallery. However, if individuals of different stages encountered each other they
removed the fungal layer by grazing, which kept them perfectly clean.
An adult
female grazes a pupa
An adult female grazes the wall
A larva grazes the wall
A male
grazes an adult female
A larva grazes a pupa
Fig. 2.4. Cooperative grazing behaviours in the different developmental stages and sexes.
In six galleries mould invaded and overgrew everything except some millimetres
around the brood chamber (fig. 2.6.). The ambrosia fungus was able to suppress the
~
invasion for around 13 days ( X , Range = 4 –40d, N = 5). It was overgrown, however,
when the number of individuals in the brood chamber decreased from 9 individuals
~
~
( X , Range = 2 – 20, N = 5) to 2 individuals ( X , Range = 1 - 14, N = 5).
29
Selfish
Cooperative
proportion of time
1.0
*
0.8
*
0.6
*
0.4
0.2
0.0
Others:
inactive
locomotion
l rd Teneral
tf
1sstl
2 ndb/3
instars instars
♀♀
af
Adult
♀♀
m
♂♂
selfish feeding
l rd Teneral
tf
1sstl
2 ndb/3
instars instars
♀♀
af
Adult
♀♀
grazing (wall / individuals)
m
♂♂
l rd Teneral
tf
1sstl
2 ndb/3
instars instars
♀♀
af
Adult
♀♀
m
♂♂
Fig. 2.5.: Proportion of combined behaviours from different developmental stages: other
selfish, selfish feeding and cooperative grazing (Median over all galleries, 25% and 75%
quartiles). * p < 0.05 (Friedman test with post-hoc comparisons: N = 24 galleries, k = 5).
Fig. 2.6. Mould is invading the gallery from the top, but is not able to enter the brood
chamber.
30
2. Altruistic behaviours
Purely altruistic behaviours (see behaviours in fig. 2.7.) were found when larval and
teneral offspring were present (stage 2 – 5). Balling and shuffling larvae were
observed in more than 75% of the galleries, and behavioural frequencies increased
after the first moult (Wilcoxons signed-ranks test: z = -2.06, p = 0.038, N = 36) (fig.
2.8.). Altruistic behaviours were more common in females than in males (Wilcoxons
signed-ranks test: z = -2.589, p = 0.007, N = 30). In contrast to founder females and
their adult daughters, teneral females never showed “blocking” of the entrance tunnel
(Wilcoxons signed-ranks test: z = -2.81, p = 0.002, N = 36). It was usually performed
by one individual at a time (in only 5.7% of all cases there were two blocking
individuals, N = 35). In the larval period without teneral daughters present (stages 2,
3) the probability to find an adult female blocking per behavioural scan was
significantly higher than later (stages 4, 5; Mann-Whitney U-test, U = 94.00, p =
0.006, N = 9 + 37). The relative time adult females spent blocking per gallery
correlated positively with the other altruistic female behaviours “digging and shuffling”
(Kendalls τ = 0.375, p = 0.012, N=37).
A larva balls frass
An adult female digs a tunnel
A teneral female shuffles larval frass
An adult female
blocks the entrance tunnel of a field gallery
Fig. 2.7. Altruistic behaviours in larvae and females.
31
Shuffling, blocking
and digging
Balling and shuffling
0.00
0.20
0.40
0.60
0.80
1.00
0.20
0.40
0.60
0.80
1.00
0.00
0.20
0.40
0.60
0.80
1.00
**
0.00
*
0.00
0.20
0.40
0.60
0.80
1.00
Fig. 2.8.: Histogram of combined altruistic behaviours. Gallery-wise comparisons between
these behaviours. Balling and shuffling in 1st instar larvae (N = 37) compared to 2nd/3rd instar
larvae (N = 39); shuffling, blocking and digging in teneral females (N = 33) compared to
adult females (N = 37). * p < 0.05, ** p < 0.01 (Wilcoxon signed-ranks tests).
3.1. Female dispersal
Daughters did not disperse before their full sclerotization. Adult females continuously
dispersed during stages 4 -6. If the total brood size was small and the developmental
period was short, the founder female sometimes dispersed in at least eight cases
after her offspring reached adulthood (N = 93). In six cases she left the brood earlier
(stage 3) because larvae died prematurely. At least in the latter cases females were
able to rear a second brood.
The retention period (RP) between the first full sclerotization and the first dispersal
was positively correlated with the average number of offspring (eggs, larvae, pupae)
present during this period (Spearman correlation: R = 0.413; p = 0.015; N = 34). The
number of females remaining longer than two weeks after the first emergence did not
correlate with the number of totally produced females (Spearman correlation: R =
0.25; p = 0.29; N = 20). In contrast, the number of totally produced females highly
32
correlated with the number of dispersed females after two weeks (Spearman
correlation: R = 0.885; p < 0.001; N = 20). Four weeks after the last teneral female
~
sclerotized, around 3.5 ( X , N = 16) adult females still remained in their natal gallery.
3.2. Experiment – Removal of founder females
Removal of blocking founder females increased the dispersal activity of adult female
daughters (fig. 2.9.) compared to the control group, where only some medium was
removed (U-Test: z = -2.442, p = 0.024, N = 5), and to the same galleries before the
removal (U-Test: z = -2.303, p = 0.024, N = 5). In contrast, there was no significant
differences in dispersal behaviour between the galleries before and after the control
treatment (U-Test: z = -1, p = ns), i.e. when only medium was removed (U-Test: z = 1, p = ns). Other behaviours of not-emerging females remained unchanged in all
treatments (data not shown).
Number of emerging females
25
*
*
20
15
10
5
0
Control
before
Founder removal
after
before
after
Treatment
Fig. 2.9. Number of emerging females before and after experimental treatment. Medians,
25% and 75% quartiles are shown (N = 5 galleries per group). * p < 0.05 (Mann Whitney Utest).
4. Female reproduction
Founder females stopped egg production, for at least a few days, before the first
daughter reached adulthood. The age of the gallery when egg production stopped
correlated significantly with the age of the gallery when the first daughter fully
sclerotized (Spearman Correlation, R = 0.4, p = 0.008, N = 43). In 15 of 43 galleries
egg production started again later.
Females dispersed from field galleries (N = 45 females from 4 galleries) always with
immature ovaries (ovaries without oocytes). In contrast, 4 of 16 field galleries
contained at least two egg-laying females. Polygyny (multiple egg-layers) happened
only in galleries where emergence of daughters had started already (N = 9 of 16). In
five of nine cases egg-laying ceased totally at that time. Before daughters started to
33
disperse galleries were always monogynous, with only the founder female producing
eggs (N = 4; fig. 2.10.).
In all dissected galleries present offspring and egg numbers were positively
associated with each other and with the number of egg layers at that time. In
contrast, there was no correlation between the total number of females in the gallery
and the number of egg-layers and eggs (table 2.2.).
Eggs total
Offspring total
Females total
Offspring
total
Females
total
Egglayers
T
0.536(*)
0.000
0.681(*)
Sig. (2-tailed)
0.007
1.000
0.003
N
16
16
14
T
0.199
0.638(*)
Sig. (2-tailed)
0.295
0.004
N
16
14
T
0.131
Sig. (2-tailed)
0.556
N
14
Table 2.2. Correlations (Kendalls Tau) between total number of eggs, offspring, all females
and the number of egglaying females in field galleries. (*) p < 0.0083 (Bonferroni corrected
α =0.05/6) (N = 14-16 galleries)
Minimum number of
egg-layers / gallery
5
2
4
3
2
4
1
5
0
0
?
1
State of emergence
Fig. 2.10. Minimum number of egg laying females per field gallery depending on the
state of emergence of the gallery at the time of dissection. 0 – emergence not started, 1 –
emergence started, ? – start of emergence unknown. (N = 16).
34
Discussion
Our data show that in X. saxeseni all developmental stages spend a large proportion
of time with cooperative behaviours, and in addition some time with altruistic
behaviours. Under favourable lab conditions X. saxeseni has two overlapping
generations and in the field there may be even more generation overlap. After
monogynous gallery initiation colonies may switch to secondary polygyny. Obligatory
sib-mating causes grandchildren to be more closely related to the founder female
than her own daughters, which means that there is little or no reproductive conflict
among females of a gallery.
Individuals of all developmental stages spend much time grazing on the walls and
body surfaces of other individuals. This is not true for males, which hardly graze on
the walls. This suggets that most required nutrients are ingested already during the
larval stage, which probably holds for ambrosia beetles in general (Kajimura and Hijii
1992, Gebhardt et al. 2004, Peer and Taborsky 2007). Ambrosia beetles constantly
need to tend their fungi. The fungus layer covering the gallery walls has to be grazed
to not get invaded by strange parasitic fungi or other microbes. Feeding on the
mutualistic fungus does not damage the growing point of the fungus, but rather
promotes sporulation (Batra 1966b, French and Roeper 1972, Norris 1979).
Additionally, simple movements of individuals causing physical disruption can induce
ambrosial growth (Whitney 1971, Leonard and Dick 1973). Fungi benefit from their
coexistence with ambrosia beetles also because they recycle urates, the major
nitrogenous excretes of Xyleborini (Batra 1963, Abrahamson and Norris 1969, Kok
1979, Norris 1979), and further growth promoting substances produced by the
beetles for the fungi are hypothesized (Francke-Grosmann 1956). Furthermore, the
xylomycetophagous feeding habit of the larvae constantly enlarges the gallery and
wall-surface for the fungus fruiting bodies to grow. In X. saxeseni our observations
revealed that enlarging of the galleries by the adult beetles is negligible. Teneral
beetles completely specialize in mycetophagy, which is visible from their frass that is
free of woody components.
Grazing on the body surface of one another looks more like grooming, and it clearly
serves to remove fungal hyphens and spores, which grow on individuals that are
lonesome for extended periods. This layer eventually covers the whole body,
because the larvae and adult beetles cannot clean most parts of their body
themselves. This fungal layer can be lethal: for example four females were not able
to walk anymore and died, because they got stuck on the wall by the fungal layer or
fungi grew under the elytre and spread them. In another Xyleborini with solely
mycetophagous feeding larvae that are not frequently in close contact with each
other, pupae develop long, stout hairs, most likely to protect them to get overgrown
by fungi, covering the gallery walls (Schedl 1964). Grazing individuals may directly
benefit in two ways. First they may take up fungal tissues for nutrition. Second the
behaviour may serve to check if an individual is still alive and in good condition. If it
does not move in response to being groomed, the groomer will use its mandibles and
open the body of the groomed within seconds, especially if it is a larva or pupa (own
observations; see similar observations in Kalshoven (1962)). This cannibalism may
raise productivity by assimilation of diseased or dead specimens, especially when the
fungal resources decrease and only few individuals will be able to finish their
development. In addition contaminated individuals should be removed quickly to
35
prevent the spread of diseases, which are a particular threat in highly inbred
communities (e.g. Hamilton 1972, Hamilton et al. 1990).
Altruistic behaviours of larvae are: balling, by which the cordlike larval frass is formed
into balls under ventral bendings of their bodies; and shuffling, by which frass is
shifted in the tunnels. Balling has not been reported from other ambrosia beetles.
Unlike most other studied species X. saxeseni larvae enlarge the larval chamber
while feeding, producing huge amounts of frass. Clean galleries may be beneficial for
the growing fungi (e.g. Francke-Grosmann 1956, Kajimura and Hijii 1992, Mizuno
and Kajimura 2002) and may facilitate mate search and mating (Stark 1982).
Altruistic behaviours by larvae are very uncommon in insects. Passing along of wood
fibres out of the entrance tunnel as a hygienic task has been reported from the
platypodid ambrosia beetle, Doliopygus chapuisi larvae (Kirkendall et al. 1997) and
might exist in some Platypus spp. (Hubbard 1897). These behaviours may be similar
to the shuffling behaviour of larvae that we observed. In ambrosia beetles hygienic
tasks have been mainly assigned to adult beetles (e.g. Francke-Grosmann 1956,
Kirkendall et al. 1997, Costa 2006). In X. saxeseni females sometimes shuffle the
balls of frass out of the gallery, but usually they are transported there or to less
frequented tunnels simply by the movements of the individuals (own observations).
Furthermore, larval frass may sometimes adhere to the fungal beds (Hubbard 1897),
which suggest that viable fungal spores are not only present in adult female frass
(Francke-Grosmann 1975), but also larval frass.
An altruistic behaviour of adult females is blocking. This behaviour may be
widespread in ambrosia beetles ( e.g. Kirkendall et al. 1997, Peer and Taborsky
2004, 2007), but our study is the first to show that also adult, non-reproductive female
daughters are helping their mother in this important task. Foundresses and their
daughters spent on average the same proportion of time with this behaviour. Blocking
may (a) prevent the accidental loss of larvae that are moving freely in the gallery, (b)
exclude males from neighbouring galleries to prevent outbreeding of daughters,
which reduces hatching rates (Peer and Taborsky 2005) (c) hold back adult colony
members to prevent outbreeding and induce helping behaviour, (d) exclude
parasites, parasitoids, predators and strange fungi, which are a common threat in
bark and ambrosia beetles, and (e) regulate the microclimate of the gallery, by
opening and closing the entrance in response to demand for ventilation (see
Kirkendall et al. (1997) for review). Especially the first two functions are obvious in X.
saxeseni: Larvae are frequently found outside the gallery (and die quickly after they
fell out of the gallery) during times when the exit is not blocked. The number of
blocking females in periods with larvae is significantly higher than in the pre- and post
larval periods. When all females have been fertilized and production of new offspring
has ceased, males leave the gallery in search for outbreeding opportunities. Before
this is the case male dispersal might be inhibited by the blocking females to
guarantee female fertilization within the gallery. The third function is of particular
interest in the context of evolutionary mechanisms underlying cooperation. Our
removal experiment showed that the absence of a founder female strongly raises
female dispersal. This suggests that the founder female might actively inhibit the
dispersal of her daughters. Even if they would not behave cooperatively or
altruistically, the presence of daughters will promote the ambrosia fungi (e.g. by
physical contact, nitrogenous excretes,…). However, we did not observe conflict
behaviours in the entrance tunnel between founders and adult daughters, so the
decision of adult females to stay may be unsolicited. Adult daughters should be able
36
to sense the removal of the reproductive female and can then decide to either leave,
help, or reproduce in their natal gallery. As founders sometimes left the developed
offspring when productivity decreased, the absence of them may signal the
daughters that future reproduction is unlikely and help will be unrewarding to them.
In a field study the delay in adult daughter dispersal related to the number of
dependent offspring in the gallery (larvae, pupae), but not to egg numbers. Peer and
Taborsky (2007) concluded that adult female offspring forgo own reproduction in
favour of helping to raise more sisters. Our lab data confirm this finding, but here also
egg numbers correlated positively with the dispersal delay of adult daughters.
However, if offspring (eggs, larvae) production is very high, the first females disperse
earlier, probably due to quicker development. The degree of dispersal delays is
difficult to measure, if individuals cannot be distinguished by the observer. Apparently
most daughters leave the natal gallery some days after reaching adulthood, but
under favourable conditions usually about three to four females remain there for
weeks until the gallery is completely abandoned, which is independent of previous
gallery productivity. In the only known eusocial ambrosia beetle, Austroplatypus
incompertus, only 4,7 ( ± 2,7; X ± SD) daughters stay with the gamergate on average
to help her raise offspring (Kent and Simpson 1992). It may be that in ambrosia
beetles the need for help is generally rather small, and gallery and fungus
maintenance can be achieved sufficiently by only a few individuals (see also Costa
2006). Nevertheless, if environmental conditions for independent breeding are bad,
daughters should stay in their natal gallery where they are well protected, which will
at least indirectly benefit the ambrosia fungus simply by their presence (see above).
Daughters can decide between dispersal and own reproduction somewhere else,
own reproduction in their natal gallery, or helping to raise offspring of their mother
and sisters. Our data show that some female daughters reproduce in their natal
gallery, which has not been shown before (Fischer 1954, Peer and Taborsky 2007).
A few of the dissected field galleries contained more than one egg laying female.
When fungus production is high, it should be beneficial for a gallery to have more
than one egg layer to make use of the proliferating but short-lived fungal resource
quickly. In bark beetles eggs are relatively large compared to female body size, up to
>¼ of female length in X. affinis (Roeper et al. 1980). It may be more efficient if a
clutch of such large eggs is produced by more than one egg-layer.
In haplodiploid societies reproducing by obligatory sibmating, granddaughters (r =
0.75) are more closely related to a female than her own daughters are (r = 0.5).
Under such conditions, foundresses should pass on reproduction to their daughters
as soon as these are sexually mature. Our data show that foundresses indeed
terminate egg production already before their daughters have reached adulthood.
However, this result needs to be confirmed for natural colonies.
If more than one female reproduce, egg number should be controlled to avoid
overproduction of eggs and overexploitation of resources. An indication for such
regulation may be that in field galleries the total number of females and the number
of egg laying females do not relate to each other (τ = 0.000). In contrast, the number
of egg laying females correlates significantly with the number of eggs and offspring
produced (larvae and pupae). We found that very productive galleries may
accommodate up to four egg-laying females.
Under obligatory sibmating all members of a gallery are virtually genetically identical,
so the fitness interests regarding optimal allocation of colony resources should not
diverge. Except for rare mutations the only source of genetic heterogeneity, is mating
37
between members from different galleries, which appears to be rare in X. saxeseni.
However, paradoxically the higher the levels of inbreeding the less likely is the
evolution of eusocial behaviours (Trivers and Hare 1976). High relatedness may help
to initiate social behaviour, but the absence of reproductive conflict will select for
polygyny (Hamilton 1972, Michod 1980, 1993), which is what we observed in X.
saxeseni. Eusociality is very rare in clonal or highly related insects (Hamilton 1987,
Stern and Foster 1997).
Another factor crucial for the evolution of higher sociality is the stability of the
environment. Dead wood as inhabited by X. saxeseni is a relatively ephemeral
habitat that may force females to remain flexible and reproduce independently if
necessary. Even founders may leave the gallery after pupae have enclosed or a
disease began to spread. However, at least two to three generations in one gallery
are possible and there are indications that some individuals never reproduce: (1)
blocking of the entrance by not reproducing females is very likely a dangerous task
and (2) dead individuals that have not dispersed can be frequently found in field
galleries (Peer and Taborsky 2007).
Due to very high relatedness and the short-lived nature of habitat strict eusociality
with reproductive and non-reproductive castes might not have evolved in most
Xyleborini. This tribe offers an ideal opportunity to study the determinants of
reproductive altruism without the confounding factors of genetic heterogeneity among
individuals. We propose that different levels of cooperation, altruism and sociality
exist in this tribe, depending on certain life history characteristics and ecological
differences between the species.
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41
Appendix 1
Behaviour
Median (25% - 75%)
Max; N of galleries
1.instars
(N = 37)
0 (0 - 7,6)
Max=100; N=17
Balling (A)
2./3.instars
(N = 39)
0 (0)
Max=10; N=3
♂♂
(N = 30)
0 (0)
Max=25; N=6
0 (0 - 5,9)
Max=100; N=12
Blocking (A)
0 (0)
Max=100; N=1
Digging (A)
Grazing
from
gallery
members
Ad. ♀♀
(N = 37)
5,2 (0 - 8,2)
Max=50; N=26
0 (0)
Max=11,1; N=6
Shuffling (A)
Ten. ♀♀
(N = 33)
0 (0)
Max=25; N=5
0 (0)
Max=50; N=1
0 (0)
Max=4,1; N=1
0 (0)
Max=3,7; N=1
0 (0)
Max=10; N=2
0 (0)
Max=100; N=2
Egg (C)
0 (0)
Max=2,6; N=3
0 (0)
Max=1,1; N=3
Larva (C)
0 (0)
Max=33,3; N=7
0 (0 - 1,6)
Max=10,3; N=13
0 (0 - 1,2)
Max=50; N=8
Pupa (C)
0 (0)
Max=10; N=6
0 (0)
Max=14,3; N=8
0 (0)
Max=12,5; N=4
0 (0)
Max=0,3; N=1
0 (0)
Max=50; N=4
0 (0)
Max=100; N=5
35,3 (16,1 - 59,4) 0 (0)
Max=100; N=29 Max=100; N=7
Adult (C)
0 (0)
Max=20; N=2
0 (0 – 20)
Max=100; N=11
Grazing fungus (wall) (C)
38,7 (19,9 – 50)
Max=100; N=32
48,9 (42 - 58,4)
Max=100; N=37
40 (17,5 - 56,3)
Max=100; N=27
Grazing glass (SE)
4,6 (0 - 11,2)
Max=33,3; N=22
5,3 (0 - 8,8)
Max=25; N=26
0 (0 - 9,8)
0 (0)
Max=37,5; N=16 Max=100; N=5
0 (0)
Max=100; N=2
0 (0)
Max=11,1; N=1
0 (0)
Max=3,2; N=4
0 (0 - 6,3)
Max=50; N=10
0 (0)
Max=14,3; N=2
0 (0)
Max=33,3; N=2
0 (0)
Max=14,3; N=3
0 (0)
Max=12,5; N=2
15,4 (0 – 20,1)
Max=100; N=23
14,3 (0 – 25)
0 (0)
Max=66,7; N=23 Max=100; N=7
19,2 (8,4 - 33,3)
Max=100; N=26
12,5 (0 - 28,7)
Max=100; N=26
0 (0)
Max=16,7; N=3
0 (0)
Max=33,3; N=6
Larva (SE)
Cannibalis- Pupa (SE)
ing
Adult (SE)
0 (0)
Max=2,7; N=1
Inactive (SO)
16,7 (3,7 - 25,6)
Max=100; N=29
0 (0)
Max=1,1; N=1
0 (0)
Max=0,9; N=1
Defecating (SO)
Locomotion (SO)
Self grooming (SO)
Mating
attempt
Mating
20 (12,5 – 30,1)
Max=71,4; N=34
26,7 (17,1 – 36,4)
Max=100; N=32
12,8 (7,7 – 19)
Max=50; N=33
36,7 (0 – 60)
Max=100; N=19
Something
0 (0)
Max=100; N=4
Teneral
0 (0)
Max=25; N=5
Adult
0 (0)
Max=100; N=6
0 (0)
Max=20; N=3
Table 2.3. Proportion of time (%) that was spent by different stages and sexes in the
gallery. (A) – altruistic behaviours, (C) – cooperative / mutually beneficial behaviours, (SE) –
selfish feeding behaviours and (SO) – other selfish behaviours. Median (25% - 75% quartile)
(1st row), Max.; N of galleries with observed behavior (2nd row).
42
Appendix 2: Methodological details
Lab rearing
Foundresses out of three different field galleries were collected in the Spilwald (near
Bern/CH) in October 2005. These females were used for the establishment of a lab
population, that was carried out parallel to the observations. Beetles were reared by removing
dispersing females from their tubes and putting them solely into sterile medium-filled tubes.
Generally in ambrosia beetles beneficial fungi are transmitted to new galleries in the gut or
within the mycetangia and protected from desiccation by secretions from the beetles
(Francke-Grosmann, 1956, 1975, 1963). Therefore, in the present study, the commonly used
(Nakashima, 1971, Batra, 1963) fractional sterilization method of Francke-Grosmann
(Francke-Grosmann, 1956) was utilized to remove general fungi attached to the bodies of the
beetles. The adult females were first placed in a sterile tube in which a sterilized filter paper
was placed; after 24h these beetles were transferred to a dry sterilized tube and kept there a
further 24h. This procedure was repeated 3 times. After that they were transferred onto the
medium on a sterile bench, they were rinsed in 70% Ethanol and deionised water for a few
seconds to prevent additional microbial contamination of the medium. Gallery structure,
brood development and fungal growth were comparable to natural galleries in wood up to the
age of maximal ten weeks; after that the medium desiccated and probably leached out
(Norris, 1972). All tubes were kept in a day-cycle, with changing temperature and light:
22°/28°C (13h night/11h day). To resemble natural conditions, they were stored horizontally
at 70% humidity. The agar-based medium consisted of 0.35 g streptomycin, 1,25 g Wesson’s
43
salt mixture, 5 g yeast, 10 g casein, 5 g starch, 5 g sucrose, 30 g agar, 200 g sawdust (beech),
2.5 ml wheat germ oil, 2.5 ml peanut oil and 5 ml 95% ethanol (modified from Peer &
Taborsky, 2004, Norris & Chu, 1985). All dry ingredients were mixed, and 580 ml of
deionised water was added. The mixture was filled into tubes and autoclaved for 20 minutes
at 124°C. To facilitate the start of excavation of the foundress, the entrance tunnels were
prebored, tubes were closed by a caput, and left to dry for 4-5 days.
X. saxeseni and its ambrosia fungi respectively, are very demanding on their growing
conditions, so not more than 15% of the started tubes reared offspring successfully.
Observations
Major parts of the galleries and brood chambers are excavated adjacent to the tube wall,
facilitating observations of insect activity and development. When a gallery was established
successfully the tube was wrapped in paper to keep galleries in darkness, but letting light to
the entrance.
For observations the tubes were put under the binocular (6,4x – 16x magnification), with an
artificial light source (max. 6W). In a preliminary study larval and adult beetles behaviour
was neither changed by the light source, nor the position relative to the gravity (Arnold &
Knecht, 2006). After observations tubes were immediately wrapped in the paper again and
stored in the same position as before.
Behavioural scans were made at different stages of the gallery of all visible individuals.
The scan technique was preferred for practical reasons to focus observations of single
individuals. Only for founder females time budgets were made by focus sampling (10 min
observations), because founders could not be observed frequently. In comparing the scan and
the focus technique, in a preliminary study, I revealed no significant differences for most
behaviours of all stages, except “not moving” of 2nd / 3rd instar larvae (paired t-test: t=-5,549,
N=7; P=0,001) and “walking” of teneral beetles (paired t-test: t=-2,877, N=5; P=0,045).
However, by combining the probabilities for the different behaviours over the stages with the
formula of Sokal & Rohlf (1995): χ²(2*N) = -2*Σ ln(pi); I found no significant difference
between the observation techniques for any of the stages.
The duration of the different developmental periods were measured with 5 to 12 different
galleries that were well observable during a certain stage. This developmental periods were
then used to fill up missing phenology data for galleries that were not fully observable: e.g. if
the first larva was observed on day 8, and their was no knowledge when the first egg had
been laid, then it was assumed that it was laid the earliest on day 4, because the
developmental period of eggs is 4 days.
Galleries were usually cultivated until all individuals dispersed. When there were no
individuals visible in the tunnels for around one week, I opened the gallery and determined
the reason of abandonment if it was not clear before. Very often there were still some adults
in the gallery in a kind of hibernating stage. In the field they would probably wait for better
growing conditions for the fungi again or await the winter. I put them in the fridge and used
them later for further breeding. There were usually some dead males and females among
them, sometimes partly cannibalised. One of them was potentially the founder female, and
others starved or died from a disease.
In spring 2006 I opened a few galleries in the field (Spilwald), to get additional information
over the reasons of gallery abandonment in the field.
The sex ratio data was analysed simply by looking at the amount of sons and daughters that
were produced by the founder female or their daughters until the gallery was abandoned.
44
Removal experiments
For up to one year I tried to breed the beetles additionally in vitrines between two glass plates
or in petri-dishes to have conditions were experimental removal of female offspring and
larvae would be possible. I wanted to test the effect of female offspring on the larvae directly.
Unfortunately, X. saxeseni or its ambrosia fungi respectively, were very demanding on their
growing conditions and it was not successful at all. In the end I tried to destroy the normal
breeding tubes, remove some individuals and put the solid medium with the gallery again in a
sterile glass tube. Also this treatment was not successful, because the gallery was invaded by
strange moults within a few days and most of the individuals died. In general it appears that
this kind of intervention is much too strong compared with the slight effect of female help that
I suppose.
The only possible experiment was the removal of the foundress.
45
Chapter III: A detailed study of behavioural
observations
The aim of this Chapter was to report behavioural observations, that could not be included in
Chapter II because of limited space in the paper. In particular all behaviours are shown for the
different developmental stages, sexes of individuals and age classes of the gallery, before
these behaviours were combined to selfish, cooperative and altruistic behaviour classes. I
show also the details of the study that compared the behaviour of foundresses and their adult
daughters.
Furthermore I report the phenology of lab galleries and show causes for their abandonment.
Results for the different developmental stages and sexes
Larvae
Larvae spent most of their time with “not moving” (Mdn = 16,9%), “walking” (Mdn = 17,6%)
and “grooming/eating fungus” (Mdn = 42,5%) (Figure 3.1). Less common behaviours were
“balling” (Mdn = 4,7%) and “grooming glass” (Mdn = 6,3%).
Other behaviours appeared in a few galleries, but were of no general importance (Mdn = 0):
“defecating” (Max = 0,7%), “shuffling” (Max = 7,6%), “grooming egg” (Max = 1,6%),
“grooming larva” (Max = 8,5%), “grooming pupa” (Max = 5,3%), “grooming adult” (Max =
0,2%), “eating larva” (Max = 4%), “eating pupa” (Max = 1,1%) and “eating beetle” (Max =
0,5%).
0,6
Median of behaviour (%)
0,5
0,4
0,3
0,2
0,1
0,0
nm def
w
bal shuf ge
gl
gp
ga
gg
gef
el
ep
ea
Figure 3.1. Time budget of
all larval stages. Medians
of the time budgets shown
by different galleries (N=40
galleries; 25% and 75%
quartiles). nm – not moving,
def – defecating, w –
walking, bal – balling, shuf
– shuffling, ge – grooming
egg, gl – grooming larva, gp
– grooming pupa, ga –
grooming adult / teneral
beetle, gg – grooming glass,
gef – grooming / eating
fungus, el – eating larva, ep
– eating pupa, ea – eating
adult / teneral beetle.
46
“Not moving” was significantly negatively correlated with “walking” (τ = -,24, p = ,028,
N=40), “balling” (τ = -,25, p = ,028) and “grooming/eating fungus” (τ = -,29, p = ,013).
“Grooming/eating fungus” was also negatively correlated with “walking” (τ = -,31, p = ,005)
and “grooming glass” (τ = -,26, p = ,019). Additionally their tended to be a positive
relationship between “shuffling” and “balling” (τ = ,23, p = ,084).
“Shuffling” and “balling” tend to be correlated and are less common in galleries with
decreased activity (= lots of “not moving” larvae). In these galleries there is probably no need
for cleaning the tunnels from frass.
Larvae are “walking”, “grooming glass” or show “not moving” more often in galleries with
less “grooming/eating fungus” behaviour. Especially the first two behaviours are probably a
reaction to a small fungus productivity in the gallery: they either search for growing fungus
(“walking”), eat small fungus amounts from the glass (“grooming glass”) or starve (“not
moving”). In fact, in half of the cases when small larvae were observed “not moving”, they
died / disappeared in the following two days (N=55). In the other half they appeared either to
rest or to moult in the next instar (N=57). “Not moving” in big larvae was connected with
pupae appearing in the following 1-5 days (75%), dying / disappearing in the next 1-2 days
(21%) or had other unknown reasons (4%) (N = 163; see Figure 3.2).
Correlations with "Not moving" 2nd-3rd larval
instars
Other
Reasons
4%
Passing
21%
Figure 3.2. Possible reasons for
“not moving” in big larvae.
Pupation: pupae appeared 1-5 days
after the detection of “not moving”.
Passing: Larvae disappeared or died
in 1-2 days after “not moving”
(N=163 observations).
Pupation
75%
There were not much differences in larval behaviour depending on the instar of the larvae. 2nd
and 3rd instar larvae “walked” (Mdn = 0,128) significantly less than 1st instar larvae (Mdn =
0,267), z = -4,19, p < ,001, r = -,49. Instead 2nd and 3rd instars showed more “balling /
shuffling” (Mdn = 0, Mdn = 0,053), z = -2,06, p = ,038, r = -,24; and more “grooming/eating
fungus” (Mdn = 0,387, Mdn = 0,489), z = -3,00, p = ,002, r = -,35. There was a trend for less
“not moving” in small larvae (Mdn = 0,167, Mdn = 0,2), z = -1,79, p = ,075, r = -,21.(see
Figure 3.3).
47
0,8
median behaviour (%)
**
0,6
**
0,4
ns
0,2
*
ns
ns
gef
gef
gg
gg
grel
grel
bash
bash
w
w
nm
nm
0,0
Figure 3.3. Behavioural differences between small (grey) and big larvae (striped) (N=37;
25% and 75% quartiles). nm – not moving, w – walking, bash – balling / shuffling, grel –
grooming somebody, gg – grooming glasss, gef – grooming / eating fungus. ns – not
significant, * - p < ,05, ** - p < ,01 (Wilcoxon Signed Ranks Test).
Female behaviour
Females (teneral and adults) spent most of their time with “not moving” (Mdn = 16%),
“walking” (Mdn = 17,9%) and “grooming/eating fungus” (Mdn = 35,7%) (Figure 4). Other
behaviours appeared to be common in a few galleries, but were of no general importance
(Mdn = 0): “blocking” (Max = 100%), “digging” (Max = 50%), “shuffling” (Max = 25%),
“self-grooming“ (Max = 20%), “grooming egg” (Max = 3,1%), “grooming larva” (Max =
20%), “grooming pupa” (Max = 10%), “grooming adult” (Max = 33,3%), “eating larva” (Max
= 40%), and “eating pupa” (Max = 12,5%) (Figure 3.4.).
“Blocking” was significantly correlated with the combined “digging / shuffling” (τ = ,29, p =
,044, N=39) and negatively with “grooming / eating fungus” (τ = -,28, p = ,025). As in larvae
“grooming / eating fungus” was also negatively correlated with “walking” (τ = -,23, p = ,047).
“Grooming something (self/egg/larva/pupa/beetle)” showed a positive relationship with
“eating something (larva/pupa)” (τ = ,27, p = ,047). Additionally their tended to be a negative
relationship between “digging / shuffling” and “walking” (τ = -,23, p = ,072).
48
Figure
3.4.
Time
budget of all female
beetles. Medians of the
time budgets shown by
0,6
different
galleries
(N=39; 25% and 75%
quartiles). nm – not
moving, blo – blocking,
0,4
w – walking, dig digging,
shuf
–
shuffling, sg – self0,2
grooming,
ge
–
grooming egg, gl –
grooming larva, gp –
grooming pupa, ga –
0,0
grooming adult / teneral
nm blo w dig shuf sg ge gl gp ga gg gef el ep
beetle, gg – grooming
glass, gef – grooming / eating fungus, el – eating larva, ep – eating pupa.
Median behaviour (%)
0,8
0,8
Median behaviour (%)
ns
0,6
ns
0,4
ns
0,2
ns
**
**
*
0,0
nm nm blockblock w
w
grel grel gg
gg
gef gef erel erel
Figure 3.5. Behavioural differences between teneral (grey) and adult female beetles
(striped) (N=33; 25% and 75% quartiles). nm – not moving, block – blocking, w – walking,
grel – grooming somebody, gg – grooming glass, gef – grooming / eating fungus, erel – eating
relatives (larvae, pupae). ns – not significant, * - p < ,05, ** - p < ,01 (Wilcoxon Signed
Ranks Test).
49
There were hardly any differences between teneral and adult females time budgets (Figure
3.5). They appeared to spend similar amounts of time with the most common behaviours “not
moving”, “walking” and “grooming / eating fungus”.
Behavioural differences could be found only in rare behaviours that were not found in all
galleries (Mdn = 0). Blocking was never observed in teneral beetles, so the difference was
significant, z = -2,81, p = ,002, r = -,35. Adult beetles “groomed glass” only twice, z = -2,37,
p = ,015, r = -,29; and “eating relatives (larva/pupa)” could be observed one time, z = -2,85, p
= ,002, r = -,35.
Founder female
Founder females spent most of their time with “not moving” (Mdn = 1,3%), “walking” (Mdn
= 29,2%), “shuffling” (Mdn = 7,4%), “grooming glass” (Mdn = 6,5%) and “grooming/eating
fungus” (Mdn = 33,3%) (Figure 6). Other behaviours appeared to be common in a few
galleries, but were of no general importance (Mdn = 0): “blocking” (Max = 17,5%), “digging”
(Max = 0,8%), “grooming larva” (Max = 8%), “grooming pupa” (Max = 6%), “grooming
adult” (Max = 2%) (Figure 3.6.).
“Self-grooming”, “grooming egg” and cannibalism (“eating larva/pupa”) could never be
observed in founder females. Although these focus observations were made after the hatching
of larvae and before the appearance of other adult females. In the prä larval period founder
females frequently groomed their eggs. Also cannibalism rarely observed before the
appearance of teneral / adult offspring.
0,6
Median of behaviour (%)
0,5
0,4
0,3
0,2
0,1
0,0
nm
blo
w
dig shuf sg
ge
gl
gp
ga
gg
gef
el
ep
Figure 3.6. Time budget
of
founder
females.
Medians of the time
budgets
shown
by
different galleries (N=5;
25% and 75% quartiles).
nm – not moving, blo –
blocking, w – walking,dig
- digging, shuf – shuffling,
sg – self-grooming, ge –
grooming egg, gl –
grooming larva, gp –
grooming pupa, ga –
grooming adult / teneral
beetle, gg – grooming
glass, gef – grooming /
eating fungus, el – eating
larva, ep – eating pupa.
There were no behavioural differences between founder females and their adult daughters
(Figure 3.7.). The data comes from focus samplings out of 5 different galleries; only adult
females that were slightly brighter than their mothers were observed.
50
Median of behaviour (%)
0,8
0,6
ns
ns
0,4
ns
ns
0,2
ns
ns
ns
ns
0,0
nm nm blockblock w
w
dish dish grel grel gg
gg
gef gef erel erel
Figure 3.7. Behavioural differences between adult female beetles (grey) and the founder
females (striped) (N=5; 25% and 75% quartiles). nm – not moving, block – blocking, w –
walking, dish – digging /shuffling, grel – grooming somebody (self-, egg, larvae, pupae,
beetle), gg – grooming glass, gef – grooming / eating fungus, erel – eating relatives (larvae,
pupae). ns – not significant (Wilcoxon Signed Ranks Test).
Median (Range)
Adult ♀♀
Foundresses
Shuffling, Digging (A)
7.7 (0 – 12)
8.2 (0 – 22)
Blocking (A)
3.9 (0 – 12)
0 (0 – 18)
1 (0 – 18)
0 (0 – 14)
36.5 (20 – 55)
33.3 (26 – 54)
Grazing glass (SE)
0 (0 – 4)
2 (0 – 8)
Cannibalising (SE)
0 (0 – 5)
Behaviour
Grazing from gallery members (C)
Grazing fungus (wall) (C)
Inactive (SO)
21.2 (0 – 29)
1.3 (0 – 46)
Locomotion (SO)
12.4 (9 – 80)
29.2 (4 – 60)
Table 3.1. Proportion of time (%) that was spent by different stages and sexes in the
gallery. (A) – altruistic behaviours, (C) – cooperative / mutually beneficial behaviours, (SE) –
selfish feeding behaviours and (SO) – other selfish behaviours. Median (Range) N = 5
galleries.
51
Male behaviour
Males spent nearly all their time in most galleries with “walking” (Mdn = 36,7%). Common
in some galleries, but still with the median = 0 were: “not moving” (Max = 100%), “grooming
adult / teneral beetle” (Max = 100%) and “grooming / eating fungus” (Max = 100%) (Figure
3.8.). Other behaviours were of no general importance (Mdn = 0): “digging” (Max = 50%),
“grooming egg” (Max = 3,7%), “grooming larva” (Max = 100%), “grooming pupa” (Max =
20%), “grooming glass” (Max = 100%), “eating larva” (Max = 33,3%), “mating attempt on
something / teneral / adult” (Max = 100%) and “mating with an teneral female” (Max = 20%).
0,6
Median behaviour (%)
0,5
0,4
0,3
0,2
0,1
0,0
nm
w
dig
ge
gl
gp
ga
gg
gef
el
ma may mao maty
Figure 3.8. Time budget of
all male beetles. Medians of
the time budgets shown by
different galleries (N=30;
25% and 75% quartiles). nm –
not moving, w – walking, dig
- digging, ge – grooming egg,
gl – grooming larva, gp –
grooming
pupa,
ga
–
grooming adult / teneral
beetle, gg – grooming glass,
gef – grooming / eating
fungus, el – eating larva, ma –
mating attempt on something,
may – mating attempt on
teneral female, mao – mating
attempt on adult female, maty
– mating with teneral female.
Males were most of their time “walking” and checking for mates. When they perceived a
female they started to groom it (“grooming adult / teneral”), but quickly attempted it for
mating. Matings were solely observed with teneral females (N=2). Mating attempts could be
observed with pieces of wood, larvae or males (N=12), teneral (N=7) and adult females
(N=11). Females were usually not influenced by male mating attempts and went on with their
shown behaviour.
“Walking” was significantly negatively correlated with “not moving” (τ = -,33, p = ,034,
N=30) and showed a negative trend with “mating attempt (something / teneral / adult)” (τ = ,29, p = ,054).
Results for behavioural correlations with certain age stages of the
gallery
Larvae
I compared all larval behaviours between 4 different stages, that are usually occurring in the
following order in most galleries: (1) founder female with larvae and pupae with eggs, (2)
52
founder female with larvae and pupae without eggs, (3) all stages (adult/teneral beetles,
larvae, pupae) with eggs, and (4) all stages without eggs.
1,2
0,30
1,0
0,25
median behaviour (%)
median behaviour (%)
I could not find any significant difference between the occurrence and the absence of
adult/teneral beetles (1+2 vs. 3+4, N = 10). There was no difference between all stages with
and without eggs (3 vs. 4, N = 15) and the founder female with larvae and pupae with and
without eggs (1 vs. 2, N = 13). Only in the last case “grooming glass” is nearly significantly
less common with eggs (Mdn = 0,041) than without eggs (Mdn = 0,092), z = -1,96, p = ,051, r
= -,38.
“Eating/grooming fungus” tended to be influenced by the stage of the gallery (1, 2, 3, 4),
Kruskal-Wallis test, H(4) = 7,012, p = 0,072. Jonckheere’s test revealed a significant negative
trend following the order of the stages (1, 2, 3, 4), J = 1160, z = -2,69, p = ,007, r = -,28
(Figure 3.9.). Cannibalism (“eating larva/pupa/adult”) also tended to be influenced by gallery
stage, H(4) = 6,226, p = 0,088. Jonckheere’s test showed a positive correlation following the
order of the stages, J = 1671, z = 2,06, p = ,033, r = ,22.
0,8
0,6
0,4
0,2
0,20
0,15
0,10
0,05
0,00
0,0
1
1
2
3
2
3
4
4
Figure 3.9. Behaviour of all larvae at different stages of the gallery. “Grooming / eating
fungus” – left graph. Cannibalism (“eating larva / pupa / beetle”) – right graph. Medians of
the time budgets shown by different galleries (25% and 75% quartiles). (1) founder female
with larvae and pupae with eggs (N = 23 galleries), (2) founder female with larvae and pupae
without eggs (N = 20), (3) all stages (adult / teneral beetles, larvae, pupae) with eggs (N = 19),
and (4) all stages without eggs (N = 29).
Teneral females
I compared all behaviours of teneral females between 3 different stages, that are usually
occurring in the following order in most galleries: (1) all stages (adult / teneral beetles, larvae,
pupae) with eggs (N = 14), (2) all stages without eggs (N = 22), and (3) post larval period (no
eggs, larvae, pupae) (N = 8).
I could not find any difference between all stages with and without eggs (1 vs. 2, N = 13) and
the later against the stage without larvae and pupae (2 vs. 3, N = 6).
“Eating/grooming fungus” tended to decrease as soon as larvae were absent (1+2 vs. 3, N =
36, M = 0,4, SE = 0,04; N = 8, M = 0,2, SE = 0,1), t-test, t = 1,975, p = ,057, r = ,29.
“Grooming something (eggs, larvae, pupae, beetles)” and cannibalism (“eating larvae, pupae,
beetles) were both not observed in the post larval period, but there are only beetles left at this
time and the number of teneral beetles decreases constantly.
53
Adult females
I compared all behaviours of adult females between 7 different stages, that are usually
occurring in the following order in most galleries: (1) founder female without eggs and
offspring (N = 1), (2) founder female with eggs but without hatched offspring (N = 3), (3)
founder female with larvae and pupae with eggs (N = 5), (4) founder female with larvae and
pupae without eggs (N = 4), (5) all stages (adult / teneral beetles, larvae, pupae) with eggs (N
= 14), (6) all stages without eggs (N = 23), and (7) post larval period (no eggs, larvae, pupae)
(N = 16).
Number of adult females / Observation
I could not find any difference of adult female behaviour between all stages with and without
eggs (3 vs. 4, N = 11) and the later against the stage without larvae and pupae (4 vs. 7, N = 9).
There was only a increase of walking between all stages with eggs (M = 0,149, SE = 0,07)
and the post larval period (M = 0,35, SE = 0,15) (5 vs. 7, N = 8), t-test, t = -2,566, p = ,05, r =
,47.
“Blocking” was totally absent before the hatching of offspring (stage 1, 2) and in the post
larval period (stage 7) (Figure 3.10.). Before the hatching of offspring the founder female is
instead sitting next to the eggs “not moving” for 75% of their time (stage 1, 2, N = 4), which
is not shown at all by the founder female during the periods with not pupated offspring (stage
3, 4, N = 9). “Blocking” was usually performed by one individual (rarely 2) at the entrance
tunnel of the gallery. Until the 4th stage the founder female is the only individual that can
perform this task. Thus, “blocking” was only shown during the time with larval offspring
(stage 3 – 6), which is mainly overlapping with the occurrence of teneral, not inseminated,
females (stage 5, 6) (see also Phenology). During the stages with larvae and pupae (3, 4, N =
9) there were significantly more individuals blocking per observation (Mdn = 0,5) than in the
additional occurrence of teneral / adult offspring (stage 5, 6, N = 37, Mdn = 0), MannWhitney test, U = 94,00, p = ,006, r = -,38.
Figure 3.10. Blocking
of adult females at
1,2
different stages of the
*
gallery. Medians of the
1,0
time budgets shown by
different galleries (25%
ns
0,8
and 75% quartiles). (1)
founder female without
ns
eggs and offspring (N =
0,6
1), (2) founder female
with eggs but without
0,4
hatched offspring (N =
3), (3) founder female
0,2
with larvae and pupae
with eggs (N = 5), (4)
founder female with
0,0
larvae
and
pupae
without eggs (N = 4), (5)
all stages (adult / teneral
1
2
3
4
5
6
7
beetles, larvae, pupae)
with eggs (N = 14), (6) all stages without eggs (N = 23), and (7) post larval period (no eggs,
larvae, pupae) (N = 16). ns – not significant; * - p < ,05.
54
There was no “digging / shuffling” of the founder female in the period with eggs (stage 2, N =
3) and of adult females in the post larval period (stage 6). “Grooming something (self-, larvae,
pupae, beetles)”, “grooming glass” and cannibalism (“eating larvae, pupae, beetles) were all
not shown by the founder female during the scan samples, thus this behaviours occurred only
after individuals pupated (stage 5 – 7).
There was no trend for more female dispersal during the last three stages (5 – 7). Dispersing
behaviour is usually shown by adult offspring, however there were three cases of dispersing
founder females during the larval period (stage 3), because of an illness in the larvae. These
founders were able to successfully found new galleries. Founders cannot be discriminated
from adult offspring after a certain age, so it is possible that they disperse again with their
adult daughters. There is no knowledge about the life expectancy of females, but founders can
survive about a month without food (data from unsuccessful galleries without growing fungi).
Males
I compared all behaviours of males between 3 different stages, that are usually occurring in
the following order in most galleries: (1) all stages (adult / teneral beetles, larvae, pupae) with
eggs (N = 15), (2) all stages without eggs (N = 16), and (3) post larval period (no eggs, larvae,
pupae) (N = 11).
There were no differences between the three stages for the relative time spent “not moving”,
“walking”, “grooming glass”, “eating larvae” and “mating”. Although “mating“ was only
shown in stage 1, but because of its general rarity (N=3) the difference was not significant. It
is not surprising that it appears immediately after pupation, when females are unfertilised.
“Grooming something” (males mainly groom females) is significantly more common before
the post larval period (2 vs. 3; N = 16, Mdn = 0,15; N = 11, Mdn = 0), when there might be
still some unfertilised females; Mann-Whitney test, U = 49,5, p = ,031, r = -,31. “Mating
attempt (teneral / adult female)” was significantly influenced by the stage of the gallery (1, 2,
3), Kruskal-Wallis test, H(3) = 9,496, p = 0,006. Jonckheere’s test revealed a significant
negative trend following the order of the stages (1, 2, 3), J = 196, z = -2,84, p = ,004, r = -,44.
This is not surprising because also the number of unfertilised females should decline along
this axes. “Eating/grooming fungus” also decreases significantly along with the stage of the
gallery (1, 2, 3), Kruskal-Wallis test, H(3) = 8,151, p = 0,013. Jonckheere’s test (1, 2, 3), J =
205, z = -2,76, p = ,005, r = -,43 (Figure 3.11, left graph). This might be a result of decreasing
fungus production or the decreasing need to feed, because sclerotization is already finished.
In contrast “dispersing from the gallery” tends to increase along with the stage of the gallery
(1, 2, 3), Kruskal-Wallis test, H(3) = 7,289, p = 0,021. Jonckheere’s test (1, 2, 3), J = 337, z =
1,51, p = ,064, r = ,23. There was significant more “dispersing” shown in the post larval
period (Mdn = 0, N = 11) than before (1+2 vs. 3, Mdn = 0, N = 31); Mann-Whitney test, U =
106,5, p = ,006, r = -,39 (Figure 3.11, right graph). As soon the production of teneral females
ceases, males should leave the gallery in search for outbreeding opportunities.
55
1,2
1,0
1,0
Median behaviour (%)
Median behaviour (%)
1,2
0,8
0,6
0,4
0,2
0,0
**
0,8
0,6
0,4
0,2
0,0
1
2
3
1
2
3
Figure 3.11. Behaviour of males at different stages of the gallery. “Grooming / eating
fungus” – left graph. Dispersing behaviour – right graph. Medians of the time budgets shown
by different galleries (25% and 75% quartiles). (1) all stages (adult / teneral beetles, larvae,
pupae) with eggs (N = 15), (2) all stages without eggs (N = 16), and (3) post larval period (no
eggs, larvae, pupae) (N = 11).
Results on the phenology of galleries
In the first hour after founder females were sat in the breeding tubes with the sterile medium
they started to dig the breeding tunnel. After the appearance of growing fungus they began to
lay eggs. When the ambrosia fungi did not grow, or other strange fungi invaded the tunnels,
founders sometimes went on with digging for up to 5 weeks. Although usually they tried to
disperse before, by climbing the surface and attempted to fly away. They would have built
another breeding tunnel from the surface, if I did not take them out and put them onto a new
sterile medium.
After 1 to 51 days they laid their first egg (N = 93, Mdn = 18). Egg-laying in the first 5 days
was very uncommon and should be caused by the accidental removal of a laying female from
their natal gallery. Relatively constant eggs hatched around 4 days after laying (N = 5; see
developmental periods in Figure 3.12). This 1st instar larvae moulted to 2nd instars 3 days later
(N = 6). Then the development got more plastic, depending on the amount of fungus
accessible for the growing offspring. On average 3rd instar larvae pupated 6 days after their
first moult from 1st larvae (N = 12, Min = 4, Max = 17). In this stage they were already very
resistant to starvation, and could survive for up to 17 days without much growing. The pupal
stages was again quite constant with 5 to 6 days (N = 12, M = 6). The time for full
sclerotization depended on the amount of food accessible to the teneral beetles and took on
average 9 days (N = 11, Min = 6, Max = 15). Teneral beetles survived for a mean of 15 days
(N = 3) when taken out of the gallery as a pupae and given no access to any food. They did
not develop ovaries, showed no fat in their abdomen, but sclerotized into a medium brown
state.
The developmental periods are well reflected in the appearance of the different stages over all
observed galleries (N = 93; Figure 3.13, 3.14). Their was a high mortality during the larval
stages, especially when productivity decreased. This is visible in Figure 13, by looking at the
disappearing of 2nd/3rd instar larvae compared to pupae. The former can be observed for
longer because they never pupated.
56
the different stages. Mean of some well
observable galleries (N = 5-12, Min,
io
n
pa
ro
t
iz
at
pu
li
ns
ta
r
st
a
cl
e
ll
s
fu
2n
d/
1s
t
3r
d
la
la
rv
a
rv
al
in
eg
r
Max).
g
duration in days
Figure 3.12. Developmental periods of
18
16
14
12
10
8
6
4
2
0
Usually founder females ceased egg production after 35 days (Mdn, Min = 15, Max 65), but
in around 1/6 of the galleries females started to lay eggs a second time some days later (N=15,
Mdn = 43, Min = 32, Max = 64). In 6 of this colonies this resulted in a second generation of
big larvae, but only in one gallery a few beetles finished their development. Thus, a second
generation is usually not successful.
Dispersing males
Males
Dispersing females
Adult females
Teneral females
Pupae
2nd/3rd Instar larvae
1st instar larvae
Eggs
0
20
40
60
80
100
120
days from gallery founding
140
Figure 3.13. Phenology of the developmental stages from gallery founding. The start of
the error reflects the 25% quartile of the 1st appearance of this stage, and the end of the error
the 75% quartile of the disappearance of this stage. Solid lines show the appearance of the 1st
breeding cycle of all galleries (N = 93). The dotted lines are of individuals from the 2nd
breeding cycle only from galleries where it appeared ( N = 15).
57
In Figure 14 developmental periods are easy to see, and the differences to Figure 12 are
caused by the fact that not all individuals were observable in all galleries at each scan. Eggs
were laid for 17 days (Mdn) in the 1st breeding cycle. 5 days (Mdn) later the first larvae
hatched and the last disappeared after around 21 days (Mdn). The 1st pupation was observed
around 16 days (Mdn) after egglaying, 4 days (Mdn) later 1st teneral females, and 10 days
(Mdn) after that the 1st adult females. The 1st males appeared around two days (Mdn) after the
1st teneral females. Before dispersal female spent 5,5 days (Mdn) in their natal gallery, males
20,5 days (Mdn).
1.dispers.m
1.male
1.dispers.f
1.ad female
last teneral f
1.teneral female
last pupa
1.pupa
last 2nd/3rd instar
1.2nd/3rd instar
last 1st instar
1.1st instar
last egg
1.egg
0
20
40
60
80
100
120
days from 1. egglaying
Figure 3.14. Phenology of the developmental stages from 1st egglaying. Box-plots for the
start and the end of each stage (N = 93). Data only for the 1st breeding cycle.
Founder females appeared to terminate egg production (first egg cycle) at the latest when
other adult females developed (Figure 3.15), but usually in the days before that; Spearman
Correlation, R = ,4, p = ,008, N = 43. The stop is probably triggered by the appaerence of
teneral females. Only in one gallery an egg was laid one day after the full sclerotization of
females. However, as mentioned already 15 galleries started to produce eggs again, after a
break of some days. The second egg-cycle could be produced by a daughter already.
Around one week after the stop of egg production by the founder, the 1st females started to
disperse.
58
80
60
40
last egglaying after first appearence
20
0
20
40
1.juv female
1.ad female
1. disp.female
60
Figure 3.15. Phenology of the last
egglaying of founders compared to
the
development
of
female
daughters. In days from the 1st
egglaying (N=43 galleries). A filled
circles represents the 1st appearances
of teneral females in a gallery, an
open circle the 1st appearance of adult
female and a filled triangle the 1st
dispersing female. The red line shows
the border between egglaying before
and after the 1st appearance of a
female stage.
80
last egglaying (days)
Gallery abandonment
Gallery productivity usually ceased between day 50. and 70. after gallery founding. Only in a
few cases egg were laid after the 100th day. However, in most galleries some females stayed
after egglaying, without much activity. They probably waited for hibernation, and I usually
opened the galleries after some time and put the remaining females in the fridge. The oldest
gallery produced eggs until day 138., and it was opened two days later. It is expected that
field galleries might be productive for a longer time than lab galleries, because in the later the
medium degenerates after a few weeks. This was the main reason of gallery abandonment by
the beetles in the lab (28,1 %), followed by “invasion of green moult” (26 %), “invasion of
mites” (15,6 %), and the “invasion of a strange white fungus” (13,5 %). Other causes like
illnesses in the larvae, other strange fungi or the dead of the founder female were found in less
or around 5% of the galleries (N = 96, see Figure 3.17). In the field galleries were abandoned
mainly because of strange invading fungi (83,3 %), that had not much importance in the lab,
and predators (16,7 %) (N = 6, see Figure 3.17).
others
predator
medium degenerated
cause of abandonment
first appearance (days)
last egglaying before first appearence
founder died
field - galleries (N=6)
lab - galleries (N=96)
mites
Figure 3.17. Histogram
for the reasons of
gallery abandonment.
Compared are field and
lab galleries.
illness
green moult
brown fungus
blue fungus
white fungus
0
5
10
15
20
25
30
number
59
Literature
Arnold, C. & Knecht, S. Gallery foundation and the influence of different parameters of observation techniques
on
the
behaviour
of
the
Ambrosia
beetle
Xyleborus
saxeseni.
2006.
Ref Type: Unpublished Work
Batra, L. R. (1963). Ecology of ambrosia fungi and their dissemination by beetles. Trans. Kansas Acad. Sci. 66:
213-236.
Francke-Grosmann, H. (1956). Hautdrüsen als Träger der Pilzsymbiose bei Ambrosiakäfern. Z. Morph. u. Ökol.
Tiere 45: 275-308.
Francke-Grosmann, H. (1963). Some new aspects in forest entomology. Annual Review of Entomology 8: 415&.
Francke-Grosmann, H. (1975). Zur epizoischen und endozoischen Übertragung der symbiotischen Pilze des
Ambrosiakäfers Xyleborus saxeseni (Coleoptera: Scolitidae). Entomologica Germanica 1: 279-292.
Nakashima, T. (1971). Notes on the associated fungi and the mycetangia of the ambrosia beetle, Crossotarsus
niponicus. Appl. Ent. Zool. 6: 131-137.
Norris, D. M. (1972). Dependence of fertility and progeny development of Xyleborus ferrugineus upon
chemicals from its symbiotes. In Insect and mite nutrition: 299-310. Rodriguez, J. G. (Ed.). Amsterdam: NorthHolland.
Norris, D. M. & Chu, H.-M. (1985). Xyleborus ferrugineus. In Handbook of insect rearing Vol.I: 303-315.
Singh, P. & Moore, R. F. (Ed.). Amsterdam: Elsevier.
Peer, K. and M. Taborsky. (2004). Female ambrosia beetles adjust their offspring sex ratio according to
outbreeding opportunities for their sons. J. Evol. Biol. 17:257-264.
60
Chapter IV: Optimal allocation of
resources: The value of males
A recent studied showed that in a haplodiploid ambrosia beetle with strong local mate
competition (LMC) females adjust offspring sex ratio according to outbreeding opportunities
for their sons. Here I demonstrate that also without outbreeding opportunities sex ratios are
highly variable, potentially influenced by vertically transmitted symbionts (e.g. bacteria in the
female ovaries, the fungus cultivar). Furthermore, my detailed lab observations show that
matings occur only with teneral females, which is assured by producing the first males usually
before the females fully sclerotize. Although solely male galleries were found, their
unfertilised foundresses were from galleries with males. Foundresses from maleless galleries
were not capable to produce a successful brood.
Introduction
When breeding sites are rare and population density is low in an outbreeding species, it might
be difficult to locate a mating partner at the breeding site. This would favour ‘assurance
mating’ before dispersal. Once pre-dispersal mating has evolved, this can lead to inbreeding if
(1) colonisation density is low, so that it is unlikely that an individual will find a mating
partner that is not closely related (e.g. at breeding sites where only one family can be
sustained), and (2) gallery architecture is such that offspring of the same brood are in close
contact with each other. The species of the tribus Xyleborini (Scolytidae) might still live
under similar environmental conditions like its ancestor, where pre-dispersal mating first
61
evolved. The ambrosial complex which is cultivated by these beetles in galleries of dead trees
is very sensitive to its growing substrate (e.g. Leach et al. 1940; Stark, 1982; Borden, 1982;
Berryman, 1989). Among others it requires a certain grade of fermentation and humidity.
Such suitable hosts are sparsely distributed, and metaphorically they could be seen as islands
in a sea of unprofitable trees. Individuals that are fertilized already when arriving on such an
“island” tree, have a headstart compared to ones that have to look out for a mate then. Bark
beetle species that evolved pre-dispersal mating (i.e. inbreeding species) are especially
common on tropical islands (Jordal et al., 2001), supporting their advantage to colonize new
habitats. In Xyleborinus saxeseni only a few family groups are extraordinarily contributing
offspring to the next generations (own unpublished data). Members of these groups are
constantly colonizing sparsely distributed hosts, without much contact between each other.
Costs of inbreeding have been widely purged already (see Peer & Taborsky, 2005) and they
seem perfectly adapted to these conditions.
It is an obvious advantage for an inbreeding species to be able to control sex ratio effectively.
This is easiest when sex determination is through arrhenotoky (e.g. Hamilton, 1967). While
haplodiploidy has one origin in the Scolytidae (Normark et al. 1999), inbreeding and biased
sex ratios have evolved in at least four groups independently (Farrell et al., 2001). Two of
these groups are haplodiploid, one is functionally haplodiploid, and one is diploid
(Dendroctonus). The mechanism for sex ratio determination in the latter is still unknown
(Farrell et al., 2001).
Moderately biased sex ratios are rare in the haplodiploid tribe Xyleborini (Scolytinae)
(Kirkendall, 1993), even though they are common in other inbreeding insects. The reasons
might be that outbreeding is very infrequent. If mating occurs exclusively between siblings,
one male egg per clutch should be sufficient. However, there are several factors that would
favour more than one male egg (see also Kirkendall 1993; Peer 2003, 2004): (1) Egg to adult
survival: if brood mortality is partial (e.g. through predation), several males could be an
insurance against total lack of males. Total brood mortality (e.g. bad breeding material)
should not influence the number of male eggs laid. (2) Egg sex control: if maternal control
over sex of eggs is not precise, one male egg might not be enough. (3) Male longevity: if the
male does not live long enough to fertilize all his sisters, several male eggs should be laid. (4)
Optimal sex ratio (OSR): males must be able to cope with the highest possible OSR. Thus,
high or highly variable OSR should favour more than one male per clutch. (5) Female
dispersal: if females disperse soon and a male can not keep up with insemination of his newly
emerging sisters, more than one male should be produced.
X. saxeseni is one of the 10 native and two introduced Central European members of the
Xyleborini. Apart from these, the ecological group of ambrosia feeders contains one
introduced species from the Pityophtorini (Gnathotrichus materiarius) and one native species
from the family Platypodinae (Platypus cylindrus).
Unlike most other ambrosia beetles, X. saxeseni does not lay its eggs singly into egg niches,
but rather, the maternal beetle lays batches of eggs in the initial tunnel. Additionally larvae
show a different habit, feeding not only on the fungal layer covering the gallery walls, but
primarily also on fungal hyphens growing into the wood (“xylomycetophagous”). By this
habit and feeding in small groups they extend the parental gallery vertically in direction of the
grain (Lengerken, 1939, Roeper, 1995; see also Chapter1). That usually results in one
common “brood chamber”. Brood sizes can get quite large, and galleries with more than
hundred individuals have been dissected (Fischer, 1954; Hosking, 1972; Peer & Taborsky,
2007). Apparently, galleries from different females are sometimes found to merge (Lengerken
1939). Brood emergence can last anywhere between 3-12 weeks (Hosking, 1972), either
because the maternal beetle oviposits over an extended period of time, or because adult
62
daughters reproduce themselves (see Chapter1). As in other Xyleborine species, brood size is
proportional to the final nest surface (e.g. Kajimura & Hiji, 1994). In Europe, there are
usually two generations per year (Peer, 2004; but see Fischer, 1954). Overwintering may take
place in all stages from eggs to adults (Fischer, 1954; Francke-Grosmann, 1967), although I
never observed eggs or pupae during winter (own observations).
Sex ratios were never studied in detail in X. saxeseni. There is only one study on another
Xyleborini – Xylosandrus germanus (Peer, 2003). In a lab study they showed, that broods
adapt their sex ratio according to outbreeding opportunities for their sons. More males are
produced if there are more family groups nearby.
Hosking (1972) found in X. saxeseni that sex ratios of individual broods are extremely
variable. While in some nests the number of males and females is about equal, in others there
are only one male to more than 100 females. Since merging of different galleries has been
reported in this species, it might explain the variation in sex ratio found. Moderately biased
sex ratios should be produced (Fisher, 1930) in such cases.
The timing of male production is hardly studied in ambrosia beetles, although it could give
hints to the mating system a species has obtained. If females disperse immediately after
finished pupation, then males should be produced first in order to successfully inseminate all
their sisters. There is only one study in Xyleborini on Xyleborus dispar looking on the timing
of male production (Roeper et al. 1980). It appeared that males are not produced first.
There is also no knowledge on life expectancy of males. If males are short-lived, the maternal
beetle would have to continuously produce males in order to insure insemination of all female
offspring. For Xylosandrus compactus it has been found that virgin females are only one third
as likely to initiate a gallery as mated females (Brader 1963, in Kirkendall, 1993). The reason
might be the cost of having to raise an all-male clutch before mating and being able to
produce daughters, what has been observed in Xylosandrus compactus, but not in Xyleborinus
saxeseni. Thus, the late production of males in some Xyleborini might be a consequence of
female offspring staying in the nest for an extended period of time. Maternal beetles could
then minimise the number of males they have to produce by laying male eggs last.
In the lab study I excluded two major factors that could influence the sex ratio in the field:
There were no outbreeding opportunities available and merging of galleries could not occur,
because families were held separately.
I asked the following questions:
1. How variable is the sex ratio under conditions without outbreeding opportunities? If
more than one male is produced per brood, the female might not be able to determine
sex ratio exactly.
2. When does mating occur and which age groups are involved?
3. When do males enclose relative to their sisters? If males enclose much later females
should not disperse immediately after reaching adulthood. However, if they emerge
before brothers are present, outbreeding may be more common than expected.
Material & Methods
See ChapterII and Appendix2
63
Results
1. Sex ratio
In our lab galleries X. saxeseni showed an extremely low production of sons (Figure 4.1). The
histogram of the number of males over all galleries resembles a Poisson distribution. Maleless galleries were usually ones with low productivity (not more than 1-5 daughters emerging;
see also Figure 4.2.). While 30 % of the galleries contained no males, they produced only 12
% of the total offspring. Although most of this offspring was used for further breeding, none
of them was able to establish a (male) brood successfully. Most of the galleries contained one
male (46,4 %).
The production of males tended to correlate with the number of female offspring; Linear
regression: T = 1,947, p = ,056, R = ,23; N = 69 (Fig. 4.3.).
__
35
Number of galleries
30
25
20
effectively found
15
predicted (POISSON)
10
5
0
0
1
2
3
4
5
Number of males
Number of female daughters
Figure 4.1. Histogram for the number of males in galleries with daughters. The black
bars show the found number of males and the grey bars the ones expected under a Poissondistribution. (N = 69)
more than 20
16 to 20
3males
2 males
11 to 15
1 male
0 males
6 to 10
1 to 5
0
5
10
15
Figure 4.2. Histogram for the
number of males in relation to
the
number
of
female
daughters. The number of
female
daughters
were
separated into 5 groups.
Number of galleries (N = 72)
64
Figure 4.3. Number
of
daughters
in
relation to number of
sons. Solely male
galleries are excluded.
(N = 69)
The maximal productivity of a gallery did not influence the number of produced sons (Fig.
4.4.), what would be predicted if a female produces more sons, because it expected a higher
productivity. In the galleries with fertilized founders, sex ratios ranged between 1:1 and 1:86
(male : female), with a mean around 1:20. Galleries with high numbers of female daughters
(more than 20 females) had usually sex ratios (male / female) close to 0.
1,2
1
3
Sex-ratio (m/f)
Number of males
4
2
2
R = 0,0048
1
0,8
0,6
0,4
0,2
0
0
20
40
60
80
max number of individuals (eggs, larvae,
pupae, adults)
100
0
0
20
40
60
Number of female daughters
Figure 4.4. Number of males in relation to maximal gallery productivity (left graph).
Maximum number of individuals expresses gallery productivity at a certain point. (N = 69)
Sex ratio in comparison to the number of female daughters (right graph). (N = 69)
65
2. Mating behaviour
Males spent most of their time “walking” and checking for mates. However, “walking” was
not more common than in females (z = -1,634, p = ,104). Males mated solely with teneral
females (N=3), although mating attempts could be observed with pieces of wood, larvae or
males (N=12), teneral (N=7) and adult females (N=11) (Fig. 4.5.). The time males spent with
“mating attempt (teneral / adult female)” was significantly influenced by the stage of the
gallery (4, 5, 6); Kruskal-Wallis test, H(3) = 9,496, p = 0,006. Jonckheere’s test revealed a
significant negative trend following the order of the stages (4,5,6), J = 196, z = -2,84, p =
,004, r = -,44. All matings happened in the 4th stage, immediately after enclosion of females
(see also Chapter 3). Along these stages also the number of teneral females decreased, while
the number of adult females increased.
Figure 4.5. Histogram of the proportion of
mating and attempting for mating males per
gallery. N = 30.
3. Male phenology and dispersal
Males were produced slightly after the production of females (Fig. 6.6., left graph), Spearman
Correlation, R = ,72, p < ,001, N = 30. As the linear regression showed around 5 days later.
This should be early enough to fertilize the teneral sisters before their full sclerotization (see
Chapter 3). Only in 3 out of 93 (3,2%) galleries the founder female was not fertilized and
produced only male offspring. These females were not originated from maleless galleries.
Males started to disperse from their natal galleries when no young females enclosed anymore
(Fig. 4.6., right graph), Spearman Correlation, R = ,95, p < ,001, N = 13. This might be
triggered by the fully sclerotization of all teneral females and their complete fertilization.
Male dispersal tended to increase along with the stage of the gallery (4,5,6); Kruskal-Wallis
test, H(3) = 7,289, p = 0,021; Jonckheere’s test (4,5,6), J = 337, z = 1,51, p = ,064. There was
significantly more “dispersing” shown in the post larval period (stage 6; N = 11) than before
(stage 4,5; N = 31); Mann-Whitney test, U = 106,5, p = ,006, r = -,39. In general dispersal
started when the last teneral female sclerotized (Spearman Correlation of the last full
sclerotization event and the first dispersing male: R = ,95, p < ,001, N = 13). However, in
81,3% of the galleries with males (N = 69), they never dispersed (see also Chapter 3).
66
80
160
males disperse before the disappearence
of teneral females
Males appear after first teneral females
140
date last teneral females
70
120
date 1. male
60
100
50
40
30
80
60
40
males disperse after the disappearence
of teneral females
Males appear before first teneral females
20
20
20
30
40
50
60
date 1. teneral females
70
80
20
40
60
80
100
120
140
160
date first dispersing male
Figure 4.6. Phenology of 1st male appearance and dispersal. In days from the 1st egglaying
(N = 30 galleries). In the left graph dots represent the date when the 1st male appeared relative
to the 1st appearance of teneral females. The right graph shows the relationship between the
fully sclerotization of females and the 1st male dispersal (N = 13). All dots on the red line
stand for events that occurred on the same day. The black line is the linear regression of the
data.
Discussion
The main results I obtained from this study are:
Founders of X. saxeseni usually produce not more than one or two males, relatively
independent of the number of female offspring produced. Thus, especially small
broods, are sometimes maleless. Females dispersing from these broods appear to be
unable to found new galleries successfully.
Mating has been observed only with teneral females, immediately after their
enclosion.
Males enclose shortly after their sisters, right before their full sclerotization. Some
females disperse soon after reaching adulthood, but not before males enclosed, so
fertilization is assured. Nevertheless, a few females dispersed unfertilised from
galleries with males.
Males appear to mate only with teneral females, but copulations are generally observed rarely.
After fertilization of all sisters, when there is no further enclosion of females, males soon start
to disperse in search for outbreeding opportunities. This is around the time when the last
teneral female fully sclerotizes. Surprisingly, up to 30% of the galleries are male-less,
although only 3 % of the successful galleries housed only male offspring. Male-less galleries
are usually ones with very low productivity, that stay usually below 10 female daughters.
Why are only a few successful females unfertilised, although their number after emergence is
much higher? The first thought might be outbreeding, but I can exclude that, because family
groups were always kept separately in our study. .My explanations are: (1) These females are
mostly from unproductive galleries, probably not in good conditions and die before the
67
successful establishment of a new brood. (2) The ambrosial complex, vertically transmitted by
the females from their natal galleries to their new galleries might be in bad shape, because
productivity has been low at the first place already. (3) Females might be partly sterile if they
are not fertilized by males, because of bacterial symbionts in their ovaries. This kind of
bacteria usually impede the production of males, because of absence of fitness gains by the
males (transovarial transmission). Such bacteria are not known from Xyleborinus saxeseni,
but described for X. ferrugineus, another Xyleborini (Peleg & Norris 1973a, Norris 1972).
This bacteria has not been identified and studied in detail, but may be similar to Wolbachia
spp., which were first described some years later (e.g. Wade & Stevens, 1985).
The brood of X. saxeseni shows a strongly female biased sex ratio. Its reason is the
maximisation of productivity under local mate competition and high relatedness between
brothers (relationship coefficient R is close to 1). To lower competition between sibs, a
female should produce only as much sons as are sufficient to fertilize all daughters and
probably a few more under conditions that approve outbreeding (e.g. Hamilton, 1967 ; Peer et
al., 2004). It is surprising that such a high proportion of galleries is maleless. Outbreeding has
not been described in X. saxeseni.
The number of males produced closely resembled a normal distribution independent of the
number of daughters, as if their production is a stochastic process, rather than adjusted by the
females. This could be caused by the bacterial symbionts already mentioned: In X. ferrugineus
the usual sperm role of mature-oocyte activation in sexual reproduction is assumed by
bacterial symbionts. The remaining role of the sperm is to create diploid progeny which yields
females (Peleg & Norris 1973a, Norris 1972). Probably most haploid embryos get killed by
the bacteria, at the beginning of their development.
The vertically transmitted ambrosia fungi are a further potential reason for a female biased
sex-ratio. The spores are solely transferred in the females and males are worthless for the
fungi. Potentially fungi could (1) eliminate males through selective digestion, (2) manipulate
hormonally male developmental programs, or (3) produce nourishment that is optimised for
female development.
If sex-ratio deviates from the bias predicted under local mate condition has to be tested in
further studies.
Literature
Berryman, A. A. (1989). Adaptive pathways in scolytid-fungus associations. In Insect-Fungus Interactions: 145159. Wilding, N., Collins, N. M., Hammond, P. M. & Webber, J. F. (Ed.). London: Academic Press.
Borden, J. H. Aggregating pheromones. Mitton, J. B. and Sturgeon, K. B. Bark Beetles in North American
Conifers.
74-139.
1982.
Austin,
University
Texas
Press.
Ref Type: Generic
Farrell, B. D., Sequeira, A. S., O'Meara, B. C., Normark, B. B., Chung, J. H., & Jordal, B. H. (2001). The
evolution of agriculture in beetles (Curculionidae : Scolytinae and Platypodinae). Evolution 55: 2011-2027.
Fischer, M. (1954). Untersuchungen über den kleinen Holzbohrer (Xyleborus saxeseni). Pflanzenschutzberichte
12: 137-180.
Fisher, R. A. (1930). The Genetical Theory of Natural Selection. Oxford: Oxford University Press.
Francke-Grosmann, H. (1967). Ectosymbiosis in wood-inhabiting beetles. In Symbiosis: 141-205. Henry, S. M.
(Ed.). New York: Academic Press.
68
Hamilton, W. D. (1967). Extraordinary sex ratios. Science 156: 477-488.
Hosking, G. B. (1972). Xyleborus saxeseni, its life-history and flight behaviour in New Zealand. N. Z. J. Forest
Science 3: 37-53.
Jordal, B. H., Beaver, R. A., & Kirkendall, L. R. (2001). Breaking taboos in the tropics: incest promotes
colonization by wood-boring beetles. Global Ecology & Biogeography 10: 345-357.
KAJIMURA, H. & HIJII, N. (1994). Reproduction and resource utilization of the ambrosia beetle, Xylosandrus
mutilatus, in-field and experiment populations. Entomologia Experimentalis et Applicata 71: 121-132.
Kirkendall, L. R. (1993). Ecology and evolution of biased sex ratios in bark and ambrosia beetles. In Evolution
and diversity of sex ratio in insects and mites: 235-345. Wrensch, D. L. & Ebbert, M. A. (Ed.). New York:
Chapman & Hall.
Leach, J. G., Hodson, A. C., Chilton, S. J. P., & Christensen, C. M. (1940). Observations on two ambrosia
beetles and their associated fungi. Phytopathology 30: 227-236.
Lengerken, H. (1939). Die Brutfürsorge- und Brutpflegeinstinkte der Käfer. Leipzig: Akademische
Verlagsgesellschaft m.b.H.
Normark, B. B., Jordal, B. H., & Farrell, B. D. (1999). Origin of a haplodiploid beetle lineage. Proc. R. Soc.
Lond. B 266: 2253-2259.
Norris, D. M. 1972. Dependence of fertility and progeny development of Xyleborus ferrugineus upon chemicals
from its symbiotes, pp. 299-310 In J. G. Rodriguez [ed.], Insect and mite nutrition. North-Holland, Amsterdam.
Peer, K.
Biased sex
Ref Type: Unpublished Work
ratios
and
cooperative
breeding
in
ambrosia
beetles.
2003.
Peer, K. Inbreeding, biased sex ratios and delayed dispersal: optimal reproductive decisions in haplodiploid
ambrosia
beetles.
1-56.
2004.
University
of
Berne.
Ref Type: Thesis/Dissertation
Peer, K. & Taborsky, M. (2004). Female ambrosia beetles adjust their offspring sex ratio according to
outbreeding opportunities for their sons. J. Evol. Biol. 17:257-264.
Peer, K. & Taborsky, M. (2005). Outbreeding depression, but no inbreeding depression in haplodiploid ambrosia
beetles with regular sibling mating. Evolution 59: 317-323.
Peer, K. & Taborsky, M. (2007). Delayed dispersal as a potential route to cooperative breeding in ambrosia
beetles.
Behav.
Ecol.
Sociobiol.
61:729-739.
Peleg, B. and D. M. Norris. 1972. Symbiotic interrelationships between microbes and ambrosia beetles. J. Invert.
Pathol. 20:59-65.
Roeper, R., Treeful, L. M., O'Brien, K. M., Foote, R. A., & Bunce, M. A. (1980). Life history of the ambrosia
beetle Xyleborus affinis (Coleoptera: Scolytidae) from in vitro culture. Great Lakes Entomologist 13: 141-144.
Roeper, R. A. (1995). Patterns of mycetophagy in Michigan ambrosia beetles. Michigan Academian 27: 153161.
Stark, R. W. (1982). Generalized Ecology and Life Cycle of Bark Beetles. In Bark Beetles in North American
Conifers: Mitton, J. B. & Sturgeon, K. B. (Ed.). Austin: University of Texas Press.
Wade, M. J. & Stevens, L. (1985). Microorganism mediated reproductive isolation in flour beetles. Science 227:
527-528.
69
Chapter V: Chemical analysis of beetle
secretions and actinomycete bacteria
There has been a long discussion how insect fungus-farmers can uphold monocultures of their
cultivar against invading commensalistic or parasitic fungal lineages. Agriculture has hardly
been studied in beetles that are obligatorily dependent on ambrosia fungi they grow in
galleries of wood, the ambrosia beetles.
Members of this group, Xyleborinus saxeseni and other Xyleborini have recently been
observed to excrete secretes over their gut and mouth. These secretions have not been
analysed up to now, although they might contain (among others) fungal growth promoting
substances. On the other hand, oral secretions of the spruce beetle, Dendroctonus rufipennis
have recently been found to contain bacteria that inhibit antagonistic fungi in the galleries
{Cardoza, Klepzig, et al. 2006 1426 /id}. These bacterial symbionts should be particularly
important to maintain fungal monocultures in bark beetle species living in obligatory
relationships with fungi they feed on, the ambrosia beetles.
Samples of galleries, beetle secretions and individuals were first screened for organic and
volatile inorganic substances by coupled capillary gas chromatography-mass spectrometry
(GC-MS). The most prominent substance found was ergosterol, a major component of fungal
tissues. There were no substances found only in the secretions of the beetles, so I rejected the
hypothesis that fatty acids are used by the beetles to strengthen their fungi.
In a second study I screened beetle bodies of Xyleborinus saxeseni with rDNA primers for
actinomycete bacteria. In most of the individuals bacteria from the genus Gordonia were
found. Further studies will be conducted to look at the abundance of this genus in beetle
populations and its potential role.
Introduction
Analysis of chemical secretions
Agriculture has evolved seven times in ambrosia beetles (20-60 Mya), and once in the
Xyleborini (30-40 Mya) (Farrell et al., 2001). Beetles adapted to their mutualists by evolving
mycangia, modifications of mandibles and guts, and glandular or physiological modifications.
The ambrosia fungi modified their growth (=ambrosial growth) specifically to efficient
consumption and digestion by beetle larvae (Mueller et al., 2005). Due to sequestering their
fungal gardens on the walls of galleries in wood, they are well protected from fungivores,
wind-borne pathogens and arthropods (e.g. mites, collembolans). However, if the gardening
insects are removed or if they abandon their nests, the garden is quickly overrun by “weedy”
fungi. This fungi normally coexist at low levels along with the crop (Leach et al., 1940, Batra
& Batra, 1967, Batra, 1979, Norris, 1979). The ambrosial layer is regularly browsed by the
beetles and their larvae, but the ways how the beetles maintain the dominance of the primary
ambrosia fungus in the galleries are hardly looked at. In contrast to the other two fungus
farming insect groups, the attine ants and the termites, the physical contact (e.g. grooming and
weeding) appears to be of less importance (Hopkins, 1898; Whitney, 1971; French & Roeper,
1972). Probably more important are secretions of beetles and their larvae (Francke-Grosmann,
1956, 1963, 1975; French et al., 1972). Hypothetically the highly specialized ambrosia fungi
70
get strengthened and suppress other invading fungi by their antibiotic attributes (FranckeGrosmann, 1956).
In bark beetles fungal cells are commonly transmitted to new galleries in invaginations of the
exoskeleton, so called mycangia (or mycetangia). The mycangia of ambrosia beetles are lined
with secretory gland cells (Batra, 1963, Schneider & Rudinsky, 1969, Six, 2003). The fungi
are not just carried as resting spores in there, but they multiply while in mycangia. This
growth is under the expense of the beetles, by providing it with a high level of free amino
acids et al. (French & Roeper, 1973; Francke-Grosmann, 1956; Norris, 1979). Additionally to
this substances that are probably leaking from the mycangia, many secretional glands are
distributed at all weakly sclerotized parts of the body (Francke-Grosmann, 1956). Xyleborinus
saxeseni is unusual to transmit its primary ambrosia fungus (Ambrosiella sulphurea) over the
gut, and has only auxiliary yeasts in its mycangia (Francke-Grosmann, 1975). They can be
observed to excrete liquid substances over the gut and the mouth (own obs.). Kept on filter
paper for some time, they release some water repellent substances on the paper (own obs.,
Bischoff, 2004) for Xylosandrus germanus). Even more likely are secretions of larvae, that
have a very thin and slimy cuticula (Francke-Grosmann, 1956; own obs.).
Sapwood is poor in nutrients, but the ambrosia fungus supplies the beetles with nutrients
usually located far away from the brood (Batra, 1963). However, low nitrogen levels are
generally considered to be limiting factors for the decomposition of wood cellulose and lignin
by fungi. In several non-symbiotic Basiodiomycetes, the addition of organic nitrogen
stimulates the decay rate of wood by sometimes over 60% (Batra, 1979; Swift, 1977). Thus, it
is clear that the fungi can reutilise part of the nitrogen excreted by the beetles for further
growth, what has been judged by mycelium dry weight (Abrahamson & Norris, 1969; Batra,
1963; Kok, 1979; Batra, 1979). Additionally it has been shown that urates (major, solid
nitrogenous excretory products of Xyleborini), urea or aspartic acid caused the characteristic
ambrosial growth form by an ambrosia fungus in several media at initial pHs between 6 and 7
(Abrahamson et al., 1969; Norris, 1979).
In conclusion simply the presence of the beetles and larvae is beneficial for the growing
fungal layer, by supplying it with nutrients, that are probably mainly excretes. The critical
point appears to be the establishment of a few larvae. As soon as some are present it boosts
ambrosia growth, probably through secretions and physical contact (pers. obs.). When the
gallery gets older and number of adult beetles decreases (through emergence), less visited (or
browsed) parts of the gallery get overgrown by weedy fungi (e.g. pers. obs.; FranckeGrosmann, 1956; Gebhardt, et al.; 2005).
It has been argued that, among others, fatty acids are produced by the beetles to strengthen
their mutualistic fungi (e.g. Francke-Grosmann, 1956; Norris, 1979; Berryman, 1989). This
substances should be detectable by coupled capillary gas chromatography-mass spectrometry
(GC-MS). I predict to find some chemical compounds on the beetles, the gallery walls and
probably the frass. This compounds should lack from probes of the medium and the fungus
growing apart from the galleries.
Actinomycetal bacteria
A well studied animal-actinomycete symbiosis is that of attine ants and actinomycete bacteria
of the family Pseudonocardiaceae (Currie, 2001; Currie, et al., 1999, 2003; Poulsen, et al.,
2003). Like the ambrosia beetles they are cultivating fungi for nutrition, and they carry the
bacteria on specific regions of their cuticle (Currie et al., 1999). The actinomycetes produce
antibiotics that specifically inhibit the growth of Escovopsis, a specialized parasitic fungus of
71
the fungus gardens (Currie et al., 1999, 2003). Its widespread association among attine ants,
and the vertical mode of transmission points to a long coevolutionary relationship between
these symbionts (Currie et al., 1999). Actinomycetes are also discussed to play a role in the
third insect farmer, the fungi-growing termites (Shinzato et al., 2005). Recently Streptomyces
species have been found to protect beewolf larvae from fungal infestation (Kaltenpoth et al.,
2005). Actinomycetes, and especially streptomycetes are known to have the ability to
synthesize a huge diversity of antibacterial and antifungal secondary metabolites. In fact most
of the antibiotics used in medical application are produced by this group (Behal, 2000;
Goodfellow & Cross, 1984).
Due to the fact that most studies on ambrosia beetles have been done before or in the 70’s,
lacking the tools to identify bacteria properly, there is no knowledge of bacteria involved in
this beetle-fungus association. However, some researchers predict bacteria to be part of the
symbiotic microbial complex, together with different fungal species (Batra, 1963;
Abrahamson & Norris, 1966; Haanstad & Norris, 1985). White biofilms that resemble
bacterial layers are sometimes observed on the glass of galleries (pers. obs.). The only
bacterial studies in Xyleborini are those on X. ferrugineus, showing that a Staphylococcus sp.
on the female ovaries mediates its parthenogenetic reproduction (Peleg & Norris, 1972;
Norris & Chu, 1980). Its influence is similar to Wolbachia sp., now known from lots of
insects, spiders and nematodes.
In my study I looked for actinomycetes, common mutualistic and antibiotics producing
bacteria.
Material & Methods
The studied beetle
Xyleborinus saxeseni transmits its primary ambrosia fungus to new galleries solely
endozoically in the form of micromycelia. The mycelia develop from ascospores which are
formed in the crop (part of the foregut) of teneral beetles. Later the intestina is completely
filled with micromycelia and growing spores (Francke-Grosmann, 1975).
The mycangia of this species are only for the transmission of auxiliary yeasts (FranckeGrosmann, 1975). However, the only studied secretions in ambrosia beetles, are that of the
mycangial glands. Buchner (Buchner, 1928) was the first studying the contents of the
secretion of intersegmental sacs (later called mycangia) of a bark beetle species and found an
acid polysaccharide protein complex. More recent studies showed that mycangia content fatty
acids, phospholipids, free sterols, sterol esters and triglycerides. Very abundant are also the
free amino acids, proline, alanine and valine; and in smaller amounts arginine, histidine and
aspartic acid (Abrahamson et al., 1969, Abrahamson & Norris, 1970; French et al., 1973;
Norris, 1979; Berryman, 1989). This situation provides the beetle with the ability to keep its
symbiotes in relatively slow growing and non-parasitic states (Batra, 1963; Farris & Funk,
1965, Abrahamson et al., 1969; Barras & Perry, 1971; Happ, et al., 1971; Norris, 1979).
The studied fungus
In X. saxeseni there is usually Ambrosiella sulphurea as the dominant primary ambrosia
fungus found in the galleries. It produces a yellow (like brimstone) ambrosial layer and its
conidiospores are filled with oil. This species belongs to the genera Ophiostoma
(Ascomycota) which invade sapwood only after the tissue has ceased to conduct water – that
is, after the host has died (Hobson et al., 1994). In general ambrosia fungi a extremely
phleomorphic and environmental conditions determine the growth at any given time: (1) a
mycelial growth form when not tended by the beetles, (2) an ambrosial or “kohlrabi” growth
72
when tended on the gallery walls, and (3) a slow growing micromycelial growth in the
mycangia or guts of the beetles when transmitted from one host to the next (e.g. FranckeGrosmann, 1956, 1963, 1975; Batra, 1963, 1979; Baker & Norris, 1968; Barras et al., 1971;
Norris, 1979; Beaver, 1989; Six, 2003). If ambrosia fungi are grown on acid mediums that are
rich in amino acids, and are exposed to more than 0,5 percent carbon dioxide, then ambrosia
are formed (Batra et al., 1967). Ambrosial growth can also be induced by physical disruption
of mycelia or low temperatures (Batra et al., 1967; Leonard & Dick, 1973) larval movements
and/or abdominal flexing of pupae (Whitney 1971, 1982).
Fungal mycelia penetrate the wood for some centimetres and when they are getting older they
stain the wood with brown excretes (e.g. Leach et al., 1940; Matthiesen-Kärik, 1953; FranckeGrosmann, 1956, 1963). This stain indicates that the fungi has successfully colonized the
substrate, are maturing and are becoming dormant. In some species it excretes a dark brown
liquid that gets collected in drops on the surface of the fungal colony (Leach et al., 1940;
Francke-Grosmann, 1956; Whitney, 1982).
Chemical analysis of beetle secretions
I analyzed beetles and parts of the gallery for organic and volatile inorganic substances. I
opened 5 productive galleries and took supplements of the beetles, the fungus-free medium,
the gallery walls, the ambrosia fungus that is growing on and apart from the galleries and the
frass of the beetles (see table 1). As a control I also identified the detectable components of
sterile medium. The different probes were placed in glass vials (4 ml), and submerged in
approximately 400 µl distilled hexane or methanol. In some of the methanol probes TMSH
was added for methylation, to increase the number of detectable compounds. Chemicals out of
the different probes were extracted for a few hours at room temperature. Then the hexane (or
methanol) from the probes was filled in a sterile glass and was reduced to about 200 µl by a
gentle constant flow of nitrogen. Samples were analyzed by coupled capillary gas
chromatography-mass spectrometry (GC-MS) with an Agilent 6890N Series gas
chromatograph (Agilent Technologies, www.agilent.com/) coupled to an Agilent 5973 inert
mass selective detector. The GC was equipped with a RH-5ms+ fused silica capillary column
(JandW, 30 m x 0.25 mm ID; df = 0.25µm; temperature programme: from 60°C to 300°C at
5°C/min, held constant for 1 min at 60°C and for 10 min at 300°C). Helium was used as the
carrier gas with a constant flow of 1 ml/min. A split/splitless injector was installed at 250°C
in the splitless mode for 60 sec. The electron impact mass spectra were recorded with an
ionisation voltage of 70 eV, a source temperature of 230°C and an interface temperature of
315°C. The software MSD ChemStation for Windows was used for data acquisition.
PCR and Sequencing of beetle bacteria
Beetles were taken from three lab-bred galleries and 5 different field galleries. They were
stored in 95% Ethanol before dissection.
Bacterial DNA was extracted from the caput and the abdomen of adult beetles according to a
standard phenol-chloroform extraction protocol. The following 16S rDNA primers were used
for general screening for actinomycete bacteria: fD1 (forward) (Weisburg et al., 1991) and
Act-A19 (reverse) (Stach et al., 2003). The primers Gord268F (forward) and Gord1096R
(reverse) (Shen & Young, 2005) were used for selective amplification of Gordonia 16S
rDNA. PCR amplification was performed on Eppendorf Mastercyclers in a total reaction
volume of 25 µl containing 4µl of template, 1x PCR buffer (10 mM Tris-HCl, 50 mM KCl,
and 0.08% Nonidet P40), 2.5 mM MgCl2, 240 µM dNTPs, 20 pmol of each primer, and 1 U
of Taq DNA polymerase (MBI Fermentas).
73
Cycle parameters were as follows: 3 min at 94°C, and then 32 cycles of 94°C for 40 s, 64°C
for 1 min, and 72°C for 1 min, and a final extension time of 4 min at 72°C. For sequencing,
we used the fD1 primer. Sequencing was carried out on a Beckmann-Coulter CEQ 2000 XL
sequencer.
Results & Discussion
Analysis of beetle secretions and fungal components
In the 6 different supplements (see Material & Methods; table 5.1.) we found 35 different
chemical components. The most common were cyclic hydrocarbons, fatty acid methylesters
and ergosterol.
9 components were associated with ambrosia beetles: three types of alcohols, two aldehyds, a
keton, tricosan, pentacosan, heptacosan and nonacosan. TMSH in combination with Methanol
was the best of the three solvents.
If beetles produce some substances, then they should be found in one of the first 4
supplements and should lack from the last 2 supplements. These components are marked with
**.
Table 5.1. components found by GC-MS in different supplements. The number gives the
amount of supplements where this component was found.
A discussion of important fungal components
The most prominent component found in all supplements was Ergosterol. Insects are
dependent on a dietary source of sterol (Clayton, 1964). Ergosterol is the only sterol found in
74
ambrosia fungi (Chu, Norris & Kok, 1970), and it is one of the most suitable sterols for
insects (CLAYTON, 1964).The Ergosterol-levels of these fungi are very high, while plant
tissues like phloem and xylem have typically low sterol concentrations. Its content increased
with age (Chu et al., 1970). Ergosterol production varies considerably by fungal species and
the one with the highest concentrations are the most beneficial for the beetle hosts (Kok &
Norris, 1973). Surprisingly we found Ergosterol also in one supplement of sterile medium.
This is either a hint on fungal infection of the medium or more likely caused by fungal parts
in the saw dust used to produce the medium.
Fungal components that are necessary for female’s reproductive processes in Xyleborini, like
certain amino acids, as lysine, methionine, arginine and histidine, (Bridges & Norris, 1977a),
(Norris, 1979b) can not be detected by GC-MS.
A discussion of components associated with beetles
Of the 9 components associated with beetles, 4 are usually parts of waxes (Tricosan,
Pentacosan, Heptacosan and Nonacosan). These waxes are probably found on the cuticle of
the beetles. Waxes generally function as protection against dessication and UV-light (Kunst &
Samuels, 2003), against pathogentic microbes by preventing moistening (Jetter & Riederer,
2000) and against pathogenic fungi (JENKS et al., 1994). Some apidae use a composition of
this 4 components as male attracting pheromones (Schiestl & Ayasse, 2000; Schiestl et al.,
2000). For ambrosia beetles it is easy to assume their role in protection. However, it is not
unlikely that females try to attract their brothers because (1) their number is very small (only
1-3 males per gallery) and (2) chemical communication is probably important in the dark and
widely branched tunnels.
The hypothesis to find fatty acids, produced by the beetles to strengthen their mutualistic
fungi (e.g. Francke-Grosmann, 1956; Norris, 1979; Berryman, 1989) can be rejected. To our
knowledge none of the 9 identified components associated with the beetles, is used in this
sense by another system. They play probably only a minor role (if any) in suppressing
pathogenic fungi and/or strengthening the ambrosia fungus.
Further studies should try to clarify the role of aminoacids and proteins for fungiculture,
known to be produced by these beetles.
Beetle bacteria
The phylogenetic analysis showed that X. saxeseni has bacteria of the genus Gordonia in both
caput and abdomen. It belongs to the suborder of Corynebacterineae, a group within the order
Actinomycetales (Stackebrandt et al., 1997). They are particularly interesting because species
out of this genus have abilities to degrade xenobiotics, environmental pollutants, or otherwise
slowly biodegradable natural polymers as well as to transform or synthesize possibly useful
compounds (Arenskotter et al., 2004). However, its absence in some specimens collected in
the field is probably an indication of contamination from the outside.
It was beyond this work to look at the Gordonia species or its role in the beetle-fungus
association. It should only be a preliminary study to attract attention to this fascinating
network of various, unknown relationships.
75
Fig. 5.1. Potentially a bacterial layer that is covering the glass.
Acknowledgements
I thank Erhard Strohm for inviting me to Würzburg for the chemical and bacterial analysis.
Many thanks also to Martin Kaltenpoth and Johannes Kroiss for their hospitality and help
with the GC-MS. Martin Kalthenpoth performed the bacterial analysis for me. All of them
and my supervisor Michael Taborsky I am grateful for helpful discussions and the
Behavioural Ecology Department in Berne for financial support to go to Germany.
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79
Schlussbemerkungen
Nun ist das Werk also vollbracht! Würde ich nicht wissen, dass ich „meinen Käfern“ und
Bern weiter treu bleiben darf, würde mich wahrscheinlich eine tiefe Schwermut erfassen. So
aber sehe ich die Arbeit als ersten Lagebericht was ich bisher über das
Gruppenzusammenleben und den Ackerbau dieser kleinen Pilzgärtner herausfinden konnte.
Was hat mich aber überhaupt dazu bewogen mit Borkenkäfern zu arbeiten? Das ist eine
längere Geschichte, und ich möchte dafür ein wenig ausholen: Schon als Kind war ich von
allen Tieren und Natur total begeistert. Seit ich denken kann ist meine Lieblingsbeschäftigung
irgendwo im Grünen zu sitzen und die Welt um mich zu beobachten. Angefangen hat das
zuhause im Garten meiner Eltern am Teich zu sitzen, alles zu bewundern und im Winter vom
Fenster aus das Futterhaus zu beobachten. Ich war eigentlich nie von einer bestimmten
Tiergruppe besonders fasziniert, sondern immer vom Leben selbst. Ich weiß nicht wie sehr so
eine Faszination angeboren sein kann, aber auf jeden Fall wurde sie schon vom Kleinkindalter
sehr von meinen Eltern durch Tierbücher gefördert. Einem solchen Buch ist es auch zu
verdanken, dass ich mich erstmalig für eine bestimmte Tiergruppe zu spezialisieren begann.
Durch einen glücklichen Zufall stieß mein Vater über einen Presseartikel auf den „Atlas der
Brutvögel Österreichs“. Dieses Buch eröffnete mir das Tor zu BirdLife Österreich – zum
ersten Mal zu Leuten außerhalb meiner Familie, die genauso begeistert von der Tierwelt
waren. Dankbar bin ich dabei besonders Peter Sackl und Max Dumpelnik, die mir auf
zahlreichen Exkursionen vor allem die Stimmen der Vögel näher gebracht haben. Nach
meinen Vater sehe ich Peter Sackl auch als ersten Förderer, da er mir die Chance gab im
Museum in Graz und in zahlreichen Projekten, vor allem Vögel betreffend, mitzuarbeiten.
Eigentlich war immer klar dass ich Zoologe werden wollte, aber als die Zeit da war ein
Studium zu wählen war ich gerade sehr fasziniert von Bionik und Biophysik. Da ich auch ein
technisches Grundinteresse habe, entschied ich mich für das Studium der Physik um mich
später in Richtung Biophysik zu spezialisieren. Aber wie das so ist, wenn man nicht
hundertprozentig überzeugt von einem Gebiet ist, merkte ich schon bald dass ich nichts mit
Physik anfangen kann und auch nicht begeisterungsfähig dafür war. So wechselte ich schon
nach einem Jahr auf das Studium das eigentlich schon immer meine Passion war – Zoologie.
Nach der Wahl dieses Studiums drängte mich mein Vater von Beginn an soviel (Auslands-)
Erfahrung wie nur möglich außerhalb des Studiums zu sammeln, um später bessere Chancen
für die begrenzten Jobchancen zu haben. Tagelanges recherchieren seinerseits, hunderte Emails und dem Zufall dadurch auf die International Crane Foundation zu stoßen, ermöglichten
mir meinen ersten längeren Auslandsaufenthalt in Baraboo/Wisconsin. Dieser Aufenthalt
öffnete mir das Tor zur Vogelberingung in Kanada im darauffolgenden Jahr, zu einem
wunderschönen Projekt und unglaublichen Eindrücken ein weiteres halbes Jahr später in der
Mongolei. Diese einsamen Monate dort, Feldarbeit mit den faszinierenden
Gelbbrauenlaubsängern, waren wohl die lehrreichsten, wichtigsten und besten in meinem
bisherigem Leben.
In den Oasen der Ruhe schöpfen wir Kraft, finden wir Gelassenheit und uns selbst.
Als ich das Mongoleiprojekt dann als Bachelorarbeit einreichen wollte, erwischte ich zufällig
gerade einen guten Zeitpunkt, denn der Professor war gerade auf der Suche nach einem
Feldassistenten in Panama. Anschließend war es nicht weiter schwierig eine
Praktikantenstelle am MPI in Seewiesen zu bekommen. Zu diesem Zeitpunkt war ich, abseits
von vielen Lehrveranstaltungen über Honigbienen an der Uni und der Forschung an
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Laubheuschrecken bzw. Fledermäusen, noch immer ganz auf Vögel spezialisiert. Einige
unschöne Erfahrungen in Seewiesen, die aber bereits vorher schon vorhandene Zweifel
bestätigten, führten dazu dass ich mir aus moralischen Gründen eine Diplomarbeit im
Vogelbereich nur unter bestimmten Voraussetzungen vorstellen konnte. Durch mehrere
glückliche Zufälle lernte ich zu diesem Zeitpunkt Prof. Taborsky aus Bern kennen. Eigentlich
ist er ja auch ursprünglicher Trofaiacher, aber das wusste ich damals noch nicht. Er war gleich
sehr entgegenkommend, und ich war von Anfang an unglaublich fasziniert von Käfern die
vielleicht eusozial sind, Pilze züchten und noch dazu überall in unseren Wäldern leben, ohne
dass ihre Einzigartigkeit von jemandem wahrgenommen wird. Ich wusste sofort, dass ich
mehr darüber wissen wollte! Leider war die Anmeldefrist für das Studium in Bern bereits
abgelaufen, und ich hatte noch immer nicht mein offizielles Bachelorzeugnis. Da stand ich
kurz vor dem Abbruch und vor der Entscheidung doch nach Wien zu gehen. Wie so oft in
solchen Situation hat mich mein Vater neu motiviert und durch das großzügige
Entgegenkommen von Prof. Zettel wurde ich dann doch noch an der Uni immatrikuliert.
Nun aber zur eigentlichen Arbeit. Der Beginn dieser war nicht leicht für mich. Es gab viele
methodische Schwierigkeiten, die Käfer waren kaum zu finden und die Zucht noch nicht
erfolgreich. Zusätzlich war Kathi Peer (meine Vorgängerin) nicht mehr vor Ort, stand mir
aber telefonisch immer mit Rat und Tat zur Verfügung. Nach einer gemeinsamen Käfersuche
im Feld gelang es mir dann endlich auch selbst Käfer zu finden. Ich bin Kathi Peer für ihre
anfängliche Motivation und Diskussionen sehr dankbar. Zum Teil war es nicht einfach mich
immer wieder zu motivieren, weil ich ja allein an dem Projekt arbeitete. Zum Glück hatte ich
aber in der Baltzerstrasse schon Salome, Michael D. und Simon kennen gelernt, die mir die
Mittagspausen versüßten. Das Einleben in Bern war anfangs nicht so leicht, aber meine
Eltern, mein Bruder Thomas, mein Cousin Jörg, meine Freundin Marianne und Martin,
Gernot und Franzi zuhause waren immer über skype für mich erreichbar.
Ohne die Spieleabende mit Salome, Claudia und Stefan könnte ich wahrscheinlich noch kaum
ein Wort schwyzerdütsch sprechen. Ich danke Salome für die schönen Erlebnisse - Fasnacht
in Luzern, die Feldarbeit im Wallis und viele andere lustige Unternehmungen.
Nicht zu vergessen sind natürlich die wechselnden Mannschaften im Studentenhaus. Das
lustige Semester mit Lukas, Venu (unserem indischen Professor), Dorian, Ghassan, Rhebecca
und Sadeq. Wie oft hab ich mir gewünscht aus dem kleinen Zimmer auszuziehen, aber die
internationale Atmosphäre und Bequemlichkeit hat mich doch immer davon abgehalten. Wie
viele Freunde hab ich dort gefunden!? Kathrin, die leider viel zu kurz da war, aber umso mehr
Zauber gebracht hat. So schnell sind noch kaum 6 Monate vergangen.
Alex war der Anfang eines neuen Teams im Studentenhaus das ich sicher sehr vermissen
werde: Annerosi unsere gute Seele, Jule unsere verträumte Partymaus, Alex der für
wissenschaftliche Diskussionen genauso zu haben ist wie fürs Feiern, und Martin der uns
leider schon verlassen hat. Nicht zu vergessen sind dann natürlich auch noch Sandra und Jane.
Eine besondere Stellung nimmt auch Nadine ein, die gerade am Ende der Arbeit einen wahren
Motivationsschub in mir ausgelöst hat. Ich bin euch allen unendlich dankbar einfach da
gewesen zu sein – ihr habt mir diese Jahre wirklich zum Genuss gemacht.
Kein Weg ist lang mit einem Freund an seiner Seite (chinesische Weisheit)
Ich danke auch der ganzen Belegschaft des Hasli, insbesondere Corni, Fränzi und Andrea für
die lustigen Stunden. Daniel für das Interesse an wissenschaftlichen Diskussionen und die
Hilfe bei verschiedenen Manuskripten. Mein Dank geht auch an Dik un Jeremy, die mit ihrer
immensen Erfahrung immer mit einem Rat zur Seite standen.
Der größte Dank geht an meinen Betreuer Michael Taborsky, der mir die ganze Arbeit hier
ermöglicht hat, trotz seiner begrenzten Zeit immer für Diskussionen zur Verfügung stand.
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Durch sein unglaublich umfassendes naturwissenschaftliches Wissen kam ich sehr oft mit
ganz neuen Einsichten aus solchen Gesprächen.
Die tiefste Dankbarkeit empfinde ich gegenüber meinen Eltern. Durch ihre unterschiedliche
Art haben sie mich für die verschiedenen Seiten des Lebens geöffnet. Von Papa habe ich
gelernt wissenschaftlich, sorgfältig und konzentriert zu arbeiten. Mama hat mir dabei gezeigt
immer auf die Signale des Körpers zu achten und liebevoll mit ihm umzugehen. Finanziell
und mental haben sie mich immer unterstützt. Obwohl ich zum Teil weit weg von ihnen bin,
weiß ich sie als Rückhalt der nicht nur in Krisen immer für mich da ist.
Danke euch allen, dass ihr mein Leben begleitet bzw. dass sich unsere Lebenswege einmal
getroffen haben! Ihr habt alle einen Teil zur Fertigstellung dieser Arbeit beigetragen!
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Peter H. W. Biedermann, BSc (MSc)
Born 21st of June 1981
Nationality AUSTRIA
Königshofergasse 8,
A-8793 Trofaiach, Austria
[email protected]
Curriculum Vitae
‘Nature magically suits a man to his fortunes, by making them the fruit of his character.’
R.W.Emerson.
Name of Referees:
Prof. Dr. Michael Taborsky • [email protected]
Dept. Behavioural Ecology, Institute of Zoology, University of Bern
Wohlenstrasse 50a, CH-3032 Hinterkappelen/Bern
Phone: +41-(0)31 631 9156
Fax: +41-(0)31 631 9141
http://zoology.unibe.ch/behav/
Prof. Dr. Bart Kempenaers • [email protected]
Managing Director Max Planck Institute for Ornithology
D -82319 Starnberg-Seewiesen, Haus 5
Phone: +49-(0)8157 - 932-334
Fax: +49-(0)8157 - 932-400
http://www.ornithol.mpg.de
O.Univ.Prof. Dr. Heiner Römer • [email protected]
Group leader of the project to study the neural system of tropical bushcrickets
Institute for Zoology, Karl-Franzens University Graz
A-8010 Graz, Universitätsplatz 2
Phone:++43/ 0316/380/5596,
Fax:++43/ 0316/380/9875
http://www.kfunigraz.ac.at/zoowww/
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Education
Zoology
Since April 2005 • Master in Ecology & Evolution at University of Bern; Master-Thesis on the
social behaviour of ambrosia beetles at the Department of Behaviour Ecology (Supervisor: Prof.
Taborsky).
January 2005 • 2.Bachelor-Thesis: “Neuronal mechanisms of spatial orientation in food storing
birds”
December 2004 • 1. Bachelor-Thesis: “Investigations on the breeding biology of the Yellowbrowed Warbler in the Mongolian Khentey”
2002-2005 • Karl-Franzens University Graz and University Vienna, Austria, Bachelor in Zoology
with the main focus on Behaviour Biology and Neurobiology in Honeybees and Crickets.
General Education
2000-2001 • Karl-Franzens University, Graz, Austria, two semesters with the major in Physics.
1999-2000 • Military service in the Rohrkaserne Villach/A
1992-1999 • BRG Leoben, AUSTRIA, school graduation with honors (A-levels in German,
Maths, Biology, Physics)
1988-1991 • Elementary School in Trofaiach/A
Professional Experience
Internships
Summer/Fall 2004 • 10 weeks at the Max Planck Institute for Ornithology. Doing a small project
on the sexual selection of blue tits (theoretical work, capturing and bleeding blue tits, laboratory
analysis of blood) under the supervision of Kaspar Delhey and Dr. Anne Peters.
Spring/Summer 2004 • Project on “Drone (Apis mellifera) – Swallow interactions on Drone
congregation areas and reference areas”, funded by the Zoological Institute of the University in
Graz.
Febr./March 2004 • Working as a field assistant at the Smithsonian-Tropical-Research Institute
at Barro Colorado Island in Panama, funded by the Austrian Science Fund (FWF).
Neurophysiological and behaviour-ecological experiments with katydids. This included (1) the
monitoring of marked insects in their daytime shelter of Aechmea plants, (2) measuring the
airborne sound and vibrational communication behaviour of male katydids at different moon
phases, or (3) performing neurophysiological experiments in the context of sound and vibration
detection. In addition Behavior-ecology and mist-netting of bats.
Spring/Summer 2003 • 3-month practical training in Mongolia for the "Institute of conservation"
at the Universität in Göttingen/D funded by the DAAD. Doing an independent project on the
breeding-ecology of Yellow-browed Warblers (Phylloscopus inornatus).
Summer 2002 • 1 month at the Internat. Crane Foundation near Baraboo/WI (USA)
Banding and radio-tracking cranes. Doing a small project on sex-differences in Sandhill-cranes.
• Two months at the Thunder Cape Bird Observatory near ThunderBay/ON (Canada)
Banding and observing song-birds, raptors and owls.
Summer 2001 • 4 weeks at the Internat. Crane Found. (see above)
Banding and radio-tracking cranes. Doing plant samplings and helped prairie restoration.
• Ten days on a whale and dolphin research ship in the Mediterranean provided by the Tethy´s
organization. Helping researchers observe and taking skin-samplings of sea-mammals.
2000 to 2005 • In cooperation with a group from the Department of Zoology at the
Museum “Joanneum” in Graz working on the 'Atlas of breeding-birds of Graz' (do data
samplings and evaluations) and some other small projects (especially monitoringprojects).
Teaching experience
June, August 2006 and 2007 • Teaching assistance at the Department of Behavioural Ecology,
University Bern for the two classes: “Practical training in Behavioural Ecology I and II”
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Peer reviewed Publications
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Biedermann, PHW (2006): Hidden leks in the Yellow-browed Warbler (Phylloscopus
inornatus)? - Investigations from the Khan Khentey Reserve (Mongolia). Acrocephalus 27:
pg. 233-247.
Delhey, K, Biedermann, PHW, Kempenaers, B, & Peters, A (Biology letters, submitted):
Optical properties of the uropygial secretion: are there UV cosmetics in birds?
Kärcher, MH, Biedermann, PHW & Hrassnigg, N (Apidology, submitted): Predator-prey
interaction between drones of Apis mellifica and the swallow species Hirundo rustica and
Delichon urbica
Biedermann, PHW & Taborsky, M (submitted): Towards eusociality in Xyleborinus saxeseni
Other Publications
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-
Biedermann, PHW & Kärcher, MH (submitted): Investigations on the feeding height of
Barn Swallows (Hirundo rustica) and House Martins (Delichon urbica) in southwestern
Styria
Biedermann, PHW (2003): Die Kraniche der Welt. Zoologie Newsletter Nr.2;
Landesmuseum Joanneum Graz
Posters
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-
Biedermann, PHW & Taborsky, M (2007): Higher sociality in a beetle with mutualistic
fungi. D-Day at the University in Lausanne/CH.
Biedermann, PHW & Taborsky, M (2007): Towards eusociality in Ambrosia beetles.
Biology07 at the ETH Zurich/CH.
Biedermann, PHW, Peer, K & Taborsky, M (2006): Social behaviour in the bark beetle,
Xyleborus saxeseni. Meeting of the Ethological Society (Ethologischen Gesellschaft) in
Bielefeld/D.
Kärcher, MH, Biedermann, PHW & Hrassnigg, N (2005): Drone (Apis mellifera) – Swallow
interactions at Drone congregation areas and reference areas”. At the Annual meeting of
the German Institutes working with bees (AG der Bieneninstitute) in Halle/D.
Talks
-
Höhere Sozialität in Ambrosiakäfern. Annual Xylobionten-Meeting at University Bern/CH
(2007).
Towards eusociality in Xyleborinus saxeseni. Meeting of the graduate students of the DZG
and the Ethological Society, University Göttingen/D (2007).
Skills
Language skills
English • fluent (more than ½ year in English speaking environment)
Italian • ordinary (3 years of grammar school)
German • Mother tongue
Personal Interests
Hobbies • I love to observe nature; committee member of BirdLife-Styria, lead excursions to show
people animals especially birds; reading (Hesse H, Dawkins R, Maturana H, Lorenz K,
Quammen D, Wilson EO…); travelling to remote places; spending time with friends & my
family.
Sports • Kriya Yoga, off-piste skiing, hiking, badminton.
Bern, May 2007
yours sincerely,
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