Seasonality of gregarine parasitism in the damselfly,

Parasitol Res
DOI 10.1007/s00436-011-2478-1
ORIGINAL PAPER
Seasonality of gregarine parasitism in the damselfly,
Nehalennia irene: understanding unimodal patterns
Mark R. Forbes & Julia J. Mlynarek & Jane Allison &
Kerry R. Hecker
Received: 30 December 2010 / Accepted: 19 May 2011
# Springer-Verlag 2011
Abstract We studied parasitism by gut protozoans
(Apicomplexa: Eugregarinidae) in the damselfly, Nehalennia
irene (Hagen) (Odonata: Coenagrionidae). We tested whether
there was any seasonal pattern, as has been found for other
parasites of damselflies and which has implications for
selection on emergence and breeding. Using aggregate data
from 12 date-by-site comparisons involving five sites, we
found that both prevalence and intensity of gregarine
parasitism were seasonally unimodal. Parasitism first
increased and then declined seasonally after peaking midseason. This damselfly species has shown seasonal increases
in density followed by declines at several sites including a site
sampled in this study. Therefore, similar seasonal changes in a
directly transmitted parasite were expected and are now
confirmed. Other factors that might account for seasonal
changes in parasitism by gregarines are either unlikely or can
be discounted including sampling of older damselflies
mid-season but not late in the season, or sex biases in
parasitism and overrepresentation of the more parasitized sex
mid-season.
Introduction
Insects of many species are expected to show increased
parasitism as they age simply because they have been
around longer to encounter more infective stages of parasites (e.g., Smith and Cook 1991; Hassall et al. 2010).
Related to this phenomenon is the fact that samples of
insects can show seasonality in patterns of parasitism (Åbro
M. R. Forbes : J. J. Mlynarek (*) : J. Allison : K. R. Hecker
Department of Biology, Carleton University,
209 Nesbitt Bldg, 1125 Colonel By Drive,
Ottawa, ON K1S 5B6, Canada
e-mail: [email protected]
1971; Zuk 1987; Locklin and Vodopich 2010a). Of course,
seasonality might also occur independent of age differences
in samples of hosts. An early study by Forbes and Baker
(1991) showed that parasitic Arrenurus mites decreased on
newly emerged (same-aged) damselflies, as the season
advanced.
Seasonal changes in parasitism are important to the
extent that the parasites or pathogens under study act as
agents of selection (Altizer et al. 2006). Advantages to early
breeding could be counteracted by selection imposed by
parasites, if parasites were most common early in the
breeding or emergence period (Altizer et al. 2006). Later
emergence might be costly in terms of finding a mate, but
beneficial if late emergence was associated with less
likelihood of becoming parasitized. In comparison, later
emergence might be disadvantageous if infective stages of
parasites accumulate through time due to the abundance of
host individuals that emerged earlier and contribute to an
increased number of infective stages, as is expected for
directly transmitted parasites with single obligate hosts
(Åbro 1971, 1974).
Odonate–gregarine interactions have been studied for
many species of odonates with mixed results or opinions as
to the effect of gregarines on their odonate host (Åbro 1971,
1974, 1976, 1987, 1990, 1996; Locklin and Vodopich 2009,
2010a). Gregarines are apicomplexan protozoans that have
a one-host life cycle. The infective cysts are ingested by
damselfly hosts either along with prey or through drinking
(Locklin and Vodopich 2010a). They develop, grow, and
reproduce in the midgut of their host. Once the gametocysts
have formed, they are excreted with the feces to infect
another individual (Åbro 1987). Since these parasites are
transmitted trophically, differences in feeding behavior and
habitat use in relation to density have to be considered for
males and females and for individuals at different sites.
Parasitol Res
We studied the prevalence and intensity of infection by
septate gregarines in the damselfly Nehalennia irene
(Hagen). Nehalennia irene is a widespread damselfly
inhabiting a variety of freshwater marshes (Walker 1953).
Females exhibit two distinct color morphs; andromorphs
closely resemble males while heteromorphs are unlike
males. Andromorphs of N. irene range from 2.1% to 95%
of all females (Forbes et al. 1995; Van Gossum et al. 2007).
Andromorphs appear to experience less male harassment
and are found in greater densities at pond edges among
mate-searching males (Forbes et al. 1995), but more
recently, the density–morph relations have been challenged,
and mixed results have been found in broad surveys (Van
Gossum et al. 2007). As in many other damselfly species,
N. irene males spend much of their time mate searching
around natal pond edges, presumably foraging only long
enough to satisfy energy needs for mate searching (Anholt
1992). In contrast, females maximize foraging time to
mature clutches of eggs. Foraging differences between
males and females may lead to differential exposure to
gregarine infective stages, which are ingested along with
food (Åbro 1976). Such sex differences in parasitism have
been documented for another coenagrionid species (Hecker
et al. 2002).
We estimated prevalence and intensity (sensu Bush et al.
1997) throughout the season, in different sampling locations, and for males and two different female morphs. In the
first instance, we predict that because males forage in
different areas and forage less than females, males will be
less parasitized by gregarines than same-aged females. We
further predicted that gregarine infection should be highest
at sites when population densities were highest because
high density should increase transmission of infective
stages. Earlier work by Van Gossum et al. (2007) showed
that male density first increased at sites seasonally
(including sites in this study) and then declined after
peaking mid-season. The purpose of this study was to test
for, and explain, any seasonal patterns in parasitism of N.
irene by gregarines.
Materials and methods
Study sites
Five sites were chosen for recurrent sampling of N. irene
damselflies and assessment of parasitism levels. This
species was studied because much is known about its
natural history including how male density changes at sites
seasonally. Males and females were collected from five
flooded beaver ponds at those sites. All ponds were within
a 10-km radius of the Queen’s University Biological
Station, near Chaffey’s Lock, Ontario, Canada (44°34′ N,
76°19′ W). Lindsey Lake Marsh (LLM) is 3 ha in size at
the southern edge of Lindsey Lake. Barb’s Marsh (BM) is
approximately 3 ha in size and adjoins a large hayfield.
Elgin Pond (EP) is 0.8 ha and has a marshy circumference,
with a mature oak forest and grassy understory surrounding
it. Upper Dowsley Lake (UD) is 0.9 ha and approximately
round in shape, with a grassy margin (including sedges)
bordered by mixed deciduous forests. Indian Lake Bight
(ILB) is 16 ha in size and part of a reedy lake with shallow
water and short grassy riparian vegetation. The dominant
emergent vegetation at all sites is Typha spp. and Carex
spp., and dead trees protrude from the surface of the water,
providing additional emergence sites for eclosing N. irene.
Collection and dissections of individuals and enumeration
of gregarines
Reproductively mature adults of N. irene were sweep net
collected from 16 June–27 July, 1997. Maturity was
determined by body coloration. Adults were sexed, and
females were further classified as andromorph or heteromorph (Van Gossum et al. 2007). After decapitation, the
body was weighed, and the gut was removed by gently
pulling on the posterior abdominal segment with forceps
until the entire digestive tract emerged. The gut was
preserved in sugared ethanol (1 L H 2 O:1 L 95%
EtOH:40 g sucrose) and stored in an Eppendorf microcentrifuge tube until dissected. A drop of 2.5% pharmaceutical iodine was used to increase contrast between
gregarines and tissue. The gut was dissected, and gregarines
were separated from the gut wall using size 00 insect pins.
The gregarines were prepared using Clopton (1997)
standard protocols. The gregarines had three distinct body
parts and are likely Hoplorhynchus spp. and one as yet
unidentified genus (Clopton, personal communication).
Estimates of prevalence, defined as the number of
individuals infected with at least one gregarine compared
to all individuals collected for a sample, were compared
between males and females and between female morphs (on
a date-by-site basis). Estimates were provided with
Clopper–Pearson 95% confidence intervals (Zar 1996). If
the 95% confidence intervals overlapped between samples
of individuals (controlling for date and site), the prevalence
of gregarine infection for the sexes (or morphs) was
statistically indistinguishable. We also compared estimates
of prevalence for males and both female morphs across all
12 date-by-site comparisons (36 estimates) using a one-way
ANOVA (alpha=0.05).
Intensity data were analyzed in two ways. We first
transformed (Log10) intensity data to satisfy the assumptions of normality (this assumption was tested using
Shapiro–Wilk tests). We then calculated mean intensity
based on transformed data for males and females of each
Parasitol Res
morph for each date-by-site combination. On two occasions, no andromorphic females were collected that were
parasitized, and so, no mean intensity values were obtained
for those females in those samples. Using these values, we
could compare whether males and females of both morphs
showed similar or dissimilar mean intensity values. We also
performed a one-way ANOVA (alpha=0.05) to compare
mean intensity between males and females of one or both
morphs. In situations where no andromorphic females in a
sample were parasitized, a two-tailed t test (alpha=0.05)
was used for this analysis. We then back transformed the
mean intensity value and the upper and lower bounds of the
standard error (standard errors are asymmetrical around
back-transformed means). We used Bonferonni correction
(0.05/12 = 0.0042) to ascertain whether any particular
samples might have shown significant differences in mean
intensity between morphs and males that could not be
explained by sampling error due to multiple tests.
Results
In total, 1,109 damselflies were collected and dissected for
gregarines. Samples were collected across each date-by-site
comparison with 76–126 damselflies being collected among
sites (Table 1). This resulted in adequate representation of
males and females in order to test for sex biases in either
prevalence or intensity of parasitism.
Of the 1,109 damselflies, 486 (43.8%) were males of
which 279 were parasitized (57.4% prevalence, estimate
ranging from 52.8% to 61.8%). Of the females, most (530
or 85%) were heteromorphic females, of which 284 were
parasitized (53.6% prevalence, estimate ranging from
49.2% to 57.9%). The remaining 93 females (15%) were
andromorphs of which 38 were parasitized (40.8% prevalence, estimate ranging from 30.8% to 51.5%).
At first glance, it would appear that andromorphic
females were less likely to be parasitized. However, on a
date-by-site comparison, this was not the case. No
consistent sex or morph biases in prevalence of infection
by gregarines were observed (see individual estimates of
prevalence and associated confidence intervals, Table 1).
The grand mean prevalence for males across 12 date-by-site
samples was 44.7% (range, 20.5–76.2%) compared to
47.2% (range, 18.5–70%) for heteromorphic females and
45.9% for andromorphic females (ranging from 0 based on
single females collected to 73.9% for larger samples). Thus,
there also was no tendency for prevalence estimates to be
higher or lower in males or one of the female morphs
(F2, 33 =0.044, p>0.95).
Sample sizes were much smaller when examining
intensity because only damselflies infected by one or more
gregarines were included and sometimes no parasitized
andromorphic females were collected (Table 2). Again, no
consistent sex or morph biases were observed regarding
gregarine intensity (Table 2). Across 12 date-by-site
comparisons, 11 samples failed to detect differences
between males and females of one or both morphs in mean
intensity of parasitism (p values ranged from 0.11 to 0.95).
However, in one sample where males and females of both
morphs were well represented (BM, 3 July, Table 2),
infected heteromorphic females had significantly lower
gregarine numbers than either infected males or infected
andromorphic females (Table 2). However, the overall
Table 1 Prevalence of infection in male, heteromorphic female, and andromorphic female N. irene (Hagan) over 12 date-by-site samples, for June
and July 1997
Site
Sample date
N ♂,♀ (H, A)
Male
Heteromorph
Andromorph
Total
BM
17 June
3 July
20 July
60, 66 (65, 1)
21, 67 (44, 23)
42, 34 (33, 1)
26.6 (16.1–39.7)
76.2 (52.8–91.8)
38.1 (23.6–54.4)
18.5 (9.9–30.0)
59.1 (43.2–73.7)
30.3 (15.6–48.7)
0.0 (NA)
73.9 (51.6–89.8)
0.0 (NA)
22.2 (15.3–30.5)
67.0 (56.2–76.7)
34.2 (23.7–46.0)
UD
LLM
22 June
23 June
10 July
21 July
26 June
11 July
24 July
28 June
12 July
44,
44,
38,
40,
45,
43,
44,
27,
38,
34.1
20.5
47.4
35.0
42.2
67.4
50.0
40.7
57.9
47.8
27.7
68.9
47.3
39.5
70.0
37.5
48.8
70.5
30.0
60.0
33.3
50.0
60.0
66.7
50.0
54.5
72.2
40.0
27.7
58.1
42.4
42.0
68.6
45.0
46.8
66.0
EP
ILB
56
57
48
59
43
43
36
52
62
(46,
(47,
(45,
(55,
(38,
(40,
(32,
(41,
(44,
10)
10)
3)
4)
5)
3)
4)
11)
18)
(20.5–49.9)
(9.8–35.3)
(31.0–64.2)
(20.6–51.7)
(27.7–57.9)
(51.5–80.9)
(34.6–65.4)
(22.4–61.2)
(40.8–73.7)
(32.9–63.1)
(15.6–42.6)
(53.4–81.8)
(33.7–61.2)
(24.0–56.6)
(53.5–83.4)
(21.1–56.3)
(32.9–64.9)
(54.8–83.2)
(6.7–65.2)
(26.2–87.8)
(0.8–90.6)
(6.8–93.2)
(14.7–94.7)
(9.4–99.2)
(6.8–93.2)
(23.4–83.3)
(46.5–90.3)
(30.3–50.3)
(19.3–37.5)
(47.0–68.7)
(32.5–52.8)
(31.6–53.0)
(57.7–78.2)
(33.8–56.5)
(35.5–58.4)
(55.8–75.2)
The sample sizes (N) for males (♂) and both female (♀) morphs (H heteromorph, A andromorph) are shown. NA refers to “not applicable”
because confidence intervals could not be calculated
The site codes are as follows: BM Barb’s Marsh, UD Upper Dowsley Marsh, LLM Lake Lindsey Marsh, EP Elgin Pond, and ILB Indian Lake Bight
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Table 2 Back-transformed mean intensity of gregarine infection in males and female morphs (H heteromorph, A andromorph) of N. irene over 12 dateby-site samples for collecting dates June–July 1997
Site
BM
UD
LLM
EP
ILB
Sample date
N ♂,♀ (H, A)
Male
Mean intensity (lower and upper SE)
Heteromorph
Andromorph
17 June
16, 12, 0
5.6 (4.3–7.3)
5.8 (4.2–7.9)
NA
3 July
20 July
22 June
23 June
10 July
21 July
26 June
11 July
24 July
28 June
12 July
16, 26, 17
16, 10, 0
15, 22, 3
9, 13, 6
18, 31, 1
14, 26, 2
19, 15, 3
29, 28, 2
22, 12, 2
11, 20, 6
22, 31, 13
9.0 (7.2–11.2)
4.1 (2.5–6.4)
5.3 (4.1–6.9)
2.6 (1.5–4.2)
3.2 (2.6–3.8)
4.1 (2.9–5.6)
9.2 (7.3–11.7)
5.7 (4.6–6.9)
4.3 (3.0–6.0)
12.3 (9.7–15.6)
10.7 (8.4–13.5)
4.2
3.5
5.3
4.7
4.4
2.6
5.4
3.4
3.0
9.1
6.0
9.3 (7.4–11.7)
NA
1.5 (1–2.2)
8.6 (6.6–11.1)
24 (NA)
13.0 (2–64)
8.0 (5.0–12.5)
2.9 (2–4)
1.4 (1–2)
4.3 (3.0–6.0)
7.9 (5.7–10.9)
(3.5–5.1)
(2.6–4.8)
(4.3–6.6)
(3.6–6.0)
(3.5–5.5)
(2.1–3.2)
(4.1–6.9)
(2.6–4.3)
(2.1–4.1)
(7.3–11.3)
(5.0–7.3)
The lower and upper range of the standard error is shown in parentheses (error is asymmetrical about the back-transformed mean). NA refers to
not applicable, either because mean intensity could not be calculated (no infected individuals were present) or because standard errors could not be
calculated (only one individual was infected)
The site codes are as follows: BM Barb’s Marsh, UD Upper Dowsley Marsh, LLM Lake Lindsey Marsh, EP Elgin Pond, and ILB Indian Lake Bight
model was not significant following Bonferroni correction
(p>0.0042).
We therefore combined data to test for seasonal changes
in gregarine parasitism. Here, we found unimodal patterns
for both prevalence and intensity of infection (Figs. 1 and 2).
Prevalence of infection was relatively low early in the season
Fig. 1 Prevalence of gregarine infection in N. irene damselflies based
on season. Shown are Clopper–Pearson 95% confidence intervals
around estimates of prevalence. The trend line is also plotted
and rose by mid-season, dropping later in the season but not
as low as early-season levels. In comparison, the mean
number of gregarines for infected individuals was already
Fig. 2 Intensity of gregarine infection in N. irene damselflies in
relation to season based on log-10 transformed data (mean±2 SE).
Both prevalence and intensity peaked mid-season, although intensity
peaked slightly earlier. Note that the back-transformed means range
from a low of 29 gregarines to a high of 194 gregarines
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high early in the season, but rose to the highest levels by
mid-season and dropped to lower levels later in the season.
Both patterns were repeatable across sites where early-, mid-,
and late-season samples were obtained (e.g., Tables 1 and 2,
Fig. 1).
Discussion
There are two main findings in our study. The first finding
is that, unlike previous work on another coenagrionid
damselfly (Hecker et al. 2002), females were not more
likely than males to be parasitized by gregarines nor did
they have higher intensities of parasitism, once date and site
of sampling were controlled. Female morphs also appeared
similar in their levels of parasitism. Ultimately, parasitism is
dictated both by exposure and susceptibility. With respect
to exposure, we expected that females would forage more
than males and be more likely to ingest gregarine oocysts.
We currently have little information that this is the case. It
is possible that females die sooner than males, and thus,
males in samples tend to be older on average than females.
However, earlier work (Hecker 1999) shows just the
opposite. When individuals are collected in the field and
deprived of food, males die sooner than females. This
finding supports the argument that females have more
reserves from having foraged more and are able to survive
better than males during periods of food deprivation (e.g.,
inclement weather), but it does not resolve why males and
females have similar levels of parasitism. It is important to
note that the highest densities of individuals occur at the
edges of ponds because ponds are the rendezvous sites, and
males should be spending more time at the edge of ponds
than females (Van Gossum et al. 2007). Thus, males might
encounter more infective stages of gregarines per foraging
attempt or drinking bout even though they are expected to
forage less actively than females. Without further data on
foraging behavior and habitat use by the sexes, we cannot
know why parasitism levels are not different between males
and females.
Our second main finding was that both prevalence and
intensity of gregarine parasitism showed unimodal distributional patterns over the season. One of the underlying
reasons for this pattern could be that, like density of hosts
which peaks mid-season (Van Gossum et al. 2007), the
density of infective stages of directly transmitted parasites
like gregarines also peaked mid-season. It is therefore not
unreasonable to speculate that density-dependent transmission is highest in the middle of the season. There is no
reason to presuppose that older damselflies with higher
gregarine loads are more likely to be collected mid-season.
If anything, they should be more common towards the end
of the season. Also, the work described above rules out
disproportionate representation of one sex in the middle of the
season being the reason for greater prevalence and intensity at
that time. In the absence of other information, the most
parsimonious explanation is that density-dependent transmission explains the unimodal pattern and that males might be
more susceptible to gregarines per foraging bout. It is
important to reiterate that the unimodal pattern is repeated
across sites with recurrent sampling through the season. Åbro
(1971) and Locklin and Vodopich (2010b) have observed
this pattern in the odonate–gregarine system but had not
tested for them.
These yearly unimodal patterns are not uncommon in
other host–parasite systems, especially in infectious disease
ecology in both vertebrates and invertebrates (Altizer et al.
2006). They are usually due to yearly fluctuations of
variables such as temperature, presence of intermediate
hosts, or breeding season. Infection in macaques by directly
transmitted nematodes occurs in a cyclic yearly pattern
based on breeding season (Macintosh et al. 2010). The
trematode, Echinostephilla patellae, infection peaks during
the summer months because appropriate temperatures allow
higher densities of the host, common limpets (Prinz et al.
2010). This unimodal pattern can also be seen in Conopid
flies parasitizing wild bumblebee species. Conopid flies
parasitize more frequently during the period of the year
when the bumblebee hosts are at their highest population
densities (Gillespie 2010). In this study, density has a
putative strong role, but it is likely that it is not the sole
determinant of parasitism level. The variation around
prevalence and intensity estimates mid-season is still rather
large. As such, site-to-site variation in starting prevalence
of gregarines and/or factors favoring transmission of
oocysts (prey species) might be important explanatory
variables. In fact, earlier work (Hecker 1999) failed to
show a strong relation between density and gregarine
parasitism for the same species when density was measured
at the same time of season at four different sites. Within-site
relations between density and parasitism should be more
likely to detect because they have the advantage that a
relevant baseline is used for comparison. In fact, parasitism
by gregarines varies substantially between populations
(Åbro 1987).
That N. irene showed seasonal variation in prevalence
and intensity of infection is important. Seasonal patterns
occur for other insect–parasite associations involving
gregarines (Zuk 1987). As mentioned, gregarines are
generally accumulated over time owing to recurrent
ingestion of infective cysts and therefore should increase
with advancing age (Åbro 1990). It is likely that both
prevalence and intensity decline later in the season because
heavily infected individuals are lost from samples, newly
emerged individuals with no parasitism are added to
samples, and the density of infective stages is also
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decreased. Individual hosts which emerge early in season are
thought to have advantages because of directional selection
for breeding onset (cf. Anholt 1992) and because infective
parasite stages are not yet abundant in the population. Hosts
emerging later might be less susceptible to parasitism, but
not realize the reproductive advantages of earlier emergence.
Acknowledgments We would like to thank Frank Phelan for
assistance in the field and Richard Clopton for help with gregarine
identification. This was partly funded by a NSERC discovery grant
awarded to MRF.
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