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 Parasitol Res 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 Parasitol Res 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 Parasitol Res 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. 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