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Keywords:

  • asexual;
  • barcoding;
  • Dahlica ;
  • Hymenoptera;
  • insect phenology;
  • Naryciinae;
  • parasites;
  • Psychidae;
  • Red Queen hypothesis;
  • Siederia

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

The parasite hypothesis for sex is one of the many theories that have been suggested to solve the mystery of the widespread occurrence of sex despite its high short-term costs. It suggests that sexual lineages have an evolutionary advantage over parthenogens because they can frequently generate new genotypes that are temporarily less prone to coevolving parasites. In this study, we looked for further supporting evidence for the parasite hypothesis of sex in an attempt to understand the coexistence of sexual and parthenogenetic bagworm moths (Naryciinae). The bagworm moths and their parasitoids form one of the few natural host–parasite systems where sexual and parthenogenetic hosts are apparently not separated by ecological or geographical barriers. Furthermore, in support of the parasite hypothesis for sex, parthenogenetic presence is negatively correlated with parasitism rate. We specifically tested, by identifying the reproductive mode of the parasitized individuals, whether parasitoids preferentially attack the parthenogens in sites with both sexual and parthenogenetic forms, as predicted by the parasite hypothesis. We collected hosts from sites with different frequencies of parthenogenetic and sexual moths. A DNA barcoding approach was used to determine the reproductive mode of the parasitized hosts. Furthermore, we investigated whether differences in host and parasitoid phenology could provide an alternative explanation for the variation in parasitism rates between parthenogens and sexuals. Our results contradict the prediction of the parasite hypothesis because parthenogenetic bagworm moths were less parasitized than sexuals in sympatric sites. Our findings can be explained by differences in phenology between the parthenogenetic and sexual moths rather than genetic incompatibility between parthenogenetic hosts and parasitoids. The stable coexistence of sexual and parthenogenetic Naryciinae despite the many apparent costs of sex in this system remains a mystery. Our work adds to the list of studies were the assumptions of the parasite hypothesis for sex are not all met.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Sexual reproduction is the most common reproductive mode in higher organisms with only approximately 0.1% of animal species showing some form of parthenogenetic reproduction (Suomalainen et al., 1987). The ‘paradox of sex’ or why obligate asexual reproduction is still so rare despite its many putative advantages has puzzled many evolutionary biologists as the problem was brought under greater attention by a.o. Williams (1975), Maynard Smith (1978) and Bell (1982). Parthenogenetic organisms avoid the ‘twofold cost of sex’ which can double the effective reproductive rate due to the absence of male progeny (Maynard Smith, 1978) and may avoid costs associated with mating behaviour, for example, predation risks while searching for a partner or the risk of sexually transmitted diseases. Furthermore, they do not face the risk of remaining unmated, assuring reproduction in sparse populations and newly invaded habitat for species with low mobility (Tomlinson, 1966; Gerritsen, 1980; Bierzychudek, 1985; Schwander et al., 2010). Due to all these advantages, once a parthenogenetic strain evolves, it should quickly replace its sexual ancestor, unless there are strong negative effects associated with asexual reproduction.

Many theories have been proposed to explain the maintenance of sex (Kondrashov, 1993; Engelstadter, 2008; Schön et al., 2009; Meirmans & Strand, 2010). Most of them suggest that sexual reproduction has an advantage due to recombination in cross-fertilization. This will lead to a higher genetic variation in offspring, so that populations of sexuals may better resist environmental challenges in time or space (Burger, 1999). Alternatively, cross-fertilization will allow the purging of deleterious mutations, avoiding ‘Muller's’ ratchet' that may cause a decline of fitness in clonal organisms (Muller, 1964; Kondrashov, 1988), and enabling sexuals to cope better under stressful situations (Lively et al., 1998; Tobler & Schlupp, 2010). Recombination will also allow the combination of beneficial mutations that may arise independently in different individuals. Ultimately, the lack of recombination is hypothesized to render parthenogenetic organisms to an evolutionary dead end (Maynard Smith, 1978; Neiman et al., 2009).

One of the most influential theories to explain the ‘paradox of sex’ is the parasite hypothesis for sex, a specific case of the original Red Queen hypothesis (Van Valen, 1973; Bell, 1982). Although several different versions of the theory have been proposed, the basic idea remains the same (Lively, 2010a, b): it assumes that parasites evolve to infect the most common host genotype(s), allowing rare genotypes to temporarily escape infection. This will lead to a decline of the common genotypes allowing rare genotypes to increase in number. Subsequently the parasites evolve to the new common host genotype. The parasite hypothesis for sex suggests that sexual lineages have an evolutionary advantage over parthenogens because they can generate more frequently new genotypes. An alternative scenario is that parthenogens are less resistant to parasites due to the accumulation of deleterious mutations, perhaps in combination with Red Queen dynamics (Howard & Lively, 1994, 1998, 2002; Park et al., 2010).

The best studied system that has shown support for the parasite hypothesis is the freshwater snail (Potamopyrgus antipodarum) and its parasitic trematodes (Lively & Jokela, 2002; Lively et al., 2004; Jokela et al., 2009; Koskella & Lively, 2009). In this system, parthenogenetic snails are more common in populations where parasite pressure is low, suggesting that sexuals have a higher resistance to parasites than parthenogens (Lively, 1992). Indeed, it was shown that the parasites adapt to the most common host genotypes, thus selecting against parthenogenetic host genotypes to become common (Lively et al., 2004; Jokela et al., 2009; Koskella & Lively, 2009).

Two closely linked predictions derived from the parasite hypothesis: ‘sexual reproduction should be favoured when infection risk is high’ (prediction 1) and ‘given a lower genetic diversity in parthenogens, higher parasite loads are expected in parthenogens in sympatric populations’ (prediction 2) can only be tested when comparing competing sexual and parthenogenetic lineages (Tobler & Schlupp, 2008). However, few studies have investigated the parasite hypothesis for sex in natural systems (Salathe et al., 2008; Tobler & Schlupp, 2008; Lively, 2010b), because in most cases, sexual and parthenogenetic lineages do not compete directly due to spatial (e.g. geographic parthenogenesis, Vandel, 1928; Glesener & Tilman, 1978; Lundmark & Saura, 2006) or ecological separation (Tobler & Schlupp, 2008). Evidence for the first prediction has been found in a limited number of studies, notably the aforementioned Potamopyrgus system (Lively & Jokela, 2002) and for bagworm moths and their parasitoids (Kumpulainen et al., 2004). On the other hand, in the freshwater snail, Melanoides tuberculata and its trematode, the positive correlation between frequency of sexual reproduction and infection was absent (Ben-Ami & Heller, 2005). Evidence for the second prediction was found, for instance, in flatworms (Schmidtea polychroa) where parthenogens were more heavily parasitized by an amoeboid than sexuals (Michiels et al., 2001). Also, for sympatric populations of several fish species, parthenogens were more often infected by trematodes than sexuals (Lively et al., 1990; Hakoyama et al., 2001; Mee & Rowe, 2006). Still, for example, Weeks (1996) did not find differences in parasitism levels between sexuals and parthenogens, and also sympatric sexual and parthenogenetic mollies were similar when comparing their whole parasite community (Tobler & Schlupp, 2005). Contradicting results were also found for several gecko species and their parasitic mites (Brown et al., 1995; Hanley et al., 1995).

One of the few natural systems where support for the parasite hypothesis was found is the bagworm moths Naryciinae (Lepidoptera; Kumpulainen et al., 2004). In this system, sexual species coexist in the same habitat with a closely related obligate parthenogen (Dahlica fennicella; Elzinga et al., 2011a). Their larvae are attacked and killed by eight species of hymenopteran parasitoids, exerting a very strong selective pressure (Kumpulainen, 2004; Elzinga et al., 2011b). Kumpulainen et al. (2004) found that parthenogens were rare in sites where parasitoid prevalence was high, in support of prediction 1. This suggests that parasitoids prevent the parthenogens from outcompeting the sexual species, allowing sympatry. However, crucially, it did not show that the parthenogenetic species had a higher risk of infection in sympatric sites (prediction 2) because the identity and thus reproductive mode of the parasitized larvae remained unknown.

In this study, we specifically tested whether in sympatric sites parthenogenetic D. fennicella are more frequently attacked by parasitoids than the sexual species (prediction 2), by comparing parasitism levels of the sexuals and parthenogens through molecular identification of the parasitized hosts. We also identified all parasitoid species involved to verify whether differences in parasitism levels between sexuals and parthenogens could be caused by different parasitoid species.

Secondly, we investigated an important prerequisite for testing the parasite hypothesis, which is that ‘all else is equal’, that is, there are no differences in life history between sexual and parthenogenetic forms (Tobler & Schlupp, 2008), which may affect parasitism levels or reproductive success. Indeed, Kumpulainen et al. (2004) found no differences in clutch size or larval survival between the sexuals and parthenogens. However, one of the most important factors influencing parasitism may be the phenology of the moth larvae which could cause differential exposure to the parasitoids. We therefore investigated the natural occurrence of the larvae of sexuals and parthenogens over time.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Study system

Naryciinae (Lepidoptera: Psychidae) females are wingless with a maximum length of 5 mm and the males have a wingspan of approximately 15 mm (Pro Natura, 1997; Suomalainen, 1980). Five sexual species and two obligate parthenogenetic Naryciinae can be found in central Finland (Elzinga et al., 2011a). After overwintering, final instar larvae climb up trees and other tall objects to pupate. Larvae carry a self-made larval case consisting of fragments of lichen, plants and other small particles joined together with silk in which they pupate. Within 2–4 weeks, the adults emerge. Sexual females then release pheromones to attract males, while assuming a characteristic calling position on top of the larval case while parthenogenetic females immediately start ovipositing. Eggs are laid inside the larval case, after which the female drops and perishes. The parthenogenetic D. fennicella frequently co-occurs with sexual species in the same forest habitats (Suomalainen, 1980; Kumpulainen et al., 2004; Elzinga et al., 2011b; this study).

Morphological identification of Naryciinae is not reliable (Grapputo et al., 2005a) but from mitochondrial DNA sequences, five sexual species: Dahlica lichenella, Dahlica charlottae, Dahlica lazuri, Siederia listerella, Siederia rupicolella and two parthenogenetic species: the common D. fennicella and the locally rare Dahlica triquetrella can be distinguished (Elzinga et al., 2011a). Dahlica fennicella is closely related to the sexual species according to a phylogenetic analysis based on mitochondrial and nuclear DNA sequences (Grapputo et al., 2005a); V. Chevasco, J.A. Elzinga, J.A. Galarza and A. Grapputo, unpublished).

Naryciinae larvae are frequently parasitized by eight hymenopteran species in central Finland widely differing in timing and mode of attack (see Elzinga et al., 2011a for a detailed analysis of the ecology of each parasitoid species).

Collection and rearing

In February 2008, before the bagworm moth larvae became active, 70 forested sites around Jyväskylä, central Finland were selected (see map in Elzinga et al., 2011b) including several sites that were originally examined by Kumpulainen et al. (2004). On each site (separated from each other by at least 500 m), adhesive tape (TRENDtape; Müroll, Frastanz, Austria) was applied with the adhesive side outward around 10–25 (average 15.9 ± 0.7 SE) large trees (mainly Picea sp.) at approximately 1.5 m from the ground. The maximum distance between trees within a site was 100 m.

From 15 March 2008, each site was visited once a week for 9 weeks, after which almost no final instar larvae were found. Each week, all cases found on the tape were placed into individual rearing tubes and were placed inside a growth cabinet at 10/20 °C (16/8 h cycle) with 85% humidity and day length adjusted to natural conditions.

All tubes were checked daily for the emergence of moths or parasitoids. Reproductive mode (sexual or parthenogenetic) was determined for each adult moth based on gender (all males are sexual) and behaviour after emergence (parthenogenetic females immediately oviposit). If a parasitoid emerged, the remains of the host larva, if present, were preserved in 99% ethanol for later molecular identification. In case of a pupal parasitoid, the gender of the moth pupa was determined, based on the presence or absence of wing plates.

On the basis of the adult reproductive modes, we distinguish, throughout the manuscript, two types of sites: the ‘sexual’ sites with 100% sexuals and the ‘sympatric’ sites where at least one parthenogenetic adult emerged. In some analyses, we used data only from those sympatric sites with over 75% of parthenogens, that is, ‘parthenogenetic’ sites.

To assess variation in phenology of developing larvae between sexual and parthenogenetic moths, not fully grown larvae (i.e. ‘immature’ larvae) were sampled throughout the whole year, each week in 2007 from four parthenogenetic sites and from eight sexual sites. Subsequently, larvae were classified according to their case size (early instars: < 3 mm, larger larvae: > 3 mm). We randomly selected up to five individuals from each week and size class, including larvae from both sexual and parthenogenetic sites for molecular identification.

Identification

All parasitoids were identified based on morphology (for details see Elzinga et al., 2011b). Species identification of the moths was determined by sequencing the first 298 bp of the COII gene with Naryciinae specific markers as described in Elzinga et al. (2011a, b). Positive identification was based on the clustering position with reference sequences from Grapputo et al. (2005a; max 0.3% bp divergence within a species, compared to 1.9–6.6% between species).

To determine the reproductive mode and species identity of the moth larvae that were killed by parasitoids, we extracted DNA from all available larval remains from the sympatric sites. To check for a potential bias in identification success, we compared the success of DNA barcoding of larval remains (n = 288) from parthenogenetic sites with 255 larval remains randomly selected from sexual sites. We could extract and analyse sequences from 49% of the larval remains, due to degradation or limited amounts of DNA available, but no bias between sexual and parthenogenetic sites was found.

Statistical analyses

For each site, we calculated the frequency of parthenogens (D. fennicella) among the adult (nonparasitized) moths that hatched (D. triquetrella was excluded from the analysis because of its rarity and clearly larger size). Parasitism rates were calculated per site from the total number of cases from which an adult moth or parasitoid emerged. Correlations between parasitism rates and the frequency of parthenogenetic adults were examined with a generalized linear model (GLM) with a quasibinomial error distribution.

To investigate whether parthenogenetic larvae are relatively more parasitized than sexuals, we calculated, for each sympatric site separately, the frequency of parthenogens among the identified hosts. These values were subsequently compared to the frequencies of parthenogens among the adult moths in a GLM (with site and sample type as explaining factors). Similarly, we investigated whether the frequency of females (which may include parthenogens) was higher among the pupae parasitized by pupal parasitoids than the frequency of females among nonparasitized emerged pupae. To investigate whether certain parasitoid species attack more parthenogens than sexuals, we compared the total frequency of parthenogens among the identified hosts with the total frequency of parthenogens among the adults with a two-sided binomial test, for each parasitoid species separately; this analysis was carried out only for data resulting from parthenogenetic sites.

GLM was performed in R 2.10.1 (R Foundation for Statistical Computing, Vienna, Austria) and all other analyses with IBM SPSS Statistics 19 (IBM Corporation, Armonk, NY, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Correlation between parasitism rates and reproductive strategy

We collected on average 147 (± 14 SE) cases of final instar larvae of Naryciinae per site. In total, from 77% either an adult moth or a parasitoid emerged. Emergence rate decreased over time from more than 95% in the first week to < 50% in the last week (Elzinga et al., 2011b). We assume that this is mainly due to greater activity of small predators (e.g. spiders, ants and shield bugs; J.A. Elzinga, personal observation), because most undeveloped larvae were damaged or were altogether missing from their cases. However, no differences (GLM, F1,54 = 1.12, P = 0.23) were found between sexual sites and parthenogenetic sites (78% vs. 76% average emergence, respectively). Of the emerged moths (n = 5942), 23% were parthenogenetic. In 27 sampling sites, parthenogenetic and sexual species occurred in sympatry with frequencies ranging from 0.6% to 97.6% (with 20 sites over 75%) parthenogens. In one site, all moths were parthenogenetic (n = 7) and 43 sites had only sexual moths.

We compared data from four parthenogenetic sites (HN, PIH, SIP2, SIP3) that were sampled between 1999 and 2001 (originally nrs 2, 24, 6 and 3, respectively, in Kumpulainen et al., 2004). Parthenogen prevalence was never significantly lower than in the previous sampling period (HN: 96% vs. 82%, 96% and 97% in 1999, 2000 and 2001, respectively; PIH: 93% vs. 70%, 70% and 50%; SIP2: 82% vs. 82%, 82% and 85% and SIP3: 97% vs. 93% and 95% in 2000 and 2001, respectively.).

Overall parasitism rate was 26%, and increased from < 20% in March to over 80% in May in both sympatric and sexual sites (Figs 1c and S1). Trachyarus borealis was the most common parasitoid (total parasitism rate of 9.2%), followed by Orthizema flavicorne (5.4%), Diadegma incompletum (4.3%), Macrus parvulus (3.1%), Trachyarus fuscipes (1.3%), Gelis fuscicornis (1.2%), Meteorus affinis (0.6%) and Trachyarus solyanikovi (0.5%).

image

Figure 1. Activity of sexual (a) and parthenogenetic (b) Naryciinae and their parasitoids (c) during the collection period in the spring of 2008 in sympatric sites. For each collection week, the number of emerged adult moths and the parasitism rates are shown. See Fig. S1 for data on sexual sites.

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The total parasitism rate was negatively correlated with the frequency of parthenogenetic adults (GLM, F1,68 = 21.955, P < 0.001), going from an average of 30% in completely sexual sites to 15% in sites dominated by parthenogens (Figs 2 and S2). Significant negative correlations (GLM, P < 0.05) between parasitism rate and the frequency of parthenogens were found for most parasitoid species (T. borealis, O. flavicorne, D. incompletum, G. fuscicornis, T. solyanikovi), but positive correlations (GLM, P < 0.05, M. affinis, M. parvulus) or no significant correlation (GLM, P > 0.1, T. fuscipes) was also observed (Fig. 2). All parasitoid species were found in sexual sites as well as in parthenogenetic sites, except for the relatively rare T. solyanikovi that was not found in parthenogenetic sites (Fig. 2).

image

Figure 2. Correlations between parasitism rates and frequency of parthenogenetic Naryciinae in 70 sites in spring 2008. Lines represent estimates from logistic regressions. P-values were derived from F-tests on generalized linear models with quasibinomial error distributions. Symbol size indicates the sample size in each site (from small to large: < 25, 25–100, > 100 larvae that developed into an adult moth or parasitoid). Note that the x-axis is expanded (between broken lines) at the 0% parthenogens point to improve data visibility.

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Differential parasitism between sexuals and parthenogens in sympatric sites

We could identify 164 larval remains from 20 sympatric sites and determine their reproductive mode. In these sites, the frequency of the parthenogenetic species D. fennicella among the parasitized hosts was significantly lower than their frequency among the hatched moths (GLM, F1,19 = 12.39, P = 0.002, Fig. 3a). In total, 12% of the hatched moths were sexuals in the parthenogenetic sites but 53% of the parasitized and identified hosts were sexual. Also, the frequency of females (which may include parthenogens) in the pupae attacked by larval–pupal parasitoids was significantly lower than their frequency among the hatched moths (GLM, F1,20 = 4.98, P = 0.037, Fig. 3b).

image

Figure 3. (a) The frequency of parthenogenetic Dahlica fennicella among nonparasitized adult Naryciinae and among identified parasitized larvae in sympatric sites in spring 2008. Sites in the grey area suggest that sexuals are overrepresented in the parasitized larvae. (b) The frequency of females among nonparasitized adult Naryciinae and among pupae parasitized by larval–pupal parasitoids in sympatric sites (closed dots) and the average (± SE, open dot) in sexual sites in spring 2008. Sites in the grey area suggest that males are overrepresented in the parasitized larvae.

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All parasitoid species (except perhaps T. solyanikovi) can develop on both sexual and parthenogenetic hosts (Fig. 4). However, the majority of parasitoids had a disproportionally higher attack rate on sexual host species (Fig. 4a). The pooled data on identified hosts (including the data from sexual sites) indicates that T. borealis is mainly associated with S. listerella, whereas other parasitoids are found from all sexual species (Fig. 4b).

image

Figure 4. Naryciinae host species for each parasitoid species from (a) sites where the frequency of parthenogenetic Dahlica fennicella is at least 75% among nonparasitized moths and (b) all sites. Numbers on top of each bar indicate the number of identified hosts. The horizontal line in (a) indicates the frequency of sexuals among the nonparasitized moths (n = 1267) found from the sites with > 75% of parthenogens. Significant deviations from the proportion of sexuals among the nonparasitized moths (12%) are shown (two-sided binomial tests, ***P < 0.001, *P < 0.05).

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Differences in phenology between sexuals and parthenogens

The final instar larvae of the parthenogenetic D. fennicella were collected from the trees on average 2 days earlier than larvae of sexual species (unequal variances t-test on all hatched moths, N = 4519 sexuals and 1381 parthenogens, t = − 8.348, P < 0.001). In fact, larvae of sexual species can be found over a longer time than parthenogens (Figs 1a,b and S1a) which can be explained by variation in phenology among the five sexual species (Elzinga et al., 2011a). This difference in phenology of final instar larvae in spring between sexuals and parthenogens could hypothetically lead to a reduction of parasitism in sympatric sites due to the increased parasitoid activity in late spring (Figs 1c and S1). However, this hypothetical decrease in parasitism was much smaller than what is actually observed in the sympatric sites (Fig. S2).

The phenology of immature larvae was different between sexual and parthenogenetic sites (Fig. 5). In the sexual sites, a relatively large first peak of early instar larvae was observed in May and June, whereas a second peak of early instar larvae in July and August was small or absent. The opposite was found for the parthenogenetic sites where the first peak was small or absent, and the second peak of early instar larvae was relatively large. Interestingly, in the parthenogenetic sites all identified individuals (n = 7) from the small first peak were sexuals. They all belonged to S. listerella however, which is consistent with the fact that the majority of the identified remains of parasitized larvae in parthenogenetic sites are also from this species (Fig. 4). In the sexual sites, all (n = 41) early instar larvae identified from the first peak were sexuals from four species, only D. charlottae was not found. From the large second peak of early instars in the parthenogenetic sites, only the parthenogenetic D. fennicella were found (n = 38). In the sexual sites, identified larvae (n = 5) from the small second peak were all sexual species (S. listerella and D. lichenella). In all sites, immature larvae with cases larger than 3 mm were found from June onwards until October, increasing in size over time. From those larger larvae, all five sexual species were found in the sexual sites (n = 65), but only the parthenogenetic D. fennicella could be detected in the parthenogenetic sites (n = 25).

image

Figure 5. The number of early instar Naryciinae larvae (case size < 3 mm) in 100% sexual sites (grey graphs) and in sympatric sites with over 75% of parthenogenetic Dahlica fennicella (white graphs) during 2007. Broken lines (open symbols) indicate the first peak in early instar larvae (May and June). The unbroken lines (filled symbols) indicate the second peak (July and August) of early instar larvae.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Parasitoids do not explain coexistence of sexual and parthenogenetic bagworm moths

The prediction derived from the parasite hypothesis states that in sympatric populations, parthenogens should be more prone to being parasitized than sexuals (Tobler & Schlupp, 2008). Our results reveal the opposite: in sites where both sexual and parthenogenetic bagworm moths are present, sexual larvae were proportionally more likely to be killed by parasitoids, most likely due to differences in larval phenology and development times. This suggests that the negative correlation between parasitism and presence of parthenogens that is repetitively observed in this system (Kumpulainen et al., 2004; this study) is the result of higher parasitism rates of sexuals and not due to a higher mortality of parthenogens as predicted. If parasitoids would indeed impact upon the abundance of parthenogens, we would expect to observe a change in the proportion of parthenogens on a site over time. This seems not to be the case. Parthenogen prevalence was not lower than in the previous sampling period in the four parthenogenetic sites where we could compare our data with the data collected 7–9 years earlier.

Our study confirms that there is a negative correlation between the frequency of parthenogenetic moths and the parasitism rate (Kumpulainen et al., 2004), but this correlation was not observed for all species of parasitoids that are involved and may reflect the level of host preference. Indeed, the parthenogenetic species are rarely attacked by T. borealis that is responsible for the majority of parasitism. Correspondingly, this species drives the strong negative correlation between the proportion of parthenogenetic moths and parasitism rate. While most other parasitoids are more common on sexuals, M. affinis and M. parvulus showed a positive correlation between their density and the proportion of parthenogens. Still, in none of the parasitoids, a clear preference for parthenogens was found as would be expected if at least one of the parasitoids would cause a decline in parthenogen presence.

In line with the parasite hypothesis, parthenogens may persist if the parasitoids would be absent or would not (yet) be locally adapted to them (Jokela et al., 2009). However, our results show that most species of parasitoids are already present in sites with parthenogens, but with relatively low parasitism rates. Furthermore, most of the parasitoid species are clearly capable of developing on the parthenogenetic hosts. Only one species, T. solyanikovi, was not found at all from parthenogenetic sites, but this was one of the rarest parasitoids in the species complex.

Little is known about the level of host specialization of the parasitoids because for most species, host information is absent or scarce (Elzinga et al., 2011b). This study presents the first data on host use for T. borealis and O. flavicorne and implies a high level of specialization. On the other hand, for D. incompletum, M. parvulus, T. fuscipes, M. affinis and G. fuscicornis several Psychidae subfamilies, or even other lepidopteran families, have been observed as hosts, confirming that these species are generalists and that D. fennicella is clearly within their host range. Several of these parasitoids apparently do not exhibit host specialization but rather utilize hosts that have similar lifestyles (such as being concealed in a case like bagworm moths). Their presence and parasitism rates could be correlated with the presence of other host species, and not just to the presence of the sexual or parthenogenetic Naryciinae.

The parasite hypothesis assumes that new parasite genotypes evolve that are adapted to recently evolved host genotypes (Salathe et al., 2008). This suggests that in the parasites, there should be some level of host genotype specificity. There is currently little evidence for this in hymenopteran parasitoids. We are aware of only two studies that investigated genotype specificity in different parasitoid genotypes and both reported the absence of genotype by genotype interactions on parasitism (Kraaijeveld & Godfray, 2009; Sandrock et al., 2010). Apparently, this is due to the fact that increased resistance in one host to a certain parasitoid genotype provides a higher resistance to all parasitoid genotypes. Similarly, a higher infectivity of a certain parasitoid genotype to one host genotype applies to all host genotypes. However, the few studies on genotype specificity in parasitoids only evaluate direct physiological host resistance mechanisms that lead to higher survival after an attack. Genotypic changes in types of host defence that may actually affect attack risk such as behavioural or timing-related changes could play an even more important role in the coevolution of hosts and parasitoids. It has been suggested that in a host–parasite/multi-parasite system, sexual reproduction is less likely to be stimulated than in a system with just one coevolving parasite (Kouyos et al., 2009; Mostowy et al., 2010). In one of the very few empirical studies with hosts in a diverse parasite community, no differences were found for parasite load between sexual and parthenogenetic mollies (Tobler & Schlupp, 2005). In host–parasitoid systems, evolutionary pressures are expected to be highly asymmetrical because the host has to defend itself against many parasitoid species, but the parasitoid may only need to adapt to a specific host.

‘All else is not quite equal’ between sexual and parthenogenetic Naryciinae hosts

An important prerequisite to test the parasite hypothesis by comparing parasitism rates between sexuals and parthenogens is the ‘all else equal’ assumption (Tobler & Schlupp, 2008). Sexuals and parthenogens should not differ in physiology, ecology or life history that could affect parasite attack in other ways than through the genetic effects of different reproductive modes. Our data suggest that this prerequisite is not fully met in our study system. Our study shows for the first time important differences in larval phenology that can strongly affect the risk of parasitism.

It seems that sexuals, or at least the most common species, such as S. listerella and D. lichenella have a very different timing with a peak of first or second instars in spring, before eggs laid that year could have hatched. Thus, in our system, sexuals seem to be mostly semivoltine (one generation per 2 years), a common feature of many Lepidoptera and previously observed for other Psychidae (Rhainds, 2008). On the other hand, we did not observe an early peak of small larvae in the parthenogenetic D. fennicella suggesting that D. fennicella is mostly univoltine (one generation per year). The timing of immature larvae is very important considering the biology of the koinobiont parasitoids, such as the most common species T. borealis, which attack immature larvae (Elzinga et al., 2011b). Also, under laboratory conditions, these parasitoids emerged before the moth's eggs hatched (Elzinga et al., 2011b). Unfortunately, no information is available for the stage at which the larvae are attacked, nor whether multiple parasitoid generations occur during the year. However, it seems likely that the (mainly sexual) larvae present during parasitoid emergence are the ones that are attacked which would explain the observed differences in parasitism rates between sexuals and parthenogens.

Many parthenogenetic animals are polyploids (Lundmark & Saura, 2006; Tobler & Schlupp, 2008) and thus, it is impossible to know whether reproductive mode (sexual vs. parthenogenetic) or ploidy level per se influence parasitism. Indeed, the parthenogenetic D. fennicella is tetraploid, whereas the sexual species are diploid (Suomalainen et al., 1981). It is unclear what effects tetraploidy may have on resistance to parasitoids, but it may affect cell physiology, and thus also the immune system (Otto & Whitton, 2000).

Coexistence of sexuals and parthenogenetic Naryciinae remains unexplained

In contrast to what we expected, parasitoids do not explain the coexistence of sexual and parthenogenetic Naryciinae species. On the contrary, sexuals are more likely to be attacked by the majority of the parasitoid species and are semivoltine. This suggests that sexuals in this system do not only suffer from the general twofold cost of sex and mating costs, but also from additional costs of parasitism and extended development time. Therefore, we would expect that the parthenogens would outcompete the sexuals, but this does not seem to occur in this system and other forces should be investigated to explain the coexistence of these species.

Almost all theories considering the paradox of sex suggest that a reduction in genetic variation is costly. As all Lepidoptera are believed to have achiasmatic meiosis without recombination in females (Sharma & Sobti, 2002), it seems that parthenogenetic bagworm moths can only acquire genetic variation through mutation. Indeed, cytological observations suggest that the closely related parthenogenetic D. triquetrella reproduces clonally and genetic variation in offspring must be extremely limited (Lokki et al., 1975). An analysis of a nuclear gene (CAD) shows that all parthenogenetic D. fennicella samples that were examined had the same haplotype despite their widespread provenance (southern Finland, central Finland and the Russian Moscow region), whereas in all five sexual species, haplotype variation was found in all genes within central Finland (J.A. Elzinga, unpublished). On the other hand, a study of allozyme markers by Grapputo et al. (2005b) showed that within D. fennicella populations, a relatively high level of genetic variation exists, although it was still lower than in the sexual species. Furthermore, a recent study of microsatellite markers revealed genetic variation in D. fennicella (Chevasco et al., 2011). It is unclear what genetic mechanism is responsible for the maintenance of genetic variability, but this genetic variation could help parthenogenetic species to adapt to their environment and may help them to escape parasitoid attacks. Indeed, if there are many different clonal lineages or if the parthenogens can somehow retain genetic diversity, there are no common clones for the parasitoids to adapt to.

Ecological niche differences could cause differences in the distribution of sexuals and parthenogens, as well as differences in parasitism levels due to variation in the presence of alternative hosts, but have not been found previously (Kumpulainen, 2004). We observed weak correlations with deciduous tree presence or proximity to open water (J.A. Elzinga, unpublished) suggesting that the parthenogenetic D. fennicella may have some small differences in ecology from the sexual species. Within the sexuals, differences in ecology may also exist, as the relatively common species D. lichenella (Elzinga et al., 2011a) was not found from the sympatric sites.

Although many aspects in our study system are still unknown, it appears that not all assumptions of the parasite hypothesis for sex to operate are met. First, exposure to the parasites should be equal for different genotypes allowing the parasites to adapt to the most common (parthenogenetic) genotypes. Unequal parasitoid exposure rates may lead to differences in coevolutionary potential. In our system, parasitoid exposure seems to be higher for the sexuals than for the parthenogens, due to differences in life history, in particular, phenology and development time. Our study shows that it is indeed essential to study potential differences in exposure to parasites between sexuals and parthenogens when parasitism rates suggest evidence for the parasite hypothesis for sex. Second, an important assumption is that genetic variance in the parthenogens should be limited leading to the appearance of a common (parthenogenetic) genotype. Unfortunately, no conclusive information about the genetic variation within or between sites is available for the parthenogenetic hosts, although most evidence points to a reduced variation in parthenogens. Thirdly, genotype specificity in infection is expected for the parasites to allow rare (sexual) genotypes to temporarily escape parasitism, but the parasitoids in our system are apparently not even species specific. Limited studies on other hymenopteran parasitoids suggest that the scope for genotype-specific interactions may be small and that parasitoids may thus in general play a limited role in the maintenance of sex in insects.

In conclusion, although it is still not clear what causes the local differences in the presence of sexual Naryciinae and parthenogenetic D. fennicella, parasitoids do not seem to prevent the parthenogens from persistence. Rather, the parasitoids preferentially attack the sexual species, most likely due to their different phenology. The question why sexuals and parthenogenetic Naryciinae can coexist in an apparently stable situation therefore remains unresolved. Our work adds to the list of studies (Tobler & Schlupp, 2008) where several assumptions of the parasite hypothesis for sex are not met.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank Sari Viinikainen for laboratory assistance. We are grateful to two anonymous reviewers for their constructive comments on an earlier version of the manuscript. This research was part of a project (no. 116892) funded by the Finnish Academy to AG, the Centre of Excellence in Evolutionary Research and the Ehrnrooth foundation to JAE. We thank Phill Watts for checking the English language.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

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Figure S1 Activity of sexual (a) Naryciinae and their parasitoids (b) during the collection period in the spring of 2008 in the sexual sites.

Figure S2 Effect of final instar larval phenology on parasitism rates in sympatric sites.

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