Polyembryonic Wasp Research Articles

Analysis of molecular mechanism for acceleration of polyembryony using gene functional annotation pipeline in Copidosoma floridanum

BackgroundPolyembryony is defined as the formation of several embryos from a single egg. This phenomenon can occur in humans, armadillo, and some endoparasitoid insects. However, the mechanism underlying polyembryogenesis in animals remains to be elucidated. The polyembryonic parasitoid wasp Copidosoma floridanum oviposits its egg into an egg of the host insect; eventually, over 2000 individuals will arise from one egg. Previously, we reported that polyembryogenesis is enhanced when the juvenile hormone (JH) added to the culture medium in the embryo culture. Hence, in the present study, we performed RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanisms controlling polyembryogenesis of C. floridanum. Functional annotation of genes is not fully available for C.floridanum; however, whole genome assembly has been archived. Hence, we constructed a pipeline for gene functional annotation in C. floridanum and performed molecular network analysis. We analyzed differentially expressed genes between control and JH-treated molura after 48 h of culture, then used the tblastx program to assign whole C. floridanum transcripts to human gene.ResultsWe obtained 11,117 transcripts in the JH treatment group and identified 217 differentially expressed genes compared with the control group. As a result, 76% of C. floridanum transcripts were assigned to human genes. Gene enrichment analysis revealed genes associated with platelet degranulation, fatty acid biosynthesis, cell morphogenesis in the differentiation and integrin signaling pathways were fluctuated following JH treatment. Furthermore, Cytoscape analysis revealed a molecular interaction that was possibly associated with polyembryogenesis .ConclusionsWe have constructed a pipeline for gene functional annotation of C. floridanum, and identified transcripts with high similarity to human genes during early embryo developmental. Additionally, this study reveals new molecular interactions associated with polyembryogenesis; these interactions could indicate the molecular mechanisms underlying polyembryony. Our results highlight the potential utility of molecular interaction analysis in human twins.

Mass killing by female soldier larvae is adaptive for the killed male larvae in a polyembryonic wasp

Self-sacrifice is very rare among organisms. Here, we report a new and astonishing case of adaptive self-sacrifice in a polyembryonic parasitic wasp, Copidosoma floridanum. This wasp is unique in terms of its larval cloning and soldier larvae. Male clone larvae have been found to be killed by female soldier larvae, which suggests intersexual conflict between male and female larvae. However, we show here that mass killing is adaptive to all the killed males as well as the female soldiers that have conducted the killing because the killing increases their indirect fitness by promoting the reproduction of their clone sibs. We construct a simple model that shows that the optimal number of surviving males for both male and female larvae is very small but not zero. We then compare this prediction with the field data. These data agree quite well with the model predictions, showing an optimal killing rate of approximately 94–98% of the males in a mixed brood. The underlying mechanism of this mass kill is almost identical to the local competition for mates that occurs in other wasp species. The maternal control of the sex ratio during oviposition, which is well known in other hymenopterans, is impossible in this polyembryonic wasp. Thus, this mass kill is necessary to maximize the fitness of the female killers and male victims, which can be seen as an analogy of programmed cell death in multicellular organisms.

Lessons from the multitudes: insights from polyembryonic wasps for behavioral ecology.

Even for parasitic Hymenoptera, polyembryonic wasps are unusual creatures. Two features in particular, allow for novel exploration of major questions in behavioral ecology: the production of multiple offspring per egg and, in some species, the production of a soldier caste. Because final brood sizes of polyembryonic species are not constrained by trade-offs between current and future parental reproductive effort, we can clearly examine the selective forces at play that drive the balance between the number of offspring and their body size. Polyembryony also provides excellent opportunities to compare the performance of identical genotypes under different environmental conditions. Finally, polyembryonic species can provide unique tests of how genetic conflicts at multiple levels are resolved.

Copidosoma floridanum (Hymenoptera: Encyrtidae) Rapidly Alters Production of Soldier Embryos in Response to Competition.

Most social insects are free living and produce castes that develop in response to extrinsic environmental cues. Caste-forming polyembryonic insects, in contrast, are all endoparasitoids that form social groups inside the bodies of host insects. The best studied polyembryonic wasp is Copidosoma floridanum (Ashmead), which produces ∼3,000 clonal offspring that develop into two castes named reproductive and soldier larvae. Caste determination in this species is mediated by whether or not embryos inherit primary germ cells (PGCs). Prior results showed that C. floridanum increases the proportion of female soldier larvae it produces per host in response to other parasitoids like Microplitis demolitor. Here we show that caste ratio shifts occur through increased formation of embryos lacking PGCs. Our results further indicated that increased soldier production was a specific response to multiparasitism elicited by the chorion of M. demolitor eggs.

Intraspecific variability in egg maturation patterns and associated life-history trade-offs in a polyembryonic parasitoid wasp

1. Life-history theory predicts a trade-off between the resources allocated to reproduction and those allocated to survival. Early maturation of eggs (pro-ovigeny) is correlated with small body size and low adult longevity in interspecific comparisons among parasitoids, demonstrating this trade-off. The handful of studies that have tested for similar correlations within species produced conflicting results. 2. Egg maturation patterns and related life-history traits were studied in the polyembryonic parasitoid wasp, Copidosoma koehleri (Hymenoptera: Encyrtidae). Although the genus Copidosoma was previously reported to be fully pro-ovigenic, mean egg loads of host-deprived females almost doubled within their first 6 days of adulthood. 3. The initial egg-loads of newly emerged females were determined and age-specific realised fecundity curves were constructed for their clone-mate twins. The females' initial egg loads increased with body size, but neither body size nor initial egg load was correlated with longevity and fecundity. 4. The variation in initial egg loads was lowest among clone-mates, intermediate among non-clone sisters and highest among non-sister females. The within-clone variability indicates environmental influences on egg maturation, while the between-clone variation may be genetically based. 5. Ovaries of host-deprived females contained fewer eggs at death (at ∼29 days) than on day 6. Their egg loads at death were negatively correlated with life span, consistent with reduced egg production and/or egg resorption. Host deprivation prolonged the wasps' life span, suggesting a survival cost to egg maturation and oviposition. 6. It is concluded that adult fecundity and longevity were not traded off with pre-adult egg maturation.

Sexual complementarity between host humoral toxicity and soldier caste in a polyembryonic wasp.

Defense against enemies is a type of natural selection considered fundamentally equivalent between the sexes. In reality, however, whether males and females differ in defense strategy is unknown. Multiparasitism necessarily leads to the problem of defense for a parasite (parasitoid). The polyembryonic parasitic wasp Copidosoma floridanum is famous for its larval soldiers’ ability to kill other parasites. This wasp also exhibits sexual differences not only with regard to the competitive ability of the soldier caste but also with regard to host immune enhancement. Female soldiers are more aggressive than male soldiers, and their numbers increase upon invasion of the host by other parasites. In this report, in vivo and in vitro competition assays were used to test whether females have a toxic humoral factor; if so, then its strength was compared with that of males. We found that females have a toxic factor that is much weaker than that of males. Our results imply sexual complementarity between host humoral toxicity and larval soldiers. We discuss how this sexual complementarity guarantees adaptive advantages for both males and females despite the one-sided killing of male reproductives by larval female soldiers in a mixed-sex brood.

The metabolic enzyme phosphoglucose isomerase (Pgi) affects the outcome of intra‐specific competition in a polyembryonic wasp

1. Polyembryonic parasitoid wasps in the family Encyrtidae (Hymenoptera) have evolved a caste system consisting of morphologically and functionally distinct larvae called soldiers and reproductives.2. Two selective pressures are thought to underlie the evolution of the soldier caste: defence against competitors and resolution of the sex ratio conflict. Previous studies also indicate that soldier development time strongly affects the outcome of intra‐specific competition in the polyembryonic encyrtid Copidosoma floridanum Ashmead. This study builds on prior findings by showing that alleles of the metabolic enzyme phosphoglucose isomerase (Pgi) differentially affect soldier development time and the outcome of competition.3. Soldier larvae with the Pgi alleles 100 or 120 emerged on average 65 h post‐parasitism, whereas soldier larvae with a third allele, 54, emerged at 67 h. In turn, C. floridanum broods homozygous for the 100 and 120 alleles outcompete broods homozygous for the 54 allele.4. Pgi allelic diversity may be maintained through a life‐history trade‐off affecting female brood sizes with homozygous broods bearing the developmentally disadvantageous 54 allele producing more adult females than broods bearing alternate common alleles.

Higher aggression towards closer relatives by soldier larvae in a polyembryonic wasp.

In the polyembryonic wasp Copidosoma floridanum, females commonly lay one male and one female egg in a lepidopteran host. Both sexes proliferate clonally within the growing host larva. Distinct larval castes develop from each wasp egg, the majority being ‘reproductives’ plus some ‘soldiers’ which sacrifice reproduction and attack competitors. Maturing mixed sex broods are usually female biased, as expected when intra-brood mating is common. Pre-mating dispersal followed by outbreeding is expected to increase sexual conflict over brood sex ratios and result in greater soldier attack rates. Owing to sexually asymmetric relatedness, intra-brood conflicts are expected to be resolved primarily via female soldier attack. We observed soldier behaviour in vitro to test whether lower intra-brood relatedness (siblings from either within-strain or between-strain crosses were presented) increased inter-sexual aggression by female as well as male soldiers. As found in prior studies, females were more aggressive than males but, contrary to expectations and previous empirical observations, soldiers of both sexes showed more aggression towards more closely related embryos. We speculate that lower intra-brood relatedness indicates maternal outbreeding and may suggest a rarity of mating opportunities for reproductives maturing from the current brood, which may enhance the value of opposite sex brood-mates, or that higher aggression towards relatives may be a side-effect of mechanisms to discriminate heterospecific competitors.

Analysis of odorant-binding protein gene family members in the polyembryonic wasp, Copidosoma floridanum: Evidence for caste bias and host interaction

The polyembryonic wasp Copidosoma floridanum produces two larval castes, soldiers and reproductives, during development within its caterpillar host. Primary structures were determined for 6 odorant-binding protein (OBP) gene family members in Copidosoma and then analyzed alongside two formerly sequenced OBP genes from this wasp. The genes were examined for caste-bias in expression patterns using reverse transcription-polymerase chain reaction (RT-PCR) and in situ expression studies. Six of the 8 genes show a clear bias in gene expression towards one or the other larval caste. Of the 3 distinct in situ probe hybridization patterns observed in this study, none lie in tissues with clear chemosensory functions. Two of the patterns suggest the majority of the Copidosoma OBP gene family members discovered thus far come into contact with host hemolymph. Most of these OBPs are expressed exclusively in the serosal membrane encompassing each of the reproductive larvae. The absence of expression in the membrane surrounding soldier larvae strongly suggests these OBPs are performing caste-specific functions in the host.

Male soldiers are functional in the Japanese strain of a polyembryonic wasp

Polyembryonic parasitoids clonally produce sterile soldier larvae in both sexes. Female soldier larvae of Copidosoma floridanum defend their siblings and host resources against heterospecific competitors as well as conspecific male embryos that results in female biased sex ratios. However, the male soldiers of the USA strain exhibit no aggressive behaviors against them, suspected to be a secondary loss of male defense function in the course of evolution. From vitro and vivo experiments, we have found functional male soldiers in the Japanese strain of C. floridanum. In vitro experiments, male soldiers exhibit aggressions against four larval competitors, though aggressiveness is much weaker than that of female soldiers. In vivo experiments, heterospecific competitors are equivocally excluded in both male and female broods. Our findings support the idea that male soldiers have evolved primarily to defend against heterospecific competitors. Further experiments against conspecific embryos may be able to confirm this hypothesis.

Sex allocation in a polyembryonic parasitoid with female soldiers: an evolutionary simulation and an experimental test.

Parasitoid wasps are convenient subjects for testing sex allocation theory. However, their intricate life histories are often insufficiently captured in simple analytical models. In the polyembryonic wasp Copidosoma koehleri, a clone of genetically identical offspring develops from each egg. Male clones contain fewer individuals than female clones. Some female larvae develop into soldiers that kill within-host competitors, while males do not form soldiers. These features complicate the prediction of Copidosoma’s sex allocation. We developed an individual-based simulation model, where numerous random starting strategies compete and recombine until a single stable sex allocation evolves. Life-history parameter values (e.g., fecundity, clone-sizes, larval survival) are estimated from experimental data. The model predicts a male-biased sex allocation, which becomes more extreme as the probability of superparasitism (hosts parasitized more than once) increases. To test this prediction, we reared adult parasitoids at either low or high density, mated them, and presented them with unlimited hosts. As predicted, wasps produced more sons than daughters in all treatments. Males reared at high density (a potential cue for superparasitism) produced a higher male bias in their offspring than low-density males. Unexpectedly, female density did not affect offspring sex ratios. We discuss possible mechanisms for paternal control over offspring sex.

Isolation of novel microsatellite markers for the social parasitoid wasp, Copidosoma floridanum (Hymenoptera: Encyrtidae)

We developed and characterized nine microsatellite loci for the polyembryonic wasp Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae). Eight of the nine loci were polymorphic and did not deviate from Hardy–Weinberg equilibrium. Our results will contribute to studies on the population genetic structure of C. floridanum.

Phenotypically plastic traits regulate caste formation and soldier function in polyembryonic wasps

Polyembryonic encyrtid wasps are parasitoids that have evolved a clonal form of embryogenesis and a caste system where some progeny become reproducing wasps whereas others develop into a sterile soldier caste. Theory based on the biology of Copidosoma floridanum predicts that the primary role of soldier larvae is to mediate conflict over sex ratio, which also favours female-biased soldier production. Other data, however, suggest that female-biased soldier production reflects a developmental constraint. Here, we assessed whether female-biased soldier function by polyembryonic wasps reflects sex-specific adaptation or constraint by conducting comparative studies with Copidosoma bakeri, a species that produces clutch sizes similar to C.floridanum yet rarely produces broods associated with sex ratio conflict. Our results indicate that the oviposition behaviour of adults, development of progeny and function of soldier larvae differ greatly between C.bakeri and C.floridanum. These findings indicate that caste formation and soldier function in polyembryonic encyrtid wasps are regulated by phenotypically plastic traits. Our results further suggest that the primary function of the soldier caste in some species is defence of host resources from competitors whereas in others it is the resolution of sex ratio conflict.

REVIEW: The evolution of polyembryony in parasitoid wasps

AbstractPolyembryony has evolved independently in four families of parasitoid wasps. We review three main hypotheses for the selective forces favouring this developmental mode in parasitoids: polyembryony (i) reduces the costs of egg limitation; (ii) reduces the genetic conflict among offspring; and (iii) allows offspring to adjust their numbers to the quality of the host. Using comparative data and verbal and mathematical arguments, we evaluate the relative importance of the different selective forces through different evolutionary stages and in the different groups of polyembryonic wasps. We conclude that reducing the cost of egg limitation is especially important when large broods are favoured. Reducing genetic conflict may be most important when broods are small, thus might have been important during, or immediately following, the initial transition from monoembryony to polyembryony. Empirical data provide little support for the brood‐size adjustment hypothesis, although it is likely to interact with other selective forces favouring polyembryony.

Host choice decisions in the polyembryonic waspCopidosoma koehleri(Hymenoptera: Encyrtidae)

Female parasitoids often reject hosts of poor quality, where the survival and fitness of their offspring are expected to be low. In polyembryonic parasitoid wasps, a clone of genetically identical embryos develops from one egg in a host. In the wasp Copidosoma koehleri, each female clone produces one soldier larva that attacks competing clones inside the host. Aggression by soldiers is directed usually towards unrelated clones. Accordingly, it may be predicted that females will prefer nonparasitized over parasitized hosts, especially if the latter have been parasitized previously by a mated unrelated female, as a result of the reduced chances of survival for their offspring inside these hosts. In accordance with these predictions, females prefer nonparasitized hosts over self-parasitized hosts when they are presented simultaneously. By contrast to the predictions, females prefer hosts parasitized by an unrelated conspecific over nonparasitized hosts when presented simultaneously. Females do not distinguish hosts parasitized by conspecifics from self-parasitized hosts when presented simultaneously. They reject self-parasitized hosts significantly more often than hosts parasitized by conspecifics when each host type is presented alone. Females faced with two previously parasitized hosts are not affected in their choice by the mating status (i.e. virgin or mated) of the previous parasitizing females. The combined results suggest that females are limited in their ability to assess the risk that their offspring will be attacked by a soldier, or that this risk is balanced by the relative advantages of ovipositing in a host parasitized by conspecifics. A possible advantage may be increased out-breeding opportunities for the emerging offspring.

The polyembryonic wasp Copidosoma floridanum produces two castes by differentially parceling the germ line to daughter embryos during embryo proliferation

Eggs of the polyembryonic wasp Copidosoma floridanum undergo a clonal phase of proliferation, which results in the formation of thousands of embryos called secondary morulae and two castes called reproductive and soldier larvae. C. floridanum establishes the germ line early in development, and prior studies indicate that embryos with primordial germ cells (PGCs) develop into reproductive larvae while embryos without PGCs develop into soldiers. However, it is unclear how embryos lacking PGCs form and whether all or only some morulae contribute to the proliferation process. Here, we report that most embryos lacking PGCs form by division of a secondary morula into one daughter embryo that inherits the germ line and another that does not. C. floridanum embryos also incorporate 5-bromo-2'-deoxyuridine (BrdU), which allows PGCs and other cell types to be labeled during the S phase of the cell cycle. Continuous BrdU labeling indicated that all secondary morulae cycle during the proliferation phase of embryogenesis. Double labeling with BrdU and the mitosis marker anti-phospho-histone H3 indicated that the median length of the G2 phase of the cell cycle was 18 h with a minimum duration of 4 h. Mitosis of PGCs and presumptive somatic stem cells in secondary morulae was asynchronous, but cells of the inner membrane exhibited synchronous mitosis. Overall, our results suggest that all secondary morulae contribute to the formation of new embryos during the proliferation phase of embryogenesis and that PGCs are involved in regulating both proliferation and caste formation.

Host Handling Time in a Polyembryonic Wasp is Affected both by Previous Experience and by Host State (Parasitized or Not)

Foraging behavior for hosts in parasitoids resembles that of predators with respect to finding, evaluating and manipulating of the prey. Host handling time may depend on the life history of the parasitoid and can be affected by oviposition experience. Additionally, handling time can be affected by host aggregation, species, size and state (parasitized or not). We studied handling times in the egg-larval parasitoid wasp Copidosoma koehleri. We allowed naive female wasps to oviposit into three consecutive unparasitized hosts, and measured time until oviposition, and the duration of ovipositor insertion. We recorded the same data for naive females ovipositing into already parasitized hosts. We found that both previous experience by females and previous parasitism of hosts reduced handling time. The results suggest that host handling durations reflect the interplay between host state and parasitoid internal state.

Limited kin discrimination abilities mediate tolerance toward relatives in polyembryonic parasitoid wasps

We modeled the possible effect of limited kin discrimination on larval aggression in polyembryonic wasps. In these organisms, each egg divides clonally to produce several genetically identical embryos inside an arthropod host. If more than one egg is laid inside a host (superparasitism), several clones compete for the host resources. In some species, a proportion of embryos develop into sterile soldier larvae that attack competitors inside the host. Soldiers were shown to attack according to relatedness, with higher levels of aggression toward less related individuals. Yet, the tolerance of soldiers toward nonclonal relatives is puzzling, given the intense competition for host resources, which is not offset by inclusive fitness. Using a decision tree model, we looked for conditions that justify soldier tolerance. We assumed 2 possible strategies for a soldier: A tolerant soldier does not attack a competing clone and an aggressive soldier attacks any clone that it identifies as a competitor. We calculated the expected payoffs of each strategy under different conditions of relatedness and probabilities for discrimination errors. The model predicts that when discrimination ability is perfect, a soldier should attack any competitor, regardless of relatedness. However, when discrimination ability is restricted, soldiers should switch from aggression to tolerance with increasing relatedness to competitors. The model demonstrates that limited discrimination abilities may shift the threshold of relatedness required to induce tolerance. This may apply to other systems of kin recognition that are prone to discrimination errors. Key words: Copidosoma koehleri, decision tree model, discrimination ability, kin recognition, polyembryonic parasitoid wasps, soldier larva. [Behav Ecol]

Brood size in a polyembryonic parasitoid wasp is affected by relatedness among competing larvae

Brood size has important implications for the fitness of both parents and offspring. In polyembryonic parasitoid wasps, each egg develops into many genetically identical embryos through clonal division inside the host. Thus, offspring may have the potential to affect brood size by adjusting the degree of embryonic division. In some species, a proportion of embryos develop into soldier larvae, which attack competitors inside the host. This may be another mechanism for offspring to affect final brood size. We investigated the effect of relatedness between competing clones on brood size in the polyembryonic wasp Copidosoma koehleri. We predicted that final brood size would be affected by the number and relatedness between competing clones inside the host. Additionally, we predicted that due to a competitive asymmetry between male and female clones (apparently only female clones produce a soldier larva), this effect would depend on the sex composition of wasps inside the host. We allowed 2 wasp eggs (laid either by 1 female or by different females) to develop in a host and counted the emerging adults. Relatedness between male clones did not affect brood size. However, female-containing broods of related clones were larger than broods of nonrelated clones, suggesting higher aggression of the soldier toward less related individuals. Dissections of hosts parasitized by 2 clones indicate that normally only 1 soldier survives and that it often eliminates unrelated clones. Thus, offspring control over brood size in response to relatedness is probably mediated by soldier aggression and not by clonal division.

Developmental patterns in the polyembryonic parasitoid wasp Copidosoma koehleri

Polyembryony is a unique mode of development in which multiple genetically identical embryos develop from a single egg. In some polyembryonic species a proportion of the embryos develop into soldier larvae, which attack competitors in the host. We studied the development of the polyembryonic wasp Copidosoma koehleri in its host Phthorimaea opercullela. We dissected hosts parasitized by either virgin or mated female wasps at 2 day intervals from hatching to the final instars. We documented host mass and head width, the number and size of developing wasps and the presence of a soldier larva. Additionally, we kept a sample of parasitized hosts until emergence of wasps and measured the head width of emerging adults. We characterized wasp development in relation to host development. One half of the broods produced by mated wasps contained one soldier larva throughout development. This suggests that in C. koehleri each female brood produces a single soldier larva, and that the soldier probably survives and grows gradually during host development. Additionally, we found that female broods were larger than male broods during development and also upon emergence. Accordingly, body size was larger for males during development as well as upon emergence. These findings may extend the existing knowledge on polyembryonic development in general, and serve as a baseline for further experiments.