Soldier Larvae Research Articles

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.

Environmental and genetic controls of soldier caste in a parasitic social wasp.

A larval army caste is found in some parasitic wasps with polyembryonic or clonal proliferation, where many clone larvae emerge from a single egg. In contrast to non-parasitic eusocial Hymenoptera, sterile soldier larvae that protect their clonal reproductives are found in both females and males. Recently, the proportion of soldier larvae has been found to vary radically, depending on the internal conditions of the host, such as multiparasitism by other larval parasites. However, the proportion of male soldier larvae is constant, irrespective of the host internal environment. It is unknown if these traits are heritable. Here we show that a high heritability is found in both sexes, while, in the 6th instar hosts, substantially lower heritability is found in females. These results imply that the structure of the larval caste is determined genetically by both female and male embryonic cells, but more likely modified environmentally in females.

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.

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.

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.

Presence of soldier larvae determines the outcome of competition in a polyembryonic wasp

Soldier-producing polyembryonic waSPS are the only social animals that develop as parasites inside the bodies of other insects. Characterizing the kin composition of broods is central to understanding the evolution of the soldier caste in these unique social insects. Here we studied the role of soldiers in mediating the outcome of competition among clones of the polyembryonic wasp Copidosoma floridanum. Soldier-producing female clones usually monopolized host resources, whereas soldierless male clones usually coexisted in hosts. Behavioural experiments further indicated that early-emerging soldiers are specialized to combat intraspecific competitors and later-emerging soldiers are specialized for defence against interspecific competitors. Taken together, our results point to intraspecific competition as a major selective force in the evolution of the soldier caste. Our data also present an evolutionary conundrum: given the benefit of soldiers, why are male clones functionally soldierless?

Caste-based differences in gene expression in the polyembryonic wasp Copidosoma floridanum

The polyembryonic parasitoid Copidosoma floridanum produces two larval castes, soldiers and reproductives, during development within its host. Soldier larvae defend the brood against competitors while reproductive larvae develop into adult wasps. As with other caste-forming insects, the distinct morphological and behavioral features of soldier and reproductive larvae likely involve differential gene expression. In this study we used a bi-directional suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes from C. floridanum soldier and reproductive larvae. We isolated 230 novel expressed sequence tags (ESTs) from the two subtractions (114 soldier/116 reproductive ESTs). Among these ESTs were sequences with significant similarity to genes coding for serine proteinases, proteinase inhibitors, odorant-binding and chemosensory proteins, and cuticular proteins. Also, three novel genes were isolated that resemble one another in conceptual translation and share the cysteine spacing pattern of short scorpion toxins and insect defensins. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of 20 ESTs from the two libraries indicated that 85% were differentially expressed in one caste or the other. We conclude that our SSH strategy was effective in identifying a number of genes differentially expressed in soldier and reproductive larvae and that several of these genes will be useful in characterizing caste-specific gene networks in C. floridanum.

Both endogenous and environmental factors affect embryo proliferation in the polyembryonic wasp Copidosoma floridanum

Copidosoma floridanum is a polyembryonic, parasitic wasp of the moth Trichoplusia ni. Following oviposition into a host, the C. floridanum egg initially undergoes complete (holoblastic) cleavage to form a single morula stage embryo. This embryo then undergoes a proliferation phase in which multiple, secondary morulae develop. C. floridanum has also evolved a caste system whereby some secondary morulae develop into soldier larvae whose function is defense whereas others develop into reproductive larvae that become adult wasps. In the current study, we conducted manipulative and candidate gene studies to identify factors affecting the proliferation phase of C. floridanum development. Transplantation of morulae of different ages into different host stages indicated that both embryo age and host environment affected the total number of offspring produced per morula. Morula age and brood size also significantly affected whether offspring of one or both castes were produced in a brood. In contrast, the host environment did not significantly affect caste formation. A putative homolog of the gene hedgehog (Cf-hh) was partially cloned from C. floridanum. In situ hybridization studies indicated that Cf-hh was expressed in secondary morulae during the proliferation phase of development, suggesting a possible role for the Hh signaling pathway in the evolution of polyembryony.

Host resistance and the evolution of kin recognition in polyembryonic wasps

Recognition of relatives is considered a key factor in the evolution of sociality as it ensures that the benefits of altruism flow to those who share the altruist's genes. However, theory predicts that genetically based recognition systems will not persist if the only selection maintaining them derives from the recognition system itself. Kin-recognition systems, therefore, are hypothesized to involve genetic variation maintained by other functions. Polyembryonic wasps are parasites of moth larvae that clonally produce large numbers of offspring and two morphologically distinct castes. Some embryos develop into reproductive larvae that mature into adult wasps, whereas others develop into soldier larvae whose function is defence. Soldiers from Copidosoma floridanum distinguish relatives from non-relatives on the basis of relatedness. Here, we report that the recognition cues used by soldiers derive from the extraembryonic membrane, which also protects these parasites from the host's immune response. This suggests that the kin-recognition system used by C. floridanum may be maintained in part by selection for resistance against the host.

Aggression by polyembryonic wasp soldiers correlates with kinship but not resource competition.

Kin selection theory predicts that individuals will show less aggression and more altruism towards relatives. However, recent theoretical developments suggest that with limited dispersal, competition between relatives can override the effects of relatedness. The predicted and opposing influences of relatedness and competition are difficult to approach experimentally because conditions that increase average relatedness among individuals also tend to increase competition. Polyembryonic wasps in the family Encyrtidae are parasites whose eggs undergo clonal division to produce large broods. These insects have also evolved a caste system: some embryos in a clone develop into reproductive larvae that mature into adults, whereas others develop into sterile soldier larvae that defend siblings from competitors. In a brood from a single egg, reproductive altruism by soldiers reflects clone-level allocation to defence at the cost of reproduction, with no conflict between individuals. When multiple eggs are laid into a host, inter-clone conflicts of interest arise. Here we report that soldier aggression in Copidosoma floridanum is inversely related to the genetic relatedness of competitors but shows no correlation with the level of resource competition.

Caste determination in a polyembryonic wasp involves inheritance of germ cells.

Social insects are characterized by the development of castes in which some colony members reproduce whereas others function as altruistic helpers. The conditional switch controlling caste formation usually involves environmental stimuli that act on processes that regulate development of individuals. Unlike other social species, embryos of polyembryonic wasps develop clonally to produce large numbers of genetically identical offspring and two morphologically distinct castes. All embryos in a clone exist in an identical environment, the host, yet develop into either reproductive larvae that mature into adult wasps or soldier larvae whose function is defense. Here, we report that caste determination in Copidosoma floridanum involves inheritance of germ cells. Expression of a C. floridanum homolog (Cf-vas) of the germ cell marker Vasa indicated that the B(4) blastomere in four cell-stage embryos is specified as a primordial germ cell. Vas expression later in development further indicated that embryos developing into reproductive larvae possess primordial germ cells whereas embryos developing into soldier larvae do not. Ablation of the B(4) blastomere resulted in most broods containing only soldiers whereas ablation of other blastomeres produced broods containing both castes. These results indicate that soldier larvae are obligately sterile and reveal a previously unknown role for germ cells in caste formation.

Imaginal wing discs in larvae of the soldier caste of Pheidole bicarinata vinelandica Forel (Hymenoptera : Formicidae)

In Pheidole bicarinata vinelandica, soldier larvae have prominent mesothoracic wing discs. Imaginal wing discs are suppressed in minor worker larvae. In soldiers, wing discs appear abruptly late in larvae life and are unusually large when compared with wing discs in worker larvae of other ant genera. Once development has been initiated, wing discs of soldier larvae grow at a rate comparable to soldier leg discs. The dynamics of development of soldier wing discs differ fundamentally from those of other holometabolous insects, worker ants and Pheidole bicarinata queens. This unusual developmental pattern may provide a clue to the physiological basis and timing of soldier determination.