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.     

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?     

Interspecific competition between the polyembryonic wasp Copidosoma floridanum and the gregarious endoparasitoid Glyptapanteles pallipes

We examined interspecific competition between the egg-larval polyembryonic parasitoid Copidosoma floridanum (Hymenoptera: Encyrtidae) and the gregarious larval endoparasitoid Glyptapanteles pallipes (Hymenoptera: Braconidae). Approximately 72% of multiparasitized Acanthoplusia agnata (Lepidoptera: Noctuidae) produced C. floridanum adults, 14% produced G. pallipes adults, and 14% died without producing any parasitoids. Development was delayed and the rate of weight gain was reduced in multiparasitized hosts that produced C. floridanum compared with singly-parasitized hosts. In contrast, only weight was reduced in multiparasitized hosts that produced G. pallipes adults. Compared with single parasitism, the brood size of wasps emerging from multiparasitized hosts was reduced in both species. The percentage of hosts containing precocious larvae of C. floridanum, which are considered to be soldiers, did not increase in response to parasitization by G. pallipes. However, developmental cessation and death of G. pallipes eggs and larvae may be closely related to the number of coexisting precocious larvae.     

Development of the polyembryonic parasitoid Copidosoma floridanum in Trichoplusia ni

Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) parasitized by the polyembryonic egg-larval parasitoid Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) attained significantly larger final weights and head capsule widths than unparasitized controls. The difference in weight between parasitized and unparasitized hosts was not entirely accounted for by the weight of the C. floridanum brood. The head capsule widths of all parasitized and unparasitized fifth instars used in the study exceeded the critical threshold of 1.66 mm previously established for T. ni metamorphosis. The critical ratios associated with each T. ni instar of: 1) maximum weight within the instar:head capsule width and 2) maximum weight within the instar:weight at the beginning of the instar differed between parasitized and unparasitized larvae. Development of C. floridanum was synchronized with that of its host. Germ band formation and gastrulation of morulae destined to produce reproductive larvae invariably coincided with the host molt to the ultimate, fifth instar. Reproductive larvae had two instars. Eclosion from the egg to the first instar occurred during day 2 of the host's fifth instar, and ecdysis from the first to the second instar was synchronized with host cocoon spinning. Conversely, embryogenesis of morulae destined to produce precocious larvae began during the host first instar, continued through the second and third instar and ceased during the penultimate, fourth instar. Precocious larvae never molted and died when the host was consumed by the reproductive larvae.

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Résumé T. ni Hübner parasité par le parasitoïde ovo-larvaire C. floridanum Ashmead à développement polyembryonnaire atteint un poids final signficativement plus élevé avec une capsule céphalique plus grosse que les témoins non parasités, sans subir de mues surnuméraires. La différence de poids entre noctuelles parasitées ou non ne correspondait pas entièrement au poids des C. floridanum. Les largeurs des capsules céphaliques de tous les T. ni du cinquième stade dépassaient toutes le seuil critique de 1,66 mm lié à la métamorphose, mais les seuils critiques de taille du corps:largeur de la capsule céphalique et/ou taille et corps, taille initiale du corps au début du stade associé à la mue, différaient chez T. ni parasités ou non. Les développements de T. ni et de C. floridanum étaient synchrones. La formation de la bande germinative et la gastrulation de la morula produisant la multiplication des larves ont coïncidé invariablement avec la mue de l'hôte donnant le dernier stade. Les larves polyembryonnaires ont présenté deux stades. L'éclosion des oeufs s'est produite le deuxième jour du cinquième stade de T. ni, et le passage du premier au second était synchrone de la formation du cocon de l'hôte. Réciproquement, l'embryogenèse de la morula qui donnait des larves précoces commençait pendant le premier stade de l'hôte et se poursuivait à travers les second et troisième, pour cesser pendant le quatrième et pénultième stade.

     

Intrinsic interspecific competition between the polyembryonic parasitoid Copidosoma floridanum and solitary endoparasitoid Microplitis demolitor in Pseudoplusia includens

Multiparasitism studies were conducted in the laboratory between the egg-larval polyembryonic parasitoid Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) and the larval endoparasitoid Microplitis demolitor Wilkinson (Hymenoptera: Braconidae) in the mutual host Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae). Most multiparasitized hosts produced either a C. floridanum brood or died without any parasitoid emergence. Most multiparasitized hosts underwent supernumerary molting; however, multiparasitized hosts that produced a C. floridanum brood exhibited growth characteristics associated with C. floridanum parasitism while multiparasitized hosts that died exhibited growth characteristics more typical of M. demolitor parasitism. Multiparasitized hosts that produced a C. floridanum brood increased in weight, and usually exhibited the behavioral and morphological characteristics associated with initiation of metamorphosis. In contrast, multiparasitized hosts that ultimately died gained little weight and exhibited no characteristics associated with metamorphosis. M. demolitor progeny died in multiparasitized hosts as eggs or first instar larvae, but no direct evidence was found to indicate that physical attack by C. floridanum precocious larvae was responsible for their elimination.

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Résumé Lors de l'étude au laboratoire du multiparasitisme de Pseudoplusia includens Walker (Lépido. Noctuidae) par le parasitoïde ovolarvaire polyembryonnaire C. floridanum Ashmead (Hyméno. Encyrtidae) et l'endoparasitoïde larvaire M. demolitor Wilkinson (Hyméno. Braconidae), soit les hôtes donnent en majorité C. floridanum, soit ils meurent sans que quoi que ce soit émerge. P. includens non parasité présente 5 stades larvaires; les hôtes parasités par C. floridanum subissent 5 ou 6 stades avant le développement complet du parasitoïde, et les hôtes parasités par M. demolitor muent en stade 5 avant émergence du parasitoïde. Par contre, les hôtes multiparasités muent en stade 7; ceux qui produisent une couvée de C. floridanum ont présenté la croissance caractéristique des hôtes parasités par C. floridanum, tandis que les hôtes multiparasités qui sont morts avaient présenté la croissance caractéristique des hôtes parasités par M. demolitor. La plupart des hôtes multiparasités desquels C. floridanum a émergé ont augmenté de poids et présenté les caractères associés au début de la métamorphose. La descendance de M. demolitor est morte dans les hôtes multiparasités au stade uf ou chenille du premier stade, mais il n'y avait aucum indice d'attaque physique par des larves de C. floridanum qui auraient été responsables de leur élimination.

     

Caste formation in the polyembryonic wasp Copidosoma floridanum (Hymenoptera: Encyrtidae): in vivo and in vitro analysis

The polyembryonic wasp Copidosoma floridanum produces two morphologically distinct types of larvae in its host Trichoplusia ni. Reproductive larvae consume the host, pupate, and form adult wasps, whereas precocious larvae manipulate the sex ratios of the reproductive caste and defend the brood against interspecific competitors. The previous study indicated that morphogenesis of the reproductive caste was associated with a 9-day competency period, and that ecdysteroids of host origin were required for completion of embryogenesis. Here we investigated whether factors associated with the host environment mediate morphogenesis of precocious larvae and caste determination. Embryogenesis of precocious larvae was found to be synchronized with specific stages of the host first-fourth instars. However, development of precocious larvae did not depend on environmental factors specifically associated with these host stages. Elevation of the host juvenoid titer using the analogue methoprene induced T. ni to undergo a supernumerary sixth instar, but did not alter the proportion of wasp embryos that developed into precocious and reproductive larvae. In contrast, embryos competent to initiate morphogenesis developed into precocious larvae when transplanted into novel host stages such as pupae. Development of precocious larvae was arrested by ablation of the host's source of ecdysteroids, but could be rescued dose-dependently by injection of 20-hydroxyecdysone. In vitro rearing studies confirmed that completion of embryogenesis of the precocious caste required an exogenous pulse of 20-hydroxyecdysone. Combined with previous studies, our results indicate that embryos forming precocious and reproductive larvae acquire the competence to undergo morphogenesis at different times. However, we find no evidence to suggest that caste determination is mediated by environmental factors associated with a specific stage of the host.     

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.     

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.     

Evolution of eusociality and the soldier caste in termites: a validation of the intrinsic benefit hypothesis

In termites the evolution of reproductive altruism is not based on a particularly high relatedness between nestmates. For the evolution and maintenance of the ancestral sterile soldier caste, the benefits generated by the soldiers' presence must compensate the loss of the soldiers' reproductive potential. To study the impact of soldiers on colony's fitness, we manipulated the proportion of soldiers to nonsoldiers in colonies of the dry-wood termite Cryptotermes secundus.'Soldier-less' colonies were obtained by removing soldiers and inhibiting their development with an extract of soldier heads. The colonies were set up for 1 year in experimental nests in the field. 'Soldier-less' colonies produced fewer soldiers. The reduction of soldiers neither affected colony survival nor helper growth, but fewer dispersing sexuals were produced in 'soldier-less' than in control colonies. This confirms what was only supposed so far, that in termites soldiers are maintained for their intrinsic benefit to cost ratio.     

Temporal polyethism in incipient colonies of the primitive termiteZootermopsis angusticollis: A single multiage caste

Division of labor was studied in incipient colonies of the dampwood termite Zootermopsis angusticollisby recording repertoire size, behavior frequencies, and time budgets of larvae. Behavioral repertoire size increased with age: first- and secondinstar larvae were mainly inactive, whereas larvae of the third through seventh instars performed 64–100% of all tasks. The increase in repertoire size from the second to the third instar was abrupt; repertoire size and composition remained more or less constant for older instars. No correlation between age (instars III–VII) and tasks was identified, suggesting that colony labor is performed by a single functional caste that spans the third to the seventh instar without any age-based division of labor. Small colony size, low oviposition rate, simple nest architecture, a lack of spatial association of tasks, and the potential for attaining reproductive status appear to be associated with the lack of age-related behavioral specialization in Z. angusticollis.In effect, the absence of temporal polyethism in this species is likely a consequence of its nesting habits and physiological and developmental constraints.     

Effect of juvenile hormone on the embryogenesis of a polyembryonic wasp,copidosoma floridanum,in vitro

Summary Single two-cell-stage embryos of a polyembryonic waspCopidosoma floridanum cultured in 20 μl droplets of culture medium developed to morulae at the same developmental rate as those in host eggs, but the subsequent development into polymorulae was inferior. This inferior development was markedly improved by addition of juvenile hormone (I, II, or III) or its analogues to the culture medium in a concentration-dependent manner.     

Soldier caste with morphological and reproductive division in the aphid tribe Nipponaphidini

Summary. We investigated demographic, morphological and histological aspects of Distylaphis foliorum, a gall-forming nipponaphidine aphid from Java, Indonesia, whose first instar nymphs had been reported to have enlarged forelegs and attack other insects. The gall inhabitants of D. foliorum consisted of two discrete populations; one developing normally and the other remaining at the first instar. Morphometric analysis identified two types of first instar nymphs; nymphs with relatively long forelegs and slender abdomen, and nymphs with relatively short forelegs and fat abdomen. The former nymphs were found from both young galls and mature galls, while the latter nymphs were from young galls only. Histological analysis strongly suggested a reproductive division in the first instar. In the former nymphs from mature galls, ovaries and mycetomes were degenerate and replaced by well-developed fat body cells. These results indicated that D. foliorum has a morphologically differentiated sterile soldier caste in the first instar. This study is the first report of a soldier caste with morphological and reproductive division in the aphid tribe Nipponaphidini, and suggests that highly specialized soldier castes have evolved at least four times in aphids.Received 25 June 2004; revised 24 September 2004; accepted 11 October 2004.     

Ecdysteroid production during caste differentiation in larvae of the ant, Plagiolepis pygmaea

ABSTRACT. Variations in ecdysteroids were measured by radio-immunoassay in worker-biased and queen-biased larvae of the ant Plagiolepis pygmaea Latr. (Hymenoptera, Formicidae) during late larval development, i.e. from the end of winter diapause up to the prepupal period. At the end of diapause, larvae are bipotential and, depending on culture conditions, can become either queens or workers. Ecdysteroid profiles revealed that there are striking differences between the two castes: worker larvae showed high titres during their development, queen larvae had low titres over the same period.     

Antifungal activity in thrips soldiers suggests a dual role for this caste

The social insect soldier is perhaps the most widely known caste, because it often exhibits spectacular weapons, such as highly enlarged jaws or reinforced appendages, which are used to defend the colony against enemies ranging in size from wasps to anteaters. We examined the function of the enlarged forelimbs of soldiers (both male and female) of the eusocial, gall-inhabiting insect Kladothrips intermedius, and discovered that they have little impact on their ability to repel the specialized invading thrips Koptothrips species. While the efficacy of the enlarged forelimb appears equivocal, we show that soldiers secrete strong antifungal compounds capable of controlling the specialized insect fungal pathogen, Cordyceps bassiana. Our data suggest that these thrips soldiers have evolved in response to selection by both macro- and micro-organisms. While it is unknown whether specialized fungal pathogens have been major selective agents in the evolution of the soldier caste in general, they were probably present when sociality first evolved and may have been the primordial enemies of social insects.     

The role of larval nutrition in pre-imaginal biasing of caste in the primitively eusocial wasp Ropalidia marginata (Hymenoptera: Vespidae)

Abstract.

• 1 When freshly eclosed females of the primitively eusocial wasp Ropalidia marginata (Lep.) are isolated, only about 50% of them build nests and lay eggs thereby suggesting a pre-imaginal biasing of caste.
• 2 Wasps that lay eggs take a very variable amount of time after eclosion to start doing so.
• 3 Females eclosing from nests where larvae are fed at a relatively higher rate are more likely to become egg-layers and are likely to take less time after eclosion to begin to lay eggs.
• 4 Thus, both forms of pre-imaginal biasing of caste, namely, differences in egg laying capacity and differences in the time taken to attain reproductive maturity, appear to be influenced by larval nutrition.

     

Proximate mechanisms and evolution of caste polyphenism in social insects: From sociality to genes

Evidence has accumulated over several decades to prove the kin selection theory of evolution of social insects, however, proximate mechanisms of social behavior, and/or caste differentiation remain obscure. Genes that regulate these mechanisms are apparently selected through kin selection, and organisms have consequently acquired sociality. Here, I will discuss several studies that were performed recently by Matsumoto Laboratory, University of Tokyo, Tokyo, Japan, in various social insects, such as termites and ants, in order to reveal the regulatory mechanisms of social behavior and the evolutionary processes of sociality. First, I will review the foraging behavior of the black marching termite Hospitalitermes medioflavus, where well-organized task allocation among castes is apparent. This suggests that regulation of postembryonic development is important in social behavior. Next, I will summarize recent progress in identifying caste-specific gene expression in the damp-wood termite Hodotermopsis sjostedti. This constitutes the basis for molecular mechanisms of caste differentiation, and moreover, the genes identified might be good markers for social evolution. Finally, the mechanism underlying winglessness in ant workers is reviewed. Apoptotic cell death was detected at the stage of pupation in wingless worker castes. Furthermore, the areas of study recently designated as sociogenomics and ecological developmental biology are discussed.     

Brain organization mirrors caste differences,colony founding and nest architecture in paper wasps (Hymenoptera: Vespidae)

The cognitive challenges that social animals face depend on species differences in social organization and may affect mosaic brain evolution. We asked whether the relative size of functionally distinct brain regions corresponds to species differences in social behaviour among paper wasps (Hymenoptera: Vespidae). We measured the volumes of targeted brain regions in eight species of paper wasps. We found species variation in functionally distinct brain regions, which was especially strong in queens. Queens from species with open-comb nests had larger central processing regions dedicated to vision (mushroom body (MB) calyx collars) than those with enclosed nests. Queens from advanced eusocial species (swarm founders), who rely on pheromones in several contexts, had larger antennal lobes than primitively eusocial independent founders. Queens from species with morphologically distinct castes had augmented central processing regions dedicated to antennal input (MB lips) relative to caste monomorphic species. Intraspecific caste differences also varied with mode of colony founding. Independent-founding queens had larger MB collars than their workers. Conversely, workers in swarm-founding species with decentralized colony regulation had larger MB calyx collars and optic lobes than their queens. Our results suggest that brain organization is affected by evolutionary transitions in social interactions and is related to the environmental stimuli group members face.     

First- and second-order sociality determine survival and reproduction in cooperative cichlids

Cooperative breeders serve as a model to study the evolution of cooperation, where costs and benefits of helping are typically scrutinized at the level of group membership. However, cooperation is often observed in multi-level social organizations involving interactions among individuals at various levels. Here, we argue that a full understanding of the adaptive value of cooperation and the evolution of complex social organization requires identifying the effect of different levels of social organization on direct and indirect fitness components. Our long-term field data show that in the cooperatively breeding, colonial cichlid fish Neolamprologus pulcher, both large group size and high colony density significantly raised group persistence. Neither group size nor density affected survival at the individual level, but they had interactive effects on reproductive output; large group size raised productivity when local population density was low, whereas in contrast, small groups were more productive at high densities. Fitness estimates of individually marked fish revealed indirect fitness benefits associated with staying in large groups. Inclusive fitness, however, was not significantly affected by group size, because the direct fitness component was not increased in larger groups. Together, our findings highlight that the reproductive output of groups may be affected in opposite directions by different levels of sociality, and that complex forms of sociality and costly cooperation may evolve in the absence of large indirect fitness benefits and the influence of kin selection.     

Random sperm use and genetic effects on worker caste fate in Atta colombica leaf-cutting ants

Sperm competition can produce fascinating adaptations with far-reaching evolutionary consequences. Social taxa make particularly interesting models, because the outcome of sexual selection determines the genetic composition of groups, with attendant sociobiological consequences. Here, we use molecular tools to uncover some of the mechanisms and consequences of sperm competition in the leaf-cutting ant Atta colombica, a species with extreme worker size polymorphism. Competitive PCR allowed quantification of the relative numbers of sperm stored by queens from different males, and offspring genotyping revealed how sperm number translated into paternity of eggs and adult workers. We demonstrate that fertilization success is directly related to sperm numbers, that stored sperm are well-mixed and that egg paternity is constant over time. Moreover, worker size was found to have a considerable genetic component, despite expectations that genetic effects on caste fate should be minor in species with a low degree of polyandry. Our data suggest that sexual conflict over paternity is largely resolved by the lifetime commitment between mates generated by long-term sperm storage, and show that genetic variation for caste can persist in societies with comparatively high relatedness.