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We evaluated the host suitability and related traits of Trichoplusia ni Hübner (Lepidoptera: Noctuidae) and Chrysodeixis chalcites Esper (Lepidoptera: Noctuidae), which is nonnative in North America, for the native parasitoids Campoletis sonorensis Cameron (Hymenoptera: Ichneumonidae) and Copidosoma floridanum Ashmead (Hymenoptera: Encyrtidae), and the nonnative parasitoid Cotesia vanessae Reinhard (Hymenoptera: Braconidae). For the larval parasitoid C. sonorensis and C. vanessae trials, three-day-old larvae of both hosts were used, whereas one-day-old eggs of both hosts were used for the egg–larval parasitoid C. floridanum trial. For suitability parameters on each host exposed separately to each of the three parasitoid species, we measured parasitoid emergence (parasitoid success), parasitoids that did not emerge (parasitoid cocoon mortality), the proportion of male offspring (parasitoid sex ratio), hosts that developed into moths (host success), hosts that died without developing into moths or producing a parasitoid (host mortality), parasitoids emerging from cocoon masses (brood size), and the developmental times of parasitoids and hosts. For C. sonorensis, the native host and the nonnative host were found to be similarly suitable. For C. vanessae, the native host was more suitable than the nonnative host. For C. floridanum, the native host was suitable, whereas the nonnative host was not; however, sublethal effects on both the native and nonnative hosts were observed. The differential suitability of the hosts observed in this study contributes to the understanding of this measure as a dynamic factor in the expansion of parasitoids into novel host species.

Herbivore populations are regulated by plant defenses and natural enemies. While plant defense can suppress herbivore populations, these defenses adversely affect natural enemies thereby releasing herbivores from top-down control. Over their lifespans, herbivores and their natural enemies may experience substantial variation in plant defense. Defense variability can suppress the growth of herbivores, but the impacts of defense variability on natural enemies and top-down control of herbivores are unknown. We independently manipulated the mean and variation of a plant toxin experienced by individual Trichoplusia ni caterpillars and its parasitoid Copidosoma floridanum. Increases in the mean toxin concentration, but not its variance, experienced by individual T. ni and C. floridanum decreased the fitness of C. floridanum, whereas both mean and variance impacted T. ni fitness. Thus, increased defense variability for individual herbivores suppressed herbivore fitness with no perceptible cost to top-down control. However, impacts of variability depend heavily on scale of variability.

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 waspCopidosoma floridanumoviposits 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 ofC. floridanum. Functional annotation of genes is not fully available forC.floridanum; however, whole genome assembly has been archived. Hence, we constructed a pipeline for gene functional annotation inC. floridanumand 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 wholeC. floridanumtranscripts 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% ofC. floridanumtranscripts 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 ofC. 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.

Copidosoma floridanum is a polyembryonic, caste-forming, wasp species. The ratio of investment in different castes changes with environmental stressors (e.g. multi-parasitism with competitors). The vasa gene was first identified in Drosophila melanogaster as a germ-cell-determining factor, and C. floridanum vasa (Cf-vas) gene positive cells have been known to develop into reproductive larvae. Cf-vas seems to control the ratio of investment in C. floridanum larval castes. In this study, we identified environmental factors that control Cf-vas mRNA expression in Japanese C. floridanum by examining Cf-vas mRNA expression under competitor (Meteorus pulchricornis) venom stress; we treated the male and female morulae with M. pulchricornis venom. We also assessed the effects of multi-parasitism of Japanese C. floridanum with M. pulchricornis and found an increasing number of female soldier larvae. The results showed that several amino acid sequences differ between the Japanese and US Cf-vas. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that Japanese Cf-vas mRNA is expressed in both male and female larvae and pupae, but mRNA expression decreases in adults. Cf-vas mRNA expression significantly decreased, while C. floridanum dronc (Cf-dronc) mRNA expression increased, in female morulae after M. pulchricornis venom treatment at 20 h and 0 h of the culture period, respectively. Females and males showed different Cf-vas or Cf-dronc mRNA expression after M. pulchricornis venom treatment. Therefore, M. pulchricornis venom could affect the ratio of investment in different female castes of Japanese C. floridanum by decreasing Cf-vas mRNA expression via apoptosis.

Aenasius bambawalei Hayat (Encyrtidae: Hymenoptera) has been synonymized with Aenasius arizonensis (Girault) is a small, newly discovered endoparasitoid of the cotton mealybug Phenacoccuss solenopsis Tinsley (Pseudococcidae: Hemiptera), which completes its life cycle inside the body of its host and it is a potential insect control tool. Despite the acquired knowledge regarding host-parasitoid interaction, little information is available on the factors of parasitoid origin able to modulate mealybug physiology. The components of A. arizonensis venom have not been well studied but venom from other parasitoids and wasps contain biologically active proteins that have potential applications in pest management or may be of medicinal importance. To provide an insight into the transcripts expressed in the venom gland of A. arizonensis, a transcriptomic database was developed utilizing high throughput RNA sequencing approaches to analyze the genes expressed in venom glands of this endoparasitic wasp. The resulting A. arizonensis RNA sequences were assembled de-novo with contigs then blasted against the NCBI non-redundant sequence database. Contigs which matched database sequences were mostly homologous to genes from hymenopteran parasitoids such as Nasonia vitripennis, Copidosoma floridanum, Fopius arsenus and Pteromalas puparium. Further analysis of the A. arizonensis database was then performed which focused on selected genes encoding proteins potentially involved in host developmental arrest, disrupting the host immune system, host paralysis, and transcripts that support these functions. Sequenced mRNAS predicted to encode full length ORFs of Calreticulin, Serine Protease Precursor and Arginine kinase proteins were identified and the tissue specific expression of these putative venom genes was analyzed by RT-PCR. In addition, results also demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database and may provide useful information for devising control tools for insect pests and other applications.

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.

Some polyembryonic parasitoid species produce two morphologically different types of larvae from a single egg. The specialized larval morph that forms in addition to the reproductive larvae has been variously described as asexual (Silvestri 1906), teratoid (Parker and Thompson 1928), and precocious (Doutt 1947, 1952) larvae and is characterized by its slender body and developed mandibles. In Copidosoma floridanum, many reproductive larvae appear at the final-instar stage of the host and eventually develop into adults, whereas a small number of precocious larvae first appear at the early-instar stage of the host and then die without undergoing metamorphosis (Fig. 4.1). Thus, the precocious larvae represent a sterile caste. There are two hypotheses for why precocious larvae are produced: (1) to act as soldier larvae that defend sibling embryos against competitors invading the same host and (2) to adjust the sex ratio in mixed-sex broods. Therefore, the function and evolution of precocious larvae have mostly been examined from the viewpoint of sociobiology.

Entry into the host body is a prerequisite for successful completion of the endoparasitoid life cycle. Most endoparasitoids achieve this by laying eggs directly inside the body cavity of the host. In most braconid and ichneumonid parasitoids, the ovipositor is inserted within the host hemocoel to lay eggs, and the hatched larvae grow and develop rapidly inside the host hemolymph as host development advances and finally consume the host tissues, leading to death of the host, which is why parasitic wasps are usually referred to as “parasitoids.” By contrast, egg–larval endoparasitoids, such as those in the genus Ascogaster (family Braconidae), lay their eggs inside the host embryo or alternatively in the yolk of the host egg, following which the newly hatched larvae enter the host embryo. However, the polyembryonic egg–larval endoparasitoid Copidosoma floridanum cannot employ this strategy due to its prolonged morula stage, so this species has evolved a novel approach for entering the host body that involves tissue-compatible invasion by the motile morula.

As part of identifying and characterizing physiological responses and adaptations by insects, it is paramount to develop non-destructive techniques to monitor individual insects over time. Such techniques can be used to optimize the timing of when in-depth (i.e., destructive sampling of insect tissue) physiological or molecular analyses should be deployed. In this article, we present evidence that hyperspectral proximal remote sensing can be used effectively in studies of host responses to parasitism. We present time series body reflectance data acquired from individual soybean loopers (Chrysodeixis includens) without parasitism (control) or parasitized by one of two species of parasitic wasps with markedly different life histories: Microplitis demolitor, a solitary larval koinobiont endoparasitoid and Copidosoma floridanum, a polyembryonic (gregarious) egg-larval koinobiont endoparasitoid. Despite considerable temporal variation in reflectance data 1-9 days post-parasitism, the two parasitoids caused uniquely different host body reflectance responses. Based on reflectance data acquired 3-5 days post-parasitism, all three treatments (control larvae, and those parasitized by either M. demolitor or C. floridanum) could be classified with >85 accuracy. We suggest that hyperspectral proximal imaging technologies represent an important frontier in insect physiology, as they are non-invasive and can be used to account for important time scale factors, such as: minutes of exposure or acclimation to abiotic factors, circadian rhythms, and seasonal effects. Although this study is based on data from a host-parasitoid system, results may be of broad relevance to insect physiologists. Described approaches provide a non-invasive and rapid method that can provide insights into when to destructively sample tissue for more detailed mechanistic studies of physiological responses to stressors and environmental conditions.

Trophic interactions and environmental conditions determine the structure of food webs and the host expansion of parasitoids into novel insect hosts. In this study, we investigate plant–insect–parasitoid food web interactions, specifically the effect of trophic resources and environmental factors on the presence of the parasitoids expanding their host range after the invasion of Chrysodeixis chalcites (Esper) (Lepidoptera: Noctuidae). We also consider potential candidates for biological control of this non‐native pest. A survey of larval stages of Plusiinae (Lepidoptera: Noctuidae) and their larval parasitoids was conducted in field and vegetable greenhouse crops in 2009 and 2010 in various locations of Essex and Chatham‐Kent counties in Ontario, Canada. Twenty‐one plant–host insect–host parasitoid associations were observed among Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae), C. chalcites, and larval parasitoids in three trophic levels of interaction. Chrysodeixis chalcites, an old‐world species that had just arrived in the region, was the most common in our samples. The larval parasitoids Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae), Cotesia vanessae (Reinhard), Cotesia sp., Microplitis alaskensis (Ashmead), and Meteorus rubens (Nees) (all Hymenoptera: Braconidae) expanded their host range into C. chalcites changing the structure of the food web. Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) was the most common parasitoid of T. ni that was not found in the invasive species. Plant species, host abundance, and agro‐ecosystem were the most common predictors for the presence of the parasitoids expanding their host range into C. chalcites. Our results indicate that C. sonorensis, C. vanessae, and C. floridanum should be evaluated for their potential use in biological control of C. chalcites and T. ni.

Insect parasitoids can play ecologically important roles in virtually all terrestrial plant-insect herbivore interactions, yet whether parasitoids alter the defensive traits that underlie interactions between plants and their herbivores remains a largely unexplored question. Here, we examined the reciprocal trophic interactions among populations of the wild cabbage Brassica oleracea that vary greatly in their production of defensive secondary compounds - glucosinolates (GSs), a generalist herbivore, Trichoplusia ni, and its polyembryonic parasitoid Copidosoma floridanum. In a greenhouse environment, plants were exposed to either healthy (unparasitized), parasitized, or no herbivores. Feeding damage by herbivores induced higher levels of the indole GSs, glucobrassicin and neoglucobrassicin, but not any of the other measured GSs. Herbivores parasitized by C. floridanum induced cabbage plants to produce 1.5 times more indole GSs than levels induced by healthy T. ni and five times more than uninduced plants. As a gregarious endoparasitoid, C. floridanum causes its host T. ni to feed more than unparasitized herbivores resulting in increased induction of indole GSs. In turn, herbivore fitness parameters (including differential effects on male and female contributions to lifetime fecundity in the herbivore) were negatively correlated with the aliphatic GSs, sinigrin and gluconapin, whereas parasitoid fitness parameters were negatively correlated with the indole GSs, glucobrassicin and neoglucobrassicin. That herbivores and their parasitoids appear to be affected by different sets of GSs was unexpected given the intimate developmental associations between host and parasitoid. This study is the first to demonstrate that parasitoids, through increasing feeding by their herbivorous hosts, can induce higher levels of non-volatile plant chemical defenses. While parasitoids are widely recognized to be ubiquitous in most terrestrial insect herbivore communities, their role in influencing plant-insect herbivore relationships is still vastly underappreciated.

Cabbage looper, Trichoplusia ni (Hübner) is an important lepidopteran pest on many vegetable and greenhouse crops, and some field crops. Although there are no commercial transgenic Bt vegetable or greenhouse crops, T. ni is a target of Bollgard II cotton, which produces Cry1Ac and Cry2Ab. We expand on previous work that examined the effect of Bt crops on parasitoids using Bt-resistant lepidopteran populations as hosts. Cry1Ac/Cry2Ab-resistant T. ni larvae were used to eliminate host quality effects and to evaluate the direct effects of Bt cotton on the parasitoids Copidosoma floridanum (Ashmead) and Cotesia marginiventris (Cresson). These tri-trophic studies confirm that Bt cotton had no significant impact on development, success of parasitism, survival and adult longevity of C. marginiventris when using Bt-resistant T. ni fed on Bt cotton. Similarly, this Bt cotton had no significant impact on the development, mummy weight and the number of progeny produced by C. floridanum. Our studies verified that lyophilized Bt crop tissue maintained its insecticidal bioactivity when incorporated into an artificial diet, demonstrating that hosts and parasitoids were exposed to active Cry proteins. The egg-larval parasitoid C. floridanum, or similar species that consume their entire host, should be considered useful surrogates in risk assessment of Bt crops to non-target arthropods.

45. Copidosoma floridanum (Ashmead, 1900) Host associations in Iran. Collected on dead wood by xylophagous beetles (Coleoptera, Scolytidae) (Lotfalizadeh 2010b). Distribution in Iran. East Azarbaijan (Lotfalizadeh 2010b). General distribution. Cosmopolitan, Published as part of Fallahzadeh, Majid & Japoshvili, George, 2017, An updated checklist of Iranian Encyrtidae (Hymenoptera, Chalcidoidea), pp. 1-46 in Zootaxa 4344 (1) on page 11, DOI: 10.11646/zootaxa.4344.1.1, http://zenodo.org/record/1042219, {"references":["Lotfalizadeh, H. (2010 b) Some new data and corrections on Iranian encyrtid wasp (Hymenoptera: Chalcidoidea, Encyrtidae) fauna. Biharean Biologist, 4 (2), 173 - 178."]}

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.

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.

Plant defensive compounds can have sometimes severe deleterious effects on both herbivores and their natural enemies. Iridoid glycosides (IGs) are defensive compounds that are well established as deterrent to several generalist herbivores and generalist predators. Trichoplusia ni Hubner (Lepidoptera: Noctuidae) is exceptional among generalist herbivores for its ability to tolerate and thrive when feeding upon IG-producing plant species; however, it is not known whether the compounds themselves have a harmful effect on T. ni and whether the effects in turn affect its oligophagous endoparasitoid Copidosoma floridanum Ashmead (Hymenoptera: Encyrtidae). To examine these effects, a semi-purified extract of the IG-containing plant Plantago lanceolata L. (Plantaginaceae), containing the IGs aucubin and catalpol, was added to artificial diets at 0, 1, 5, or 10% diet dry weight. These diets were fed to both C. floridanum-parasitized and unparasitized T. ni. Diets higher in IGs tended to be more toxic to both parasitized and unparasitized larvae: host larvae that did survive were slightly smaller and took longer to develop on higher IG diets and total clutch size and survival of the parasitoid C. floridanum were greatly reduced as the host's dietary intake of IGs increased. Only small amounts of aucubin were detected inside the T. ni hemocoel, suggesting that the negative effect of these compounds on C. floridanum is due to nutritional quality of the host being reduced rather than direct toxic effects of the compounds.

Copidosoma floridanum (Ashmead, 1900) Litomastix maculata Ishii, 1928: 115. Synonymised with Copidosoma floridanum by NOYES (1988: 200, 202). Material examined. 1 ♀, Fuchu, Tokyo, x.1955, T. Tachikawa lgt. & det.; 1 ♀, Tokyo, ex Thysanoplusia intermixta, x.1955, T. Ichinose lgt. T. Tachikawa det. (ELKU). Distribution. Japan: Honshu (ISHII 1928). Cosmopolitan (NOYES 2016). Comments. We were not able to find the type material of C. maculata., Published as part of Japoshvili, George, Higashiura, Yoshimitsu & Kamitani, Satoshi, 2016, A review of Japanese Encyrtidae (Hymenoptera), with descriptions of new species, new records and comments on the types described by Japanese authors, pp. 345-401 in Acta Entomologica Musei Nationalis Pragae 56 (1) on pages 369-370, DOI: 10.5281/zenodo.5306831, {"references":["ISHII T. 1928: The Encyrtinae of Japan. I. Bulletin of the Imperial Agricultural Experiment Station of Japan 3: 79 - 160.","NOYES J. S. 1988: Copidosoma truncatellum (Dalman) and C. floridanum (Ashmead) (Hymenoptera, Encyrtidae), two frequently misidentified polyembryonic parasitoids of caterpillars (Lepidoptera). Systematic Entomology 13: 197 - 204","NOYES J. S. 2016: Universal Chalcidoidea Database. World Wide Web electronic publication. http: // www. nhm. ac. uk / chalcidoids (Accessed: 30 May 2016)."]}

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.

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.

1. Intrinsic competition was compared in three species of braconid wasps, the solitary Meteorus pulchricornis Wesmael, and the gregarious Cotesia kariyai (Watanabe) and Cotesia ruficrus Haliday in caterpillars of their common host, the armyworm Mythimna separata Walker. Competition was determined in pair-wise contests consisting of simultaneous and subsequent parasitisms at various time intervals between the first and second attacks (

1. Plant defensive chemistry is predicted to have a more negative effect on generalist herbivores and their parasitoids than on specialist herbivores and their parasitoids. 2. This prediction was examined by comparing the effects of the wild parsnip (Pastinaca sativa L.) toxin, xanthotoxin, on a generalist herbivore–parasitoid association [the cabbage looper, Trichoplusia ni Hubner, and its polyembryonic parasitoid, Copidosoma floridanum (Ashmead)] and a specialist herbivore–parasitoid association [the parsnip webworm, Depressaria pastinacella (Duponchel), and its polyembryonic parasitoid, Copidosoma sosares (Walker)]. 3. Copidosoma floridanum brood sizes were smaller and experienced lower survivorship when reared in a host feeding on an artificial diet containing a low concentration of xanthotoxin. No T. ni hosts, parasitised or unparasitised, survived on a diet high in xanthotoxin. In contrast, C. sosares brood size and survivorship were unaffected by the presence of low levels of xanthotoxin in the host diet. Copidosoma sosares experienced reduced brood size and survivorship only when its host consumed a diet containing 15 times the level of xanthotoxin as the diet adversely affecting its congener. 4. The differences in response to xanthotoxin exhibited by C. floridanum and C. sosares are explained partly by a differential reduction in host quality and partly by differential exposure to xanthotoxin in host haemolymph. Unlike D. pastinacella, T. ni experienced reduced pupal weight and survivorship and prolonged developmental time on a low-xanthotoxin diet. More xanthotoxin passed unmetabolised into the haemolymph of T. ni than into the haemolymph of D. pastinacella.

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.

Diet of herbivorous insects can influence both the herbivores and their natural enemies. We examined the direct and indirect effects of diet on the interactions between the polyphagous herbivore Trichoplusia ni Hubner (Lepidoptera: Noctuidae) and its polyembryonic parasitoid Copidosoma floridanum Ashmead (Hymenoptera: Encyrtidae). To determine how host plant species and host plant iridoid glycoside content affect host caterpillars and their parasitoids, parasitized and unparasitized T. ni were given leaves of either Plantago lanceolata L., which contains the iridoid glycosides aucubin and catalpol, Plantago major L. (Plantaginaceae), which contains only aucubin, or Taraxacum officinale F.H. Wigg (Asteraceae), which contains neither. Survival of unparasitized T. ni was much lower when fed P. major compared with the other two host plants, whereas pupae were smallest when fed T. officinale and developed most slowly when fed P. lanceolata as larvae. Neither aucubin nor catalpol were detected in intact Plantago-fed T. ni larvae or their hemolymph, and only trace amounts of aucubin were detected in frass, suggesting that these compounds are mostly metabolized in the mid-gut and are not encountered by the parasitoid. Copidosoma floridanum clutch size was almost doubled when reared from P. lanceolata-fed T. ni compared with T. officinale-fed larvae and tripled compared with P. major-fed larvae, although the percent of parasitoids surviving to adulthood was uniformly high regardless of host diet. The observed variation in C. floridanum fitness among host diets is likely mediated by the effect of the diets on host quality, which in turn may be influenced more by other factors in the host plants than their iridoid glycoside profiles. Interactions between plant metabolites, generalist herbivores like T. ni, and their parasitoids may be predominantly indirect.

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.

Polyembryonic development, where multiple embryos are formed from a single zygote, evolved at least 15 times in six different phyla in animals. The mechanisms leading to polyembryony and the forces that shaped the evolution of the polyembryonic developmental program have remained poorly understood. Recent studies of the polyembryonic development in the endoparasitic wasp Copidosoma floridanum have revealed that the evolution of polyembryony is associated with the evolution of developmental novelties such as total cleavage, early specification of embryonic and extra-embryonic fates, and a specific cell proliferation phase. These changes cumulatively result in the formation of thousands of embryos from a single egg. Laser ablation studies and analysis of early cell fate specification have revealed that a single blastomere representing the progenitor of the primordial germ cell regulates the proliferation of the embryos. We propose that evolutionary changes in cell cleavage, cell interactions, and the cell-differentiation program, reminiscent of interactions between the germinal stem cell and stem cell niche in fly ovaries, underlies the evolution of polyembryony.

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?

Embryonic invasion into the tissue of genetically different organisms has been known only in mother-embryo interactions of viviparous organisms. Hence, embryonic invasions have been thought to occur only within the same or closely related species. For endoparasitic Hymenoptera, which are oviposited in their host egg but complete their development in the later stages, entry into the host embryo is essential. To date, the entry of these parasitoids is known to be accomplished by either egg deposition directly into the embryo or by the newly hatched larva boring into the embryo. However, Copidosoma floridanum is a polyembryonic parasitoid whose development is characterized by a prolonged embryonic stage, and which lacks a larval form during its host embryogenesis. We have analyzed the behavior and fate of C. floridanum embryos co-cultured with their host embryo in vitro. Here, we show that the morula-stage embryo of C. floridanum actively invades the host embryo. Histological analyses have demonstrated that C. floridanum embryonic invasion is associated with adherent junction to host cells rather than causing an obvious wound on the host cells. These findings provide a novel case of embryonic invasion into a phylogenetically distant host embryo, ensuring cellular compatibility with host tissues.

Summary 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.