Functional endogenous viral elements in the genome of the parasitoid wasp Cotesia congregata: insights into the evolutionary dynamics of bracoviruses

Bracoviruses represent the most complex endogenous viral elements (EVEs) described to date. Nudiviral genes have been hosted within parasitoid wasp genomes since approximately 100 Ma. They play a crucial role in the wasp life cycle as they produce bracovirus particles, which are injected into parasitized lepidopteran hosts during wasp oviposition. Bracovirus particles encapsidate multiple dsDNA circles encoding virulence genes. Their expression in parasitized caterpillars is essential for wasp parasitism success. Here, we report on the genomic organization of the proviral segments (i.e. master sequences used to produce the encapsidated dsDNA circles) present in the Cotesia congregata parasitoid wasp genome. The provirus is composed of a macrolocus, comprising two-thirds of the proviral segments and of seven dispersed loci, each containing one to three segments. Comparative genomic analyses with closely related species gave insights into the evolutionary dynamics of bracovirus genomes. Conserved synteny in the different wasp genomes showed the orthology of the proviral macrolocus across different species. The nudiviral gene odv-e66-like1 is conserved within the macrolocus, suggesting an ancient co-localization of the nudiviral genome and bracovirus proviral segments. By contrast, the evolution of proviral segments within the macrolocus has involved a series of lineage-specific duplications.     

Transfer of a chromosomal <Emphasis Type="Italic">Maverick</Emphasis> to endogenous bracovirus in a parasitoid wasp

Bracoviruses are used by parasitoid wasps to allow development of their progeny within the body of lepidopteran hosts. In parasitoid wasps, the bracovirus exists as a provirus, integrated in a wasp chromosome. Viral replication occurs in wasp ovaries and leads to formation of particles containing dsDNA circles (segments) that are injected into the host body during wasp oviposition. We identified a large DNA transposon Maverick in a parasitoid wasp bracovirus. Closely related elements are present in parasitoid wasp genomes indicating that the element in CcBV corresponds to the insertion of an endogenous wasp Maverick in CcBV provirus. The presence of the Maverick in a bracovirus genome suggests the possibility of transposon transfers from parasitoids to lepidoptera via bracoviruses.     

When parasitic wasps hijacked viruses: genomic and functional evolution of polydnaviruses

The Polydnaviridae (PDV), including the Bracovirus (BV) and Ichnovirus genera, originated from the integration of unrelated viruses in the genomes of two parasitoid wasp lineages, in a remarkable example of convergent evolution. Functionally active PDVs represent the most compelling evolutionary success among endogenous viral elements (EVEs). BV evolved from the domestication by braconid wasps of a nudivirus 100 Ma. The nudivirus genome has become an EVE involved in BV particle production but is not encapsidated. Instead, BV genomes have co-opted virulence genes, used by the wasps to control the immunity and development of their hosts. Gene transfers and duplications have shaped BV genomes, now encoding hundreds of genes. Phylogenomic studies suggest that BVs contribute largely to wasp diversification and adaptation to their hosts. A genome evolution model explains how multidirectional wasp adaptation to different host species could have fostered PDV genome extension. Integrative studies linking ecological data on the wasp to genomic analyses should provide new insights into the adaptive role of particular BV genes. Forthcoming genomic advances should also indicate if the associations between endoparasitoid wasps and symbiotic viruses evolved because of their particularly intimate interactions with their hosts, or if similar domesticated EVEs could be uncovered in other parasites.     

Phoresy in the field: natural occurrence of Trichogramma egg parasitoids on butterflies and moths

Phoretic insects utilize other animals to disperse to new environments. We recently discovered how egg parasitoids use an exciting phoretic strategy to reach egg-laying sites of their butterfly hosts. In the laboratory, female Trichogramma wasps detect and mount mated female cabbage white butterflies that emit an anti-aphrodisiac pheromone. Hardly any information exists about the natural occurrence of phoresy in wasps of this genus. Therefore, we monitored the presence of phoretic Trichogramma wasps on lepidopteran hosts in the field. Only female wasps were found at low prevalence on six lepidopteran species. Wasps were mostly found on female hosts and mainly on abundant solitary host species. This is the first report of phoretic Trichogramma wasps on butterflies in nature. We suggest that phoresy is only one of several strategies used by these polyphagous egg parasitoids. The evolution of phoresy is discussed in relation to the nutritional ecology of egg parasitoids.     

Effects of Intraspecific Competition and Host-Parasitoid Developmental Timing on Foraging Behaviour of a Parasitoid Wasp

In a context where hosts are distributed in patches and susceptible to parasitism for a limited time, female parasitoids foraging for hosts might experience intraspecific competition. We investigated the effects of host and parasitoid developmental stage and intraspecific competition among foraging females on host-searching behaviour in the parasitoid wasp Hyposoter horticola. We found that H. horticola females have a pre-reproductive adult stage during which their eggs are not mature yet and they forage very little for hosts. The wasps foraged for hosts more once they were mature. Behavioural experiments showed that wasps’ foraging activity also increased as host eggs aged and became susceptible to parasitism, and as competition among foraging wasps increased.     

Plasticity in life features,parasitism and super-parasitism behavior of Bracon hebetor,an important natural enemy of Galleria mellonella and other lepidopteran host species

The wasp, Bracon hebetor Say, is an important potential biocontrol agent of a wide range of lepidopteran insect species. The current study was subjected to compare these ectoparasitoid fitness traits on different host species belonging to the order Lepidoptera. Moreover, we determine the population dynamic with different host densities, sex ratio patterns, super-parasitism, longevity, paralysis success, and reproductive potential under laboratory conditions. Our results revealed that oviposition increased with an increase in host density, while the adult emergence and egg hatching were decreased due to the super-parasitism on host larvae. A higher male and female-biased population were observed when virgin and mated females offered fresh hosts. Adults' longevity was recorded more in females than males when kept only with bee honey + royal jelly + host larvae. The mean duration of egg-adult development was recorded higher on Galleria mellonella and lowest on Busseola fusca. The parasitization rate, super-parasitism, and cumulative fecundity of mated and virgin female wasps with different host species were observed higher on specific host Galleria mellonella while it was lower on Phthorimaea operculella. Furthermore, the parasitoids having mating experience preferred fresh, while the parasitoids' lack of mating experiences preferred paralyzed host under olfactometer test. Besides, this research has produced novel facts on the biology of parasitic wasp, B. hebetor that may guide the advancement of sustainable biological control programs to control lepidopteran pests.     

Polydnavirus genome: integrated vs. free virus

Polydnaviruses are unique because of their obligatory association with thousands of parasitoid wasp species from the braconid and ichneumonid families of hymenopterans. PDVs are injected into the parasitized hosts and are essential for parasitism success. However, polydnaviruses are also unique because of their genome composed of multiple dsDNA segments. Cytological evidence has recently confirmed the results of genetic and molecular analyses indicating that PDV segments were integrated in the wasp genome. Moreover a phylogenetic study performed using the age of available fossils to calibrate the molecular clock indicated that the polydnaviruses harboured by braconid wasps have resided within the wasp genome for approximately 70 million years. In the absence of horizontal transmission, the evolution of the PDV genomes has been driven exclusively by the reproductive success they have offered the wasps. The consequences of this particular selection pressure can be observed in the gene content of certain PDV genomes from which increasing sequence data are available. Molecular mechanisms already identified could be involved in the acquisition and loss of genes by the PDV genomes and lead us to speculate on the definition of the virus genome.     

Development and encapsulation of the endoparasitoid,Microplitis croceipes (Hym.: Braconidae), in six candidate host species (Lep.)

Encapsulation and development of the endoparasitoid,Microplitis croceipes (Cresson), were studied in six atypical lepidopteran host species whose usual host isHelicoverpa zea (Boddie). The candidate hosts examined were: the fall armywormSpodoptera frugiperda (J. E. Smith); the beet armyworm,Spodoptera exigua (Hübner); the cabbage looper,Trichoplusia ni (Hübner); the greater wax moth,Galleria mellonella (L.); the Indian meal moth,Plodia interpunctella (Hübner); and the diamondback moth,Plutella xylostella (L.). BothS. exigua andT. ni were completely unsuitable forM. croceipes development due to the high rate of eggs that were encapsulated within three days after parasitism. Encapsulation inS. frugiperda included mainly parasitoid eggs and was first detected six days after parasitization at 25°C and two days at 30°C. Encapsulation inG. mellonella occurred only in the larval stage of the parasitoid. InP. interpunctella, parasitoid larvae reached the 3rd stadium, but none of them pupated. OnlyS. frugiperda andG. mellonella supported successful development ofM. croceipes from egg to adult. The percentage of parasitoids reaching the adult stage in these hosts was higher at 30°C than at 25°C (13% vs. 4% inS. frugiperda, and 21% vs. 3% inG. mellonella, respectively). However, these percentages were too low to substitute them as a more economical host for rearingM. croceipes. This biological information will be useful in additional laboratory studies directed toward reducing the rate of encapsulation (e.g., manipulation of host rearing temperature) to increase production ofM. croceipes on these hosts.     

Widespread Genome Reorganization of an Obligate Virus Mutualist

The family Polydnaviridae is of interest because it provides the best example of viruses that have evolved a mutualistic association with their animal hosts. Polydnaviruses in the genus Bracovirus are strictly associated with parasitoid wasps in the family Braconidae, and evolved ∼100 million years ago from a nudivirus. Each wasp species relies on its associated bracovirus to parasitize hosts, while each bracovirus relies on its wasp for vertical transmission. Prior studies establish that bracovirus genomes consist of proviral segments and nudivirus-like replication genes, but how these components are organized in the genomes of wasps is unknown. Here, we sequenced the genome of the wasp Microplitis demolitor to characterize the proviral genome of M. demolitor bracovirus (MdBV). Unlike nudiviruses, bracoviruses produce virions that package multiple circular, double-stranded DNAs. DNA segments packaged into MdBV virions resided in eight dispersed loci in the M. demolitor genome. Each proviral segment was bounded by homologous motifs that guide processing to form mature viral DNAs. Rapid evolution of proviral segments obscured homology between other bracovirus-carrying wasps and MdBV. However, some domains flanking MdBV proviral loci were shared with other species. All MdBV genes previously identified to encode proteins required for replication were identified. Some of these genes resided in a multigene cluster but others, including subunits of the RNA polymerase that transcribes structural genes and integrases that process proviral segments, were widely dispersed in the M. demolitor genome. Overall, our results indicate that genome dispersal is a key feature in the evolution of bracoviruses into mutualists.     

Venom gland extract is not required for successful parasitism in the polydnavirus-associated endoparasitoid Hyposoter didymator (Hym. Ichneumonidae) despite the presence of numerous novel and conserved venom proteins

The venom gland is a conserved organ in Hymenoptera that shows adaptations associated with life-style diversification. Few studies have investigated venom components and function in the highly diverse parasitic wasps and all suggest that the venom regulates host physiology. We explored the venom of the endoparasitoid Hyposoter didymator (Campopleginae), a species with an associated polydnavirus produced in the ovarian tissue. We investigated the effects of the H. didymator venom on two physiological traits of the host Spodoptera frugiperda (Noctuidae): encapsulation response and growth rate. We found that H. didymator venom had no significant effect on host cellular immunity or development, suggesting that it does not contribute to parasitism success. The host physiology seemed to be modified essentially by the ovarian fluid containing the symbiotic polydnaviruses. Proteomic analyses indicated that the H. didymator venom gland produces a large variety of proteins, consistent with the classical hymenopteran venom protein signature, including: reprolysin-like, dipeptidyl peptidase IV, hyaluronidase, arginine kinase or allergen proteins. The venom extracts also contained novel proteins, encoded by venom genes conserved in Campopleginae ichneumonids, and proteins with similarities to active molecules identified in other parasitoid species, such as calreticulin, reprolysin, superoxide dismutase and serpin. However, some of these proteins appear to be produced only in small amounts or to not be secreted. Possibly, in Campopleginae carrying polydnaviruses, the host-modifying activities of venom became redundant following the acquisition of polydnaviruses by the lineage.     

Interspecific discrimination and larval competition amongMicroplitis croceipes,Microplitis demolitor,Cotesia kazak (Hym.: Braconidae), andHyposoter didymator (Hym.: Ichneumonidae), parasitoids ofHeliothis virescens (Lep.: Noctuidae)

Interspecific host discrimination by adults, and larval competition among the endoparasitoidsMicroplitis croceipes (Cresson),Microplitis demolitor Wilkinson,Cotesia kazak (Telenga) andHyposoter didymator (Thunberg) were investigated usingHeliothis virescens (F.) as the host. In ovipositional choice tests, the mean number of encounters and ovipositions for unparasitized hosts was not significantly different from the mean number of encounters and ovipositions for parasitized hosts for each treatment combination (P>0.05). Thus, none of the parasitoid species discriminated between host larvae recently parasitized once by a female of another species und unparasitized hosts. However, in all but two cases, females did discriminate between unparasitized hosts and hosts in which an early first instar of the first-attacking species was developing.Cotesia kazak andH. didymator did not discriminate between unparasitized hosts and hosts parasitized by an early first instar ofM. demolitor. Larval competition among these parasitoid species was studied for three time intervals between the first and second species parasitization: 1) second species attack immediately (5–15 sec) after the first; 2) second species attack 24 h after the first; and 3) second species attack 48 h after the first. Time until egg eclosion was shortest forM. demolitor, thenC. kazak, thenM. croceipes, and longest forH. didymator. When the second parasitoid species attacked a host immediately after the first species, the species in which egg eclosion occurred first was the victor more frequently, except whenM. demolitor competed withC. kazak andH. didymator. With a 24 h delay between the first and second species to attack, the older first instar from the first parasitization usually outcompeted the younger first instar from the second attack. A first instar from the second species to attack generally outcompeted the second instar of the first species when the second parasitization had been delayed 48 h. Competiors were eliminated mainly by physical attack, butC. kazak andM. croceipes apparently also killedH. didymator eggs by physiological processes.     

The Bracovirus Genome of the Parasitoid Wasp Cotesia congregata Is Amplified within 13 Replication Units,Including Sequences Not Packaged in the Particles

The relationship between parasitoid wasps and polydnaviruses constitutes one of the few known mutualisms between viruses and eukaryotes. Viral particles are injected with the wasp eggs into parasitized larvae, and the viral genes thus introduced are used to manipulate lepidopteran host physiology. The genome packaged in the particles is composed of 35 double-stranded DNA (dsDNA) circles produced in wasp ovaries by amplification of viral sequences from proviral segments integrated in tandem arrays in the wasp genome. These segments and their flanking regions within the genome of the wasp Cotesia congregata were recently isolated, allowing extensive mapping of amplified sequences. The bracovirus DNAs packaged in the particles were found to be amplified within more than 12 replication units. Strikingly, the nudiviral cluster, the genes of which encode particle structural components, was also amplified, although not encapsidated. Amplification of bracoviral sequences was shown to involve successive head-to-head and tail-to-tail concatemers, which was not expected given the nudiviral origin of bracoviruses.     

Host color preferences and short-range searching behavior of the egg parasitoid Trichogramma ostriniae

The color of lepidopteran eggs often varies by species or egg condition, and parasitoids that attack lepidopteran eggs could therefore potentially use color to obtain information about host identity or quality. The objective of our study was to determine whether females of the egg parasitoid Trichogramma ostriniae Pang & Chen (Hymenoptera: Trichogrammatidae) showed differential responses to egg color when searching for hosts over short distances and when evaluating the suitability of encountered eggs. We examined the wasps’ host‐selection behavior in a Petri dish arena using white, yellow, green, and black clay beads as egg models presented against a green background (to mimic leaf color). In no‐choice tests, bead color had a significant effect on the proportion of tested wasps that accepted a bead for further examination, on the time it took wasps to find and begin examining a bead, and on the time that wasps spent examining the beads. However, bead color had only a marginally significant effect on the proportion of wasps attempting to drill into a bead with their ovipositors, and no significant effect on the amount of time they spent drilling. The wasps also showed significant color preferences when given a choice between two adjacent beads of different colors. The results of the no‐choice and choice trials taken together indicated a color preference ranking of yellow > white > green > black. The wasps’ higher preference for the yellow and white egg models generally corresponds to the white or yellowish‐white egg color of T. ostriniae's target host, the European corn borer moth, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae). The wasps’ strong rejection of black egg models is likely to be an adaptive response that reflects the fact that eggs that are wholly or partially black are often unsuitable for parasitization due to advanced caterpillar development, damage to the egg, or previous parasitization.     

Relationships between polydnavirus gene expression and host range of the parasitoid wasp Campoletis sonorensis

To evaluate the relationship between immune suppression and host range six lepidopteran species were parasitized by the ichneumonid parasitoid Campoletis sonorensis. Parasitism inhibited the growth of permissive hosts (Heliothis virescens, Helicoverpa zea, and Trichoplusia ni), whereas growth of semi-permissive (Spodoptera exigua, Agrotis ipsilon) and non-permissive hosts (Manduca sexta) was not significantly affected. The 29-36 kDa ovarian protein (OP), responsible for transient immunosuppression in the permissive host H. virescens, bound to and was endocytosed by hemocytes of permissive and non-permissive hosts. Expression of the cysteine-rich polydnavirus gene, VHv1.4, was detected in all the hosts, but declined only in semi- and non-permissive hosts at later times after parasitization. The VHv1.4 protein bound to hemocytes of permissive and semi-permissive hosts, but did not bind to hemocytes of the non-permissive host, M. sexta. Melanization of larval hemolymph was severely inhibited by parasitism in permissive hosts, but was unaffected in M. sexta. In the semi-permissive host, A. ipsilon, hemolymph melanization was transiently inhibited while viral genes were expressed. In conclusion, C. sonorensis OP transiently inhibits encapsulation in all hosts that were tested. The host range of C. sonorensis seems to be determined by whether or not the C. sonorensis ichnovirus (CsIV) is able to establish persistent infections of parasitized larvae to provide long-term suppression of host immunity.     

Relationships between polydnavirus genomes and viral gene expression

Polydnavirus genomes and viral gene functions are atypical for viruses. Polydnaviruses are the only group of viruses with segmented DNA genomes and have an unusual obligate mutualistic association with parasitic Hymenoptera, in which the virus is required for survival of the wasp host and vice versa. The virus replicates asymptomatically in the wasp host but severely disrupts lepidopteran host physiology in the absence of viral DNA replication. It is not surprising then that viral gene expression is divergent in its two insect hosts and that differences in viral gene expression are linked to these divergent functions. Some viral genes are expressed only in the wasp host while other viral genes are expressed only in the lepidopteran host and are presumed to be involved in the disruption of host physiological systems. Our laboratory has described the expression and regulation of a family of viral genes implicated in suppressing the lepidopteran immune system, the cys-motif genes. In conjunction with these studies we have described the physical organization of additional viral gene segments. We have cloned, mapped and begun the sequence analysis of selected viral DNA segments. We have noted that some viral DNA segments are nested and that nested viral DNA segments encode the abundantly expressed, secreted cys-motif genes. Conversely, other viral segments are not nested, encode less abundantly expressed genes and may be targeted intra-cellularly. These results suggest that nesting of segments in polydnavirus genomes may be linked to the levels of gene expression. By extension, the unique, segmented organization of polydnavirus genomes may be associated, in part, with the requirement for divergent levels of viral gene expression in lepidopteran hosts in the absence of viral DNA replication.