Host refractoriness of the tobacco hornworm, Manduca sexta, to the braconid endoparasitoid Cotesia flavipes

Cotesia flavipes (Hymenoptera:Braconidae) is a gregarious endoparasitoid of several pyralid stemborer larvae of economic significance including the sugarcane borer, Diatraea saccharalis. In this study, the ability of this parasitoid to develop in a sphingid host, Manduca sexta, was tested. First, second, third, fourth, and even pharate fifth instar host tobacco hornworm larvae were readily parasitized by the female C. flavipes parasitoids but no wasp larvae hatched from the eggs in this refractory host. Instead, the parasitoid eggs were invariably encapsulated by the host's hemocytes and, ultimately, no parasitoids emerged from tobacco hornworm hosts. The first stages of encapsulation were evident at 2 h post-parasitization of the host M. sexta larvae, when the beginning stages of capsule formation were seen. The developmental fate of the host larvae with encapsulated parasitoids was variable. Most succumbed as abnormally small fifth instars or as post-wandering prepupal animals, while a few developed normally to the pupal stage. Dissection of all the larvae or pupae with encapsulated wasp eggs showed evidence of hemocytic encapsulation and melanization of the C. flavipes eggs. This report describes the association between C. flavipes and M. sexta, which appears to be an excellent model system for studying the physiological processes accompanying wasp egg encapsulation that result in death of the host as well as the parasitoid. Since the parasitoid egg never hatches, the system offers an excellent opportunity to identify and study the effects of parasitoid-injected polydnavirus and venom on host physiology.     

Differential expression of the CrV1 haemocyte inactivation-associated polydnavirus gene in the African maize stem borer Busseola fusca (Fuller) parasitized by two biotypes of the endoparasitoid Cotesia sesamiae (Cameron)

Polydnaviruses are rarely studied for their natural variation in immune suppressive abilities. The polydnavirus harboring braconid Cotesia sesamiae, a widespread endoparasitoid of Busseola fusca and Sesamia calamistis in sub-Saharan Africa exists as two biotypes. In Kenya, the western biotype completes development in B. fusca larvae. However, eggs of the coastal C. sesamiae are encapsulated in this host and ultimately, no parasitoids emerge from parasitized B. fusca larvae. Both biotypes develop successfully in S. calamistis larvae. Encapsulation activity by B. fusca larvae towards eggs of the avirulent C. sesamiae was detectable six hours post-parasitization. The differences in encapsulation of virulent and avirulent strains were associated with differences in nucleotide sequences and expression of a CrV1 polydnavirus (PDV) gene, which is associated with haemocyte inactivation in the Cotesia rubecula/Pieris rapae system. CrV1 expression was faint or absent in fat body and haemolymph samples from B. fusca parasitized by the avirulent C. sesamiae, which exhibited encapsulation of eggs. Expression was high in fat body and haemolymph samples from both B. fusca and S. calamistis larvae parasitized by the virulent C. sesamiae, encapsulation in the former peaking at the same time points as CrV1 expression in the latter. Non synonymous difference in CrV1 gene sequences between virulent and avirulent wasp suggests that variations in B. fusca parasitism by C. sesamiae may be due to qualitative differences in CrV1-haemocyte interactions.     

Inhibition of testicular growth and development in Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata

Tobacco hornworm larvae parasitized by the gregarious larval endoparasitoid Cotesia congregata exhibited an inhibition in testicular growth and development, the extent of which was determined by the age and developmental stage of the host at the time of parasitization. The degree of parasitic castration, as assessed by measurements of testicular volume, was correlated with the stadium in which parasitization occurred. A mathematical formula requiring the measurement of testicular length, width and depth was used to calculate testicular volume. The use of the depth parameter revealed a negative correlation between host weight and testicular volume in parasitized larvae. Testicular volumes of fifth instar hosts, which had been parasitized in the first stadium, were significantly smaller than those originally parasitized as fourth or fifth instar larvae and were not correlated with parasitoid load. Effects of natural parasitism were not duplicated by injections of C. congregata polydnavirus and venom, topical treatment with the juvenile hormone analog methoprene, or starvation of nonparasitized larvae. Larvae receiving virus plus venom or methoprene grew larger due to delayed wandering and had larger testes than controls. Deleterious effects on host testes may be due to the effects of nutrient competition between the developing parasitoid progeny and the gonads, combined with the juvenilizing effects believed to be caused by the polydnavirus.     

Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host,Pieris rapae

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host (Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of approximately 14 and approximately 17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts.     

Mechanism of reduction in the number of the circulating hemocytes in the Pseudaletia separata host parasitized by Cotesia kariyai

Larval endoparasitoids can avoid the immune response of the host by the function of polydnavirus (PDV) and venom. PDV infects hemocytes and affects the hemocyte function of the host. In this paper, we investigated how PDV and venom affect the hemocyte population of the host. Cotesia kariyai, the larval endoparasitoid, lowers the hemocyte population of the noctuid host larvae soon after parasitization. The reduction in the number of circulating hemocytes is caused by the breakdown of the circulating hemocytes and of the hematopoietic organ which generates the circulating hemocytes. The decrease in the number of hemocytes shortly after parasitization is a response to the venom. However, the decrease in hemocyte population on and after 6 h post-parasitization appears to be caused by the PDV. Apoptosis in circulating hemocytes was observed on and after 6 h post-injection of PDV plus venom. It was revealed through cytometry that mitosis of circulating hemocytes was halted within 24 h after the injection of PDV plus venom. Apoptosis in the hematopoietic organ was induced 12 h after the injection of PDV plus venom. Furthermore, the plasma from the hosts injected with PDV plus venom depressed the number of hemocytes released from the hemotopoiteic organs.     

Co-infection of Manduca sexta larvae with polydnavirus from Cotesia congregata increases susceptibility to fatal infection by Autographa californica M Nucleopolyhedrovirus

We investigated pathogenesis of Autographa californica M Nucleopolyhedrovirus in the semipermissive host, Manduca sexta, using a lacZ recombinant virus (AcMNPV-hsp70/lacZ) to track the temporal progression of infection. Results from time course studies monitoring infections initiated orally in fourth instars demonstrated that primary infection of midgut columnar cells began at 3 h post inoculation (hpi). We observed secondary infections in midgut-associated tracheae as early as 9 hpi, showing that the early events of pathogenesis in M. sexta are similar to those of permissive noctuid larvae. In M. sexta, however, unlike in permissive hosts, hemocytes rapidly surrounded infected tracheal cells and formed capsules. Subsequently, baculovirus infections failed to spread and ultimately were cleared, suggesting that a cellular immune response had been triggered. To assess the effects of immunosuppression on baculovirus-induced disease, we compared the outcome of infections in immunocompetent hosts with those that were immunocompromised either by parasitization with the braconid, Cotesia congregata, or by injection of the parasitoid's polydnavirus. During the first 9 days after inoculation, parasitized and polydnavirus-inoculated M. sexta larvae died more quickly and at higher levels than nonparasitized and sham-injected controls, suggesting that the cellular immune response was a factor in conferring resistance to fatal infection by AcMNPV-hsp70/lacZ.     

Isolation of an imaginal disc growth factor homologue from Pieris rapae and its expression following parasitization by Cotesia rubecula

Endoparasitoid insects introduce maternal factors into the body of their host at oviposition to suppress cellular defences for the protection of the developing parasitoid. We have shown that transient expression of polydnavirus genes from a hymenopteran parasitoid Cotesia rubecula (CrPDV) is responsible for the inactivation of hemocytes from the lepidopteran host Pieris rapae. Since the observed downregulation of CrPDV genes in infected host tissues is not due to cis-regulatory elements at the CrV1 gene locus, we speculated that the termination of CrPDV gene expression may be due to cellular inactivation caused by the CrV1-mediated immune suppression of infected tissues. To test this assumption, we isolated an imaginal disc growth factor (IDGF) that is expressed in fat body and hemocytes, the target of viral infection and expression of CrPDV genes. Time-course experiments showed that the level of P. rapae IDGF is not affected by parasitization and polydnavirus infection. However, the amount of highly expressed genes, such as storage proteins, arylphorin and lipophorin, are significantly reduced following parasitization.     

Effects of Parasitism by the Braconid Wasp Cotesia congregata on Metabolic Rate in Host Larvae of the Tobacco Hornworm,Manduca sexta

We examined growth rates, gas exchange patterns and energy metabolism of tobacco hornworm (Manduca sexta) larvae parasitized by the braconid wasp Cotesia congragata. Larvae parasitized at the beginning of the fourth-instar had reduced growth compared to unparasitized larvae of the same age and short-term differences in metabolism (measured as rates of CO(2) production, Vdot; CO(2)) were apparent almost immediately after wasp oviposition. However, over the growth period between parasitization and the last part of the fifth-instar, there was no significant difference between parasitized and unparasitized hosts as seen in the relationship between mass and Vdot; CO(2). One day prior to parasitoid emergence, host larvae stopped eating, ceased spontaneous locomotor activity and showed a dramatic decline in metabolism. The 60% decline of Vdot; CO(2) at this time is consistent with lack of specific dynamic action because the animals were not feeding. Gas exchange became highly cyclical on the day of parasitoid emergence, but the cause and significance of this phenomenon, which disappeared by the third day following emergence, are not clear. This pattern of cycling was not induced by starving nonparasitized larvae for 6days, nor by immobilizing nonparasitized larvae with tetrodotoxin. Ecdysteroid levels in the host's hemolymph significantly increased on the day when parasitoids completed their L2-L3 molt and began emerging, but not during the wasps' L1-L2 molt which occurred a few days earlier. Contrary to our initial expectation that hemolymph ecdysteroid titers might be linked to alterations in the host's metabolic rate, we observed no such correlation.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Evolution of a polydnavirus gene in relation to parasitoid-host species immune resistance

CrV1, a polydisperse DNA virus (polydnavirus or PDV) gene contributes to the suppression of host immunity in Cotesia genus parasitoids. Its molecular evolution was analyzed in relation to levels of resistance in the sympatric host species. Natural selection for nonsynonymous substitutions (positive Darwinian selection) was observed at specific amino acid sites among CrV1 variants; particularly, between parasitoid strains immune suppressive and nonimmune suppressive to the main resistant stem borer host, Busseola fusca. In Cotesia sesamiae, geographic distribution of CrV1 alleles in Kenya was correlated to the relative abundance of B. fusca. These results suggest that PDV genes evolve through natural selection and are genetically linked to factors of suppression of local host resistance. We discuss the forces driving the evolution of CrV1 and its use as a marker to understand parasitoid adaptation to host resistance in biological control.     

Comparing the physiological effects and function of larval feeding in closely‐related endoparasitoids (Braconidae: Microgastrinae)

Abstract The larvae of most endoparasitoid wasps consume virtually all host tissues before pupation. However, in some clades, the parasitoid larvae primarily consume haemolymph and fat body and emerge through the side of the host, which remains alive and active for up to several days. The evolutionary significance of this host‐usage strategy has attracted attention in recent years. Recent empirical studies suggest that the surviving larva guards the parasitoid broods against natural enemies such as predators and hyperparasitoids. Known as the ‘usurpation hypothesis’, the surviving larvae bite, regurgitate fluids from the gut, and thrash the head capsule when disturbed. In the present study, the ‘usurpation hypothesis’ is tested in the association involving Manduca sexta, its parasitoid Cotesia congregata, and a secondary hyperparasitoid Lysibia nana. Percentage parasitoid survival is higher and hyperparasitism lower when cocoons of C. congregata are attached to the dorsum of M. sexta caterpillars. Fat body contents in several associations involving solitary and gregarious parasitoids feeding on haemolymph and fat body are also compared. The amount of fat body retained in parasitized caterpillars varies considerably from one association to another. In M. sexta and Pieris brassicae, considerable amounts of fat body remain after parasitoid emergence whereas, in Cotesia kariyai and Cotesia rufricus, virtually all of the fat body is consumed by the parsasitoid larvae. The length of post‐egression survival of parasitized caterpillars differs considerably in several tested associations. In Pseudeletia separata, most larvae die within a few hours of parasitoid emergence whereas, in M. sexta, parasitized larvae live up to 2 weeks after parasitoid emergence. Larvae in other associations parasitized by gregarious and solitary endoparasitoids live for intermediate periods. The results are discussed in relation to the adaptive significance of different feeding strategies of immature parasitoids and of the costs and benefits of retaining the parasitized caterpillar in close proximity with the parasitoid cocoons.     

Inhibitory effects of parasitism by the gregarious endoparasitoid Cotesia congregata on host testicular development

Cotesia congregata is a gregarious larval endoparasitoid of the tobacco hornworm, Manduca sexta. Parasitized larvae exhibit a variety of physiological and developmental aberrations, the most obvious of which is the induction of developmental arrest characterized by the absence of wandering behavior and suppression of pupation. This arrest appears attributable to continued maintenance of an elevated titer of juvenile hormone and reduced levels of hemolymph juvenile hormone esterase activity. Injection of the wasp's polydnavirus into nonparasitized larvae also causes arrest and the larvae eventually form larval-pupal intermediates instead of normal pupae, indicating the virus may be partially responsible. Aside from causing arrested host development, parasitism also inhibits the normal development and differentiation of testes in male host larvae, so that the testes atrophy instead of growing synchronously with other larval tissues. Here we report that parasitism has pronounced disruptive cytological effects on the developing reproductive organs of male hosts, in addition to causing them to atrophy. Parasitism results in a reduction in testicular volume attributable to a reduction in the number of developing germ cells. Microscopy revealed that the structural integrity of the sheaths surrounding the testicular follicles also is disrupted, so that the tissues appear grossly abnormal compared to those of nonparasitized larvae. Intrahemocoelic injection of purified C. congregata polydnavirus in combination with venom into nonparasitized fourth instar larvae, or topical application of 100 μg of methoprene to fourth instar larvae, also alters sheath integrity and reduces the numbers of developing germ cells, but not to the same degree as the pattern observed in truly parasitized hosts. The occurrence of cell death in the male gonad was documented using the vital dyes acridine orange and ethidium bromide. Arch. Insect Biochem. Physiol. 36:95–114, 1997. © 1997 Wiley-Liss, Inc.     

A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes

Polydnaviruses are associated with certain parasitoid wasps and are introduced into the body cavity of the host caterpillar during oviposition. Some of the viral genes are expressed in host tissues and corresponding proteins are secreted into the hemocoel causing suppression of the host immune system. The Cotesia rubecula polydnavirus gene product, CrV1, effectively inactivates hemocytes by mediating cytoskeleton breakdown. A precondition for the CrV1 function is the incorporation of the extracellular protein by hemocytes. Here, we show that a coiled-coil domain containing a putative leucine zipper is required for CrV1 function, since removal of this domain abolishes binding and uptake of the CrV1 protein by hemocytes.     

Parasitism-induced Effects on Host Growth and Metabolic Efficiency in Tobacco Hornworm Larvae Parasitized by Cotesia congregata

Parasitism by the braconid wasp Cotesia congregata affects the growth of Manduca sexta larvae in a parasitoid 'dose-dependent' fashion. Following parasitization of fourth-instar larvae, more heavily parasitized larvae grew larger compared to those containing fewer parasitoids due to an increase in host dry weight. The differences in host mass appeared to arise after oviposition. A 'dose-dependent' enhancement of host dry weight would appear nutritionally beneficial for the parasitoids developing in more 'crowded' hosts. The efficiencies of conversion of ingested and digested food to body mass and the approximate digestibility of the diet ingested by the host caterpillar did not vary significantly with clutch size although parasitoids took slightly longer to develop in the more heavily parasitized hosts. Larval parasitoids developing in the presence of many competitors weighed up to 50% less than those developing in hosts with fewer endoparasitoids, although the weight of adult female parasitoids did not vary significantly with wasp clutch size. The maximum number of emerging wasps was 200 parasitoids, possibly representing the host's 'carrying capacity' for larvae parasitized in the fourth-instar. The ratio of emerging to non-emerging parasitoids decreased as parasitoid clutch size increased, with few or none emerging from very heavily parasitized hosts containing more than 400 parasitoids. Copyright 1997 Elsevier Science Ltd. All right reserved     

Changes in hemocytes of Plutella xylostella after parasitism by Diadegma semiclausum

We examined the changes of hemocytes in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), due to parasitism by the endoparasitoid Diadegma semiclausum (Hymenoptera: Ichneumonidae). Necrosis of prohemocytes in different stages was observed while cell death was absent in the mature hemocytes in the parasitized larvae, which was related to the declined total hemocyte count per microliter (THC). THC in the host hemolymph declined sharply by 12 h post-parasitization and then remained at a low level. When hemocytes of the parasitized larvae were cultured in vitro, encapsulation ability was suppressed coincidently with the inhibited spreading ability; however, such effects were transient. Simultaneously, activation of the prophenoloxidae from the hemocytes was inhibited. Unlike the results of previous studies, the decrease in hemocytes, which was due to the necrosis of the prohemocytes instead of the mature hemocytes in our study, was not responsible for the impaired encapsulation. Our studies suggest that parasitism by D. semiclausum have some effects on hematopoietic regulation and on hemocyte immune reaction of P. xylostella larvae.     

Immunoevasive property of a polydnaviral product, CpBV-lectin, protects the parasitoid egg from hemocytic encapsulation of Plutella xylostella (Lepidoptera: Yponomeutidae)

Immunosuppression is the main pathological symptom of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae), parasitized by an endoparasitoid wasp, Cotesia plutellae (vestalis, Hymenoptera: Braconidae). C. plutellae bracovirus (CpBV), which is a symbiotic virus of C. plutellae, has been known to be the main parasitic factor in the host-parasitoid interaction. CpBV-lectin, encoded in the viral genome and expressed in P. xylostella during early parasitization stage, was suspected to play a role in immunoevasion of defense response. Here we expressed CpBV-lectin in Sf9 cells using a recombinant baculovirus for subsequent functional assays. The recombinant CpBV-lectin exhibited hemagglutination against vertebrate erythrocytes. Its hemagglutinating activity increased with calcium, but inhibited by adding EDTA, indicating its C-type lectin property. CpBV-lectin showed specific carbohydrate-binding affinity against N-acetyl glucosamine and N-acetyl neuraminic acid. The role of this CpBV-lectin in immunosuppression was analyzed by exposing hemocytes of nonparasitized P. xylostella to rat erythrocytes or FITC-labeled bacteria pretreated with recombinant CpBV-lectin, which resulted in significant reduction in adhesion or phagocytosis, respectively. The immunosuppressive activity of CpBV-lectin was further analyzed under in vitro encapsulation response of hemocytes against parasitoid eggs collected at 1- or 24-h post-parasitization. Hemocytic encapsulation was observed against 1-h eggs but not against 24-h eggs. When the 1-h eggs were pretreated with the recombinant CpBV-lectin, encapsulation response was completely inhibited, where CpBV-lectin bound to the parasitoid eggs, but not to hemocytes. These results suggest that CpBV-lectin interferes with hemocyte recognition by masking hemocyte-binding sites on the parasitoid eggs.