Parasitism-induced hemolymph polypeptides in Manduca sexta (L.) larvae parasitized by the braconid wasp Cotesia congregata (Say)

Parasitization of newly ecdysed third, fourth, or fifth instar Manduca sexta larvae by the gregarious braconid wasp Cotesia congregata induces synthesis of new hemolymph proteins in the host. Analysis of hemolymph from parasitized and unparasitized control larvae using SDS gel electrophoresis showed that a major 33 kd band, plus several minor bands, were synthesized in parasitized but not control larvae; autoradiograms of proteins labeled in vivo for 1 hr with [³⁵S]methionine indicated that synthesis of the 33 kd polypeptide began a few hours following oviposition by the wasp. Synthesis of the 33 kd parasitism-specific polypeptide was induced in unparasitized larvae by the injection of ovarian calyx fluid from adult female wasps; this fluid is known to contain two morphologically distinct types of virus particles that are normally injected into the host along with eggs during parasitization. Exposure of calyx fluid to psoralen in the presence of long-wave u.v. light destroyed its capacity to induce synthesis of the 33 kd protein, suggesting that synthesis of this polypeptide may be mediated by viral nucleic acid.     

Developmental changes in teratocytes of the braconid wasp Cotesia congregata in larvae of the tobacco hornworm,Manduca sexta

The endoparasitic wasp Cotesia congregata develops in the hemocoel of larval stages of the tobacco hornworm, Manduca sexta. Teratocytes were released from the serosal membrane during hatching of the first instar wasp larva at 2-3days after oviposition; about 160 cells were released per embryo. The cells increased in diameter from about 10 to >200&mgr;m prior to wasp emergence. Nascent microvilli, visible on the cell surface before hatching of the first instar larva, rapidly increased in length and number following release of the cells. Irrespective of when the wasps were due to emerge, or how many parasitoids were present in the host, dramatic cytological changes occurred in the cells during the last instar of the host's development. Many of these morphological and ultrastructural changes were symptomatic of the cytological features of degenerating or apoptotic cells, and large numbers of vesicles appeared interspersed amongst the microvilli. The nucleus developed extensive dentritic ramifications, and the chromatin condensed in large clumps on the inner nuclear membrane. At the final stages of the wasps' development, the nucleus occupied the bulk of the interior of the cell. The cytoplasm gradually grew dramatically more electronluscent and less granular, as did the nucleoplasm, which is also indicative of impending cell death. Following the parasites' emergence, many of the cells underwent extensive blebbing of the cell surface. Teratocytes within a host appeared heterogeneous with respect to their morphological appearance. Analysis of the proteins secreted by teratocytes in vitro following labelling with (35)S-methionine showed that many (>30) polypeptides were synthesized de novo and secreted by the cells; some proteins were clearly targeted for secretion. We presume that the cells likely secrete a large number of proteins in vivo as well as in vitro.     

Tyrosine and catecholamine levels in the hemolymph of tobacco hornworm larvae,Manduca sexta,parasitized by the braconid wasp,Cotesia congregata,and in the developing parasitoids

Parasitism of fifth instar Manduca sexta larvae by the gregarious parasitoid Cotesia congregata prevented normal storage of tyrosine in the hemolymph, whereas total tyrosine levels increased over eight times in the hemolymph of unparasitized larvae by day 4. Tyrosine glucoside, the hemolymph storage form of tyrosine and the precursor for pupal cuticle sclerotizing agents, was found only in trace amounts in parasitized larvae at the time of parasitoid emergence, but had increased to over 6 mM in hemolymph of unparasitized larvae. Concentrations of dopamine and N-β-alanyldopamine (NBAD), precursors for melanization and sclerotization of cuticle, respectively, had approximately doubled in the hemolymph of parasitized larvae by the day of parasitoid emergence, but not in unparasitized larvae. Catecholamine biosynthesis may be transiently stimulated for wound-healing, as black melanic pigmentation appeared around the wasp emergence holes in the host integument. C. congregata larvae accumulate tyrosine, dopamine, and NBAD by the time of emergence and cocoon spinning, either by direct uptake or by synthesis from precursors obtained from the host. NBAD increased in parasitoid larvae close to pupation, suggesting it functions as the main precursor for pupal cuticle tanning. Both dopamine and NBAD increased dramatically in pharate adult wasps just before eclosion and N-acetyldopamine (NADA) appeared for the first time. Dopamine was highest in concentration and total amount, and it can serve both as a precursor for black melanic pigmentation of adult wasp cuticle and for synthesis of NADA and NBAD, the precursors for cuticle sclerotization. Arch. Insect Biochem. Physiol. 38:193–201, 1998. © 1998 Wiley-Liss, Inc.     

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host, the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes from newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host.     

Manipulation of fifth-instar host (Manduca sexta) ecdysteroid levels by the parasitoid wasp Cotesia congregata

Although 5th (last) instar parasitized Manduca sexta larvae undergo developmental arrest and do not wander, they exhibit a small hemolymph ecdysteroid peak (300-400pg/&mgr;l) which begins one day prior to the parasitoid's molt to the 3rd (last) instar and concomitant emergence from the host. Ecdysteroids present in this peak were 20-hydroxyecdysone, 20,26-dihydroxyecdysone and one or more very polar ecdysteroids, as well as small amounts of 26-hydroxyecdysone and ecdysone. In parasitized day-1 and -2 5th instars ligated just behind the 1st abdominal proleg, hemolymph ecdysteroid levels increased in both anterior and posterior portions (100-500pg/&mgr;l), while in unparasitized larvae, hormone levels only increased in the anterior portion (100-350pg/&mgr;l). Thus, the ecdysteroid peak observed in host 5th instars was probably produced, at least in part, by the parasitoids. It may serve to promote Cotesia congregata's molt from the second to the third instar and/or to facilitate parasitoid emergence from the host. In parasitized day-1 and -2 5th instars ligated between the last thoracic and 1st abdominal segments, hemolymph ecdysteroid titers reached much higher levels (500-3500pg/&mgr;l) in the anterior portion (no parasitoids present) than in the posterior portion (150-450pg/&mgr;l). Therefore, it appears that the parasitoid's regulation of hemolymph ecdysteroid titers occurs at two levels. First, parasitization neutralizes the host's ability to maintain its normal hemolymph ecdysteroid levels. Second, in a separate action, the parasitoid manipulates the ecdysteroid-producing machinery so that hemolymph levels are maintained at the 200-400pg/&mgr;l characteristic of day 3-4 hosts. This is the first report of a parasitoid's ability to interfere with the normal inhibitory mechanisms which prevent prothoracic gland production of ecdysteroid at inappropriate periods of insect growth and development.     

Alteration of hemolymph polypeptides in Manduca sexta larvae parasitized by Cotesia congregata: A two-dimensional electrophoretic analysis and comparison with major bacteria-induced proteins

Analyses using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) previously demonstrated that parasitization by the braconid wasp Cotesia congregata significantly alters the normal hemolymph polypeptide profile of host Manduca sexta larvae. In the present study two-dimensional gel analyses corroborated our earlier findings and provided additional evidence that multiple parasitism-specific polypeptides were induced, which varied according to the stage of development of the wasps. Parasitization additionally elicited changes in the total protein concentration detected in the blood. Initially an elevation was observed, with newly parasitized larvae exhibiting a twofold elevation in hemolymph protein concentration by 12–24 h postoviposition. In contrast, terminal-stage hosts with second instar parasites had significantly less protein in the hemolymph, likely due to reduced growth and inhibition of arylphorin synthesis by the fat body during the final stages of parasitism. Comparison of the array of hemolymph polypeptides produced in unparasitized larvae injected with 10⁶cells of the gram-negative bacterium Enterobacter cloacae with those proteins induced by parasitization indicated the two classes are different. Our findings confirm that the hostresponse to parasitism is a specific one, and not mimicked by bacterial challenge. Duringshort-term in vitro culture of wasp larvae dissected from the host hemocoel, several proteins were detected in the medium using SDS - PAGE, with their appearance in vitro suggestive of secretion by the wasps in vivo. Moreover, hemolymph from the parasites had significant amounts of putative host proteins, including an arylphorin - like polypeptide and a protein with a mobility similar to that of insecticyanin. Thus, a dynamic interchange of proteins may occur, with the parasites accumulating host proteins while simultaneously secreting a variety of factors into the host hemocoel.     

Host hemolymph monophenoloxidase activity in parasitized Manduca sexta larvae and evidence for inhibition by wasp polydnavirus

In insects, one of the primary routes of defense against parasites is encapsulation by hemocytes followed by melanization, in which tyrosine and DOPA are converted to melanin via toxic quinone intermediates. This report describes the use of an in vitro radiochemical assay to monitor hemolymph monophenoloxidase (MPO) conversion of [³H]tyrosine to [³H]DOPA, using a method utilized previously for dipteran and mammalian enzymes. Parasitism of fifth instar tobacco hornworm larvae by the braconid wasp Cotesia congregata depresses the rate of hemolymph monophenoloxidase activity in the host. Significant inhibition of hemolymph MPO was detectable in newly parasitized larvae and terminal stage hosts. A similar effect was seen in unparasitized larvae following injection of sucrose-gradient purified wasp polydnavirus (PDV) particles, which are normally injected by the female wasps into the host, suggesting the inhibition may be virally mediated. Hemolymph MPO activity was assayed in vitro 24 h after injection of PDV in vivo, and intercalation of viral DNA by exposure to psoralen and long-wave u.v. light eliminated its inhibitory effect on MPO. Despite inhibition of hemolymph MPO activity by parasitism, host cuticular enzymes appear unimpaired, since cuticular melanization occurs at sites of integumental wounding during emergence of the wasps from the host. Red pigments appear in the dorsal vessel and integument of parasitized and virus-injected larvae; whether these pigmentation changes are related to effects of parasitism on tyrosine metabolism and ommochrome biosynthesis remains to be determined.     

Retardation of testis development in the armyworm,Pseudaletia separata,parasitized by the braconid wasp,Cotesia kariyai

Summary

In order to complete growth and development, the endoparasitoid wasp, Cotesia (=Apanteles) kariyai, inhibits pupation of its armyworm host, Pseudaletia (=Leucania) separata. In host larvae retardation of testis and spermatocyst development caused by the parasitoid was also observed. The agents causing the retardation were found in the ovaries and venom of the female adult parasitoid. When an unparasitized male host larva was artificially injected with calyx fluid obtained from ovaries together with venom, it showed the same degree of developmental retardation of testes and spermatocysts as in natural parasitization. Testes implanted in isolated abdomens of healthy larvae did not increase in size by ecdysteroid stimulation after exposure to calyx fluid plus venom. It is suggested that both symbiotic polydnavirus existing in calyx fluid and venom in the parasitoid, C. kariyai, are responsible for the parasitic retardation of the male reproductive organs in the host, P. separata.     

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.     

Effects of parasitization by Cotesia congregata on the brain-prothoracic gland axis of its host,Manduca sexta

The ability of prothoracic glands (PTGs) from parasitized and unparasitized Manduca sexta 5th-instars to respond to ecdysiotropic extracts prepared from day-5 5th instar brains was compared. An in vitro bioassay revealed that PTGs from parasitized animals were much less responsive to brain PTTH than glands from unparasitized larvae. However, when incubated in Grace's medium in the absence of brain extract, glands from day-3 and -4 hosts remained active for a much longer period of time than did those dissected from their unparasitized counterparts. Rather than exhibiting reduced (basal) levels of synthesis after the 3rd hour of incubation, glands from these parasitized larvae continued to synthesize/release ecdysteroid into the medium at relatively high rates. The timing of this enhanced secretory activity is coincident with the ecdysteroid peak that occurs just prior to and during wasp emergence. Following parasite emergence, gland activity decreased, and by the third day after emergence, was reduced to low levels. Results suggest that the requirement for PTTH to stimulate ecdysteroid production has been bypassed, i.e. that the parasite has uncoupled the normal mechanisms that permit brain regulation of PTG activity. The ability of brains from parasitized M. sexta to stimulate PTGs from unparasitized day-2 5th instars was also examined. Dose-response analyses performed for the first 7 days of the 5th instar showed that on a per brain basis ecdysiotropic activity in brains from parasitized and unparasitized animals was similar. However, when differences in brain size were considered, ecdysiotropic activity appeared to be more concentrated in brains from day-7 parasitized larvae than in brains from similarly aged unparasitized larvae. Analysis of the size distribution of the ecdysiotropic activity in brains from parasitized larvae revealed a unique form that was larger than the 29kDa standard. This suggests that parasitization may inhibit neuropeptide processing, particularly during the final stages preceding emergence of the wasps from the host. Thus, both an inhibition of prothoracicotropic hormone processing and the inability to respond to this neurohormone may contribute to the developmental arrest characteristic of parasitized 5th instars.     

Rearing temperature and parasitoid load determine host and parasitoid performance in Manduca sexta and Cotesia congregata

1. Temperature strongly influences the rates of physiological processes in insects, including the herbivore Manduca sexta and its larval endoparasitoid Cotesia congregata. Parasitisation by C. congregata decreases the growth and consumption of food by larval M. sexta. However, the effects of temperature on parasitised caterpillars and the developing wasp larvae are largely unknown. 2. In this study, parasitised and unparasitised caterpillars were reared at three constant temperatures (20, 25 and 30 °C) throughout larval development. Caterpillar mass gain and consumption were monitored daily until wandering (unparasitised control group) or wasp emergence (parasitised group) was observed. Development time and survival to emergence were measured as metrics of parasitoid performance. 3. Parasitised M. sexta developed more slowly than unparasitised controls, but had similar cumulative consumption until the terminal instar. Parasitised caterpillars with relatively large parasitoid loads had higher rates of consumption and growth than those with smaller loads. Both temperature and parasitoid load strongly affected wasp success. Mean development time to wasp emergence increased with low temperatures and with large loads. The combination of warm temperature and large parasitoid loads greatly reduced wasp survival. 4. These results demonstrate the interactive effects of rearing temperature and parasitisation on host consumption and growth rates throughout larval development. In addition, wasp performance was affected by the interaction of temperature and parasitoid load size. High temperatures alter the dynamics of the interaction between the parasitoid and its caterpillar host, which could have far-reaching impacts as the global temperatures continue to rise.     

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.     

Immunoanalysis of unique protein in Trichoplusia ni larvae parasitized by the braconid wasp Chelonus near curvimaculatus

Parasitization in insects brings about profound biochemical and physiological effects in the host which may include complete overriding of the normal endocrinological program, resulting in precocious metamorphosis and in blockage of pupal development. The subtle effects of parasitization include changes in the expression of hemolymph proteins and the appearance of proteins which are unique to parasitized hosts. One such protein has been identified in the hemolymph of Trichoplusia ni larvae parasitized by the braconid wasp Chelonus near curvimaculatus. In this study, purified preparations of the parasitism-specific protein were used to generate polyconal antibodies against the protein. Results from the immunocharacterization indicate the antibodies obtained are highly specific for the protein and are present in a high titer (1:8000 antiserum dilution yielded strong signals in analysis of the protein in 0.25 μl hemolymph). Subsequently, the expression of the parasitism-specific protein in the hemolymph and tissues was analyzed by immunoblotting during the entire course of development in normal and parasitized insects. The parasitism-specific protein was not detected in normal, unparasitized larvae. In parasitized insects, expression of the parasitism-specific protein appears to be stage-specific in that it is only detected during the last larval stadium of precociously metamorphosing larvae, but is absent from all earlier stages of development.     

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.     

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.     

Disruptive developmental effects of benzyl-1,3-benzodioxole derivatives on unparasitized and parasitized Manduca sexta larvae

Treatment of tobacco hornworm larvae with the benzyl-1,3-benzodioxole derivative J-2710 immediately after ecdysis to the fourth instar disrupted development either during the moult to the fifth instar or shortly thereafter. Larvae given topical applications of 100 μg J-2710 in 1 μl acetone suffered 100% mortality, often after secreting moulting fluid in large pockets between the epidermis and the cuticle later in the fourth instar. Larvae that successfully ecdysed had abnormalities of the mouthparts and cervix that interfered with normal feeding, inhibiting growth in the fifth instar. Larvae of the gregarious endoparasitic wasp Cotesia congregata (=Apanteles congregatus) frequently failed to emerge from host Manduca sexta larvae treated with high doses of J-2710, particularly when the host failed to feed normally. Less potent disruptive effects on Manduca and Cotesia were seen after treatment of larvae with the derivatives J-3370 and J-2581.No anti-juvenile hormone action of J-2710 was observed. J-2710-treated M. sexta larvae showed no precocious metamorphosis and the developmental effects of J-2710 were not prevented by co-application of the juvenile hormone analogue methoprene in doses ranging from 1 to 100 μg/larva. Moreover, J-2710 had no effect on the action of methoprene in the black larval assay for juvenile hormone-like activity, unlike results reported to occur using the Galleria wax wound assay.