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     

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.     

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.     

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.     

Polyamine Regulation of Protein Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta

An analysis of the effects of polyamines on protein phosphorylation in cytosolic fractions of the pupal brain of Manduca sexta showed that spermine elicited an increase in casein phosphorylation in a dose-dependent manner (maximum three- to fourfold at 2.0 mM), whereas spermidine was less effective and putrescine was without effect. In contrast, with phosvitin as the exogenous substrate, higher doses of polyamines, especially spermine, inhibited phosphorylation. High salt conditions abolished the polyamine response. Cytosol protein kinase activity eluted from DEAE-cellulose at 0.2-0.3 M NaCl. This activity was enhanced in the presence of spermine, and inhibited in the presence of heparin (IC50 approximately equal to 30 ng/ml). The enzyme was characterized by a sedimentation coefficient of 6.5S, and a Stokes radius of 49 A, consistent with a Mr of 130,000. Both GTP (Km, 55 microM) and ATP (Km, 34 microM) were utilized as phosphoryl donors (Vmax for ATP being four-fold higher than that observed for GTP). These results indicate the presence in the insect brain of an enzyme very similar to vertebrate casein kinase II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated that low concentrations of spermine (100 microM) strongly enhanced the phosphorylation of three high-molecular-weight cytosolic proteins (305,000, 340,000, and 360,000) localized in the insect nervous system.     

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.     

Defensive Sound Production in the Tobacco Hornworm, Manduca sexta (Bombycoidea: Sphingidae)

The tobacco hornworm (Manduca sexta) is a model organism extensively studied for many aspects of its biology, including its anti-predator strategies. We report on a novel component of this caterpillar’s defence repertoire: sound production. Late instar caterpillars produce discrete clicking sounds in response to disturbance. Click trains range in duration from 0.3–20.0 s (mean 3.3 ± 4.8 s) and contain 2–41 clicks (mean 7.1 ± 9.5). Sounds are broadband with a dominant frequency of 29.8 ± 4.9 kHz. We investigated the mechanism of sound production by selectively ablating three identified sets of ridges on the mandibles, and determined that ridges on the inner face strike the outer and incisor ridges on the opposing mandible to produce multi-component clicks. We tested the hypothesis that clicks function in defence using simulated attacks with blunt forceps. In single attack trials 77% of larvae produced sound and this increased to 100% in sequential attacks. Clicks preceded or accompanied regurgitation in 93% of multiple attack trials, indicating that sound production may function in acoustic aposematism. Sound production is also accompanied by other behaviours including directed thrashing, head curling, and biting, suggesting that sounds may also function as a general warning of unprofitability.     

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.     

Isolation of Plasmid-Harboring Serratia plymuthica from Facultative Gut Microflora of the Tobacco Hornworm, Manduca sexta

Aseptic isolation of the facultative gut microflora of the tobacco hornworm, Manduca sexta, yielded four microorganisms. Two were gram-positive Bacillus spp., one was Serratia plymuthica, and another was the yeast Candida guilliermondii. The three bacterial species were screened for extrachromosomal DNA, and S. plymuthica was found to have a 6.4-kilobase plasmid, which was designated pCP-1.     

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.     

Polyamines Differentially Inhibit Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta

The effects of the naturally occurring polyamines spermine and spermidine on phosphorylation promoted by cyclic AMP (cAMP)-dependent protein kinase (PK) (cAMP-PK; EC 2.7.1.37) were studied using the brain of the tobacco hornworm, Manduca sexta. Four particulate-associated peptides (280, 34, 21, and 19 kilodaltons) in day 1 pupal brains are endogenous substrates for a particulate type II cAMP-PK. These phosphoproteins are present in brain synaptosomal, as well as microsomal, particulate fractions but are not present in the cytosol. They are distributed throughout the CNS and PNS and are present in several nonneuronal tissues as well. Phosphorylation of these proteins via cAMP-PK was inhibited markedly by micromolar concentrations of spermine and spermidine. Other particulate-associated peptides phosphorylated via a Ca2+/calmodulin-PK or Ca2+ and cAMP-independent PKs were unaffected by polyamines, whereas the phosphorylation of a 260-kilodalton peptide was markedly enhanced. Spermine did not exert its inhibitory effect indirectly by enhancement of cAMP or ATP hydrolysis or via proteolysis, but its action appears to involve a substrate-directed inhibition of cAMP-PK-promoted phosphorylation as well as enhanced dephosphorylation. Although addition of spermine resulted in marked ribosome aggregation in synaptosomal and microsomal particulate fractions, this phenomenon was not involved in the inhibition of cAMP-PK-promoted phosphorylation.     

Supply-Side Constraints Are Insufficient to Explain the Ontogenetic Scaling of Metabolic Rate in the Tobacco Hornworm,Manduca sexta

Explanations for the hypoallometric scaling of metabolic rate through ontogeny generally fall into two categories: supply-side constraints on delivery of oxygen, or decreased mass-specific intrinsic demand for oxygen. In many animals, supply and demand increase together as the body grows, thus making it impossible to tease apart the relative contributions of changing supply and demand to the observed scaling of metabolic rate. In larval insects, the large components of the tracheal system are set in size at each molt, but then remain constant in size until the next molt. Larvae of Manduca sexta increase up to ten-fold in mass between molts, leading to increased oxygen need without a concomitant increase in supply. At the molt, the tracheal system is shed and replaced with a new, larger one. Due to this discontinuous growth of the tracheal system, insect larvae present an ideal system in which to examine the relative contributions of supply and demand of oxygen to the ontogenetic scaling of metabolic rate. We observed that the metabolic rate at the beginning of successive instars scales hypoallometrically. This decrease in specific intrinsic demand could be due to a decrease in the proportion of highly metabolically active tissues (the midgut) or to a decrease in mitochondrial activity in individual cells. We found that decreased intrinsic demand, mediated by a decrease in the proportion of highly metabolically active tissues in the fifth instar, along with a decrease in the specific mitochondrial activity, contribute to the hypoallometric scaling of metabolic rate.     

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.     

Gut Bacteria Are Not Required for the Insecticidal Activity of Bacillus thuringiensis toward the Tobacco Hornworm,Manduca sexta

It was recently proposed that gut bacteria are required for the insecticidal activity of the Bacillus thuringiensis-based insecticide, DiPel, toward the lepidopterans Manduca sexta, Pieris rapae, Vanessa cardui, and Lymantria dispar. Using a similar methodology, it was found that gut bacteria were not required for the toxicity of DiPel or Cry1Ac or for the synergism of an otherwise sublethal concentration of Cry1Ac toward M. sexta. The toxicities of DiPel and of B. thuringiensis HD73 Cry⁻ spore/Cry1Ac synergism were attenuated by continuously exposing larvae to antibiotics before bioassays. Attenuation could be eliminated by exposing larvae to antibiotics only during the first instar without altering larval sterility. Prior antibiotic exposure did not attenuate Cry1Ac toxicity. The presence of enterococci in larval guts slowed mortality resulting from DiPel exposure and halved Cry1Ac toxicity but had little effect on B. thuringiensis HD73 Cry⁻ spore/Cry1Ac synergism. B. thuringiensis Cry⁻ cells killed larvae after intrahemocoelic inoculation of M. sexta, Galleria mellonella, and Spodoptera litura and grew rapidly in plasma from M. sexta, S. litura, and Tenebrio molitor. These findings suggest that gut bacteria are not required for B. thuringiensis insecticidal activity toward M. sexta but that B. thuringiensis lethality is reduced in larvae that are continuously exposed to antibiotics before bioassay.Bacillus thuringiensis has long been regarded as a bona fide entomopathogen that can produce an array of virulence factors including insecticidal parasporal crystal (Cry) toxins, vegetative insecticidal proteins, phospholipases, immune inhibitors, and antibiotics (31). B. thuringiensis establishes lethal infections in many insect species after intrahemocoelic inoculation (9, 10, 14, 26, 31), and the insecticidal activity of Cry toxins, which lyse the intestinal epithelium, can be synergized by the presence of viable B. thuringiensis spores (31). In each instance, synergism has been attributed to hemocoelic infection by B. thuringiensis.A novel hypothesis (6, 7) proposed that B. thuringiensis is incapable of killing Lymantria dispar, Manduca sexta, Pieris rapae, or Vanessa cardui in the absence of gut bacteria. Prior exposure of L. dispar larvae to a combination of four antibiotics severely reduced the subsequent toxicity of the B. thuringiensis-based (spores and Cry toxins) bioinsecticide, DiPel (Valent BioSciences) (6). Both larval susceptibility to B. thuringiensis and the number of culturable gut bacteria were found to be negatively correlated with the concentration of antibiotics to which larvae were previously exposed. Furthermore, a total reduction in larval susceptibility was coincident with the elimination of any detectable gut bacteria. Experimental reinfection with Enterobacter sp. strain NAB3, found in the guts of some populations of L. dispar larvae, was found to rescue the toxicity of B. thuringiensis, whereas reinfection with Enterococcus casseliflavus and Staphylococcus xylosus did not. It was also shown that while Escherichia coli, Enterobacter sp. strain NAB3, and B. thuringiensis could all grow in tryptic soy broth, B. thuringiensis alone could not grow in filter-sterilized plasma from L. dispar larvae. Finally, it was shown that the toxicity of Cry1Aa-expressing E. coli JM103 to L. dispar larvae was reduced by the prior exposure of larvae to antibiotics and could be eliminated when E. coli was also heat killed before use. It was concluded that B. thuringiensis-induced mortality results from a mixed infection of the hemocoel that must include bacteria capable of growth within the L. dispar larval hemolymph (6).Using the same methods, it was subsequently reported that prior exposure of Vanessa cardui, M. sexta, Pieris rapae, and Heliothis virescens larvae to antibiotics eliminated culturable bacteria and rendered larvae resistant to DiPel (7). Experimental reinfection of larvae with Enterobacter sp. strain NAB3 rescued DiPel toxicity in V. cardui, M. sexta, and P. rapae but not in H. virescens larvae. Using a continuous-exposure bioassay, the susceptibility of Pectinophora gossypiella to the Cry1Ac-based bioinsecticide MVPII was found to be increased by prior exposure to antibiotics. Toxicity from a 48-h exposure of L. dispar larvae to MVPII was reduced, but not eliminated, by prior antibiotic exposure and could be rescued by reinfection with Enterobacter sp. strain NAB3. It was concluded that “enteric bacteria have important roles in B. thuringiensis-induced killing of Lepidoptera across a range of taxonomy, feeding breadth, and relative susceptibility to B. thuringiensis” (7).The present work shows that gut bacteria are not required for the insecticidal activity of B. thuringiensis or Cry1Ac toxin toward M. sexta but that prior antibiotic exposure reduces larval susceptibility to B. thuringiensis.     

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.     

Parasitism enhances the induction of glucogenesis by the insect, Manduca sexta L

Metabolic alterations that accompany parasitism of invertebrate animals can play an important role in parasite development. Employing 13C NMR, this study examined pyruvate cycling from (2-(13)C)pyruvate in the lepidopteran insect Manduca sexta, and the effects of parasitism by the hymenopteran Cotesia congregata on the gluconeogenic formation of trehalose, the haemolymph or blood sugar of insects. Larvae were maintained on a semi-synthetic sucrose-free diet, or on the same diet with sucrose at 8.5 g/l. Pyruvate cycling was evident from the 13C enrichment in C3 of alanine, derived following carboxylation to oxaloacetate, and was similar in parasitized and normal insects regardless of diet. Trehalose was formed following de novo synthesis of glucose, and net synthesis was estimated from the 13C distribution in trehalose and alanine. The 13C-enrichment ratio [2trehalose C6/alanine C3] is an indicator of the level of gluconeogenesis relative to glycolysis, both enrichments were derived from (2-(13)C)pyruvate in the same manner. The ratio was greater than unity in all insects, regardless of diet, but was significantly greater in parasitized larvae, demonstrating an enhanced level of gluconeogenesis. This was confirmed by analysis of the 13C distribution in trehalose and glutamine derived from (3-(13)C)alanine. Despite enhanced de novo trehalose formation in parasitized insects, the haemolymph sugar level was similar to that of normal larvae. Because haemolymph trehalose regulates dietary carbohydrate intake, but not gluconeogenesis, the results suggest that accelerated induction of gluconeogenesis is an adaptive response to parasitism that provides increased carbohydrate for parasite growth and simultaneously maintains nutrient intake.     

Pharmacological Study of Intracellular Second Messengers that Affect Active Ion Transport in the Midgut of Tobacco Hornworm, Manduca sexta

ABSTRACT Midgut active ion transport changes during the final larval stage of the tobacco hornworm. The short-circuit current ( I _sc) of the posterior midguts dissected from feeding fifth instars (day 2) is higher than that of midguts from wandering larvae (day 5). The I _sc of midguts from day 2 larvae is inhibited by 1 mM cAMP and 0.1 mM cGMP, whereas the midguts from day 5 larvae are stimulated by cAMP but unaffected by cGMP. A similar pattern is observed if the midguts are exposed to the cyclic nucleotide derivative, 8-bromo-cGMP. Exposure to the calcium ionophore, A23187, or the endoplasmic calcium ATPase inhibitor, thapsigargin, slightly inhibited the I _sc of day 2 larval midguts, but this inhibition was not significant. Pharmacological agents known to modulate the activities of protein kinase A, protein kinase C, or protein phosphatases did not change the I _sc. These results indicate that midgut active ion transport is modulated by cyclic nucleotides, but the manner of the response depends on the developmental status of the insect.     

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.