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.     

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.     

Developmental attenuation of the pre-ecdysis motor pattern in the tobacco hornworm,Manduca sexta

Summary At the culmination of each molt, the larval tobacco hornworm exhibits a pre-ecdysis behavior prior to shedding its old cuticle at ecdysis. Both pre-ecdysis and ecdysis behaviors are triggered by the peptide, eclosion hormone (EH). Pre-ecdysis behavior consists of rhythmic abdominal compressions that loosen the old larval cuticle. This behavior is robust at larval molts, but at the larval-pupal molt the only comparable behavior consists of rhythmic dorso-ventral flexions of the anterior body. These flexions appear to be an attenuated version of the larval pre-ecdysis behavior because (1) they show the same EH dependence, and (2) the motor patterns recorded from EH treated, deafferented larval and pupal preparations are similar except that the pupal pattern is much weaker. Both patterns are characterized by rhythmic, synaptically-driven bursts of action potentials in motoneurons MN-2 and MN-3, which occur synchronously in all segments. However, the synaptic drive to the motoneurons and their resultant levels of activity are reduced during the pupal pre-ecdysis motor pattern, especially in posterior abdominal segments. Although the dendritic arbors of both motoneurons regress somewhat during the larval-pupal transformation, this does not appear to be the primary source of diminished synaptic drive because regression is greatest in the segments in which synaptic inputs remain the strongest. The developmental weakening of the pre-ecdysis motor pattern thus may be due to changes at the interneuronal level.Abbreviations A2, A3... abdominal segments 2, 3, etc. - ALE anterior lateral external muscle - day L3 third day of the 5th larval instar - day P0 the day of pupal ecdysis - DN _a anterior branch of the dorsal nerve - EH eclosion hormone - HPLC high performance liquid chromatography - TP tergopleural muscle     

Studies on vitellogenin from the tobacco hornworm,Manduca sexta

In the tobacco hornworm moth, Manduca sexta, vitellogenin (Vg) is a very high-density (1.29 g/ml) phosphoglycolipoprotein containing 13% lipids, 3% carbohydrates, and 0.6% protein-bound phosphorus. Vitellogenin (M_r～500,000) has two apoproteins designated apoVg-l (M_r 177,000 ± 3,600) and apoVg-ll (M_r45,000 ± 5,000). ApoVg-l and apoVg-II can be dissociated with 6 M guanidine HCI and separated from each other by gel permeation chromatography. Immunoblotting experiments using antibodies against the apoproteins showed that apoVg-l and apoVg-II antigens were immunologically distinct polypeptides. Antibodies against Vg reacted only with apoVg-l. Antibodies against Vg and apoVg-l reacted with Vg in double immunodiffusion experiments, whereas antibodies against apoVg-II did not. These results suggest that in the native Vg molecule, apoVg-II is positioned inside the molecule away from the aqueous environment. Only apoVg-I contained covalently bound carbohydrate as shown by fluorescein isothiocyanateconjugated concanavalin A, periodate-Schiff reagent, and in vivo labeling with ³H-Man. In vivo labeling with ³²P-inorganic phosphate and chemical determination showed that apoproteins of both Vg and vitellin contain covalently bound phosphate groups.     

Developmental expression of three genes for larval cuticular proteins of the tobacco hornworm, Manduca sexta

Three cDNA clones coding for the 12.8, 13.3, and 14.6 kDa larval cuticular proteins of the tobacco hornworm, Manduca sexta, were isolated and characterized. Hybridization to abdominal epidermal RNA from different stages showed that the genes for the 12.8 and 13.3 kDa proteins were expressed only during larval life. By contrast, the gene for the 14.6 kDa protein was expressed throughout the segment during the feeding, growing larval stages, then only in the flexible intersegmental regions during the deposition of endocuticle in the pharate pupa and adult. Quantitative RNA dot blot hybridizations showed that the RNA for each protein disappeared during the larval molt when the ecdysteroid titer was high, then reappeared during the preecdysial deposition of endocuticle. All disappeared when the epidermis became pupally committed at the onset of wandering. Exposure of the fourth instar epidermis to 20-hydroxyecdysone (20HE) in vitro under conditions that lead to the formation of a new larval cuticle by 48 hr caused the disappearance of these RNAs by 18 hr. Exposure of Day 2 fifth instar epidermis to 20HE in vitro caused a depression of these RNAs which in the case of the RNAs coding for the 12.8 and 13.3 kDa proteins was partially prevented by simultaneous exposure to methoprene, a juvenile hormone (JH) mimic. By contrast, the RNA for the 14.6 kDa protein was suppressed by exposure to methoprene alone. Thus, each of these larval cuticular genes is turned off by high ecdysteroid; the presence or absence of JH determines whether or not this suppression is permanent in some or all cells.     

Microstructure of feeding by tobacco hornworm caterpillars, Manduca sexta

The microstructure of the feeding activity of tobacco hornworm caterpillars (Manduca sexta Johansson) on tomato leaf was examined by means of an automated cafeteria. In this device each activity of the caterpillar generates a characteristic slow electrical change which can be recorded. The apparatus is therefore both accurate and sensitive. Examination of the activity records indicated that larger animals ate more than smaller ones by increasing both bite frequency and the lengths of meals. Meal frequency did not increase. Correlations amongst a variety of measures indicated that there was regulation of feeding both between and within meals.     

Proteinases in molting fluid of the tobacco hornworm,Manduca sexta

Manduca sexta pharate pupal molting fluid contains more than 10 proteolytic enzymes that differ in relative mobility during electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and gelatin. The major gelatin digesting enzyme was an endoprotease with an apparent molecular weight of 100 kDa. Gel filtration on a Sephacryl S-300 column resolved another endoprotease of similar size that digests azocoll and [³H]casein. In addition we found an aminopeptidase-like enzyme (MW_app 500 kDa) and at least three carboxypeptidase-like enzymes (MW_app 10–60 kDa). Use of pseudosubstrates and inhibitors suggested the presence of both trypsin-like and chymotrypsin-like enzymes with the former activity approx. 10-fold greater than the latter. However, none of the proteolytic enzymes were substantially inhibited by diisopropylphosphorofluoridate or phenylmethylsulfonyl fluoride which are poteint inhibitors of trypsin and chymotrypsin. No carboxyl or sulfhydryl proteases were detected. The enzymes were most active in the neutral to alkaline pH range, but they were relatively unstable during storage which precluded their purification to homogeneity. Proteolysis of Manduca cuticular protein appears to involve a rather complex and unique mixture of endo- and exo-cleaving proteolytic enzymes.     

Larval cuticular morphogenesis in the tobacco hornworm, Manduca sexta, and its hormonal regulation

The sequential synthesis and deposition of larval cuticular proteins was followed during the final larval molt and the final larval instar of the tobacco hornworm Manduca sexta and correlated with changes in cuticular structure. On the final day of feeding (Day 3) before the onset of metamorphosis many endocuticular proteins were no longer synthesized and new isoelectric variants of 27,000-Da polypeptides were deposited into the cuticle coincident with the formation of lamellae 5- to 10-fold thinner than those previously deposited. Application of a juvenile hormone analog methoprene on Day 1 prevented this change in protein synthesis and in lamellar structure by preventing the observed rise in the intermolt ecdysteroid titer on Day 2. These changes could be induced in vitro by 25-100 ng/ml 20-hydroxyecdysone in the absence of juvenile hormone. Thus, the intermolt change in the lamellar assembly process appears to result from hormone-induced changes in cuticular protein synthesis.     

Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Manduca sexta

Lipoprotein biosynthesis in larvae of the tobacco hornworm (Manduca sexta) was investigated. By immunoblotting, it was shown that the apoproteins are present in the fat body, but not in the midgut. Fat body incubated in vitro with [35S]methionine secreted labeled apoproteins. However, when the density of the secreted particle was determined, it was found at 1.24-1.28 g/ml instead of 1.15 g/ml, which is the density of the circulating lipoprotein. Lipid analysis of immunoprecipitated lipoprotein secreted by the fat body showed a phospholipid/diacylglycerol ratio of 8.3 rather than 0.9, the ratio found in the circulating lipoprotein. When labeled oleic acid or triolein was fed to larvae, it was found that greater than 98% of the label in the circulating lipoprotein was in diacylglycerol. In studies using animals raised on a fat-free diet, it was shown that the circulating lipoprotein has properties comparable to those of the material secreted in vitro by the fat body and that this diacylglycerol-poor particle can be converted to the normal lipoprotein by feeding a bolus of triolein. These data support the hypothesis that the fat body makes and secretes a "nascent" lipoprotein which contains apoproteins and phospholipid, but is devoid of diacylglycerol. The diacylglycerol is then picked up from the midgut to complete assembly of the mature circulating lipoprotein.     

Phospholipase A2 in hemocytes of the tobacco hornworm,Manduca sexta

On the hypothesis that prostaglandins and other eicosanoids mediate nodulation responses to bacterial infections in insects, we describe an intracellular phospholipase A₂ (PLA₂) in homogenates prepared from hemocytes collected from the tobacco hornworm, Manduca sexta. PLA₂ hydrolyzes fatty acids from the sn-2 position of phospholipids. Some PLA₂s are thought to be the first and rate-limiting step in biosynthesis of prostaglandins and other eicosanoids. The hemocyte PLA₂ activity was sensitive to hemocyte homogenate protein concentration (up to 250 μg protein/reaction), pH (optimal activity at pH 8.0), and the presence of a Ca²⁺ chelator. Like PLA₂s from mammalian sources, the hemocyte PLA₂ was inhibited by the phospholipid analog oleyoxyethyl phosphorylcholine. Whereas most intracellular PLA₂s require Ca²⁺ for catalytic activity, some PLA₂s, including the hemocyte enzyme, are Ca²⁺-independent. The hemocyte PLA₂ exhibited a preference for arachidonyl-associated substrate over palmitoyl-associated substrate. These findings show that M. sexta hemocytes express a PLA₂ that shows a marked preference for hydrolyzing arachidonic acid from phospholipids. The biological significance of this enzyme relates to cellular immune responses to bacterial infections. The hemocyte PLA₂ may be the first biochemical step in synthesis of the eicosanoids that mediate cellular immunity in insects. © 1996 Wiley-Liss, Inc.     

Regulation of ecdysteroidogenesis in prothoracic glands of the tobacco hornworm Manduca sexta

Ecdysteroidogenesis in Manduca sexta prothoracic glands is regulated by a set of bioregulatory molecules, including prothoracicotropic hormone (PTTH) and a protein factor present in larval hemolymph, and by the competence of the glands to synthesize ecdysteroids in response to those molecules. A larval molting bioassay was used to assess the in vivo activity of Manduca PTTHs. Crude PTTH, big PTTH, and small PTTH each elicited a larval molt in head-ligated larvae. However, big PTTH was approximately 10-fold more potent than crude PTTH, which was, in turn, several orders of magnitude more potent than small PTTH. When big and small PTTH were combined, the molting response was similar to that elicited with crude PTTH. The chemical nature of the hemolymph protein factor was also investigated. Injection of [3H]cholesterol into last-instar larvae and fractionation of the radiolabeled hemolymph by gel filtration chromatography revealed three peaks of radioactivity. One peak eluted in fractions containing the hemolymph protein factor, a result consistent with the notion that the factor transports a sterol substrate. The possibility that the factor is a 3(2)-ketoreductase was investigated by assessing the effect of the factor on the accumulation of RIA-detectable ecdysteroids in prothoracic-gland-conditioned medium. Three of five preparations of the factor significantly enhanced the amount of RIA-detectable ecdysteroids in conditioned medium, indicating that at least some preparations of the factor may contain ketoreductase activity. The above findings are discussed in the context of current hypotheses of how bioregulatory molecules interact with the prothoracic glands to regulate ecdysteroidogenesis in Manduca.     

Tritrophic interactions reinforce a negative preference–performance relationship in the tobacco hornworm (Manduca sexta)

1. Host plants that promote development of insect herbivores are sometimes less preferred to more toxic plants, which are co-opted for protection from natural enemies, resulting in higher fitness in communities with strong top-down control. However, the degree to which variation in growth rate and risk of natural enemy attack drive insect plant preferences is an open question, with little field data available across diverse plant families.
2. The present study investigated the preference–performance relationship and tritrophic interactions involving the hornworm Manduca sexta, its natural enemies, and plants in the nightshade family (Solanaceae) using a 2-year common garden containing 18 wild and domesticated species. The degree to which natural enemy pressure explained field patterns in the laboratory was then tested using targeted assays involving parasitism by the wasp Cotesia congregata.
3. In the field, the most preferred plants for female oviposition tended to be inversely correlated with the species providing optimal larval growth. Hawkmoths preferred plants in the subgenus Potatoe, Nicotiana, and Datura compared with Capsicum, Physalis, and the other Solanum subgenera. However, larval parasitism by C. congregata was only significant for hornworms on Potatoe /Datura and not Nicotiana (i.e. 33% vs. 12% vs. 4% parasitism on Potatoe, Datura, and Nicotiana, respectively). Experimental laboratory rearing confirmed that wasp survival is lower on Nicotiana sp. than Solanum lycopersicum, which could be driven by nicotine.
4. The data obtained in the present study show that the negative preference-performance relationship in hornworms across solanaceous plants is maintained in part because by utilising noxious food plants M. sexta gains protection against parasitism.

     

Behavioral and genomic characterization of molt-sleep in the tobacco hornworm,Manduca sexta

During the transition from feeding to molting, larval insects undergo profound changes in behavior and patterns of gene expression regulated by the neuroendocrine system. For some species, a distinctive characteristic of molting larvae is presence of a quiescent state sometimes referred to as “molt-sleep”. Here, observations of 4th instar Manduca sexta larvae indicate the molting period involves a predominantly quiescent state that shares behavioral properties of adult insect sleep in that it is rapidly reversible and accompanied by a reduced responsiveness to both mildly arousing and noxious stimuli. When subjected to noxious stimuli, molting larvae exhibit locomotory and avoidance behaviors similar to those of inter-molt larvae. Although less consolidated, inter-molt quiescence shares many of the same behavioral traits with molting quiescence. However, when subjected to deprivation of quiescence, inter-molt larvae display a compensatory rebound behavior that is not detected in molting larvae. This suggests that molting quiescence is a specialized form of inactivity that affords survival advantages to molting larvae. RNA-seq analysis of molting larvae shows general reduction in expression of genes encoding GPCRs and down regulation of genes connected with cyclic nucleotide signaling. On the other hand, certain ion channel genes are up-regulated, including transient receptor potential (TRP) channels, chloride channels and a voltage-dependent calcium channel. These findings suggest patterns of gene expression consistent with elevation of quiescent state characteristic of the molt in a model holometabolous insect.     

Isolation of ommochromes and 3-hydroxykynurenine from the tobacco hornworm,Manduca sexta

A complex of the two red pigments was prepared from epidermis of the fifth instar larvae of the black mutant of the tobacco hornworm according to the method generally available for ommochromes. The primary component, a brick-red pigment (II), was shown to be identical with synthetic l-dihydroxanthommatin by various physico-chemical tests. The other red pigment (I) was similar to pigment (II), except in the i.r. spectra in 1000–1150 cm⁻¹ region and is thought to be also an ommatin. A red pigment identical with pigment (I) was obtained during the chemical oxidation of 3-hydroxy-l-kynurenine. These same two pigments were found in the red epidermis underlying the black cuticle of larvae either allatectomized or neck-ligatured before the critical period for juvenile hormone action during the last larval molt. Also, they were found in the dorsal epidermis of wandering stage larvae. 3-Hydroxykynurenine, the immediate precursor of the ommochrome pigments, was found to accumulate in the epidermis before the synthesis of the ommochromes began.     

Developmental and regional-specific expression of FLRFamide peptides in the tobacco hornworm,Manduca sexta,suggests functions at ecdysis

The developmental profile of a family of three FLRFamide (Phe-Leu-Arg-Phe-NH₂) peptides in the tobacco hornworm, Manduca sexta, revealed regional-specific expression patterns within the segmental ganglia. Levels of the three peptides—F7G (GNSFLRFamide), F7D (DPSFLRFamide), and F10 (pEDVVHSFLRFamide)—were always higher in the thoracic than abdominal ganglia. The predominant peptide also differed regionally, with F7G being highest in the thoracic ganglia and F7G and F10 being equivalent in the abdominal ganglia. Furthermore, we found regional-specific transient declines in ganglion peptide levels temporally correlated to ecdysis. Thoracic ganglion peptide levels declined at each molt, while abdominal ganglion levels declined over a period of 2 days after ecdysis. The decline in central levels was accompanied by an increase in levels in peripheral neurohemal sites, the transverse nerves (TNs). These observations suggest peptides were released from neurosecretory cells (NSCs) at ecdysis. Distinct sets of thoracic and abdominal NSCs and their processes in peripheral neurohemal sites were immunoreactive, supporting the biochemical data. These results also suggest the regional differences may arise from cellular-specific expression patterns for this family of peptides. In addition, fine immunoreactive processes were observed traveling between TNs and skeletal muscles, suggestive of myotropic actions. We propose that the release of different M. sexta FLRFamides from regionally distinct NSCs leads to a coordinated modulation of skeletal and visceral muscles that facilitate ecdysis. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 469–485, 1998     

Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.     

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm,Manduca sexta

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.