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     

Carbohydrate metabolism during the pupal molt of the tobacco hornworm,Manduca sexta

During the pupal molt of the tobacco hornworm, Manduca sexta, the percentage of active fat body glycogen phosphorylase increased from 5–10 to 20%, but only for a period of 5 h prior to the molt. From the time of the appearance of two sclerotized dorsal bars to the time of the molt, the concentration of total hemolymph carbohydrates doubled to 100 mM trehalose. Initially, the glucose level was high (16 mM) when compared with feeding larvae (approximately 1 mM) but decreased to zero just prior to the molt. The amount of cuticular chitosan decreased from approximately 100 mg to 10 mg at pupation; the exuvia contained approximately 7 mg. While the levels of total lipids in hemolymph were not affected, the lipid content of the fat body decreased significantly prior to the molt but increased sharply thereafter. Fat body glycogen phosphorylase in pharate pupae and pupae of M. sexta was substantially activated by the Manduca adipokinetic peptide hormone, which in pharate pupae, produced the same response at 2 and 20 pmol per insect as in ligated larval abdomens. In pupae the response was clearly reduced. Using chilling to stimulate glycogen phosphorylase, it was found that the enzyme in pharate pupae and pupae responded both in vivo and in vitro as in ligated abdomens of larvae. Thus, a transition to the adult response seems to occur during the pupal and pharate adult development. © 1995 Wiley-Liss, Inc.     

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.     

Regulation of translation during oogenesis and embryogenesis in the tobacco hornworm,Manduca sexta

Summary

Translational activity in the oocyte and early embryo of Manduca sexta may be regulated by a number of mechanisms including availability of ribosomal subunits, the protein complement of the maternal mRNP, and the presence of a functional 5′ cap structure on the maternal mRNA. We present preliminary experiments concerning the role of intracellular pH in the activation of protein synthesis and the nature of the cortical cytoskeleton of the mature oocyte and its possible role in immobilizing maternal mRNA.     

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.     

Heat sensitivity and protein synthesis during heat-shock in the tobacco hornworm,Manduca sexta

Summary Fifth instar larvae of the tobacco hornworm,Manduca sexta, tolerate 1-h exposures to temperatures as high as 42°C. Above 42°C, survival declines rapidly to 18% at 44°C and 0% at 48°C. As in other insects, the heat-shock response ofManduca sexta involves the induction of synthesis of heat-shock proteins very similar in size to theDrosophila heat-shock proteins (84, 73, 71, 27, 25, 23, and 22 kd). In the epidermis, heat-shock protein synthesis peaks at 42°C, correlating with the heat sensitivity of both the tissue itself and the intact larva. Some heat-shock proteins have different isoelectric forms depending on tissue. Also, the heat-shock proteins are synthesized over a wider range of temperatures in the imaginal discs and the fat body as compared to the epidermis. In contrast to dipteran insects,Manduca sexta does not exhibit a strong repression of non-heat-shock protein synthesis under tolerable conditions.Abbreviations TCA trichloroacetic acid - PAGE polyacrylamide gel electrophoresis - AZT arbitrary Zeitgeber time - kd kilodaltons     

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.     

Acid phosphatases of Metarhizium anisopliae during infection of the tobacco hornworm Manduca sexta

Three acid phosphatase (AcP) isozymes, pI 8.1, 8.0 and 7.8, were isolated, purified and partially characterised from optimised cultures of the entomopathogenic fungus Metarhizium anisopliae. The enzymes had similar molecular masses (approximately 44.0 kDa), and could degrade sugar phosphates found in the haemolymph of a host insect, the tobacco hornworm Manduca sexta. The AcP activity in haemolymph of mycosed insects increased significantly over controls, and some new isozymes were present. The infection-related isoforms were similar in molecular mass and pI to some of the in vitro AcP isozymes of M. anisopliae. Results of dot blot and Western blot analyses using anti-AcP antibodies suggested that at least one Metarhizium phosphatase isoform was present in haemolymph of infected caterpillars. Antibodies did not cross-react with immune (chemically stimulated) or non-immune haemolymph from Manduca sexta. Consistent with the appearance of highly active fungal phosphatase in caterpillar blood, free phosphate concentration increased dramatically during the late stages of infection to a level two to five times that of controls. Phosphate was limiting to growth of the fungus at the concentration found in control haemolymph and supplementation of phosphate significantly increased fungal growth in vitro in haemolymph. These results suggest that Metarhizium AcP may play a key role in providing phosphorus for fungal growth at the expense of the insect.     

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.     

The development of identified neurosecretory cells in the tobacco hornworm, Manduca sexta

The prothoracicotropic hormone (PTTH) is a principal neuropeptide regulator of insect postembryonic molting and metamorphosis. In the tobacco hornworm, Manduca sexta, PTTH is produced by two neurosecretory cells (NSC) located in each protocerebral lobe of the brain. The development of these neurons, the L-NSC III, has been investigated immunocytologically to establish the time course of their morphological differentiation. PTTH may be one of the earliest neuropeptides expressed in insect embryos. PTTH-immunoreactivity was initially detected in the somata at 24 to 30% of embryonic development. Neurites sprouted shortly thereafter and began to grow medially through the brain anlage. By 42% embryonic development, the neurites had decussated to the contralateral brain lobe. As development progressed, the L-NSC III neurites grew along specific tracts through the contralateral brain lobe reaching the ventrolateral regions of the brain by approximately 60% development. The axons exited the brain through a retrocerebral nerve, the nervi corporis cardiaci I + II. At approximately 63% development, the axons innervated the corpus allatum and began branching to form neurohemal terminals for PTTH release. At 60% development, short collaterals began extending in the protocerebral neuropil. During the remainder of embryogenesis, both the dendritic collaterals and the terminal neurohemal varicosities continued to elongate and arborize. By 85% embryonic development, the basic architecture of the L-NSC III was established.     

Carbohydrate and lipid metabolism during the last larval moult of the tobacco hornworm, Manduca sexta

Abstract. At 25°C and with a light regime of 17 h light and 7h dark, the last larval moult of the tobacco hornworm, Manduca sexta , lasts approximately 32 h, during which profound changes of metabolism were observed. At the onset of the moult, which coincides with the cessation of feeding, the proportion of active fat body glycogen phosphorylase increased from 10 (-2h) to 25–30% (Oh). A biphasic pattern with peak activities of 45–50% after t – 12 h and again just prior to the shedding of the cuticle (32 h) was subsequently observed. Haemolymph trehalose concentration decreased significantly from c. 35 (Oh) to 20mM (8h), but then recovered to an intermediate level (30mM; 12h). After completion of the moult, the trehalose concentration was 35–40 mM. The haemolymph glucose level in feeding fourth instar larvae was 4–5 mM, but decreased sharply before the onset of the moult to c. 1 mM, followed by a slow 6-fold increase over the next 20h. Prior to the shedding of the cuticle, the glucose level dropped again dramatically. The haemolymph lipid level increased slowly from an initial level of 1.2–1.4mg/ml during the early part of the moult, reaching a maximum of 1.8mg/ml after /= 16 h. Afterwards, a decrease of c. 50% was observed until ecdysis occurred. Oxygen consumption per animal decreased steadily from 30–35 μl/min pre-moult by approximately 70% to c. 10 μl/min but started to increase about 5 h before the animals resumed feeding.     

Organization of the larval pre-ecdysis motor pattern in the tobacco hornworm,Manduca sexta

The tobacco hornworm, Manduca sexta, undergoes several larval molts before transforming into a pupa and then an adult moth. Each molt culminates in ecdysis, when the old cuticle is shed. Prior to each larval ecdysis, the old cuticle is loosened by pre-ecdysis behavior, which consists of rhythmic compressions that are synchronous along the abdomen and on both body sides, and rhythmic retractions of the abdominal prolegs. Both pre-ecdysis and ecdysis behaviors are triggered by a peptide, eclosion hormone. The aim of the present study was to investigate the neural circuitry underlying larval preecdysis behavior. The pre-ecdysis motor pattern was recorded in isolated nerve cords from eclosion hormone-treated larvae, and the effects of connective transections and ionic manipulations were tested. Our results suggest that the larval pre-ecdysis compression motor pattern is coordinated and maintained by interneurons in the terminal abdominal ganglion that ascend the nerve cord without chemical synaptic relays; these interneurons make bilateral, probably monosynaptic, excitatory connections with identified pre-ecdysis motor neurons throughout the abdominal nerve cord. This model of the organization of the larval pre-ecdysis motor pattern should facilitate identification of the relevant interneurons, allowing future investigation of the neural basis of the developmental weakening of the pre-ecdysis motor pattern that accompanies the larval-pupal transformation.Abbreviations A3, A4... abdominal ganglia 3, 4... - AT terminal abdominal ganglion - ALE anterior lateral external muscle - DN dorsal nerve - DN_A anterior branch of the dorsal nerve - DN_L lateral branch of the dorsal nerve - DN_P posterior branch of the dorsal nerve - EH eclosion hormone - TP tergopleural muscle - VN ventral nerve - VN_A anterior branch of the ventral nerve - VN_L lateral branch of the ventral nerve - VN_P posterior branch of the ventral nerve     

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     

Alterations in DNA-dependent RNA polymerase activity during the development of the tobacco hornworm, Manduca sexta.

RNA polymerases I, II and III have been detected in the extracts of fat body and integument of the tobacco hornworm, and their activity during larval-pupal-adult metamorphosis has been measured. Total RNA polymerase activity of both tissues reaches a peak just prior to the wandering stage of the fifth instar larva. The enzyme activity of the integument declines thereafter while, in the fat body, a change in the cellular compartmentalization of enzyme activity occurs during development. This is indicated by the observations that RNA polymerase activity, which was predominantly in the soluble fraction up until the onset of the wandering stage, declines rapidly during the wandering stage while RNA polymerase activity in the insoluble-pellet fraction increases. A steady-state level is reached just prior to pupation, and the enzyme activity remains at that level during pharate adult development. The α-amanitin-sensitive enzyme appears to be responsible for most of the RNA polymerase activity during larval life. The findings that the peak of RNA polymerase activity in both tissues and the subsequent changes in compartmentalization in the fat body occur coincidentally with the first surge of α-ecdysone release by the prothoracic glands raises the possibility that control of RNA polymerase activity may be humorally mediated.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Toxicity of Bacillus thuringiensis spores to the tobacco hornworm, Manduca sexta.

Toxicity of Bacillus thuringiensis spores to the tobacco hornworm, Manduca sexta, is described. The numbers of larvae killed were in relation to spore dry weight. At a surface application of 6.8 ng/cm2, there was an 85 percent survival, but less than 50 percent survived at 68.2 ng/cm2. Striking similarity of spores to parasporal crystals is revealed by slope of mortality curves, inhibition of stadial growth, and 50 percent lethal dose values based on protein content.     

Prothoracicotropic hormone activity in the embryonic brain of the tobacco hornworm,Manduca sexta

Summary Head segments and brains were extirpated from embryos of the tobacco hornworm,Manduca sexta, extracted and the resulting extracts assayed for prothoracicotropic hormone (PTTH) activity on prothoracic glands from day 3 fifth instar larvae and day 0 pupae. Dose-response curves were generated and indicated the presence of PTTH activity in embryonic brains and head segments, suggesting a role(s) for this neurohormone during embryogenesis. Maximal PTTH activity was found in brains from embryos 117 h post-oviposition, just prior to hatching, but activity was also noted in head segments as early as 24 h postoviposition. These data on PTTH and those on ecdysteroids and juvenile hormones in embryos suggest that these 3 classes of hormones which control insect post-embryonic development, may also be involved in the regulation of developmental processes in the embryo.     

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.     

Life cycle expression of a bombyxin-like neuropeptide in the tobacco hornworm,Manduca sexta

Immunocytochemistry was used to investigate the developmental expression of the insulin-like neuropeptide bombyxin in the tobacco hornworm, Manduca sexta. A mouse monoclonal antibody raised against a synthetic peptide corresponding to bombyxin's A-chain N-terminus was used to localize a bombyxin-like peptide to a group of cerebral medial neurosecretory cells, the M-NSC IIa(2). Immunostaining was first detected on day 0 of the second larval instar, localized in the M-NSC IIa(2) somata and in the neurohemal organ, the corpora allata (CA). By day 0 of the fourth larval instar, the peptide was present throughout the M-NSC IIa(2) somata, axons, dendritic fields and CA. Between days 7 and 9 of the fifth instar, a dramatic reduction in the dendritic fields and CA staining occurred, suggesting the peptide is released. After day 2 of the pupal period, only M-NSC IIa(2) somata immunostained, a pattern that persisted through day 2 of the adult stage. The specificity of immunostaining was demonstrated by using a synthetic bombyxin peptide to block staining. These developmental data reveal times of potential Manduca bombyxin-like peptide release which should provide insight into the peptide's function.