Ultrastructure of neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta(L)

Ultrastructural evidence is presented to indicate the synthesis, storage, and packaging of neurosecretory material in the frontal ganglion of Mandaca sera. Two morphologically distinct stages in the development of NSG were observed in the NSC. The immature granule contains granular electron dense material and arises from dilations of the rough endoplasmic reticulum. In diapause pupae the immature granules accumulate, whereas in developing larvae and pupae, they fuse with the proximal face of the Golgi apparatus. The mature granule contains opaque electron dense material and is seen in close association with the distal cisternae of the Golgi apparatus in developing pupae and larvae. The mature granules presumably contain the material from the immature granules which has been chemically altered and condensed by the Golgi apparatus and probably represent the mature NSG.The second process in neurosecretory cells of developing larvae is cyclic and the various stages are described. A comparison of neurosecretion in photoperiodically induced diapause and developing (non-diapause) larvae and pupae is presented.     

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.     

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.     

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.     

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.     

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.     

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.     

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.     

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.     

Structure-activity relationship of ETH during ecdysis in the tobacco hornworm, Manduca sexta

In insects, ecdysis or shedding of the old cuticle, consists of a series of behaviors that are regulated by the coordinated actions of a number of neuropeptides, one of which is ecdysis triggering hormone (ETH). ETH acts directly on central pattern generators of the abdominal ganglia to trigger onset of pre-ecdysis behaviors, as well as indirectly to activate release of eclosion hormone, thereby inducing onset of ecdysis behaviors through a cGMP-mediated mechanism. We assessed the minimal C-terminal amino acids required for biological activity of ETH, by assessing: (i) onset of pre-ecdysis and ecdysis behaviors in vivo, after injection of peptide analogs, (ii) onset of fictive pre-ecdysis and ecdysis motor patterns in vitro, as recorded extracellularly, after incubation of the CNS with the peptide analogs, and (iii) accumulation of cGMP within cells of the abdominal ganglia, as assessed immunohistochemically. Amidation of ETH at the C-terminus was required to elicit a biological response in vivo and in vitro, as well as an accumulation of cGMP within the CNS. The five amino acid amidated C-terminus of ETH (NIPRMamide) was the minimal moiety able to induce a robust pre-ecdysis response in vivo and in vitro, while a seven amino acid core (NKNIPRMa) was required for induction of ecdysis, including accumulation of cGMP immunoreactivity within the CNS. Analogs smaller than 12 amino acids in length were only active at very high concentrations in vivo, suggesting that smaller fragments might be susceptible to hemolymph degradation. Some alanine substitutions or removal of internal amino acids altered the activity of ETH, as well as the time of onset of ecdysis behaviors, suggesting that internal amino acids play a role in maintaining proper folding of the peptide for successful binding or activity at the ETH receptor.     

Identification of neuroendocrine cells producing a diuretic hormone in the tobacco hornworm moth,Manduca sexta

Summary Separate antisera were raised to the N- and C-terminal half of the diuretic hormone from Manduca sexta. Antisera against the two halves of this peptide recognized the same cells in M. sexta, and preabsorption of the antisera with the peptides used as antigens abolished the immunoreactivity, confirming their specificity. The antisera reacted with two median neurosecretory cells on each side of the protocerebral groove in larvae, and with a group of about 80 small median neurosecretory cells in the adult, as well as their axons to, and their axon terminals in, the corpora cardiaca. During the early pupal stages, small cells, which are possibly derived from a common neuroblast, differentiate into immunoreactive neurosecretory cells, which explains the large increase in cell numbers in the adult. In the sleepy sulphur butterfly, Eurema nicippe, homologous median neurosecretory cells in the adult were immunoreactive with both antisera.     

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.     

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.     

Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.     

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     

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.