Programmed cell death in the tobacco hornworm, Manduca sexta: alteration in protein synthesis

The metamorphic death of the labial glands of the tobacco hornworm, Manduca sexta, occurs during a 4 day period during larva-to-pupa metamorphosis. The earliest changes marking the death of the cell, all occurring on the first day, are a sharp drop in protein synthesis, coupled with the selective survival or upregulation of a few messages. An early rearrangement of the rough endoplasmic reticulum is presumably related to the generalized decrease in protein synthesis. Lysosomal acid phosphatase also begins to increase very early, and ultimately the bulk of the cytoplasm is destroyed in autophagic vacuoles, but activation of lysosomes does not account for the decreased rate of synthesis. The mechanism by which most protein synthesis is depressed remains under investigation.     

Incorporation of polyunsaturated fatty acids into phospholipids of hemocytes from the tobacco hornworm, Manduca sexta

We examined the incorporation of four radioactive fatty acids, 18:1n-9, 18:2n-6, 20:4n-6 and 20:5n-3, into cellular lipids of hemocytes from tobacco hornworms, Manduca sexta. Most of the radioactivity associated with 18:1n-9 was recovered from triacylglycerols (TGs), and the radioactivity associated with 18:2n-6 was heavily incorporated into phospholipids (PLs) and TGs. Most of the radioactivity associated with the two eicosanoid-precursor polyunsaturated fatty acids (PUFAs), 20:4n-6 and 20:5n-3, was incorporated into PLs. The incorporated fatty acids were redistributed among the lipid classes during 2 h incubations. The two C20 PUFAs were moved from PLs to TGs. While 18:2n-6 underwent little change, 18:1n-9 was redistributed from TGs to PLs. Within PLs, each of the fatty acids were incorporated into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PtG) and phosphatidylserine/inositol (PS/PI). The incorporation patterns changed over time, indicating that the incorporated fatty acids were redistributed among the four PL fractions. The radioactivity associated with 18:1n-9 was mostly recovered from the sn-1 position of PC (59%) and PE (83%). Most of the radioactivity associated with 18:2n-6 was found in the sn-2 position of PC (88%) and PE (67%). Over 90% of the radioactivity associated with 20:5n-3 was recovered from the sn-2 position of PC and PE. Incorporation of 20:4n-6 differed from 20:5n-3 because more radioactivity was recovered from the sn-2 position of PC (93%) than PE (69%). These findings are in line with the general background of lipid biochemistry, from which incorporation of 20:4n-6 into PE marks a notable departure: 31% of the radioactivity associated with this acid was recovered from the sn-1 position of PE. These findings indicate that hemocytes from the tobacco hornworm elaborate a fatty acid incorporation system, which exhibits specificity with respect to fatty acid structure and lipid class.     

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 (GNSFLRF-amide), F7D (DPSFLRFamide), and F10 (pEDVVHS-FLRFamide)-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 F100 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.     

Ultrastructure of the neurosecretory cells in the brain of diapausing pupae of the tobacco hornworm, Manduca sexta (L)

The ultrastructure of seven different types of neurosecretory cells (NSC) found in the medial and lateral areas of the brain of diapausing Manduca sexta is described. The five different types of NSC in the medial area have characteristic differences in their shape, size, neurosecretory granules (NSG), and the morphology of their organelles. The cell types of the medial area accumulated the NSG, but did not appear to be synthesizing and packaging new NSG, whereas the NSC in the lateral region were synthesizing and packaging NSG during diapause. The possible significance of the relationship between the lateral and medial cells is discussed.     

Allatostatin-like-immunoreactive neurons of the tobacco hornworm, Manduca sexta, and isolation and identification of a new neuropeptide related to cockroach allatostatins

The YXFGLamide C-terminus serves to define most members of a family of structurally related neuropeptides, the YXFGLamides. These peptides have been identified from the nervous system of various insects and include the allatostatins of cockroaches and crickets, the schistostatins of locusts, and the callatostatins of blowflies. The YXFGLamides have been shown to have various functions, including inhibition of juvenile hormone biosynthesis in cockroaches and crickets and inhibition of contraction of certain insect visceral muscles. We wanted to know if these peptides occur in Manduca sexta and what functions they might have. A new peptide, AKSYNFGLamide, was isolated and identified from M. sexta and has been named "lepidostatin-1"; this is the first YXFGLamide to be found in a lepidopteran, and there are indications that additional YXFGLamides occur in M. sexta. An antiserum to cockroach allatostatins (YXFGLamides) was shown to recognize lepidostatin-1 of M. sexta and was used to map YXFGLamide-immunoreactive neurons in larvae. Because immunoreactive interneurons were found to form an extensive neuropil, YXFGLamides probably function as neuromodulators in M. sexta. Neuroendocrine cells in the brain, abdominal ganglia, and their respective neurohemal organs were YXFGLamide immunoreactive and appear to release YXFGLamides as neurohormones. Immunoreactivity to YXFGLamides and M. sexta diuretic hormone were found to be colocalized and appear to be coreleased in these neuroendocrine cells, indicating that YXFGLamides may be involved in regulation of fluid transport. Innervation of the corpora allata by YXFGLamide-immunoreactive processes was very sparse, suggesting that this innervation does not play an important role in allatostasis. Many thoracic motor neurons were YXFGLamide immunoreactive, suggesting that YXFGLamides may have a myomodulatory or myotrophic function in larvae. However, this immunoreactivity disappeared early in metamorphosis and did not reappear in the adult. The YXFGLamide-immunoreactive neurons in the terminal abdominal ganglion were found to innervate the hindgut, indicating that YXFGLamides may be involved in the control of the rate of myogenic contractions of the larval hindgut.     

Postembryonic neurogenesis in the central nervous system of the tobacco hornworm, Manduca sexta. III. Spatial and temporal patterns of proliferation

Postembryonic neurogenesis leads to a dramatic increase in the number of functional neurons within the segmental ganglia of the moth, Manduca sexta. These adult-specific neurons are generated during larval life by segment-specific arrays of individually identifiable stem cells, or neuroblasts (Nbs). By the end of the feeding larval stage, each Nb has generated a discrete nest of progeny, which ranges in size from less than 10 to more than 70 progeny. The sizes of these identifiable nests of progeny vary in a segment-specific manner, with the thoracic nests containing a greater number of progeny compared with their homologues in the simpler abdominal ganglia. In order to describe those factors that influence the size of the post-embryonic neuronal lineages, we examined the spatial and temporal pattern of postembryonic neurogenesis in the segmental ganglia of Manduca. The rates at which the identifiable nests accumulated progeny were estimated by counting the number of progeny within the nests, using sectioned material isolated from animals at stages ranging from embryonic hatching until the end of the feeding larval stage. All of the postembryonic Nbs began to generate progeny at around the time of the molt to the third larval instar. Each nest added progeny at a rate that was a characteristic of its identity and segment of origin. Although all of the nests within the thorax continued to accumulate progeny throughout the feeding larval stage, several of the abdominal nests showed little or no growth following the molt to the fifth larval instar. The thymidine analog 5-bromo 2-deoxyuridine (5-BrdU) was used to estimate the mitotic rates of the identifiable Nbs. The number of labeled progeny within a nest 24 h after application of 5-BrdU ranged from a low of 1 to 2 to a high of 11 to 13 labeled cells. In some instances there was a good correlation between the estimated mitotic rate of an identified Nb and the rate of growth of its associated nest of progeny. However, several of the identifiable nests accumulated progeny at a slower rate than predicted based on the estimated mitotic rate of the Nb. Cell death appears to be responsible for slowing the growth of the nests during the feeding larval stage. We estimate that 10% to 70% of the neurons generated during the feeding larval stage degenerate within 24 h of their birth. The level of cell death observed within a nest was dependent on both its identity and its segment of origin.     

Pollination-enhanced expression of a receptor-like protein kinase related gene in tobacco styles

We have isolated a putative serine/threonine receptor kinase gene with an expression pattern indicating that it may play a role in the stylar response to pollination. Differential display PCR was used to select tobacco mRNAs with increased accumulation following pollination. NTS16, a cDNA identified by this method, is homologous to a ca. 2.4 kb mRNA primarily expressed in pistil tissues. Levels of this mRNA increase during floral development and are further increased by pollination reaching maximal accumulation 12-18 hours after pollination and then declining. mRNA levels can also be increased by the application of ethylene to unpollinated flowers. A polypeptide encoded by the NTS 16 open reading frame has sequence similarity to the catalytic domain of several receptor protein kinases from plants including the S-receptor kinases implicated in the rejection of self-pollen in Brassica species and the Pto gene product of tomato which confers resistance to a bacterial pathogen.     

Partial characterization of allatinhibin, a neurohormone of Manduca sexta

During the last larval stage, corpora allata (CA) of Manduca sexta are inactivated by a factor from the brain. Apparently the same factor (allatinhibin, AI) is secreted by day 4 Vth instar brains kept overnight in Grace's medium. AI is rapidly inactivated by heat or acid but withstands exposure to alkali and can be recovered after freezing and lyophilization. Exposure to pronase, chymotrypsin, carboxypeptidases-A and -Y, as well as leucine aminopeptidase eliminated AI activity completely, whereas after exposure to trypsin and protease XVII-S, some residual activity remained. Inactivation by pyroglutamate aminopeptidase is interpreted as being due to prolinase activity of this enzyme. Incubation of CA with gentamicin, an aminoglycoside antibiotic, affects neither their ability to produce JH in vitro nor their viability in implantation assays. However, AI did not inactivate CA in the presence of low concentrations of gentamicin. This effect was used to guard against false positive assay results possibly produced by allatotoxic contamination. AI was purified by chromatography on Sephadex G-25. All activity recovered emerged from the columns in intermediate fractions with an apparent M(r) of 1,000-2,000.     

Innervation regulates the metamorphic fates of larval abdominal muscles in the moth, Manduca sexta

With the onset of metamorphosis, the abdominal muscles of the moth, Manduca sexta, follow one of three developmental fates: maintenance, respecification, or death. The maintained muscles retain their larval size and morphology throughout adult development. The respecified and dying muscles dedifferentiate, which involves regression, nuclear degeneration, and myofibril breakdown. Nuclei in both dying and respecified muscles also proliferate. The amount of nuclear degeneration is greater in the dying muscle fibers, and the amount of nuclear proliferation is greater in the respecified muscles. Four to ten days after pupation, the sizes of the respecified muscles stabilize while the dying muscles are lost. During regression, a subset of the respecified muscle fibers die. The surviving respecified muscle fibers grow and differentiate during the last half of adult development. In respecified muscles, denervation triggers an increased amount of nuclear degeneration and a decreased amount of nuclear proliferation. As a result, denervated respecified fibers experience increased muscle regression including an increased loss of muscle fibers and sometimes muscle death. Surviving respecified fibers still grow and differentiate yet are only 5 to 12% of the control size. Denervation triggers dedifferentiation in maintained muscles, resulting in fiber loss and occasionally muscle death. The percentage of fibers which dedifferentiate varies between different muscles. Denervation also triggers nuclear proliferation, with the amount of nuclear proliferation correlated with the extent of dedifferentiation of the individual muscle fibers. The dedifferentiated maintained fibers subsequently undergo differentiation in the absence of muscle growth.     

Thermal sensitivity of growth and feeding in Manduca sexta caterpillars

We explore how the thermal sensitivity of organismic performance emerges from the thermal sensitivity of the underlying component processes involved, using growth and feeding of Manduca sexta caterpillars as a model system. We measured thermal performance curves for the short-term rates of growth, consumption, protein (casein) digestion, amino acid (methionine) uptake, and respiration in fifth-instar caterpillars over a biologically realistic temperature range from 14 degrees to 42 degrees C. Growth and consumption rates increased between 14 degrees and 26 degrees C, reached a maximum value near 34 degrees C, and declined rapidly above 38 degrees C. In contrast, protein digestion rate and respiration rate increased monotonically over the entire temperature range, and amino acid uptake rate increased with temperatures up to 38 degrees C and then leveled off between 38 degrees and 42 degrees C. These results suggest that the shape and position of the thermal performance curve for growth rate--in particular the maximum at 34 degrees C and rapid decline above 38 degrees C--was most closely correlated with the thermal sensitivity of consumption rate; the declining growth performance above 38 degrees C was not associated with declines in digestion or uptake rates or with accelerated respiration rates at these temperatures.     

Prothoracicotropic hormone in Manduca sexta: localization by a larval assay

1. A new method for the assay of insect prothoracicotropic hormone (PTTH) is described, using fourth instar larvae of Manduca sexta. Larvae neck-ligated at a critical time to prevent release of PTTH from the head fail to undergo the next larval moult. Such ligated larvae moult to fifth instar larvae or larval-pupal intermediates after injection of brain homogenates from Manduca larvae, pupae or pharate adults. The degree of response is proportional to the concentration of brain homogenate injected. 2. The source of PTTH in the pupal brain is the dorsal region of the protocerebrum containing the lateral neurosecretory cells. Microhomogennates of single pieces of brain showed activity with this method. 3. PTTH activity in partially purified extracts is water soluable, stable to boiling for 10 min, and is destroyed by Pronase or trypsin.     

Programmed cell death of identified peptidergic neurons involved in ecdysis behavior in the Moth, Manduca sexta

Molecular dynamics simulation in explicit solvent and continuum solvent models are applied to investigate the relative stability of A- and B-form helices for two DNA sequences, dA10-dT10 and dG10-dC10 in three structural forms. One structural form is based on an unrestrained molecular dynamics (MD) trajectory starting from a canonical B-DNA structure, the second is based on a MD trajectory starting in a canonical B-DNA structure with the sugars constrained to be C2'-endo and the third simulation started from a canonical A-DNA structure with the sugars constrained to C3'-endo puckers. For the energetic analysis, structures were taken as snapshots from nanosecond length molecular dynamics simulations computed in a consistent fashion in explicit solvent, applying the particle mesh Ewald method and the Cornell et al. force field. The electrostatic contributions to solvation free energies are computed using both a finite-difference Poisson-Boltzmann model and a pairwise Generalized Born model. The non-electrostatic contributions to the solvation free energies are estimated with a solvent accessible surface area dependent term. To estimate the gas phase component of the relative free energy between the various structures, the mean solute internal energies (determined with the Cornell et al. molecular mechanics potential including all pairwise interactions within the solute) and estimates of the solute entropy (using a harmonic approximation) were used. Consistent with experiment, the polyG-polyC (GC) structures are found to be much more A-phillic than the polyA-polyT (AT) structures, the latter being quite A-phobic. The dominant energy components responsible for this difference comes from the internal and van der Waal energies. A perhaps less appreciated difference between the GC and AT rich sequences is suggested by the calculated salt dependence which demonstrates a significantly enhanced ability to drive GC rich sequences towards an A-form structure compared to AT rich sequences. In addition to being A-phobic, the AT structure also has a noticably larger helical repeat than GC and other mixed sequence duplexes, consistent with experiment. Analysis of the average solvent density from the trajectories shows hydration patterns in qualitative agreement with experiment and previous theoretical treatments.     

Development and organization of a nitric-oxide-sensitive peripheral neural plexus in larvae of the moth, Manduca sexta

Each hemisegment of the Manduca sexta larva is supplied with a subepidermal plexus of approximately 350 multidendritic neurons. An initial set of neurons, the primary plexus neurons, arise at 35-45% of embryogenesis. These neurons comprise 12-16 uniquely identifiable neurons per hemisegment that have homologues in other insect larvae. Each spreads processes across a characteristic portion of the body wall and has an axon that projects into the central nervous system. Secondary plexus neurons are born in two waves: the first between 70% and 80% of embryogenesis and the second during the molt to the second larval stage. The secondary plexus neurons are multidendritic, spread uniformly across the body wall, and appear to make contacts with the primary plexus neurons. Each secondary plexus cell arises as part of a five-cell cluster; the other cells produce a sensory bristle and socket along with the bristle sensory neuron and a glial cell. Application of nitric oxide (NO) donors induces plexus neurons to produce cyclic 3',5' guanosine monophosphate (cGMP), suggesting the presence of soluble guanylate cyclase. With few exceptions, plexus neurons become sensitive to NO stimulation approximately 10 hours after their birth and remain so throughout larval life. Cyclic GMP is detected primarily in the cytoplasm of plexus neurons and extends into the finest peripheral dendrites. Our results suggest that cGMP participates in the development and/or physiology of this peripheral neural plexus.     

Absence of short-term regulation over gluconeogenesis by glucose in the insect Manduca sexta L

In vivo gluconeogenesis from (3-13 C)alanine was evident in terminal instar Manduca sexta larvae from the selective fractional 13C enrichment in trehalose, a disaccharide of glucose and the major blood sugar of insects. De novo glucose synthesis was observed in insects fed a low carbohydrate diet for 1 or more days. Gluconeogenesis was not inhibited by a single injection of glucose nor by short-term feeding on glucose-supplemented diet. Reduced fractional 13C enrichment in trehalose was demonstrated upon glucose administration, but was explained by isotopic dilution following direct synthesis of trehalose from the unlabeled glucose. Isotopic dilution was also quantified by analysis of the 13C labeling pattern in trehalose synthesized following injection of (1,2-13C2)glucose. The results suggest the absence of short-term regulation over gluconeogenesis by glucose and may partially explain why blood sugar level in M. sexta and other insects fluctuates over a wide concentration range. Although glucose had no observable effects on gluconeogenesis, injection of or feeding glucose resulted in a significantly increased activity of the pentose phosphate pathway.     

Adipokinetic hormone-induced lipolysis in the fat body of an insect, Manduca sexta: synthesis of sn-1,2-diacylglycerols

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed.     

20-Hydroxyecdysone stimulates proliferation of glial cells in the developing brain of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone (20-HE) controls diverse aspects of neuronal differentiation during metamorphosis in the hawkmoth Manduca sexta. In the present study we have examined the effect of 20-HE on glial cells of the brain during the metamorphic period. The antennal (olfactory) lobe of Manduca provides an ideal system in which to study effects of hormones on glial cells, since three known classes of glial cells participate in its development, and at least one type is critically important for establishment of normal neuronal morphology. These glial cells, associated with the neuropil, form boundaries for developing olfactory glomeruli as a result of proliferation and migration. We determined whether glial cells proliferate in response to 20-HE by injecting a pulse of 20-HE into the hemolymph at different stages of development and monitoring proliferation of all three types of glial cells. Hormone injections at the beginning and end of metamorphic development, when hormone titers are normally low, did not stimulate proliferation of neuropil-associated glial cells. Injections during the period when hormone titers are normally rising produced significant increases in their proliferation. Injections when hormone titers are normally high were ineffective at enhancing their proliferation. One other class of glial cells, the perineurial cells, also proliferate in response to 20-HE. Thus, glial proliferation in the brain is under the control of steroid hormones during metamorphic development.