Adult Manduca Sexta Research Articles

Ramification pattern and ultrastructural characteristics of the serotonin-immunoreactive neuron in the antennal lobe of the moth Manduca sexta: a laser scanning confocal and electron microscopic study.

The two antennal lobes, the primary olfactory centers of the brain, of the moth Manduca sexta each contain one neuron that displays serotonin immunoreactivity. The neuron projects out of the antennal lobe and sends branches into ipsi- and contralateral protocerebral areas. An axon-like process extends from the contralateral protocerebrum to, and terminates in, the contralateral antennal lobe. In order to begin to investigate the possible role of this unique neuron in olfactory information processing, we have used laser scanning confocal microscopic and electron microscopic immunocytochemical techniques to study the ramification pattern, ultrastructural characteristics, and synaptic connections of the neuron in the antennal lobes of female adult Manduca sexta. The neuron ramifies extensively in the antennal lobe contralateral to the cell body. The ramifications, mainly in the base and center of each glomerulus, do not overlap with those of the sensory axons from the antenna. This finding suggests that the serotonin-immunoreactive neuron may not receive direct input from sensory neurons, and that it may modulate the activity of the neurons of the antennal lobe rather than that of the sensory neurons. In the electron microscope, the neuron exhibits large dense-cored vesicles and small, clear round vesicles. In the antennal lobe ipsilateral to the cell body, the primary neurite of the serotonin-immunoreactive neuron is unbranched and lacks detectable synaptic connections. The ramifications in the contralateral antennal lobe, however, participate in synaptic connections. At very low frequency, contralateral branches form synapses onto unlabeled processes and also receive synapses from unidentified neurons in the glomeruli, indicating that the neuron may participate directly in synaptic processing of olfactory information. The high ratio of output to input synapses made by the serotonin-immunoreactive processes in the contralateral antennal lobe is consistent with the idea that this neuron may receive synaptic input via its bilateral branches in the protocerebrum and then send information to the contralateral antennal lobe where the neuron may exert feedback or modulatory influences on olfactory information processing in the glomeruli.

Organisation of the thoracic ganglia of the adult sphinx moth Manduca sexta (Insecta, Lepidoptera)

The present work describes the neuroanatomy of the thoracic ganglia in the adult sphinx moth Manduca sexta from transverse, horizontal and sagittal sections in order to provide a basis for a more precise description of identified neurons and their connections within the thoracic ganglia. The prothoracic ganglion of M. sexta derives from a single neuromere, whereas the pterothoracic ganglion results from the fusion of the meso- and metathoracic and the first and second abdominal neuromeres. Each hemiganglion contains nine longitudinal tracts (MDT, LDT, DMT, DIT, VLT, VIT, LVT, MVT and VMT) and six dorsal (DC I–VI) and five ventral commissures (VC I, dVCL II, vVCL II, SMC and PVC). Additionally there are vertical or oblique tracts such as the T-tract, C-tract or I-tract. The ring tract could not be found, LDT, DIT, DMT and MVT1 are compact and well defined, whereas VMT and MVT and also VLT and VIT are difficult to separate in many sections. MDT and especially LVT are quite inconspicuous in transverse sections. In horizontal or sagittal sections it is much easier to identify these tracts. As in Locusta migratoria and Carausius morosus, the second dorsal commissure (DC II) is subdivided. DC IV and DC V are difficult to separate in all cutting planes. The results are compared with published data of other insects. The present work supports the hypothesis of a common basic pattern of all insect ganglia.

Conformational, thermodynamic, and stability properties of Manduca sexta apolipophorin III.

Apolipophorin III (apoLp-III) is a major protein in hemolymph of adult Manduca sexta. Although it normally exists in a lipid-free state, during sustained flight, apoLp-III functions as an apolipoprotein, reversibly associating with the surface of lipoprotein particles. In an effort to gain a fuller understanding of this dual existence, we have investigated its solution properties using spectroscopic methods. The UV absorption spectrum of apoLp-III is distinctive owing to the absence of tryptophan and the presence of a single tyrosine residue. Circular dichroism experiments revealed an enhancement of apoLp-III alpha-helical content when spectra were obtained in 50% trifluoroethanol versus aqueous buffer. The helical content in buffer was unaffected by protein concentration, suggesting that apoLp-III exists in solution as a monomeric species. At pH values > 10 and < 4, there was a marked loss of helical content. Increasing the temperature of apoLp-III solutions also caused a loss of secondary structure, with a temperature-induced denaturation midpoint of 52 degrees C. Upon recooling of heat-denatured apoLp-III, approximately 95% of the secondary structure was restored. In guanidine HCl denaturation studies monitored by CD, a 50% transition midpoint of 0.355 M was determined, corresponding to a delta GDH2O of 1.29 kcal/mol. Fluorescence studies indicated that guanidine HCl induced an enhancement of tyrosine fluorescence emission at 300 nm when excited at 277 nm. In native apoLp-III, we propose that tyrosine fluorescence is quenched to a large extent due to a hydrophobic stacking interaction of its side chain with that of a neighboring phenylalanine residue. delta GDH2O was determined from the fluorescence data to be 2.1 kcal/mol, with a transition midpoint occurring at 0.25 M guanidine HCl. The lower concentration of guanidine HCl required to induce half-maximal tyrosine fluorescence enhancement versus the transition midpoint detected by CD may be a reflection of the fact that this residue is located near the COOH-terminal end of the protein and as such may be more susceptible to denaturation. The results presented indicate that apoLp-III assumes a relatively labile conformation in solution that appears to be partially stabilized by side chain charge-charge interactions within predicted alpha-helical segments.

Demonstration of motoneuron-12 sparing in cultured Manduca sexta ventral nerve cords.

The emergence of the adult Manduca sexta moth is accompanied by the death of half of the neurons present in the pupal abdominal nervous system (Truman, 1983). This developmental neuronal death is highly selective, so that the same neurons die at the same time relative to emergence in every moth. In the case of the MN-12 motoneurons, this cell death is regulated both by hemolymph concentrations of a steroid hormone, 20-hydroxyecdysone, and by actions exerted by adjacent ganglia (Truman and Schwartz, 1984; Fahrbach and Truman, 1987). This latter effect, which has been previously described in isolated abdomens and in moths with transected ventral nerve cords, has now been reproduced under controlled culture conditions in which the selectivity and extent of postemergence neuronal death is comparable to that seen in vivo. With respect to the MN-12 neurons found in the most anterior unfused abdominal ganglion, A3, the pterothoracic ganglion appears to be the source of a factor that permits these neurons to die according to their usual developmental schedule.

Regulation of fat body glycogen phosphorylase activity in adult Manduca sexta

Fat body glycogen phosphorylase was activated very slowly when adult tobacco hornworms, Manduca sexta, were starved. By contrast, chilling led to a significant activation of the enzyme within 2 h. This is a direct effect, since enzyme activation can also be induced in vitro. Inactivation of glycogen phosphorylase occured very rapidly in intact animals and in vitro showing that the fine-tuning of the glycogen breakdown/synthesis enzyme system favours glycogen synthesis in these animals. Activation of fat body glycogen phosphorylase in adult M. sexta at 0°C in vitro could be prevented by supplementing the saline with glucose; trehalose was ineffective. This effect of glucose was dose-dependent between 2 and 6mg/ml. The same action could not, however, be detected in larval fat body. When adult fat body was incubated at 0°C for up to 4h, no progressive activation of glycogen phosphorylase was observed in the absence of glucose suggesting that glucose is not the main regulator of enzyme activity. Glucose, however, may be a potent modulator of the glycogen phosphorylase/glycogen synthase system.

Mode of action studies on a Manduca sexta diuretic hormone

AbstractThe mode of action of a diuretic hormone from pharate adult Manduca Sexta heads, which triggers fluid loss in M. sexta larvae and Pieris rapae adults, was studied. In vivo, Mas‐DH (M. sexta diuretic hormone) decreased fluid absorption from larval recta, and increased levels of the second messenger cAMP in recta and Malpighian tubules (Mt) from larvae, and in fat body of larvae and adult M. sexta. In vitro, Mas‐DH triggered minor changes in fluid loss from adult Mt, but did not affect levels of cAMP in Mt from larvae, pharate adults, or adults, though it elevated cAMP levels in fat body of these stages. © 1992 Wiley‐Liss, Inc.

Allatotropin‐Induced formation of inositol phosphates in the corpora allata of the moth, Manduca sexta

AbstractAllatotropin (AT) is a 13 amino acid neuropeptide isolated from the tobacco hornworm, Manduca sexta, which stimulates the biosynthesis of juvenile hormone (JH) in adult females of this insect. In this paper we report that AT stimulates inositol phosphate formation in both female and male corpora allata (CA) of adult Manduca sexta. Furthermore, thapsigargin, a potent Ca2+‐ATPase inhibitor, staurosporine, a potent inhibitor of protein kinases A and C, and 2,5‐di‐(tert‐butyl)‐1,4‐hydroquinone (DBHQ), a potent inhibitor of liver microsomal ATP‐dependent Ca2+ sequestration, all stimulate JH biosynthesis without changing the basal level of inositol phosphates. These data suggest that AT activates an inositol 1,4,5‐trisphosphate (IP3) pathway. Furthermore, increases in the free intracellular Ca2+ concentration in the CA stimulate JH biosynthesis. © 1992 Wiley‐Liss, Inc.

Lipophorin structure analyzed by in vitro treatment with lipases.

Adult Manduca sexta high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-I, -II, and -III) and 52% lipid. The flight-specific low density lipophorin (LDLp) contains 62% lipid and is associated with several additional molecules of apoLp-III. The amount of phospholipid remains constant in lipophorin (140 mol/mol of lipophorin), while the diacylglycerol content varies between different lipophorin species (310 mol/mol HDLp up to 1160 mol/mol LDLp). Both lipophorin particles were enzymatically depleted of phospholipid or diacylglycerol by in vitro incubation with either phospholipase A2 or triacylglycerol lipase. Albumin was used to remove free fatty acids generated during the reaction. Treatment with phospholipase A2 removed all phospholipids (except sphingomyelin) and the resulting particles were stable. Triacylglycerol lipase hydrolyzed large fractions of diacylglycerol. The resulting particles were smaller in size, higher in density, and devoid of apoLp-III. The particles retained apoLp-I and -II and the other lipid components, including a substantial amount of diacylglycerol. Structural integrity of diacylglycerol-depleted lipophorin was confirmed by electron microscopical analysis. When treated with both phospholipase A2 and triacylglycerol lipase, lipophorin precipitated. From these results we conclude that: 1) all phospholipid and apoLp-III are located at the surface of lipophorin, whereas diacylglycerol is partitioned between the sublayers and the surface of the particle; 2) both diacylglycerol and phospholipid play a role in stabilizing lipophorin in the aqueous medium; and 3) lipophorin can be extensively unloaded and still retain its basic structure, a necessary feature for its function as a reusable lipid shuttle.

Changes in lipid and carbohydrate metabolism during starvation in adult Manduca sexta

Adult Manduca sexta feed very irregularly in the laboratory, and many adult males never feed. Feeding adults live longer and feeding females lay many more eggs; however, in both feeding (sugar water) and starving adults a decrease of metabolic reserves is observed. Carbohydrates disappear from hemolymph and from fat body. Fat body lipid also decreases, while hemolymph lipid concentration increases strongly in starving adults. The activity of fat body glycogen phosphorylase increases strongly in starving adult M. sexta. The activity of glycogen phosphorylase is correlated inversely with hemolymph sugar concentration. Injected trehalose inactivates glycogen phosphorylase within 2 h. and lowers the hemolymph lipid level within 6 h. In starving adult M. sexta, neither the activation of glycogen phosphorylase nor the increase of hemolymph lipid concentration depends on adipokinetic hormone, since cardiacectomy does not prevent the activation of glycogen phosphorylase nor the increase of hemolymph lipid level.

A comparison of adult and larval Manduca sexta apolipophorin-III

Apolipophorin-III (apoLp-III) is known to be present in the hemolymph of larval and adult Manduca sexta, but is associated with lipophorin only in the adult. We tested two hypotheses which might account for the lack of apoLp-III in larval lipophorin: (1) there might be a difference in processing of the prepro precursor between adult and larval fat body; (2) there might be larval and adult specific transcription of the apoLp-III gene. ApoLp-III isolated from adult or larval hemolymph has exactly the same amino acid composition and therefore there is not a significant amount of the pro precursor present in hemolymph in either case. We did confirm the presence of the pro precursor in the fat body and showed the site of cleavage of the prepro precursor is between ser (−6) and ala (−5). Restriction mapping, cDNA sequencing and primer extension analysis all indicated that both larval and adult apoLp-III have the same mRNA transcripts and utilize the same coding sequences of a single apoLp-III gene. The data show that neither hypothesis can explain the lack of apoLp-III in larval lipophorin and that other explanations, perhaps involving the mechanism of lipophorin assembly in the fat body, will have to be sought. It was shown that the apoLp-III gene is expressed in a cyclic manner during larval development with maximum expression occurring during the feeding period and that adult fat body contains 2- to 10-fold higher levels of the apoLp-III mRNA.

Identification of an Allatotropin from Adult Manduca Sexta

A peptide that strongly stimulates the secretion of juvenile hormone from corpora allata in vitro (allatotropin) has been purified from extracts of heads of pharate adult Manduca sexta. The primary structure of this 13-residue peptide has been determined: H-Gly-Phe-Lys-Asn-Val-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-NH(2). This neurohormone has no sequence similarity with any known neuropeptide from other organisms. Synthetic allatotropin, as well as truncation fragments, including one with the five amino terminal residues deleted, showed in vitro activity indistinguishable from that of native allatotropin.

Sexual Dimorphism in Juvenile Hormone Synthesis by Corpora Allata and in Juvenile Hormone Acid Methyltransferase Activity in Corpora Allata and Accessory Sex Glands of Some Lepidoptera

Summary 1. Corpora allata of adult Manduca sexta and Heliothis zea are sexually dimorphic in that females produce juvenile hormones (JH) while males lack JH acid methyltransferase and therefore produce JH acid. Male and female CA of Vanessa cardui contain JH acid methyltransferase.2. Male but not female reproductive tract tissues of M. sexta, H. zea, and V. cardui contain JH acid methyltransferase activity. The enzyme is absent from male and female reproductive tracts of Blaberus discoidalis, Oncopeltus fasciatus and other non-lepidopteran species.3. It is suggested that sexual dimorphism with regard to JH acid methyltransferase in CA of adults is restricted to moths and that the ability to methylate JH acids in the male reproductive tract is a characteristic of Lepidoptera.

Possible interactions of a steroid hormone and neural inputs in controlling the death of an identified neuron in the moth Manduca sexta.

The emergence of the adult Manduca sexta moth is followed by the loss of almost half of this insect's abdominal motoneurons and interneurons (Truman, 1983). This programmed cell death completes the transformation of the nervous system of the caterpillar into that of the moth. The death of these neurons has been previously shown to be a response to an endocrine signal: the decline in ecdysteroids that occurs at the end of metamorphosis (Truman and Schwartz, 1984). Our current research is focussed on the regulation of the fate of a pair of identified motoneurons, the MN-12 cells, in the third abdominal ganglion. Isolation of this ganglion from anterior parts of the nervous system can prevent the death of these cells at the time when they would normally die in response to the decline in ecdysteroids. Transection of the ventral nerve cord at various levels revealed that the source of this regulatory "death signal" is the fused pterothoracic ganglion and that it is transmitted via the interganglionic connectives. We hypothesize that the factors mediating this effect may act in concert with the ecdysteroid decline to specify the exact time of death for individual neurons.

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph.

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.

Octopamine enhances neuromuscular transmission in developing and adult moths, Manduca sexta.

The effect of octopamine on neuromuscular transmission was examined in developing and adult Manduca sexta. Intracellular recordings were made from the dorsal longitudinal muscle (DLM), superfused with solutions containing DL-octopamine or other amines. In untreated adult moths and pharate adults nearly ready to enclose (stage Day 19), stimulation of the motor nerve evokes a large excitatory junction potential (EJP), an active membrane response, and a twitch. In adults and Day 19 animals DL-octopamine (10(-7) to 10(-4)M) has no effect on the amplitude and rise-time of the electrical response in normal saline, but 10(-6) to 10(-4) M DL-octopamine increases the amplitude of the excitatory junction potential recorded in saline containing one-third the normal calcium concentration. Immature (Day 16) muscle, which normally produces only small EJPs following stimulation of its motor nerve, responds to 10(-6) to 10(-4) M DL-octopamine by an increase in the EJP above threshold for an active membrane response and a contraction. When the muscle has developed sufficiently to spike and contract in response to nerve stimulation in the absence of exogenous octopamine (Days 17 and 18), application of DL-octopamine increases the maximum rate at which the muscle contracts in response to each stimulus in a train (designated the maximum following frequency, MFF). The threshold dose for an effect on the MFF of Day 18 immature moths is less than 10(-10) M. At this stage 10(-8) M DL-octopamine increases the MFF four-fold. The effect on the MFF is dose-dependent over the range 10(-10) M to 10(-6) M. The biogenic amines DL-epinephrine, DL-norepinephrine, tyramine, DL-phenylethanolamine, 2-phenylethylamine, and dopamine, applied at concentrations of 10(-8) or 10(-4) M, do not change the MFF. Both DL-synephrine (10(-8) M) and serotonin (10(-7) M) mimic the action of 10(-10) M DL-octopamine on the MFF. The action of DL-octopamine (10(-7) M) is blocked by phentolamine (10(-4)M) but not by propranolol (10(-4)M). The octopamine content of hemolymph was determined with a radioenzymtic assay. The concentration of octopamine in the hemolymph increases 3.6-fold, from 5 X 10(-8) M on Day 18 (duration of adult development is 19 days) to 1.85 X 10(-7) M one day following eclosion.(ABSTRACT TRUNCATED AT 400 WORDS)

Relative hemolymph juvenile hormone binding capacity during larval, pupal and adult development in Manduca sexta

Hemolymph from larval, pupal and adult Manduca sexta was analyzed for juvenile hormone binding protein activity by a hydroxylapatite adsorption assay. During each of the larval instars studied, the relative binding capacity followed a cyclical fluctuation with the early part of each instar displaying relatively lower capacities than the midpart of the instar. Binding levels declined in the period preceding ecdysis to the next instar or stage. Analysis of total hemolymph protein indicated that the binding titers generally followed the increase in hemolymph protein concentration. During the pupal and adult stages, sexual dimorphism was observed, with females displaying significantly more binding per unit hemolymph than males. The highest levels of binding at any time during development were observed in the adult female hemolymph 36 hr post eclosion. Adult male hemolymph showed no corresponding increase during the same period.

Insect apolipophorin III. Purification and properties.

The hemolymph of adult Manduca sexta (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin III. Apolipophorin III was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin III was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin III is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr = 34,000.

Octopamine and chlordimeform enhance sensory responsiveness and production of the flight motor pattern in developing and adult moths

Octopamine and an agonist, chlordimeform, increase the responsiveness of adult and pharate adult Manduca sexta to gentle mechanical stimulation of the wing. Higher doses of chlordimeform elicit almost continuous production of the flight motor pattern in both adults and pharate adults, and the effect persists for more than 24 h. The dose of chlordimeform necessary for this effect increases with age. Mechanical stimulation of the wing of pharate adults elicits several cycles of flight motor pattern, but with repeated stimulation the animal habituates. Habituation is slower in chlordimeform-treated animals than in controls. Injection of octopamine (1-8 X 10(-8) mol) or chlordimeform (3 X 10(-9) mol) into the mesothoracic ganglion elicits the flight motor pattern. The excitatory actions of both compounds can be blocked by cyproheptidine. Chlordimeform (5 X 10(-8) mol) in acetone applied to the wing does not cause a noticeably greater increase in teh electrical activity of sensory neurons than does acetone applied alone; this result suggests that chlordimeform does not act on these peripheral sites or on axonal membranes in general. We suggest that chlordimeform and octopamine act on the thoracic ganglia to alter the level of excitation or effectiveness of synaptic transmission among central neurons, including those involved in producing the flight motor pattern.

Developmental changes in the response of the fat body of Manduca sexta to injections of Corpora cardiaca extracts

The Corpora cardiaca of larval and adult Manduca sexta contain glycogen phosphorylase-activating and adipokinetic activity. Depending on the age of the injected animal, injection of Corpora cardiaca extracts induces different responses. In larvae of the 1st and 2nd day of the Vth instar, phosphorylase is activated strongly by injections of Corpora cardiaca extracts, whereas in older animals it is only slightly activated. Mobilization of lipids in response to Corpora cardiaca extract injections is found only in animals injected after the imaginal molt; in younger animals this response is absent. It is argued that this difference in responsiveness may be due to a change in the properties of the target organ fat body.

Steroid regulation of neuronal death in the moth nervous system

The emergence of the adult Manduca sexta moth is followed by the programmed degeneration of about 50% of the insect's abdominal interneurons and motoneurons. Neurons in implanted ganglia undergo degeneration in concert with neurons of the host, thereby indicating that a hormonal cue is important. The neuronal death follows the normal decline in the levels of the steroid hormones, ecdysteroids, that occurs at the end of metamorphosis. Manipulations that cause a precocious ecdysteroid decline result in an early death of the neurons. Also, prolongation of the ecdysteroid titer extend the life of the neurons. By properly timed steroid application it was possible to dissociate the neuronal degeneration from the behavioral events and muscle degeneration that also occur at emergence. Also the stereotyped sequence of death among identified motoneurons could be stopped at various points in the sequence by the carefully timed application of steroid. It was concluded that the signal for neuronal death was the withdrawal of ecdysteroids at the end of metamorphosis and that the death was probably a direct response of the individual neurons to the hormone withdrawal.