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PubMed2000

Octopamine mimics the effects of parasitism on the foregut of the tobacco hornworm Manduca sexta.

The parasitic braconid wasp Cotesia congregata lays its eggs inside the body of the larval stage of its host, the moth Manduca sexta. The Cotesia congregata larvae develop within the hemocoel of their host until their third instar, when they emerge and spin cocoons and pupate on the outer surface of the caterpillar. From this time until their death approximately 2 weeks later, the Manduca sexta larvae show striking behavioral changes that include dramatic declines in spontaneous activity and in the time spent feeding. Coincident with these behavioral changes, it is known that octopamine titers in the hemolymph of the host become elevated by approximately 6.5-fold. Octopamine is an important modulator of neural function and behavior in insects, so we examined hosts for neural correlates to the behavioral changes that occur at parasite emergence. We found that, in addition to the changes reported earlier, after parasite emergence (post-emergence), Manduca sexta larvae also showed marked deficits in their ability to ingest food because of a disruption in the function of the frontal ganglion that results in a significant slowing or the absence of peristaltic activity in the foregut. This effect could be produced in unparasitized fifth-instar larvae by application of blood from post-emergence parasitized larvae or of 10(-6)mol l(-1)d,l-octopamine (approximately the level in the hemolymph of post-emergence larvae). In contrast, blood from parasitized larvae before their parasites emerge or from unparasitized fifth-instar larvae typically had no effect on foregut activity. The effects of either post-emergence parasitized blood or 10(-6)mol l(-1) octopamine could be blocked by the octopamine antagonists phentolamine (at 10(-5)mol l(-1)) or mianserin (at 10(-7)mol l(-1)).

Animals↗
PubMed2001

The role of low levels of juvenile hormone esterase in the metamorphosis of Manduca sexta.

The activity of juvenile hormone esterase (JHE) in feeding fifth instar larvae of Manduca sexta increases gradually with larval weight and rises to a peak after larvae pass the critical weight when juvenile hormone secretion ceases. Starvation of larvae of Manduca sexta (L.) that had exceeded the critical weight inhibited peak levels of JHE, but did not delay entry into the wandering stage when larvae leave the plant in search of a pupation site. This suggests that peak levels of JHE may not be essential for the normal timing of metamorphosis. Starved larvae pupated normally, indicating the peak of JHE was not necessary for a morphologically normal pupation. Treatments of larvae with the selective JHE inhibitor O-ethyl-S-phenyl phosphoramidothiolate (EPPAT) that began immediately after larvae achieved the critical weight (6.0 to 6.5 grams for our strain of Manduca) delayed entry into the wandering stage. By contrast, EPPAT treatment of larvae at weights above 8.0 g had no effect on the subsequent timing of the onset of wandering. Therefore, although the normal timing of the onset of wandering does not require peak levels of JHE, it requires low to moderate levels of JHE to be present until larvae reach a weight of about 8.0 g.

Animal Nutritional Physiological Phenomena↗
PubMed1998

The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta.

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCalpha1 and MsGCbeta1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NO-sGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.

Animal Structures↗
PubMed1990

Biological effects of synthetic AKH in Manduca sexta and estimates of the amount of AKH in corpora cardiaca.

Dose-response curves were measured with synthetic Manduca adipokinetic hormone (AKH) for glycogen phosphorylase activation in larvae and for lipid mobilization in adults. Both responses are known hormonal functions in Manduca sexta. In ligated larvae, full activation of glycogen phosphorylase was achieved with 0.1 pmol and half-maximal activation with 0.03-0.04 pmol. Maximal lipid mobilization in adults required 10 pmol and half-maximal mobilization 0.15 to 0.2 pmol, respectively. An estimate of AKH content of corpora cardiaca from M. sexta was gained by comparing the dose-response curves for synthetic Manduca AKH with curves from gland extracts. Corpora cardiaca extracts were also quantitated by high performance liquid chromatography. According to both estimates corpora cardiaca of adults contain 10-20 pmol AKH per pair, while a pair of larval corpora cardiaca contains 0.7-2 pmol.

Adipose Tissue↗
PubMed1991

Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N-acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle.

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N-acetyldopamine (NADA) to N-acetylnorepinephrine (NANE). Either heat treatment (70 degrees C for 10 min) or addition of phenylthiourea (2.5 microM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N-acetylcysteine in the NADA-cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA-quinone-N-acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of beta-methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N-beta-alanyldopamine (NBAD)-Manduca enzyme reaction was carried out in the presence of L-kynurenine, two diastereoisomers of NBAD quinone methide-kynurenine adduct (= papiliochrome IIa and IIb) could be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Localization of immunoreactive ubiquitin in the nervous system of the Manduca sexta moth.

Selective neuronal death is a normal component of metamorphosis in the moth, Manduca sexta. In particular, the three unfused abdominal ganglia of the ventral nerve cord serve as a useful experimental preparation in which to study the regulation of the molecular mechanisms that mediate programmed cell death. Ubiquitin, a highly conserved 76-amino acid protein found in all eukaryotic cells, has previously been shown to be present in increased amounts in some tissues undergoing programmed cell death (e.g., larval intersegmental muscles in Manduca sexta moths, dying cells in developing tunicates), but not in others (T-cells, Drosophila ommatidial cells, cultured sympathetic neurons deprived of nerve growth factor). It has been hypothesized that the need for ubiquitin-dependent proteolysis is increased in dying cells, and that the accumulation of ubiquitin might serve as an early marker for cells committed to die. Immunohistochemical localization of ubiquitin at the light microscopic level in the abdominal ganglia of Manduca sexta suggests that this protein plays a number of important roles in neuronal physiology and may be associated with the death of some neurons in this tissue. The most intense staining of neuronal cytoplasm, however, was found not in dying neurons, but instead in sets of persisting neurons that may serve a primarily neurosecretory or neuromodulatory function. The staining obtained in these cells with antibodies directed against ubiquitin was developmentally regulated.

Abdomen↗
PubMed1989

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.

Animals↗
PubMed1993

Ultrastructure of prothoracic glands during larval-pupal development of the tobacco hornworm, Manduca sexta: a reappraisal.

The structure of Manduca sexta prothoracic glands was investigated using a protocol that preserves membranes. During the last larval stadium, prothoracic gland cells increase in diameter, volume, protein content, and perhaps number, enhancing their capacity to produce ecdysteroids. The glands' strand-of-cells morphology, their in situ location, the presence of gap junctions between cells, and junctional foot-like structures within cells support previous findings that prothoracicotropic hormone stimulates ecdysteroidogenesis via Ca(2+)-induced Ca2+ release. A different method of tissue fixation from that previously used to investigate the ultrastructure of Manduca sexta prothoracic glands has revealed a significantly different ultrastructure. These new findings begin to define roles for endoplasmic reticulum and mitochondria in ecdysteroid synthesis and support the hypothesis that the glands secrete the steroid hormone via exocytosis. The structural dynamics of the glands are discussed in the context of the glands' function during Manduca sexta larval-pupal development.

Animals↗
PubMed1990

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

Fifth instar larvae of the tobacco hornworm, Manduca sexta, tolerate 1-h exposures to temperatures as high as 42 degrees C. Above 42 degrees C, survival declines rapidly to 18% at 44 degrees C and 0% at 48 degrees C. As in other insects, the heat-shock response of Manduca sexta involves the induction of synthesis of heat-shock proteins very similar in size to the Drosophila heat-shock proteins (84, 73, 71, 27, 25, 23, and 22 kd). In the epidermis, heat-shock protein synthesis peaks at 42 degrees 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.

Animals↗
PubMed1994

Physical characteristics of the cerebral big prothoracicotropic hormone from Manduca sexta.

The prothoracicotropic hormones (PTTHs) are cerebral peptides that control insect postembryonic development by stimulating the prothoracic glands to synthesize ecdysteroids. In Manduca sexta, the tobacco hornworm, two classes of PTTH are distinguished by their M(r), small (ca. 7 kDa) and big PTTH (ca. 25-30 kDa). Little is known about the physical nature of the PTTHs and this study takes a first step towards defining characteristics of the Manduca big PTTH. The neurohormone has a Stokes radius of 2.59 nm and a sedimentation coefficient of 2.76 S. Based on these data, an M(r) of 29,443.7 and an f/fo of 1.27 were calculated. Combined, the physical data reveal Manduca big PTTH is an asymmetrical acidic homodimeric peptide with intra- and intermolecular disulfide bonds.

Animals↗
PubMed1988

Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes.

A genomic clone was isolated from the tobacco hornworm, Manduca sexta, by virtue of its similarity to a Drosophila larval cuticle gene. RNA analysis shows that this clone, B311, is expressed at times appropriate for a larval cuticle gene. Hybrid-selection experiments using B311 DNA show that it encodes a 14 x 10(3) Mr protein, LCP-14, which is precipitated by an antiserum to Manduca larval cuticle. We have sequenced both genomic and cDNA clones for the LCP-14 gene. A conceptual translation of the cDNA sequence shows that the LCP-14 protein is similar not only to another Manduca cuticle protein, but also to Drosophila, Sarcophaga and Hyalophora cecropia cuticle proteins. Since these proteins are found in flexible cuticle and have similar sequences, we conclude they are encoded by homologous genes.

Animals↗
PubMedDate not supplied

Immunoaffinity purification of the neuropeptide prothoracicotropic hormone from Manduca sexta.

The prothoracicotropic hormones (PTTH) are cerebral peptides that control insect postembryonic development by stimulating the prothoracic glands to synthesize ecdysteroids. Using immunoaffinity chromatography and SDS-PAGE, a 25.5 kDa big PTTH has been purified from Manduca sexta. Based upon SDS-PAGE and Western blot analysis, the native form of big PTTH appears to be a dimer with monomers of 16.5 kDa. Four HPLC-separated fragments of this acidic peptide were sequenced and exhibited no sequence similarity with Bombyx mori PTTH. In agreement with this finding, the basic Bombyx PTTH had no PTTH bioactivity in Manduca. One sequenced fragment of the Manduca PTTH is approximately 70% similar to the vertebrate cellular retinoid binding proteins, suggesting these binding proteins may be present in insects.

Amino Acid Sequence↗
PubMed1998

Intracellular calcium in prothoracic glands of Manduca sexta.

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.

Journal Article↗
PubMed2002

Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta.

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.

Animals↗
PubMed1985

Insect cardioactive peptides. I. Distribution and molecular characteristics of two cardioacceleratory peptides in the tobacco hawkmoth, Manduca sexta.

Using an in vitro heart bioassay, the pharmacological and biochemical properties of two cardioactive peptides derived from neural tissues of the moth, Manduca sexta, were analysed. Gel filtration of ventral nerve cords (VNC) from pharate adults identified two cardioacceleratory peptides (CAP1 and CAP2) with apparent molecular weights of 1000 and 500 Da, respectively. Both CAPs were localized to the abdominal perivisceral organs, the major neurohaemal release sites in the insect VNC. Pulse application of CAP1 or CAP2 on the in vitro Manduca heart produced a dose-dependent increase in rate but had no effect on beat amplitude. The threshold dose for the action of each peptide on the isolated heart bioassay was less than 0.05 abdominal nerve cord equivalents. Both CAPs were present in the pharate adult VNC of several other Lepidopteran species. Neither CAP1 nor CAP2 was detected in the prepupal VNC of Manduca sexta.

Animals↗
PubMed1995

Feeding behavior in the nocturnal moth Manduca sexta is mediated mainly by blue receptors, but where are they located in the retina?

The spectral sensitivity of nectar feeding by adults of the tobacco hawkmoth Manduca sexta was measured in free-choice experiments. The action spectrum displayed a narrow peak at 450 nm and a low secondary maximum at 560 nm. Thus, the feeding response is mediated primarily by blue-sensitive receptors containing the Manduca sexta photopigment P450, while green-sensitive receptors containing P520 play a minor role. A minimum at 500 nm separating the two peaks suggests mutual inhibition between green and blue receptors or negative interaction more proximally in the visual system. The action spectrum drops off abruptly at 400 nm, in accordance with an earlier finding that ultraviolet wavelengths, discerned by receptors containing P357, obstruct the feeding response. The spectral sensitivity of the Manduca sexta compound eye, determined by electroretinogram recordings, and earlier visual pigment measurements indicate that approximately 75 % of the receptors are green-sensitive, with the remainder divided between blue- and ultraviolet-sensitive cells. The distribution of receptor types in small areas of the retina was measured by their ultrastructural response to light. Green and ultraviolet receptors were found, but not the blue receptors that dominate the feeding response. Possibly they are concentrated in a particular region of the retina that has not yet been found.

Journal Article↗
PubMed1988

Monoclonal antibodies reveal cell-type-specific antigens in the sexually dimorphic olfactory system of Manduca sexta. I. Generation of monoclonal antibodies and partial characterization of the antigens.

The olfactory system of the moth Manduca sexta is sexually dimorphic. Male moths possess a male-specific olfactory "subsystem," comprising olfactory receptor cells (ORCs) and CNS neurons and synaptic areas associated with the detection of female sex pheromones, in addition to elements common to males and females. In order to explore the molecular differences between cells that subserve the sexual dimorphism and odor-specificity of components of the olfactory system, we generated monoclonal antibodies (Mabs) against tissue of the olfactory system of the moth. In 2 fusions, we screened 1105 hybridoma lines and obtained 272 lines that secreted antibodies against Manduca nervous tissue, as assayed immunocytochemically on sections of the primary olfactory center (the antennal lobe) in the brain of Manduca. We describe here 3 classes of Mabs exemplifying the several cell-type-specific antibodies obtained through the screening procedure. Seven hybridoma lines secrete antibodies that specifically recognize cell bodies, axons, and initial segments of dendrites of many or all ORCs of both males and females (classified as olfactory-specific antibodies, OSAs). Electron-microscopic studies of 2 of the Mabs in this class showed that they recognize antigens associated with the cell membrane and that the immunoreactive ORC axons are bundled together in fascicles in the antennal nerve. On immunoblots, one of the OSA Mabs recognizes 3 distinct protein bands of apparent Mrs 42,000, 59,000, and 66,000 Da. When tissue samples enriched in either receptor cell bodies, dendrites, and initial segments of axons or in distal segments of axons and their terminals and synapses were extracted separately, different patterns of bands were detected--42,000 and 59,000 Da bands from cell bodies and initial segments of axons and dendrites, and 42,000 and 66,000 Da bands from distal segments of axons and their terminals--suggesting that the 59,000 Da protein is modified to the 66,000 Da protein during axonal transport. The second Mab we describe here, the male olfactory-specific antibody (MOSA), selectively recognizes the sexually dimorphic ORCs that are present only in males. The antigen recognized by this antibody is found in cell bodies, dendrites, axons, and axon terminals. By electron-microscopic immunocytochemistry, the MOSA immunoreactivity is found in the cytoplasm and appears not to be associated with particular subcellular organelles. This antibody demonstrates that male-specific ORCs are molecularly distinct from other types of ORCs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Imaginal cell-specific accumulation of the multicatalytic proteinase complex (proteasome) during post-embryonic development in the tobacco hornworm, Manduca sexta.

The multicatalytic proteinase complex is a multi-subunit, high molecular weight proteinase present in the nucleus and cytoplasm of eukaryotic cells. This catalytic complex is involved in diverse cellular functions as part of the ubiquitin proteolysis system, including non-lysosomal proteolysis, antigen presentation, cell cycle progression, and cell proliferation, and in the programmed death of intersegmental muscles after adult eclosion in the tobacco hornworm moth, Manduca sexta. We have investigated the distribution of the multicatalytic proteinase complex in the central nervous system of this moth. At all stages of post-embryonic development, most cell types exhibited consistent, low levels of cytoplasmic and nuclear immunoreactivity for the multicatalytic proteinase complex. High levels of cell-specific accumulation of the complex were, however, demonstrated in abdominal neurosecretory cells and in imaginal cells in the larval brain, the larval segmental ganglia, and the developing wing discs. Imaginal cells exhibited intense immunoreactivity for the multicatalytic proteinase complex only until the onset of terminal differentiation. Intersegmental muscles undergoing programmed cell death exhibited intense cytoplasmic immunoreactivity for the multicatalytic proteinase, while persisting flight muscles and dying neurons were characterized by basal levels of staining. These staining patterns suggest that the multicatalytic proteinase of Manduca sexta serves multiple functions and is associated with the period of developmental arrest displayed by imaginal cells prior to metamorphosis.

Animals↗