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PubMed2001

The novel guanylyl cyclase MsGC-I is strongly expressed in higher-order neuropils in the brain of Manduca sexta.

Guanylyl cyclases are usually characterized as being either soluble (sGCs) or receptor (rGCs). We have recently cloned a novel guanylyl cyclase, MsGC-I, from the developing nervous system of the hawkmoth Manduca sexta that cannot be classified as either an sGC or an rGC. MsGC-I shows highest sequence identity with receptor guanylyl cyclases throughout its catalytic and dimerization domains, but does not contain the ligand-binding, transmembrane or kinase-like domains characteristic of receptor guanylyl cyclases. In addition, MsGC-I contains a C-terminal extension of 149 amino acid residues. In this paper, we report the expression of MsGC-I in the adult. Northern blots show that it is expressed preferentially in the nervous system, with high levels in the pharate adult brain and antennae. In the antennae, immunohistochemical analyses show that it is expressed in the cell bodies and dendrites, but not axons, of olfactory receptor neurons. In the brain, it is expressed in a variety of sensory neuropils including the antennal and optic lobes. It is also expressed in structures involved in higher-order processing including the mushroom bodies and central complex. This complicated expression pattern suggests that this novel guanylyl cyclase plays an important role in mediating cyclic GMP levels in the nervous system of Manduca sexta.

Animals↗
PubMed2001

A potential dual apical pathway in polarized regenerative cells of the midgut of Manduca sexta.

Two novel proteins with apparent molecular weight of 38 (Manduca sexta midgut MsM38) and 46kDa (MsM46) were isolated from midgut homogenates in wandering stage Manduca sexta larvae and both of them were found to be present exclusively in this tissue on Western blots. Immunocytochemical studies revealed that both proteins are expressed in the regenerative cells however, their distribution pattern is clearly different. MsM38 is localized in the cytoplasm of resting regenerative cells during the feeding period, and is accumulated in the calcospherits at the beginning of the wandering period. Along with the delamination of the larval epithelium, this protein is released apically from these vesicles. The antiserum labels an additional 76 kDa protein in the wandering larval midgut homogenates. The appearance of this 76 kDa protein coincides with the accumulation of the immunopositive material in the calcospherits. MsM46 is similarly distributed during the feeding period in the cytoplasm of regenerative cells. At the beginning of the wandering period it accumulates around the newly forming large apical vacuoles, that are released at the time of complete delamination of the larval epithelium. In parallel with this process MsM46, and another 40 kDa protein, that becomes labeled from this period on Western blots appeares on the apical microvillar projections. Thus both isolated proteins are directed apically from different compartments, that raises the possibility of a dual apical routing pathway in regenerative cells.

Animals↗
PubMed1995

Effect of cycloheximide on eclosion hormone sensitivity and the developmental appearance of the eclosion hormone and cGMP regulated phosphoproteins in the CNS of the tobacco hornworm, Manduca sexta.

The neuropeptide, eclosion hormone (EH), triggers ecdysis behavior at the end of each molt in Manduca sexta. Previous studies have shown that the action of EH is mediated by an increase in cGMP and is associated with the phosphorylation of two proteins, named the EGPs. The ability of insects to respond to EH is developmentally regulated with sensitivity being first seen at about 8 hr prior to the normal time of ecdysis. The EGPs are also first detectable in the CNS at 8 hr prior to ecdysis, suggesting that it is their synthesis which determines EH sensitivity. The protein synthesis inhibitor, cycloheximide was used to study the development of the events leading to pupal ecdysis in Manduca. The results of these experiments suggest that protein synthesis is necessary about 10 hr before ecdysis for both the development of EH sensitivity and for the appearance of the EGPs.

Animals↗
PubMed1999

Contribution of different bitter-sensitive taste cells to feeding inhibition in a caterpillar (Manduca sexta).

Many compounds that taste bitter to humans also inhibit feeding in insects. Caterpillars (e.g., Manduca sexta) detect these compounds with a few bitter-sensitive taste cells. This study examined the role of these taste cells in feeding inhibition. Behavioral studies demonstrated that 3 bitter compounds (caffeine, salicin, and aristolochic acid) all inhibited feeding rapidly in Manduca sexta. Electrophysiological studies revealed that each pair of bitter-sensitive taste cell differs in responsiveness to the bitter compounds. Ablation studies indicated that (a) those pairs of bitter-sensitive taste cells that responded vigorously to a particular bitter compound were sufficient to inhibit feeding on diets containing the same compound, but that (b) no pair of bitter-sensitive taste cells was necessary for inhibiting feeding. Thus, the different pairs of bitter-sensitive taste cells appear to make partially redundant contributions to feeding inhibition.

Animals↗
PubMed2000

Neurons involved in nitric oxide-mediated cGMP signaling in the tobacco hornworm, Manduca sexta.

Recently, both nitric oxide synthase (NOS), and nitric oxide (NO)-sensitive guanylyl cyclase were cloned in Manduca sexta and implicated in several cellular, developmental, and behavioral processes (Nighorn et al. [1998] J Neurosci 18:7244-7255). However, NO is a highly diffusive gas, and little is known about the range and specificity of its actions on neurons. To begin examining the role of NO as a neurotransmitter in the central nervous system (CNS) of larval Manduca, we have mapped potential NO-producing neurons using fixation-resistant NADPH-diaphorase staining and antisera that recognize a NOS-specific epitope. In addition, to detect NO-responsive neurons, we treated the CNS with NO donors and used antibodies that recognize elevated levels of cyclic 3;,5;-guanosine monophosphate (cGMP). Many potential NO-producing neurons were mapped, including the ventral unpaired median cells and three pairs of lateral cells in each abdominal ganglion. Additional neurons in the dorsal midline of ganglia A5-7 (PM2) appear to express NOS in a segment-specific manner. At the larval-to-pupal transition, this staining pattern changes; the PM2 neurons stain weakly or are undetectable and there is novel expression of NOS in cell 27. In response to NO donors, a small number of neurons produce detectable cGMP accumulation in a segment-specific pattern. These include a pair of posteriodorsally positioned interneurons (IN505) in all the abdominal ganglia, PM2 neurons in A5, and PM1 and PM2 neurons in A7. Hence, PM2 neurons in A5 and A7 are potentially capable of producing and responding to NO. These identified NO-producing and responding neurons provide a tractable model system for studying the dynamics and specificity of NO signaling in the CNS.

Animals↗
PubMed2003

Interaxonal Eph-ephrin signaling may mediate sorting of olfactory sensory axons in Manduca sexta.

We have investigated possible roles of the Eph family receptor tyrosine kinases and their ligand ephrins in the developing primary olfactory nerve pathway in the moth Manduca sexta. The Manduca homologs of the Eph receptor (MsEph) and ephrin ligand (MsEphrin) are most closely related to Drosophila Eph and ephrin, respectively. In situ labeling with Fc-fusion probes, in which IgG Fc was linked to the extracellular domain of MsEph (Eph-Fc) or MsEphrin (ephrin-Fc), reveals that both Eph receptors and ephrins are expressed on axons of olfactory receptor cells (ORCs) during their ingrowth to the primary center, the antennal lobe (AL). Interestingly, Eph receptors and ephrins are differentially distributed among identifiable glomeruli such that glomeruli with high receptor staining show little or no ligand staining, and vice versa, suggesting a complementary Eph-ephrin expression by subsets of ORC axons innervating a particular set of glomeruli. In contrast, neither Eph receptors nor ephrins are detectable in intrinsic components of the AL. In vitro, ephrin-Fc and Eph-Fc, when present homogeneously in the substratum, inhibit neurite outgrowth from olfactory epithelial explants. Moreover, in patterned substratum, neurites growing on the standard substratum turn or stop after encountering the test substratum containing ephrin-Fc. These in vitro observations indicate that MsEphrin can act as an inhibitor/repulsive cue for ORC axons. Based on results from in situ and in vitro experiments, we hypothesize that Eph receptors and ephrins mediate axon sorting and fasciculation through repulsive axon-axon interactions.

Amino Acid Sequence↗
PubMed1996

Habituation and dishabituation of the proleg withdrawal reflex in larvae of the sphinx hawk, Manduca sexta.

The neural circuit for the proleg withdrawal reflex (PWR) of Manduca sexta larvae exhibits activity-dependent plasticity. This study demonstrates habituation and dishabituation of PWR behavior. Repeated deflection of 1 or 5 mechanosensory hairs on a proleg at a 60-s interstimulus interval (ISI) for 20 trials produced a significant decrease in evoked force of proleg withdrawal. Habituation was similar with 30-s and 60-s ISIs and nonsignificant with a 5-min ISI or without repeated stimulation. Habituated responses recovered after cessation of stimulation or pinch of the body wall (dishabituation). The role of intersegmental influences was tested by severing connectives to leave different numbers of ganglia connected to the test segment. Habituation was robust even in test segments that were disconnected from the rest of the central nervous system. The PWR of Manduca is appropriate for further analysis of cellular mechanisms underlying learning.

Animals↗
PubMed1991

Structure-activity studies on adipokinetic hormones in Manduca sexta.

Structure-activity studies were performed for adipokinetic hormone (AKH) in Manduca sexta. Seven naturally occurring and four synthetic peptides of the red pigment concentrating hormone (RPCH)/AKH family were tested in larvae of M. sexta for activation of glycogen phosphorylase in fat body. pGlu at the N-terminal was found to be important for activity of peptides; however, Manduca AcGly1AKH is partially active. The amino acids at all positions appear to be of importance for activity, with the possible exception of the two serine residues in positions six and seven. Generally, the more amino acids are exchanged, the less the peptide will bind to the receptor. In M. sexta a beta-bend appears not to be important for the binding of peptides. Peptides ten amino acids long appear to be more active than shorter ones.

Amino Acid Sequence↗
PubMed1981

Anatomical and neurochemical consequences of deafferentation in the development of the visual system of the moth Manduca sexta.

The primordium of the compound eye of the moth Manduca sexta was removed by surgery within 12 hours after the molt to the pupa. When the operated animals were examined as pharate adults just prior to emergence from the pupal cuticle, no eye tissue was present on the operated side. Histological examination of the brain at successive developmental stages showed that the volume of laminar neuropil on the operated side increased very little after deafferentation while the volume of the control-side laminar neuropil increased more than 20-fold. The impairment of development of the lamina was accompanied by a reduction in the synthesis and storage by the optic lobe of two neurotransmitter candidates, acetylcholine and 5-hydroxytryptamine. The endogenous content of 5-hydroxytryptamine was also reduced in these preparations. Excision of a section of the stemmatal nerve, which connects the primordium of the compound eye to the brain, also arrested the development of the lamina, but in this case the compound eye itself developed apparently normally without making contact with the brain. This finding supports the hypothesis that the centripetal growth of photoreceptor-cell axons normally is contact-guided. Results of neurochemical experiments on retinal tissue argue against the idea that acetylcholine, 5-hydroxytryptamine, or gamma-aminobutyric acid is a likely photoreceptor-cell neurotransmitter. From these and our previous studies of the metamorphosing brain in Manduca we conclude that different classes of neurons in the same central nervous system can exhibit widely different degrees of dependency on their normal innervation for their survival and differentiation.

Acetylcholine↗
PubMed1993

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.

Animals↗
PubMed1986

Effects of octopamine, dopamine, and serotonin on production of flight motor output by thoracic ganglia of Manduca sexta.

Effects of biogenic amines on a centrally generated motor pattern in Manduca sexta were examined by pressure injecting nanomole to micromole amounts of octopamine, dopamine or serotonin into thoracic ganglia. Motor output was recorded extracellularly from a pair of antagonistic flight muscles and their motor neurons. The monoamines were found to alter production of a motor pattern that produces rhythmic wing flapping (10 Hz) and exhibits phase relationships similar to those in the flight pattern of intact moths. In mesothoracic ganglia with sensory nerves intact, octopamine (4 X 10(-9) mol) injected into lateral regions evoked regular firing of a single motor neuron, whereas a higher dose (4 X 10(-8) mol) often elicited the flight motor pattern. In the absence of sensory input, these doses of octopamine had little effect. Low doses (10(-10) mol) greatly enhanced motor responses to electrical stimulation of a wing sensory nerve. Dopamine (2 X 10(-10) mol) injected into the medial region of the mesothoracic ganglion elicited the flight motor pattern in the presence or absence of sensory input. Rhythmic output induced by dopamine (5 X 10(-10) mol) was suppressed by injecting serotonin (5 X 10(-10) mol) into the same region. These findings demonstrate that dopamine, octopamine, and serotonin have different effects on motor output in Manduca and suggest that these amines are involved in initiating, maintaining and terminating flight behavior, respectively. Octopamine may elicit flight production by enhancing the efficacy of sensory transmission thereby increasing excitability or arousal. Dopamine may act on interneurons involved in generating the flight motor pattern.

Animals↗
PubMed1989

Respecification of larval proleg motoneurons during metamorphosis of the tobacco hornworm, Manduca sexta: segmental dependence and hormonal regulation.

The principal locomotory appendages of the Manduca sexta caterpillar, the prolegs, are present on the third through sixth abdominal segments (anal prolegs located on the terminal segment were not included in this study). Previous studies have characterized some of the proleg retractor muscles and their motoneurons. In the present study we identified additional proleg motoneurons and their putative homologs in the non-proleg-bearing segments. One of the motoneurons present in the proleg-bearing segments is absent in the non-proleg-bearing segments. At pupation the prolegs are lost, their muscles degenerate, and some of their motoneurons regress structurally. Subsequently, subsets of the proleg motoneurons and their homologs in other segments die in a segment-specific pattern. This is the first report of segment-specific motoneurons, and of segment-specific death of identified motoneurons, in Manduca. During adult development the surviving proleg motoneurons innervate the tergosternal muscle (TSM) and grow bilateral dendritic arbors. Dendritic growth is completed by about the 12th of the 18 days of adult development. Following adult emergence all but one of the respecified proleg motoneurons dies. The hormonal dependence of dendritic outgrowth was tested by isolating abdomens to eliminate the ecdysteroid-secreting glands in the thorax. Between the second and fifth days after pupation the motoneurons became progressively more competent to undergo dendritic outgrowth following abdomen isolation. The extent of dendritic outgrowth paralleled the degree of morphological development attained by isolated abdomens. It is concluded that ecdysteroids are required for motoneuron outgrowth, but our findings suggest that, unless an abdominal source of ecdysteroids exists in pupae, a relatively small exposure may be sufficient.

Animals↗
PubMed1994

Isolation and partial characterization of a gene from trachea of Manduca sexta that requires and is negatively regulated by ecdysteroids.

The molting cycle in insects is regulated by the fluctuating levels of ecdysteroids. Twenty-four hours prior to the pupal molt in Manduca sexta steroid titers are relatively high and they fall to very low levels at 4 hr before the molt. Several physiological events accompanying molts have been shown to require the rise and then the subsequent decline of ecdysteroid levels. These events also require the expression of a new set of genes. We used subtractive hybridization to isolate genes that are expressed 4 hr, but not 24 hr, prior to the pupal molt in the nervous system of Manduca. These genes are expected to play a role either in ecdysis behavior or in the development of pupal/adult structures. The first such gene we have identified, esr20 (EcdySteroid Regulated) encodes a 20-kDa protein. The deduced amino acid sequence of the protein shows similarity to acidic domains in three urelated proteins, chick nucleolin, rat heavy-molecular-weight neurofilament, and the Drosophila steroid orphan receptor, knirps-related. The similarity among the four proteins appears to be in regions implicated in protein-protein interactions. The accumulation of esr20 mRNA starts 16 hr prior to the pupal molt and abruptly stops at ecdysis. Expression of esr20 was localized to tracheal epithelial cells within all tissue types, and the transcript was present prior to the larval, pupal, and adult molts only after the molting peak of ecdysteroids had declined. Its expression was inhibited in the presence of ecdysteroids in vitro and required protein synthesis.

Amino Acid Sequence↗
PubMed1986

An accessory olfactory pathway in Lepidoptera: the labial pit organ and its central projections in Manduca sexta and certain other sphinx moths and silk moths.

In the hawkmoth, Manduca sexta, the third segment of each labial palp contains a pit, which houses a densely packed array of sensilla. We have named this structure the labial pit organ (LPO). The sensilla within the pit are typical of olfactory receptors, characterized by a grooved surface, wall pores, and pore tubules. Axons arising from receptor cells that innervate these sensilla project bilaterally to a single glomerulus in each antennal lobe. We have compared this central projection with that in three other species of Manduca (M. quinquemaculata, M. dilucida, and M. lanuginosa) and in the silkmoths Antheraea polyphemus and Bombyx mori. A bilateral projection to a single glomerulus in each antennal lobe is present in all cases. We suggest that the LPO serves as an accessory olfactory organ in adult Lepidoptera.

Animals↗
PubMed1988

Iron binding proteins and their roles in the tobacco hornworm, Manduca sexta (L.).

Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When 59Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle. Studies of the hemolymph disclosed two iron-containing proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 micrograms of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin in Manduca sexta, the latter serving a storage function during development and possibly also a transport function.

Animals↗
PubMed1993

A 28-kDa cerebral neuropeptide from Manduca sexta: relationship to the insect prothoracicotropic hormone.

1. A 28-kDa peptide from the brain of the tobacco hornworm, Manduca sexta, was purified via HPLC. The peptide copurified with the insect neurohormone, prothoracicotropic hormone (PTTH), through two HPLC columns. 2. Immunocytochemistry using polyclonal antibodies against the 28-kDa peptide revealed that the peptide was produced in the same protocerebral neurons that produce PTTH. Western blot analysis demonstrated that the 28-kDa peptide and big PTTH are different molecules. 3. A PTTH in vitro bioassay indicated that despite having chromatographic properties similar to those of big PTTH and being produced by the same neurons, the 28-kDa peptide did not have PTTH activity. 4. Amino acid sequence analysis yielded a 27 N-terminal amino acid sequence that had no similarity with known peptides. 5. Immunocytochemical studies revealed that the 28-kDa peptide is present as early as 30% embryonic development and is absent by adult eclosion. This is in contrast to big PTTH, which is expressed throughout the Manduca life cycle. 6. These data suggest that the 28-kDa peptide is another secretory phenotype of the lateral neurosecretory cell group III (L-NSC III) which may have functions distinct from those for big PTTH or may act synergistically with big PTTH.

Animals↗
PubMed1975

Properties of cyclic nucleotide phosphodiesterase in the central nervous system of Manduca sexta.

The properties of cyclic nucleotide phosphodiesterase were studied in soluble and particulate fractions from the central nervous system of Manduca sexta (Lepidoptera: Sphingidae). It was determined that: (1) The highest levels of phosphodiesterase occur in nervous tissue. (2) The total and specific enzyme activities of larval and adult brains are greater than those of the remaining ganglia. (3) Specific central nervous sy stem phosphodiesterase activities of the adult are lower than those of the larva, but both protein and total phosphodiesterase contents are considerably greater in the adult central nervous system. (4) Mg2+ is not absolutely required for either cyclic AMP-phosphodiesterase or cyclic GMP-phosphodiesterase activity. (5) Phosphodiesterase is inhibited by a variety of physiological and non-physiological compounds, nucleoside triphosphates being particularly effective; Some potent inhibitors of mammalian phosphodiesterase are comparatively ineffective toward Manduca sexta phosphodiesterase. (6) Kinetic analyses of soluble and particulate phosphodiesterase revealed non-linear double-reciprocal plots for the hydrolysis of both cyclic AMP and cyclic GMP, with Michaelis constants of approximately 10 mu M and 20 mu M; (7) The hydrolysis of both cyclic nucleotides appears in part to be the function of a single enzyme or related enzymes in the insect central nervous system. It follows that the intracellular level of one cyclic nucleotide may influence the concentration of the other by inhibiting its DEGRADATION.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Phosphoprotein phosphatase in the central nervous system of Manduca sexta.

The existence and some enzymological properties of phosphoprotein phosphatase (EC 3.1.3.16) have been established in the larval central nervous system of the tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae). A simple, sensitive and reproducible assay employing 32-P-labeled protamine as a phosphoprotein substrate was employed to measure phosphatase activity in both soluble and particulate fractions of the insect nerve cord. The specific activity of soluble phosphatase in the Manduca sexta central nervous system is of the same order of magnitude as that in mammalian brain. Nerve cord phosphoprotamine phosphatase activity may be stimulated by a variety of monovalent salts, the optimal concentration of NaCl or KCl being 0.2 molar. Activity does not appear to be dependent on bivalent metals and is stimulated by EDTA. A reduced sulfhydryl group is obligatory for maximum activity. Phosphatase could be greatly inhibited by sodium fluoride, ATP and GTP. Cyclic AMP and cyclic GMP are without effect on enzyme activity. Although most of the phosphatase activity in the insect nerve cord appears to be of cytosolic origin, much latent activity can be unmasked by incubating membranous fractions with Triton X-100. In contrast to soluble phosphatase, the detergent-solubilized activity is moderately stimulated by Mn-2+.?

Animals↗