Steroid regulation of octopamine expression during metamorphic development of the moth Manduca sexta.

Octopamine (OA), a biogenic amine similar to norepinephrine, has profound and well-documented actions on the nervous systems of invertebrates. In the insect, Manduca sexta, we examined the developmental plasticity of OA synthesis, studied its endocrine regulation, and observed previously undescribed OA-immunoreactive (ir) neurons. We found that levels of tyramine beta-hydroxylase (TbetaH), an essential enzyme for the biosynthesis of OA, increase during metamorphosis. Based on the established and influential roles of the steroid hormone 20-hydroxyecdysone (20-HE) during development, we tested the hypothesis that increases in TbetaH levels and OA immunoreactivity are regulated by the rise in 20-HE occurring during pupal-adult development. We determined that the levels of TbetaH in the terminal abdominal ganglion (neuromeres 6-9) remain at a constant level during pupal development and the early stages of adult development. Beginning at ca. pupal stage 8, however, the levels of TbetaH begin to rise, reaching a maximum level by pupal stage 12. By removing the source of ecdysteroid hormone through ligation, and by subsequent replacement of 20-HE via infusion, we found evidence indicating that the preadult rise of 20-HE is both necessary and sufficient for the increased levels of TbetaH. During the course of our study, we also identified previously unreported OA-ir neurons. In particular, adult-specific OA-ir lateral cells were found, as were relatively small OA-ir dorsal median pairs that doubled in size during adult development. Abdominal ganglia not exposed to the preadult rise in 20-HE possessed neither the OA-ir lateral neurons nor the somatic growth of the smaller OA-ir median neurons. These newly described OA-ir neurons probably contribute to the steroid-induced elevations of TbetaH observed at the end of metamorphosis.

Characterization and developmental regulation of tyramine-beta-hydroxylase in the CNS of the moth, Manduca sexta.

Octopamine (OA) is present in insect nervous tissue, but little is known about its biosynthesis. In the CNS of Manduca sexta, OA levels increase markedly during postembryonic adult development. To study this increase, we developed an assay for tyramine-beta-hydroxylase, the putatively rate-limiting enzyme for OA biosynthesis. Tyramine-beta-hydroxylase activity in extracts of M. sexta CNS tissue: (1) was time- and protein-dependent, and with protein concentrations up to 2 microg/microl, was linear for 20 min; (2) had a pH optimum of 7.0 for conversion of tyramine to OA; (3) required ascorbate, copper, and catalase; and (4) had an apparent K(M, tyramine) of 0.22+/-0.04 mM. These characteristics resemble those of the mammalian enzyme dopamine-beta-hydroxylase, suggesting that these two enzymes are functionally related. During adult development, tyramine-beta-hydroxylase activity increased 11-fold in the brain and 9-fold in the abdominal ganglia, paralleling increases in OA levels in those CNS structures during metamorphosis. The apparent kinetic constants of tyramine-beta-hydroxylase suggested that the amount of this enzyme present in the tissues increases. The increase in OA levels during adult development thus appears to be due to an increase in the level of enzyme available for OA synthesis and may reflect an increase in the number of octopaminergic neurons.

Characterization of crustacean cardioactive peptide-like immunoreactivity in the horseshoe crab, Limulus polyphemus.

The localization of crustacean cardioactive peptide-like immunoreactivity in the horseshoe crab Limulus polyphemus was investigated with enzyme-linked immunosorbent assay and fluorescence microscopy. Immunoreactivity was quantified in the opisthosomal nervous system (67.7 +/- 11.4 ng/g), cardiac ganglion (45.0 +/- 10.3 ng/g), prosomal nervous system (28.5 +/- 6.6 ng/g), and midgut (24.6 +/- 6.7 ng/g). In the brain, immunoreactive somata were observed in ganglion cells of the central body, in the medullary group and within the ventral medial group. Clusters of immunoreactive cells were found in each of the circumesophageal, pedal ganglia, and in the opisthosomal, abdominal ganglia. In the periphery, immunoreactive varicose fibers were observed in branches of the intestinal nerves, and near longitudinal and circular muscle fibers of the midgut. Immunoreactivity was observed in the cardiac ganglion and myocardium of the neurogenic heart. Synthetic crustacean cardioactive peptide had slight excitatory effects on the cardiac rhythm at doses up to 10(-6) M. This peptide had excitatory effects on the midgut at nanomolar doses. Ventral nerve cord extracts were partially purified with reverse phase high performance liquid chromatography. Two regions of immunoreactivity were detected, one of which coeluted with the authentic peptide. The distribution of crustacean cardioactive peptide immunoreactivity is compared with other transmitter systems in the Limulus nervous system, and myotropic actions of this peptide are discussed with respect to peptidergic modulation of intestinal motility.

Crustacean cardioactive peptide in the sphinx moth, Manduca sexta.

The isolation, identification, and actions of crustacean cardiactive peptide (CCAP) have been examined in the sphinx moth Manduca sexta. A sensitive and specific enzyme-linked immunosorbent assay (ELISA) was used to quantify CCAP-like immunoreactivity in the nervous system. The CCAP-like immunoreactivity from the abdominal CNS was then purified, and its sequence was ascertained by amino acid analysis, mass spectral analysis, and HPLC. These studies showed that the nervous system of M. sexta contains a peptide with the sequence Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, identical to CCAP originally isolated and sequenced from the shore crab Carcinus maenas. The actions of CCAP on the isolated heart of M. sexta and the extensor-tibia muscle of Schistocerca americana were tested. Crustacean cardioactive peptide had excitatory actions on both preparations: a dose-dependent increase in the rate of contractions was observed on the heart, and an increase in the rate of the myogenic rhythm was observed in the leg muscle. Moreover, purified and synthetic CCAP had identical effects on the isolated heart. We conclude that CCAP occurs in M. sexta and exerts potent neurotransmitter or neurohormonal actions on a variety of muscles.

Immunoreactivity in Limulus: III. Morphological and biochemical studies of FMRFamide-like immunoreactivity and colocalized substance P-like immunoreactivity in the brain and lateral eye.

FMRFamide-like immunoreactivity (FLI) and the colocalization of FMRFamide and substance P-like (SPLI) immunoreactivities were examined in the brain and lateral eye of the horseshoe crab with FITC- and TRITC-labeled secondary antibody techniques. In the brain, fibers with FLI were localized in the neuropils of the lamina, medulla, central body, corpus pedunculatum, optic tract, circumesophageal connective, and central neuropil. An extensive network of reactive fibers innervatives the brain's vascular sheath. Somata with FLI were found in the dorsal medial group, dorsal lateral posterior groups #1 and #2, and ventral posterior lateral groups #1 and #2. Several distinct subgroups of reactive somata were noted in both the medullar and ventral medial groups. The distribution of fibers in the brain with colocalized FLI and SPLI includes those which innervate the vascular sheath and widespread populations of small-diameter beaded fibers in the central neuropil and circumesophageal connective. Somata with colocalized FLI and SPLI constitute minority populations in the medullar and dorsal medial groups but form the majority population of a subgroup in the ventral medial group. Overall localization of SPLI was reevaluated and is reported here according to the nomenclature of the new Chamberlain and Wyse brain atlas. In addition to those previously reported, somata with SPLI were found in the dorsal lateral posterior groups #1 and #2, the ventral lateral posterior groups #1 and #2, and several distinct subgroups of the medial and ventral medial groups. In the retina of the lateral eye, fibers with both FLI and SPLI ramify in the lateral plexus and ultimately innervate the corneal epidermis. Brain homogenates were examined for immunoreactive (ir) FMRFamide and ir-substance P with radioimmunoassay techniques. Ir-FMRFamide and ir-substance P eluted in different fractions from both gel filtration chromatography and HPLC. Furthermore, the binding curves for both substances were similar to those of the corresponding synthetic compounds. Brain homogenates were also bioassayed on the lateral eye. Three gel filtration fractions mimic natural circadian activity by increasing the sensitivity of the lateral eye, but they were not coincident with ir-FMRFamide or ir-substance P. Although it is not completely resolved what the active molecules in these fractions are, it is clear that neither ir-FMRFamide nor ir-substance P is a possible candidate.

Localization of FMRFamide-like peptides in the snail Helix aspersa.

The distribution of FMRFamide-like material in the gastropod mollusc, Helix aspersa, was studied by radioimmunoassay (RIA) and immunocytochemistry. Most of the RIA activity was concentrated in the central nervous system, the male reproductive tract, the tentacles and the posterior digestive system (Table 1). The density of FMRFamidergic perikarya, nerves and nerve varicosities in the muscle tissue of all these regions, as indicated immunocytochemically (Fig. 2), was well correlated with the distribution as determined by RIA. Gel chromatography of each extract resolved two peaks of FMRFamide-like immunoreactivity (Fig. 3). The first of these was further analysed by high-pressure liquid chromatography (HPLC), and the components included two major immunoreactive peaks identifiable, both by their retention times and their effects on the radula protractor muscle of Busycon contrarium, as the known peptides FMRFamide and pQDPFLRFamide (Figs 4-6). The second peak from gel chromatography gave only a single peak, distinct from that of FMRFamide and pQDPFLRFamide, in two HPLC systems (Fig. 7), but it did not behave like a competitive ligand in the FMRFamide RIA. Moreover, its immunoreactivity, unlike any peptides we tested, was not affected by carboxypeptidase Y (Fig. 8), and it was not active on the radula protractor muscle. Thus, it is certainly not an FMRFamide-like peptide. We conclude that Helix aspersa contains at least two FMRFamide-like peptides, FMRFamide and pQDPFLRFamide. These peptides appear to act both as neurohormones and as neurotransmitters or modulators in the central ganglia, reproductive, digestive, muscular and circulatory systems.

The actions of FMRFamide-like peptides on visceral and somatic muscles of the snail Helix aspersa.

Phe-Met-Arg-Phe-NH2 (FMRFamide) and pyroGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (pQDPFLRFamide) occur in the ganglia and tissues of the snail, Helix aspersa. This report describes the effects of these two neuropeptides on five visceral organs or somatic muscles isolated from the snail (Table 1). The epiphallus, as well as the rest of the male reproductive tract, was contracted by both FMRFamide and pQDPFLRFamide, and the threshold was usually below 5 X 10(-9) mol l-1 (Fig. 1). Both peptides also reduced the resting tone of the crop and decreased the force and frequency of its rhythmic activity; FMRFamide is about 10 times more potent (Fig. 4). In contrast, pQDPFLRFamide was about 100 times more potent than FMRFamide as a cardioexcitatory agent (Fig. 5). The actions of the peptides on the pharyngeal and tentacle retractor muscles were markedly different: FMRFamide primarily contracted these muscles; and pQDPFLRFamide usually had no effect alone, but relaxed or diminished contractions induced by FMRFamide and acetylcholine (ACh) (Figs 6, 8, 9). Other analogues of FMRFamide were tested, but none was as effective a relaxing agent as pQDPFLRFamide. The effects of FMRFamide and pQDPFLRFamide on all of the preparations could be distinguished from those produced by ACh and 5-hydroxytryptamine (5-HT); thus the actions of the neuropeptides were not mediated by cholinergic or serotonergic neurones. The stimulation of the musculature in the male reproductive tract and the inhibition of motility of the digestive system by FMRFamide and pQDPFLRFamide implicate these peptides in the control of reproductive behaviour. The effectiveness of pQDPFLRFamide in relaxing the retractor muscles and as a cardioexcitatory agent led to the hypothesis that this heptapeptide and FMRFamide, acting at distinct receptors, cooperate to regulate the excitability and contractility of the snail's musculature between the extremes of aestivation and active locomotion.

Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family.

Two structurally related arthropod neuropeptides, red pigment concentrating hormone (RPCH) and adipokinetic hormone (AKH), are potent excitors of the heart of the clam Mercenaria mercenaria. The response is bimodal: whereas the threshold for affected hearts is 1-3 X 10(-9) M, about 40% of the preparations are virtually unresponsive. Aqueous extracts of Mercenaria ganglia contain a substance which concentrates the red pigment in the erythrophores of intact destalked Uca pugilator and even of its isolated legs. This substance is retained on Sephadex G-15 and co-elutes with synthetic shrimp RPCH. The active fractions also concentrate the erythrophores and the leucophores of destalked shrimp (Penaeus). Neither dopamine nor the molluscan neuropeptide FMRFamide had any chromatophorotropic effect in these assays. The activity of the ganglion extracts was abolished by digestion with chymotrypsin. In conclusion, molluscan ganglion extracts contain a peptide factor, possibly an analog of RPCH, that concentrates the pigments of crustacean chromatophores by a direct action on the cells.

THE FMRFamide-LIKE NEUROPEPTIDE OF APLYSIA IS FMRFamide.

The head ganglia from 350 Aplysia brasiliana were extracted and purified by gel (Sephadex G-15) and cation exchange (CM-Sephadex) chromatography; the fractions were examined with radioimmunoassays (RIA) for the molluscan neuropeptides, FMRFamide and SCPB. Immunoreactive (ir-) FMRFamide (but not ir-SCPB) coeluted with authentic FMRFamide from both chromatographic columns. The amino acid composition of the purified peptide was: Phe 2: Arg 1: Met 1. Digestion of purified ir-FMRFamide with carboxypeptidase Y indicated that the four residues were in the same sequence as occurs in FMRFamide. The dose-response curves for purified and synthetic FMRFamide on the radula protractor muscle of Busycon contrarium were coincident, as were their inhibition binding curves in the FMRFamide RIA. The highest concentrations of ir-FMRFamide were in the pedal and pleural ganglia; but SCPB was concentrated in the buccal ganglion. Synthetic SCPB has no effect on the radula protractor muscle of Busycon or the isolated heart of Mercenaria. In conclusion, the FMRFamide-like peptide in the gastropod Aplysia is FMRFamide, so this peptide has now been identified in two molluscan classes. Moreover, the proposed structural relationship between FMRFamide and SCPB is fortuitous, and these two peptides should have different physiological functions in Aplysia.

The molluscan neurosecretory peptide FMRFamide: comparative pharmacology and relationship to the enkephalins.

The molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) has diverse actions on excitable tissues of molluscs, including hearts, noncardiac muscles, complex organs, and neurons. The intracellular transducing mechanisms are also diverse and are not readily correlated with particular responses. FMRFamide increases cyclic AMP levels concomitant with both cardioexcitation and inhibition, but not with muscle contraction. In the same tissues, the effects of 5-hydroxytryptamine are dissimilar and are always accompanied by a cyclic AMP increase. FMRFamide and acetylcholine cause similar tonic contractions of the Busycon radula protractor muscle and identical catch contractures of the mytilid anterior byssus retractor muscle, but the ionic basis of excitation and the sources of activator calcium for contraction are not the same for the two agonists. A comparative study of structure-activity relations showed that FMRFamide receptors are heterogeneous. Helix aspersa ganglia contain no FMRFamide, but a close analog occurs and has been tentatively identified. Evidence supporting a proposed homology between FMRFamide-like and opioid peptides is summarized. The effects of the amphiactive heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 on the venus clam rectum support this hypothesis.

Aspects of copulatory behavior and peptide control of egg laying in Aplysia.

The atrial gland of the marine mollusc Aplysia is associated with the large hermaphroditic duct of the reproductive system and contains several peptides capable of inducing egg laying. The structure and function of these peptides are briefly reviewed. It has been hypothesized that during copulation the atrial gland of the female is stimulated by penile insertion to release its peptides, which in turn initiate events leading to egg deposition. To test this hypothesis we monitored reproductive activity over periods of weeks in individual, paired, and grouped A. brasiliana. It was found that copulation is not a necessary stimulus for egg laying, because individually housed Aplysia lay more eggs than when they are paired and allowed to copulate. Nor is copulation a sufficient stimulus, because the vast majority of copulations are not followed by egg laying. Simultaneous egg laying and female copulatory behavior were often observed with grouped and paired animals, but these events are probably not causally related. It is concluded that although the atrial gland contains at least three peptides that can induce egg laying, stimulation of this gland during copulation does not normally serve to initiate egg laying.

Competitive inhibition by dimethylsulfoxide of molluscan and vertebrate acetylcholinesterase.

Anticholinesterase-like effects of dimethylsulfoxide (DMSO) were demonstrated on a variety of invertebrate muscles. The excitatory effects of acetylcholine (ACh) on the isolated preparations of the Geukensia demissa heart and anterior byssus retractor muscle (ABRM), and of the Busycon contrarium radula protractor muscle, were potentiated by DMSO (1-5 microliters/ml; 1 microliter/ml = 14 mM). The negative chronotropic effects of ACh, but not of 4-ketoamyltrimethylammonium, were potentiated by DMSO (1-5 microliters/ml) on the isolated heart of the oyster Crassostrea virginica. These four muscles have acetylcholinesterase enzymes of high activity. In contrast, Mercenaria mercenaria hearts have weak cholinesterase activity, and the effects of ACh on this isolated myocardium were not potentiated by DMSO (2-20 microliters/ml). DMSO (0.1-15 microliters/ml) was a competitive inhibitor of both a crude preparation of oyster heart acetylcholinesterase (AChE) (the Km increased 24-fold with DMSO at 15 microliters/ml; the I50 was 1.3 microliters/ml DMSO when [ACh] = Km) and a purified Electrophorus AChE (the Km increased 4.5-fold when DMSO was 10 microliters/ml; the I50 was 10 microliters/ml DMSO near [ACh] = Km). The same doses of DMSO were needed to potentiate the pharmacological effects of ACh on the oyster heart, as to inhibit the AChE of this tissue.

Effects of sevin on development of experimental estuarine communities.

The composition of animal communities developing from planktonic larvae in aquariums. A marked increase in the abundance of the annelid Polydora ligni in aquariums containing sand and flowing estuarine water was altered in the presence of the carbamate insecticide Sevin (carbaryl). Treatments were control and concentrations of Sevin that averaged 1.1, 11.1, and 103 micrograms/l; each treatment was replicated 8 times. Animals that colonized aquarium sand were collected in a 1-mm mesh sieve after 10 wk of exposure. Mollusks' arthropods, annelids, and nemerteans were the numerically dominant phyla. The average number of species per aquarium was significantly less (alpha = 0.05) in aquariums containing 11.1 or 103 micrograms/l than in those containing 1.1 micrograms/l or in control aquariums. The abundant clam Ensis minor grew significantly less in length at the higher concentrations of Sevin. The amphipod Corophium acherusicum was particularly affected; significantly fewer were found at all concentrations than in the control aquariums containing 103 micrograms/l corresponded to a marked decrease in the number of other annelids and to a significant absence of nemerteans.