Suppression of grasshopper sound production by nitric oxide-releasing neurons of the central complex

The central complex of acridid grasshoppers integrates sensory information pertinent to reproduction-related acoustic communication. Activation of nitric oxide (NO)/cyclic GMP-signaling by injection of NO donors into the central complex of restrained Chorthippus biguttulus females suppresses muscarine-stimulated sound production. In contrast, sound production is released by aminoguanidine (AG)-mediated inhibition of nitric oxide synthase (NOS) in the central body, suggesting a basal release of NO that suppresses singing in this situation. Using anti-citrulline immunocytochemistry to detect recent NO production, subtypes of columnar neurons with somata located in the pars intercerebralis and tangential neurons with somata in the ventro-median protocerebrum were distinctly labeled. Their arborizations in the central body upper division overlap with expression patterns for NOS and with the site of injection where NO donors suppress sound production. Systemic application of AG increases the responsiveness of unrestrained females to male calling songs. Identical treatment with the NOS inhibitor that increased male song-stimulated sound production in females induced a marked reduction of citrulline accumulation in central complex columnar and tangential neurons. We conclude that behavioral situations that are unfavorable for sound production (like being restrained) activate NOS-expressing central body neurons to release NO and elevate the behavioral threshold for sound production in female grasshoppers.     

Suppression of allatotropin simulates reproductive diapause in the mosquito Culex pipiens

The cessation of juvenile hormone (JH) production is a key endocrine event that halts ovarian development and hence initiates diapause in females of the mosquito, Culex pipiens. The shutdown in endocrine activity of the corpora allata (CA), the source of JH, was manifested in the smaller size of CA in females reared under short daylengths (diapause) compared to those reared under long daylengths (nondiapause), as well as in low expression of the mRNA encoding allatotropin, the neuropeptide that promotes JH biosynthesis in the CA. Genes encoding both allatotropin and allatostatin were identified in C. pipiens, but only expression levels of allatotropin differed in the two types of females. Knockdown of allatotropin mRNA using RNA interference in females programmed for nondiapause resulted in a cessation of ovarian development akin to diapause. This arrest in development could be reversed with an application of JH. Our results thus suggest that suppression of allatotropin is a critical link in regulating the shutdown of the CA during diapause.     

Role of juvenile hormone and allatotropin on nutrient allocation, ovarian development and survivorship in mosquitoes

Teneral reserves are utilized to initiate previtellogenic ovarian development in mosquitoes. Females having emerged with low teneral reserves have reduced juvenile hormone (JH) synthesis and previtellogenic development. We investigated what role JH, allatotropin (AT) and other head-factors play in the regulation of previtellogenic ovarian development and adult survivorship. Factors from the head are essential for corpora allata (CA) activation and reproductive maturation. We have shown that decapitation of females within 9-12h after adult ecdysis prevented normal development of the previtellogenic follicles; however maximum previtellogenic ovarian development could be induced in decapitated females by topically applying a JH analog. When females were decapitated 12 or more hours after emergence nutritional resources had been committed to ovarian development and survivorship was significantly reduced. To study if allatotropin levels correlated with teneral reserves, we measured AT titers in the heads of two adult phenotypes (large and small females) generated by raising larvae under different nutritional diets. In large mosquitoes AT levels increased to a maximum of 45 fmol in day 4; in contrast, the levels of allatotropin in the heads of small mosquitoes remained below 9 fmol during the 7 days evaluated. These results suggest that only when nutrients are appropriate, factors released from the brain induce the CA to synthesize enough JH to activate reproductive maturation.     

Allatotropin-like peptide in Heliothis virescens: tissue localization and quantification

The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in the central nervous system. Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of AT acting on the CA.     

Regulation of juvenile hormone biosynthesis in Heliothis virescens by Manduca sexta allatotropin

In Heliothis virescens, reproduction is strictly dependent on juvenile hormone (JH). In females, mating induces a sharp increase in JH titers, which stimulates increased vitellogenin biosynthesis and higher rates of egg production. JH biosynthesis is presumably stimulated by production and/or release of stimulatory neuropeptides such as allatotropins. There is evidence that allatotropin of H. virescens may be structurally related to Manduca sexta allatotropin (Manse-AT). In a radiochemical in vitro assay, synthetic Manse-AT stimulated JH biosynthesis by corpora allata (CA) of virgin H. virescens females in a dose-dependent manner, but had no effect on CA activity in H. virescens males. In females, the CA showed a transient increase in sensitivity to Manse-AT shortly after mating. Several structurally related peptides stimulated CA activity to a similar extent as Manse-AT. Corpora allata activity was stimulated by a Ca2+ ionophore, A23187. A membrane-permeable Ca2+ chelator, BAPTA/AM, antagonized the stimulatory effects of Manse-AT, suggesting that Manse-AT may enhance CA activity by increasing intracellular Ca2+ concentration.     

Allatostatins and allatotropin in the corpus cardiacum/corpus allatum complex of larval and adult lepidopterans studied by confocal laser scanning microscopy: correlation to juvenile hormone biosynthesis

Peptidergic innervation of the corpus cardiacum/corpus allatum (CC/CA) retrocerebral complex, and neurosecretory areas of the brain of the lepidopterans Lacanobia oleracea, Heliothis virescens and Manduca sexta was studied by immunocytochemistry linked to confocal laser scanning microscopy. The patterns of immunostaining resulting from the simultaneous application of fluorochrome-conjugated antibodies against Manduca sexta allatostatin (Mas-AS), M. sexta allatotropin (Mas-AT), and a representative of the –Y/FXFGL-NH₂ superfamily of allatostatins was correlated with the physiological effects of these putative allatoregulatory peptides on juvenile hormone (JH) biosynthesis by the corpora allata. Whereas the two types of allatostatin immunoreactivity are present in both larval and adult CA of the three species, allatotropin immunoreactivity occurs only in the adult gland. The conclusion that withdrawal of the stimulatory effect of allatotropin is unlikely to be involved in the downregulation of CA activity prior to the onset of metamorphosis, but that an inhibitory influence of at least Mas-AS is important, is borne out in physiological experiments on JH biosynthesis in M. sexta larvae (Mas-AS inhibitory, Mas-AT without effect). Immunoreactivity to the Y/FXFGL-NH₂ allatostatins is present in both larval and adult CA and CC, frequently co-localised with Mas-AS. The function of this peptide family in the retrocerebral complex remains enigmatic since experiments on JH biosynthesis, either when the peptide is administered alone, or together with Mas-AS, show no effect on JH biosynthesis.Financial support was provided by The Wellcome Trust (063367/Z/00) (to A.T.) and by the Pesticide Safety Directorate of the Department for Environment, Food and Rural Affairs (to N.A. and R.J.W.)     

Stimulation of juvenile hormone biosynthesis by analogues of a Manduca sexta allatotropin: in vitro studies.

Two analogues of a Manduca sexta allatotropin (Mas-AT) were synthesized. They correspond to the active fragment, amino acids 5-13, of the natural Mas-AT with substitution of norleucine for methionine. ATANA has the structure Val-Glu-Nle-Nle-Thr-Ala-Arg-Gly-Phe-NH2, ATAA is acetylated at the N-terminus. Allatotropic potency was evaluated by measuring the in vitro rates of juvenile hormone (JH) biosynthesis in corpora allata (CA) of M. Sexta. At a concentration of 20 nM, ATANA and ATAA increased JH production in day 0, day 1, and day 3 adult female CA by a factor of 3-8. Larval CA were not affected. These results correspond to activities reported for the natural Mas-At. ATANA did not stimulate pharate adult female CA to produce JH. Stimulation of female CA with ATANA was reversed when the CA were transferred to fresh medium while stimulation with ATAA under the same condition persisted. Exogenous farnesoate was converted to JH-III at a rate exceeding the highest ATANA-stimulated rate. ATANA in addition to farnesoate did not increase JH-III production, but increased JH-II production in addition to the already high production rate for JH-III. It is inferred that Mas-AT stimulates a rate-limiting enzyme in the biosynthesis of farnesoate and its homologues but does not affect epoxidation and methylation.     

Allatotropic and allatostatic activities in extracts of brain and the effects of Manduca sexta allatotropin and Manduca sexta allatostatin on juvenile hormone synthesis in vitro by corpora allata from the Eri silkworm, Samia cynthia ricini

Both allatotropic and allatostatic activities were found in crude extracts of brain from adult and larval Eri silkworm, Samia cynthia ricini, but it seems that allatotropic activity dominates in each stage. There was a high level of allatotropic activity in the crude extract of brain from newly emerged female adults, but allatostatic activity appeared in the bioassay when excessive amounts of crude extracts of brain were added. Crude extracts of brain from premoulting fourth‐instar larvae and from newly ecdysed fifth‐instar larvae exhibited allatotropic activities, whereas extracts of brain from the second and third day of the fifth‐instar larvae inhibited juvenile hormone (JH) release slightly. Allatotropic activity from the brains of adults and larvae stimulated both adult and larval corpora allata (CA) to synthesize JH. Manduca sexta allatotropin (AT) (Mas‐AT) and M. sexta allatostatin (AST) (Mas‐AST) also stimulated and inhibited both adult and larval S. cynthia ricini CA to synthesize JH, respectively. Higher concentrations of Mas‐AT (10^?4 or 10^?3 M) showed an inhibitory effect on adult CA. CA from newly emerged female adults were the most sensitive to inhibition by Mas‐AST, whereas CA from female pharate adults at about 6 h before adult emergence were the most sensitive to stimulation by Mas‐AT and S. cynthia ricini brain allatotropic activity. An extract of brain and Mas‐AT induced some of the non‐active female pharate adult CA at 12 h before emergence to synthesize a small amount of JH.     

Juvenile hormone biosynthesis in diapause and nondiapause females of the adult blow fly Protophormia terraenovae

In vitro synthetic activities of juvenile hormones (JH) were examined using a radiochemical assay in diapause females and reproductive females of the blow fly, Protophormia terraenovae. Thin layer chromatography showed that products of the corpus allatum (CA) comigrated with a synthetic sample of JH III bisepoxide but neither with JH III nor methylfarnesoate. JH synthetic activities increased in females reared under LD 18:6 at 25 degrees C, as the ovaries developed. The synthetic activities remained low in previtellogenic females reared under LD 12:12 at 20 degrees C. Removal of the pars intercerebralis completely prevented ovaries from development under reproductive conditions, and removal of the pars lateralis caused partial or full development of ovaries under diapause-inducing conditions. In these operated animals, the JH synthetic activities were not significantly different from those of the intact and sham-operated animals. The results indicate that the CA in P. terraenovae produces mainly JH III bisepoxide and a decrease in the JH production rate is a cause of diapause induction. PI neurons and PL neurons in the brain do not directly mediate changes in the JH production rate, but regulate ovarian development cooperatively with some unknown allatostatic and allatotropic factors.     

Chilling affects allatal cell proliferation via antennae and protocerebral neurons in the cockroach Diploptera punctata

The corpora allata (CA) cells in a mated female of the cockroach Diploptera punctata undergo numerous mitotic divisions before an increase in juvenile hormone synthesis. A previous study demonstrated that this mitotic wave could be suppressed by exposure of the mated female to melting ice. Herein, we report that chilling suppresses CA mitosis via antennal perception. Cell proliferation-suppressing stimuli from chilling were acquired in proportion to the length of time of exposure to the low temperature and the physical length of the antennae exposed to chilling. Sixty basal antennal annuli should remain exposed to chilling for at least 1.5 h in order to suppress mitotic divisions in CA. Mitotic divisions in corpus allatum are suppressed by stimuli from contralateral antenna, predominantly via pars intercerebralis neurons. Selective disconnection of pars intercerebralis neurons from CA, prior to chilling, restored the mitotic wave in CA. Cellular divisions did not occur in CA of chilled females if either pars lateralis neurons were severed or left intact.     

Citrulline immunohistochemistry for demonstration of NOS activity in vivo and in vitro.

Nitric oxide (NO), a biomolecule with major cytotoxic potency, is generated by NO synthases (NOS) utilizing l-arginine as substrate and citrulline is formed as a "side product." In brain tissue, citrulline is considered to be produced exclusively by NOS, due to the incomplete urea cycle in the brain. We aimed to characterize NOS activity by citrulline immunostaining in different cell types of the brain under in situ conditions and in slice and culture experiments. NOS-positive neurons and activated microglial cells were the most prominent citrulline-positive structures. Lack of citrulline immunoreaction in neurons of nNOS knockout mice emphasizes the dependency of citrulline positivity on NOS activity, and likewise there was no citrulline staining after application of the NOS inhibitors 7-nitroindazole and NIL. Interestingly, only a portion of NOS-containing neurons costained for citrulline. The inhibition of argininosuccinate synthetase by alpha-methyl-dl-aspartate increased the number of citrulline-positive cells, apparently due to reduction of the turnover rate of citrulline. Cells positive for NOS but negative for citrulline may indicate that the enzyme is either not activated or inhibited by cellular control mechanisms. The fact that not all citrulline-positive cells were NOS positive may be explained by an insufficient detection sensitivity or by disparate sites of citrulline production and recycling. The present results show that citrulline immunocytochemistry offers a viable and convenient means for studying NOS activity at the single-cell level to elicit its posttranslational control under physiological and pathophysiological conditions.     

Allatotropin regulation of juvenile hormone synthesis by the corpora allata from the lubber grasshopper, Romalea microptera

The in vitro synthesis of juvenile hormone (JH) by corpora allata (CA) from the lubber grasshopper (Romalea microptera) was stimulated by low concentrations of brain extract and this effect was reduced at higher concentrations, suggesting the presence of allatotropin (AT) and allatostatin (AST) factors in the brain. The AT activity of brain extracts caused a rapid and reversible stimulation and appeared to be a peptide(s). Reversed phase (C18) HPLC analysis of brain extracts disclosed two peaks of AT activity but no significant AST activity. Manse-AT, Schgr-NPF, and Locmi-FLRF had no effect on JH synthesis by lubber CA, indicating that the Rommi-AT factors are distinct from these peptides. High concentrations of Dippu-AST-7 and Grybi-AST-1 inhibited JH synthesis, implying that AST factors might be present in lubber grasshoppers. CA response to AT activity of brain extracts varied during the oviposition cycle ( approximately 35 days), with the maximum response occurring on days 16-18. AT activity of brain extracts also varied during the cycle, being highest on day 25. Our data suggest that the lubber CA is largely regulated by AT activity, and that JH synthesis reflects both CA response to AT activity and the level of AT activity in the brain.     

Juvenile hormone biosynthesis by corpora allata of larval tomato moth, Lacanobia oleracea, and regulation by Manduca sexta allatostatin and allatotropin

Juvenile hormone (JH) biosynthesis and the effects of synthetic Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin (Mas-AT) were investigated in isolated corpora allata (CA) of Vth stadium larvae of the tomato moth, Lacanobia oleracea. Reversed-phase high-performance liquid chromatography (RP-HPLC) of JH extracted from CA shows that larvae produce predominantly JH II and its corresponding acid. It appears that the acid homologue is a result of JH esterase activity in the CA (and other tissues) rather than the lack of JH acid methyltransferase. Mean rates of synthesis (100-200fmol/pr/h) were inhibited ca. 70% by Mas-AS and stimulated in a dose-dependent manner up to three times by Mas-AT. However, Mas-AS had no significant effect on Mas-AT-stimulated rates of JH biosynthesis. Using RP-HPLC and an enzyme-linked immunosorbent assay (ELISA) to Mas-AT, a peak of Mas-AT-like immunoreactivity was detected in larval L. oleracea brain homogenates. Co-elution of this immunoreactive peak with synthetic Mas-AT suggests that this neuropeptide is also present in L. oleracea.     

Interaction between Manduca sexta allatotropin and manduca sexta allatostatin in the fall armyworm Spodoptera frugiperda

A peptide that strongly stimulates juvenile hormone (JH) biosynthesis in vitro by the corpora allata (CA) was purified from methanolic brain extracts of adult Spodoptera frugiperda. Using HPLC separation followed by Edman degradation and mass spectrometry, the peptide was identified as Manduca sexta allatotropin (Mas-AT). Treating the CA from adult S. frugiperda with synthetic Mas-AT (at 10(-6) M) caused an up to sevenfold increase in JH biosynthesis. The stimulation of JH synthesis was dose-dependent and reversible. Synthetic M. sexta allatostatin (Mas-AS) (10(-6) M) did not affect the spontaneous rate of JH secretion from CA of adult S. frugiperda, nor did any of the allatostatins of the Phe-Gly-Leu-amide peptide family tested. However, when CA had been activated by Mas-AT (10(-6) M), addition of synthetic Mas-AS (10(-6) M) reduced JH synthesis by about 70%. This allatostatic effect of Mas-AS on allatotropin-activated glands was also reversible. When CA were incubated in the presence of both Mas-AT (10(-6) M) and various concentrations of Mas-AS (from 10(-8) to 10(-5) M), the stimulation of JH-biosynthesis observed was inhibited in a dose-dependent manner. The experiments demonstrate a novel mechanism of allatostatin action. In S. frugiperda JH synthesis was inhibited only in those glands which had previously been activated by an allatotropin.     

Allatotropic activity in the brain of female Phormia regina (Diptera: Calliphoridae)

In Phormia regina, the rate of juvenile hormone (JH) synthesis rises rapidly after the ingestion of an adequate protein meal. In a previous publication we have localized the neurons containing Manduca sexta allatotropin (Mas-AT)-like substances in the brain of P. regina and demonstrated the allatotropic effect of synthetic Mas-AT in sugar-fed flies in vitro. In this current study, we examined the possible role of the brain of P. regina after the fly received a protein meal. In vitro studies showed that the brain releases, at 8 h after a protein meal, a factor(s) with a strong allatotropic effect. This factor(s) stimulates the corpus allatum (CA) to produce 6.9 times more juvenile hormones (JHs) than the control CA. Time course studies showed that the release of this allatotropic factor(s) is temporally controlled. Only the brains collected from flies at 6 and 8 h after the onset of a liver meal release allatotropic factor(s). Injection of anti-Mas-AT antiserum partially suppressed the fly follicle development in vivo. Presence of anti-Mas-AT antiserum decreased the allatotropic effect of the brain released allatotropic factor(s) in vitro. In addition to a Mas-AT-like substance, it is possible that the brains of liver-fed P. regina females may synthesize other allatotropic factors that are chemically unrelated or partially related to Mas-AT, which cannot be recognized/neutralized by our anti-Mas-AT antiserum.     

Cell size control by ovarian factors regulates juvenile hormone synthesis in corpora allata of the cockroach, Diploptera punctata

The corpora allata synthesize and release juvenile hormone (JH) that in turn regulates insect growth, metamorphosis and reproduction. In the corpus allatum (CA) of the female adult cockroach Diploptera punctata, cyclic rise and decline in JH synthesis rates occur concurrently with cyclic growth and atrophy during an ovarian cycle. Here, we report that protein content decreases, whereas Golgi population, lysosomal content and autophagic activities increase with decrease in CA cell size. Also, the concentration of cyclic GMP (cGMP) is low in large cells and high in small cells. Results of treating CA with ovarian tissue suggest that a putative peptidergic growth regulator released from mature ovaries acts directly on active CA cells and induces the elevation of intracellular cGMP content. Consequently, elevated cGMP may inhibit protein synthesis or trigger massive and synchronous autophagic activities, resulting in cell atrophy and reduction of protein content. As a result of the depletion of cellular machinery, CA glands exhibit long-term depression in JH synthesis.     

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.     

Role of endothelin type B receptor in NO/cGMP signaling pathway in rat median eminence

We studied the effect of endothelins (ETs) on receptor-mediated NO/cGMP signaling in rat arcuate nucleus–median eminence (AN-ME) fragments, an hypothalamic structure known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers together with densely arranged ET_B-receptor-like immunoreactive fibers. NOS activity was determined measuring the conversion of [³H] arginine to [³H] citrulline, as an index of NO produced. cGMP production was determined by radio immunoassay. ET-1, ET-3, and the selective ET_B receptor agonist, IRL1620, significantly increased cGMP formation and NOS activity. Preincubation of AN-ME fragment with L-arginine analog, N-nitro-L-arginine (L-NAME), inhibited ET-1 or IRL1620-stimulated cGMP formation. The addition of the selective ET_B receptor antagonist, BQ788, blocked ET-1-, ET-3-, or IRL1620-induced increase in NOS activity and cGMP generation, while BQ123, a selective ET_A receptor antagonist, was ineffective. Our results demonstrate that in whole rat AN-ME fragments, ETs stimulate NO/cGMP signaling pathway through the interaction with the ET_B receptor subtype, supporting the concept that ETs may represent an important regulator of reproductive and neuroendocrine function.     

Development of the nitric oxide/cGMP system in the embryonic and juvenile pond snail, Lymnaea stagnalis L. A comparative in situ hybridization, histochemical and immunohistochemical study

Recent studies have indicated that nitric oxide (NO)-induced cGMP synthesis is involved in different steps of neurogenesis in invertebrates. The development of putative NO synthetising elements was described earlier in the embryonic and juvenile pond snail, Lymnaea stagnalis, applying NADPH-diaphorase histochemistry (Serf?z? et al., 1998). In the present study, we examined the distribution of NO synthase (NOS) during Lymnaea development by in situ hybridization for Lymnaea-NOS mRNA, histochemical, and immunohistochemical techniques for the NOS and NO-stimulated cGMP. Peripheral fibers projecting to the CNS and terminating in the ganglionic neuropils showed NOS immunoreactivity from 85% of embryonic development. At the same time, a fine dot-like, immunostaining indicated the presence of cGMP in the neuropil area. In the CNS, Lymnaea-NOS mRNA positive, as well as NOS and cGMP immunoreactive perikarya were detected first during postembryonic development; their number significantly increased from P3 juvenile stage. Some of the cell groups in the CNS containing NOS immunoreactive material also displayed Lymnaea-NOS mRNA hybridization signal and were cGMP-positive. However, in the subesophageal ganglia, the distribution of Lymnaea-NOS mRNA positive cell groups did not correspond to that of the NOS immunoreactive cells. Neurons revealing transient NOS and cGMP immunoreactivity, respectively, could also be detected in this part of the CNS. In most of the ganglia the number of Lymnaea-NOS mRNA containing and cGMP immunopositive neurons, respectively, exceeded that of the NOS immunoreactive cells from P4 juvenile stage. The localization of NADPH-diaphorase reaction also correlated well with that of the NOS immunoreactivity in the developing CNS. At the periphery, colocalization of Lymnaea-NOS mRNA signal, NOS and cGMP immunoreactivities were observed in the epithelial cells of the esophagus and mantle after hatching. The findings suggest the functional maturity of the NO/cGMP signal transduction pathway at both central and peripheral levels during the development of the snail, Lymnaea stagnalis. The differences in the localization of Lymnaea-NOS mRNA labeling and NOS immunoreactivity in the CNS and PNS can be explained by the existence of different NOS isoforms, posttranslational regulation of NOS, and/or some non-specific antibody labeling.