Juvenile hormone controls ovarian development in female Anopheles albimanus mosquitoes.

Anophelinae mosquitoes are vectors of human malaria, a disease that infects hundreds of millions of people and causes almost 600,000 fatalities annually. Despite their medical importance, laboratory studies on key aspects of Anophelinae reproductive biology have been limited, and in particular, relatively little is known about the role of juvenile hormone (JH) in the control of female reproduction. The study presented here attempts to fill a gap of knowledge in our understanding of the JH control of ovarian development in female Anophelinae mosquitoes, using Anopheles albimanus as a model. Our studies revealed that JH controls the tempo of maturation of primary follicles in An. albimanus in a similar manner to that previously described in Aedes aegypti. At adult eclosion JH hemolymph titer was low, increased in 1-day old sugar-fed insects, and decreased in blood fed individuals. JH titers decreased if An. albimanus females were starved, and were reduced if insects emerged with low teneral reserves, precluding previtellogenic ovarian development. However, absolute hemolymph titers were lower than Ae. aegypti. Decapitation experiments suggested that if teneral reserves are sufficient, factors from the head activate JH synthesis by the corpora allata (CA) during the first 9-12 h after adult emergence. In conclusion, our studies support the hypothesis that JH controls previtellogenic ovarian development in female An. albimanus mosquitoes, in a similar manner that have been described in Culicinae.

Dietary sugar promotes systemic TOR activation in Drosophila through AKH-dependent selective secretion of Dilp3.

Secreted ligands of the insulin family promote cell growth and maintain sugar homeostasis. Insulin release is tightly regulated in response to dietary conditions, but how insulin-producing cells (IPCs) coordinate their responses to distinct nutrient signals is unclear. Here we show that regulation of insulin secretion in Drosophila larvae has been segregated into distinct branches-whereas amino acids promote the secretion of Drosophila insulin-like peptide 2 (Dilp2), circulating sugars promote the selective release of Dilp3. Dilp3 is uniquely required for the sugar-mediated activation of TOR signalling and suppression of autophagy in the larval fat body. Sugar levels are not sensed directly by the IPCs, but rather by the adipokinetic hormone (AKH)-producing cells of the corpora cardiaca, and we demonstrate that AKH signalling is required in the IPCs for sugar-dependent Dilp3 release. Thus, IPCs integrate multiple cues to regulate the secretion of distinct insulin subtypes under varying nutrient conditions.

Role of the brain in photoperiodic regulation of juvenile hormone biosynthesis in the brown-winged green bug Plautia stali.

We investigated the roles of the brain in photoperiodic regulation of juvenile hormone (JH) biosynthesis in Plautia stali (Heteroptera: Pentatomidae). Within 4days after transferring the adults from short-day to long-day conditions, the biosynthesis of juvenile hormone III skipped bisepoxide became higher than that under short-day conditions. When the corpus cardiacum-corpus allatum complex (CC-CA) was coincubated with different regions of the brain, JH biosynthetic activities were inhibited by a middle part of the brain, i.e., the brain excluding the optic lobes, extirpated from short-day and long-day females. Backfills from the CC-CA revealed three groups of somata in the middle part of the brain: 15-18 pairs of somata were stained in the pars intercerebralis, 9-10 pairs were stained in the pars lateralis, and five pairs were stained on the nervi corporis cardiaci 2. These results suggest that inhibitory molecules of JH biosynthesis are produced in the middle part of the brain, regardless of photoperiod, but their release is suppressed under long-day conditions, leading to an increase in JH biosynthetic activities. Further, some neurons with somata in the pars intercerebralis, in the pars lateralis, or on the nervi corporis cardiaci 2 might be involved in photoperiodic regulation of JH biosynthesis.

Decrease in juvenile hormone level as a result of genetic ablation of the corpus allatum cells affects the synthesis and metabolism of stress related hormones in Drosophila.

Previous studies have shown that juvenile hormone (JH) regulates dopamine (DA) and octopamine (OA) content in Drosophila, and we have shown the influence of an increase in JH level on DA and OA metabolism in young females of Drosophila virilis and Drosophila melanogaster. Here we investigate the effects of genetic ablation of a subset of cells in the Corpusallatum (CA, endocrine gland synthesizing JH) on the DA levels and activities of alkaline phosphatase (ALP), tyrosine hydroxylase (TH), DA-dependent arylalkylamine N-acetyltransferase (DAT) and tyrosine decarboxylase (TDC) in young D. melanogaster females under normal conditions and upon heat stress (38°Ð¡). We show that ablation of СА cells causes: (1) a decrease in ALP, TH and DAT activities, (2) an increase in DA level and (3) an increase in TDC activity in young females. The CA ablation was also found to modulate ALP, TH and TDC responses to heat stress. Mechanisms of regulation of DA and OA levels by JH in Drosophila females are discussed.

A role for juvenile hormone in the prepupal development of Drosophila melanogaster.

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met(27) larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis.

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly, Protophormia terraenovae.

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum-hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg(7)]-corazonin and [Arg(7)]-corazonin(3)(-)(11)) in the PL, 13 peptides (including myosuppressin, [Arg(7)]-corazonin and [Arg(7)]-corazonin(3-11)) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 degrees C) and diapause-averting conditions (LD 18:6 at 25 degrees C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd-10th amino acids were sequenced as -YRKPPFNGS-, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.

Changes in the corpora allata and epidermal proliferation along the fourth instar of the Chagas disease vector Triatoma infestans

Triatoma infestans, a blood-feeding insect, synchronises physiological mechanisms leading to moult with food intake. Since the corpora allata are important in moult and metamorphosis regulation, we have studied morphological changes in 4th instar nymphs (gland size, cell density, percent of animals showing mitoses and cell size). Changes were correlated with the effect of precocene II, epidermal proliferation, and with the extent of the [quot ]head critical period[quot ]. Based on morphological grounds, three stages can be defined in the gland along the 4th instar: Stage 1 (days 0-2 after feeding) showed small corpora allata, composed by a small number of cells, and in which mitoses were absent; Stage 2 (days 3-9) showed growing corpora allata, in which cell number was increasing and proliferation was apparent; and Stage 3 (days 10-13) showed no mitotic activity, and a sharply diminishing size of the gland, as a consequence of the diminishing size of their cells. The ability of precocene II to induce abnormal moulting disappeared during stage 2 correlating with the termination of the head critical period and suggesting that corpora allata are essential during days 3 to 5 to determine normal growth. Epidermal cell number was increasing as a consequence of more frequent mitotic activity, beginning after the finalization of the head critical period and after a first increment in the size of the gland.

Changes in the corpora allata and epidermal proliferation along the fourth instar of the Chagas disease vector Triatoma infestans

Triatoma infestans, a blood-feeding insect, synchronises physiological mechanisms leading to moult with food intake. Since the corpora allata are important in moult and metamorphosis regulation, we have studied morphological changes in 4th instar nymphs (gland size, cell density, percent of animals showing mitoses and cell size). Changes were correlated with the effect of precocene II, epidermal proliferation, and with the extent of the [quot ]head critical period[quot ]. Based on morphological grounds, three stages can be defined in the gland along the 4th instar: Stage 1 (days 0-2 after feeding) showed small corpora allata, composed by a small number of cells, and in which mitoses were absent; Stage 2 (days 3-9) showed growing corpora allata, in which cell number was increasing and proliferation was apparent; and Stage 3 (days 10-13) showed no mitotic activity, and a sharply diminishing size of the gland, as a consequence of the diminishing size of their cells. The ability of precocene II to induce abnormal moulting disappeared during stage 2 correlating with the termination of the head critical period and suggesting that corpora allata are essential during days 3 to 5 to determine normal growth. Epidermal cell number was increasing as a consequence of more frequent mitotic activity, beginning after the finalization of the head critical period and after a first increment in the size of the gland.(AU)

Changes in the corpora allata and epidermal proliferation along the fourth instar of the Chagas disease vector Triatoma infestans.

Triatoma infestans, a blood-feeding insect, synchronises physiological mechanisms leading to moult with food intake. Since the corpora allata are important in moult and metamorphosis regulation, we have studied morphological changes in 4th instar nymphs (gland size, cell density, percent of animals showing mitoses and cell size). Changes were correlated with the effect of precocene II, epidermal proliferation, and with the extent of the "head critical period". Based on morphological grounds, three stages can be defined in the gland along the 4th instar: Stage 1 (days 0-2 after feeding) showed small corpora allata, composed by a small number of cells, and in which mitoses were absent; Stage 2 (days 3-9) showed growing corpora allata, in which cell number was increasing and proliferation was apparent; and Stage 3 (days 10-13) showed no mitotic activity, and a sharply diminishing size of the gland, as a consequence of the diminishing size of their cells. The ability of precocene II to induce abnormal moulting disappeared during stage 2 correlating with the termination of the head critical period and suggesting that corpora allata are essential during days 3 to 5 to determine normal growth. Epidermal cell number was increasing as a consequence of more frequent mitotic activity, beginning after the finalization of the head critical period and after a first increment in the size of the gland.

The effect of a static magnetic field on the morphometric characteristics of neurosecretory neurons and corpora allata in the pupae of yellow mealworm Tenebrio molitor (Tenebrionidae).

PURPOSE: The morphometric characteristics of A1 and A2 protocerebral neurosecretory neurons (cell and nuclei size, number of nucleoli in the nuclei); corpora allata size, nuclei size, cell number, were investigated in the pupae of yellow mealworm, Tenebrio molitor (L.), exposed to a strong static magnetic field of 320 mT maximum induction (10,000 times higher than the Earth's). MATERIALS AND METHODS: The experimental groups of Tenebrio molitor pupae were: A control group exposed only to natural magnetic field and sacrificed at the eighth day of pupal development (C); and pupae kept in a strong static magnetic field for eight days and then sacrificed (MF). Serial brain cross-sections were stained using the Alcian Blue Floxin technique. All the parameters were analyzed and measurements were performed using an image processing and analysis system (Leica, Cambridge, UK) linked to a Leica DMLB light microscope (program is QWin - Leica's Quantimet Windows-based image analysis tool kit). RESULTS: The values of morphometric parameters of neurosecretory neurons and corpora allata were significantly increased after exposure of the pupae to the strong magnetic field. CONCLUSIONS: The strong magnetic field influence characteristics of protocerebral neurosecretory neurons and corpora allata in the late Tenebrio molitor pupae.

Developmental regulation and functions of the expression of the neuropeptide corazonin in Drosophila melanogaster.

Although the corazonin gene (Crz) has been molecularly characterized, little is known concerning the function of this neuropeptide in Drosophila melanogaster. To gain insight into Crz function in Drosophila, we have investigated the developmental regulation of Crz expression and the morphology of corazonergic neurons. From late embryo to larva, Crz expression is consistently detected in three neuronal groups: dorso-lateral Crz neurons (DL), dorso-medial Crz neurons (DM), and Crz neurons in the ventral nerve cord (vCrz). Both the vCrz and DM groups die via programmed cell death during metamorphosis, whereas the DL neurons persist to adulthood. In adults, Crz is expressed in a cluster of six to eight neurons per lobe in the pars lateralis (DLP), in numerous neuronal cells in the optic lobes, and in a novel group of four abdominal ganglionic neurons present only in males (ms-aCrz). The DLP group consists of two subsets of cells having different developmental origins: embryo and pupa. In the optic lobes, we have detected both Crz transcripts and Crz promoter activity, but no Crz-immunoreactive products, suggesting a post-transcriptional regulation of Crz mRNA. Projections of the ms-aCrz neurons terminate within the ventral nerve cord, implying a role as interneurons. Terminals of the DLP neurons are found in the retrocerebral complex that produces juvenile hormone and adipokinetic hormone. Significant reduction of trehalose levels in adults lacking DLP neurons suggests that DLP neurons are involved in the regulation of trehalose metabolism. Thus, the tissue-, stage-, and sex-specific expression of Crz and the association of Crz with the function of the retrocerebral complex suggest diverse roles for this neuropeptide in Drosophila.

Farnesoid secretions of dipteran ring glands: what we do know and what we can know.

Harnessing of the Drosophila genetic system toward ascertaining the molecular endocrinology of higher dipteran (cyclorrhaphan) larval development has been a goal for over 70 years, beginning with the data left to us by pioneer researchers from the classical endocrine era. The results of their experiments evidence numerous ring gland activities that are parsimoniously explained as arising from secretions of the larval corpora allatal cells. Utilization of those data toward an understanding of molecular endocrinology of cyclorrhaphan metamorphosis has not yet achieved its hoped for fruition, in part due to a perceived difficulty in identifying larval targets of the molecule "methyl epoxyfarnesoate" (=juvenile hormone III). However, as is reviewed here, it is important to maintain a conceptual distinction between "the target of JH III"Versus "the target(s) of products secreted by the larval corpora allatal cells of ring glands." Recent advances have been made on the identity, regulation and reception of ring gland farnesoid products. When these advances are evaluated together with the above data from the classical endocrine era, there is a new opportunity to frame experimental hypotheses so as to discern underlying mechanisms on cyclorrhaphan larval-pupal metamorphosis that have been heretofore intractable. This paper reconsiders a number of evidenced physiological targets of secretions of corpora allatal cells of the larval ring gland, and places them in the context of more recent biochemical and molecular advances in the field.

Partial morphological and functional characterization of the corpus allatum-corpus cardiacum complex from the two-spotted stinkbug, Perillus bioculatus (Hemiptera: Pentatomidae).

Selected morphological and physiological properties of the corpus allatum (CA)-corpus cardiacum (CC) complex from the two-spotted stinkbug, Perillus bioculatus (Hemiptera: Pentatomidae), were studied. The CAs play an important role in insect physiology because of their production of the juvenile hormones (JHs), i.e., key hormones involved in development and reproduction. We found that the P. bioculatus CA-CC complex is present in two distinct morphological forms, the more frequently observed complex containing one "fused" CA between two CCs and the more rarely observed complex having one CA laterally attached to each CC. These complexes were tested for their ability to synthesize JH-like compounds. We found that the primary lipophilic compound synthesized by the CA-CCs migrated differently from JH III (a JH found in numerous insect species) when subjected to thin-layer chromatography. Furthermore, the synthesis of this compound is stimulated by 2E,6E-farnesol, a known precursor for JH III. These data indicate that the P. bioculatus CA- CC product has chemical properties similar to that of other (as of yet unidentified) hemipteran JHs. In addition, we found that the synthesis of this product is sensitive to pH and buffer type; minimally or not affected by the absence of the CC; expressed at similar levels in days 5-30 postemergent adults; and inhibited or decreased in adults reared under low temperature-short day conditions.

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.

Evidence for brain-derived neurotrophic factor-like neuropeptide in brain of the silk moth Bombyx mori during postembryonic periods.

Brain-derived neurotrophic factor-like neuropeptide is produced in the brain of the silk moth, Bombyx mori. Immunocytochemical studies of brain and retrocerebral complex of larvae, prepupae, pupae and adults showed that four pairs of median neurosecretory cells and six pairs of lateral neurosecretory cells which had different immunoreactivities to BDNF peptide. Day-1 adult brains showed no evidence of neurons stained by anti-BDNF antibodies. Those reactivities, which were much stronger in median cells than in lateral cells, were the weakest in an earliest larval stage and a latest pupal stage but the strongest in late larval stage. Median neurosecretory cells projected their axons into the contralateral corpora allata by decussation in the median region, nerve corpora cardiaca (NCC) I, and nerve corpora allata (NCA) I, whereas lateral neurosecretory cells extended their axons to the ipsilateral corpora allata via NCC II and NCA I.

Morphology and electrophysiological properties of neurons projecting to the retrocerebral complex in the blow fly, Protophormia terraenovae.

Morphological and electrical properties of neurons with somata in the pars intercerebralis (PI) and pars lateralis (PL) were examined by intracellular recording and staining in the adult blow fly, Protophormia terraenovae. According to the location of somata and fiber distribution, two types of PI neurons (PIa and PIb) and two types of PL neurons (PLa and PLb) were identified. PIb neurons were further divided into two subgroups of PIb1 and PIb2 depending on fiber branching patterns in the retrocerebral complex. PIa neurons projected axons to the contralateral nervi corporis cardiaci, whereas PLa and PLb neurons projected axons to the ipsilateral nervi corporis cardiaci. PIb neurons characteristically showed symmetrical morphology with their somata along the midline. PLb neurons had a large branching area in the subesophageal ganglion. In the retrocerebral complex, PIb2 and PLa neurons sent fibers into the corpus allatum. PIa, PIb1 and PLb neurons projected not to the corpus allatum but to the corpus cardiacum-hypocerebral complex or visceral muscles in their vicinity. PIa, PIb and PLa neurons showed long spike durations (3-10 ms). PLb neurons were immunoreactive with antisera against corazonin, FMRFamide, or beta-pigment-dispersing hormone. This is the first report revealing the morphology of individual neurons with somata residing in PI and PL in the adult fly.

Chilling stress effects on corpus allatum proliferation in the Hawaiian cockroach, Diploptera punctata: a role for ecdysteroids.

Endocrine regulation of corpus allatum (CA) cell proliferation in response to chilling was studied in mated females of the Hawaiian cockroach, Diploptera punctata. Chilling alone, when applied 24 h post-mating, suppressed CA cell division, and elevated ecdysteroid levels in Diploptera's haemolymph. Application of 20-hydroxyecdysone (20E) at 24 h post-mating similarly suppressed CA cell division, but had no effects at 48 h or 72 h post-mating. Severance of the ventral nerve cord prior to chilling or to the application of 20E prevented suppression of CA cell division, indicating that the effects of either chilling or 20E application are mediated by the ventral nerve cord.

Single K+ channels in endocrine cells dispersed from the cricket (Gryllus bimaculatus) corpora allata.

Single channel currents were recorded from cell-attached patches of endocrine cells of the adult male cricket corpora allata. Three distinct types of K+ channels were identified; a weak inward rectifier (Type 1), a strong inward rectifier (Type 2) and a weak outward rectifier (Type 3). The type 1 channel had a slope conductance of 191 +/- 9 pS (n = 4) at negative membrane potentials (Vm) and 101 +/- 6 pS (n = 6) at positive Vm. In addition, the channel showed fast open-closed kinetics at negative Vm and slow open-closed kinetics at positive Vm. The open probability (Po) of this channel was strongly voltage-dependent at positive Vm, but less voltage-dependent at negative Vm. The reversal potential was not modified significantly by the substitution of gluconate for external Cl- but was modified after N-methyl-D-glucamine (NMDG+) was substituted for external K+, according to the Nernst equation for a K+-selective channel. The type 2 channel had a slope conductance of 44 +/- 2 pS (n = 5) at negative Vm, but no detectable outward current was observed at positive Vm. This channel showed very slow open-closed kinetics at negative Vm and its Po was not voltage-dependent. The type 3 channel had a limit conductance of 55 +/- 12 pS (n = 3) at negative Vm and 88 +/- 10 pS (n = 3) at positive Vm. This channel showed slow open-closed kinetics at negative Vm and fast open-closed kinetics at positive Vm. The Po for the channel was voltage-dependent at positive Vm but was voltage-independent at negative Vm. These three types of K+ channels may be important for the control of the resting membrane potential, and may thus participate in the regulation of Ca2+ influx and juvenile hormone secretion in corpora allata cells.

A Na+-dependent electrogenic glutamate transporter current in voltage-clamped cells of corpora allata in the cricket Gryllus bimaculatus.

Application of L-glutamate (1 mM) to corpora allata cells of the adult male cricket Gryllus bimaculatus caused a membrane depolarization of 5.9+/-0.3 mV (mean +/- SE) from a resting potential of -62.2+/-1.3 mV (n=57). The underlying mechanism for this depolarization was studied by applying the two-electrode voltage-clamp technique. Application of L-glutamate (1 mM) elicited an inward current that peaked at 8.1+/-0.7 nA (n = 73) at a holding potential of-50 mV. Both L- and D-aspartate also induced an inward current of almost the same amplitude as L-glutamate, whereas D-glutamate failed to induce an inward current. Glutamate receptor agonists, such as kainate, quisqualate, alpha-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid, and N-methyl-D-aspartate, were ineffective in eliciting inward currents. The glutamate-induced inward current did not reverse even when the holding potential was set to +40 mV. The replacement of extracellular Na+ with choline+ eliminated the inward current. These results strongly suggest that the current induced by glutamate is mediated by a glutamate transporter rather than a glutamate receptor. We further examined the effects of 12 amino acid analogs which are known to be selective inhibitors of the mammalian excitatory amino acid transporters (EAATs) on the corpora allata transporter. From the effects of these inhibitors, we conclude that the glutamate transporter expressed in corpora allata cells of the cricket is similar to the high affinity glutamate transporters cloned from human brain, especially EAAT1 and EAAT3. Unlike mammalian transporters, however, serine-O-sulfate has the most potent action, suggesting the unique feature of the glutamate transporter expressed in the corpora allata.

Nadph-diaphorase activity in corpus allatum cells of the cockroach, Diploptera punctata.

Using the fixation insensitive NADPH-diaphorase reaction as a histochemical marker for the enzyme nitric oxide synthase (NOS), we investigated the possible sites of putatively NOS-related NADPH-diaphorase in the brain and retrocerebral complex of the cockroach, Diploptera punctata. In the cerebral ganglion, NADPH-diaphorase expression was localized in antennal lobes, optic lobes, mushroom bodies and neurosecretory cells. The highest NADPH activity was detected in the corpora allata (CA). Spectrophotometric quantitation indicated that NADPH-diaphorase activity first increased and then decreased (cycled) in the CA of mated females. In addition, during the first ovarian cycle, NADPH-diaphorase activity fluctuated concurrently with cyclic changes in the size of corpus allatum cells. In virgin females, NADPH-diaphorase activity remained at a low level, but it increased if the neural connectives between CA and brain were severed, indicating that the brain inhibited NADPH-diaphorase expression in the CA. Although nerve terminals were abundant in the CA, NADPH-diaphorase was clearly endogenous and synthesized by glandular cells, as was shown by histochemical staining of the cytosol in all dissociated cells of the CA. We have also demonstrated NADPH-diaphorase activity in the CA of the American cockroach Periplaneta americana, the house cricket Acheta domesticus, the lepidopteran Leucania loreyi, and the fruit fly Drosophila melanogaster, suggesting that NOS occurs in the CA of most, if not all insects. It is therefore possible that corpus allatum cells release NO, along with juvenile hormone, which presumably can function as a messenger molecule.