Neurochemical architecture of the central complex related to its function in the control of grasshopper acoustic communication

The central complex selects and coordinates the species- and situation-specific song production in acoustically communicating grasshoppers. Control of sound production is mediated by several neurotransmitters and modulators, their receptors and intracellular signaling pathways. It has previously been shown that muscarinic cholinergic excitation in the central complex promotes sound production whereas both GABA and nitric oxide/cyclic GMP signaling suppress its performance. The present immunocytochemical and pharmacological study investigates the question whether GABA and nitric oxide mediate inhibition of sound production independently. Muscarinic ACh receptors are expressed by columnar output neurons of the central complex that innervate the lower division of the central body and terminate in the lateral accessory lobes. GABAergic tangential neurons that innervate the lower division of the central body arborize in close proximity of columnar neurons and thus may directly inhibit these central complex output neurons. A subset of these GABAergic tangential neurons accumulates cyclic GMP following the release of nitric oxide from neurites in the upper division of the central body. While sound production stimulated by muscarine injection into the central complex is suppressed by co-application of sodium nitroprusside, picrotoxin-stimulated singing was not affected by co-application of this nitric oxide donor, indicating that nitric oxide mediated inhibition requires functional GABA signaling. Hence, grasshopper sound production is controlled by processing of information in the lower division of the central body which is subject to modulation by nitric oxide released from neurons in the upper division.     

Muscarinic excitation in grasshopper song control circuits is limited by acetylcholinesterase activity

The species- and situation-specific sound production of grasshoppers can be stimulated by focal application of both nicotinic and muscarinic receptor agonists into the central body complex of the protocerebrum. Pressure injection of the intrinsic transmitter acetylcholine only elicits fast and short-lived responses related to nicotinic receptor-mediated excitation. Prolonged sound production that includes complex song patterns requires muscarinic receptor-mediated excitation. In addition, basal muscarinic excitation in the central body neuropil seems to determine the general motivation of a grasshopper to stridulate. To demonstrate that endogenous acetylcholinesterase limits the activation of muscarinic receptors by synaptically released acetylcholine in the central body of Chorthippus biguttulus, we investigated both its presence in the brain and effects on sound production resulting from inhibition of esterase activity. Acetylcholinesterase activity was detected in the upper and lower division of the central body. Both these neuropils known to be involved in the cephalic control of stridulation were also shown to contain muscarinic acetylcholine receptors expressed by columnar neurons suggested to serve as output neurons of the central complex. Pressure injection of the acetylcholinesterase inhibitor eserine into protocerebral control circuits of restrained male grasshoppers stimulated long-lasting stridulation that depended on scopolamine-sensitive muscarinic receptors. In restrained males, eserine released the typical response song by potentiating the stimulatory effect of the conspecific female song. Eserine-mediated inhibition of acetylcholinesterase in the central body prolongs the presence of synaptically released acetylcholine at its postsynaptic receptors and increases its potency to activate muscarinic receptor-initiated signaling pathways acting to promote grasshopper sound production.     

Nitric oxide/cGMP signaling in the corpora allata of female grasshoppers

The corpora allata (CA) of various insects express enzymes with fixation resistant NADPHdiaphorase activity. In female grasshoppers, juvenile hormone (JH) released from the CA is necessary to establish reproductive readiness, including sound production. Previous studies demonstrated that female sound production is also promoted by systemic inhibition of nitric oxide (NO) formation. In addition, allatotropin and allatostatin expressing central brain neurons were located in close vicinity of NO generating cells. It was therefore speculated that NO signaling may contribute to the control of juvenile hormone release from the CA.This study demonstrates the presence of NO/cGMP signaling in the CA of female Chorthippus biguttulus. CA parenchymal cells exhibit NADPHdiaphorase activity, express anti NOS immunoreactivity and accumulate citrulline, which is generated as a byproduct of NO generation. Varicose terminals from brain neurons in the dorsal pars intercerebralis and pars lateralis that accumulate cGMP upon stimulation with NO donors serve as intrinsic targets of NO in the CA. Both accumulation of citrulline and cyclic GMP were inhibited by the NOS inhibitor aminoguanidine, suggesting that NO in CA is produced by NOS. These results suggest that NO is a retrograde transmitter that provides feedback to projection neurons controlling JH production. Combined immunostainings and backfill experiments detected CA cells with processes extending into the CC and the protocerebrum that expressed immunoreactivity against the pan-neural marker anti-HRP. Allatostatin and allatotropin immunopositive brain neurons do not express NOS but subpopulations accumulate cGMP upon NO-formation. Direct innervation of CA by these peptidergic neurons was not observed.     

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.     

Nitric oxide synthase inhibitors and nitric oxide donors modulate the biosynthesis of thaxtomin A, a nitrated phytotoxin produced by Streptomyces spp.

Evidence for the involvement of a bacterial nitric oxide synthase (NOS) in the biosynthesis of a phytotoxin is presented. Several species of Streptomyces bacteria produce secondary metabolites with unusual nitrogen groups, such as thaxtomin A (ThxA), which contains a nitroindole moiety. ThxA is a phytotoxin made by three pathogenic Streptomyces species that cause common scab of potato. All three species possess a gene homologous to the oxygenase domain of murine inducible NOS, and this gene, nos, is essential for normal levels of ThxA production. We grew Streptomyces turgidiscabies in the presence of several known NOS inhibitors and a nitric oxide (NO) scavenger to determine their effect on ThxA production. The NO scavenger (CPTIO) and four NOS inhibitors (NAME, NMMA, AG, and 7-NI) reduced ThxA production without affecting bacterial growth. A strain of S. turgidiscabies from which the nos gene had been deleted was grown in the presence of three NO donors (DEANO, SIN, and SNAP), and all three partially restored ThxA production. Our data suggest that bacterial nitric oxide synthases may, at least in part, produce NO for biosynthetic purposes, rather than for cellular signaling, as they do in mammals.     

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death

The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis.     

The effects of nitric oxide on prostanoid production and release by human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) express and synthesize both constitutive and inducible nitric oxide synthase (NOS) and cyclo-oxygenase (COX) enzymes, and have been extensively used as an in vitro model to investigate the role of these enzymes in the patho-physiology of placenta-fetal circulation. In this study we investigated the role of NO in regulating prostanoid production and release from HUVEC. Both untreated and IL-1beta-treated HUVEC were exposed to various NOS inhibitors and NO donors in short-term (1 or 3 hours) experiments, and the effects on prostanoid production were evaluated through the measurement of prostaglandins (PG) I2, E2 and F2alpha released in the incubation medium. We found that the inhibition of inducible NOS but not endothelial NOS antagonizes the IL-1beta-induced increase in PGI2 release. However, NOS inhibitors do not modify baseline PGI2 production. Pharmacological levels of NO, obtained with various NO donors, inhibit basal and IL-1beta-stimulated PG release.     

Developmental expression of neuromodulators in the central complex of the grasshopper Schistocerca gregaria

The central complex is a major integrative region within the insect brain with demonstrated roles in spatial orientation, the regulation of locomotor behavior, and sound production. In the hemimetabolous grasshopper, the central complex comprises the protocerebral bridge, central body (CB), ellipsoid body, noduli, and accessory lobes, and this modular organization develops entirely during embryogenesis. From a biochemical perspective, a range of neuroactive substances has been demonstrated in these modules of the adult central complex, but little is known about their developmental expression. In this study, we use matrix‐assisted laser desorption/ionization‐imaging mass spectrometry on single brain slices to confirm the presence of several peptide families (tachykinin, allatostatin, periviscerokinin/pyrokinin, FLRFamide, and neuropeptide F) in the adult central complex and then use immunohistochemistry and histology to examine their developmental expression, together with that of the indolamin serotonin, and the endogenous messenger nitric oxide (NO; via its synthesizing enzyme). We find that each neuromodulator is expressed according to a unique, stereotypic, pattern within the various modules making up the central complex. Neuropeptides such as tachykinin (55%) and allatostatin (65%), and the NO‐synthesizing enzyme diaphorase (70%), are expressed earlier during embryonic development than the biogenic amine serotonin (80%), whereas periviscerokinin‐like peptides and FLRFamide‐like peptides begin to be expressed only postembryonically. Within the CB, these neuroactive substances are present in tangential projection neurons before they appear in columnar neurons. There is also no colocalization of serotonin‐positive and peptide‐positive projections up to the third larval instar during development, consistent with the clear dorsoventral layering of the neuropil we observe. Our results provide the first neurochemical fingerprint of the developing central complex in an hemimetabolous insect. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

一氧化氮合酶在豚鼠听觉核团的分布

为了研究一氧化氮合酶（ｎｉｔｒｉｃｏｘｉｄｅｓｙｔｈａｓｅ,ＮＯＳ）在听觉核团的分布特点,探讨一氧化氮（ｎｉｔｒｉｃｏｘｅｄｅ,ＮＯ）在听觉径路中的作用,本文采用ＮＡＤＰＨ硫辛酸胺脱氢酶（ＮＡＤＰＨ－ｄ）组织化学方法,研究了豚鼠听觉核团内ＮＯＳ的分布。结果发现,在各级听觉传入核团,均有ＮＯＳ阳性神经元,而上橄榄复合体ＮＯＳ反应阴性。耳蜗核ＮＯＳ阳性神经元主要集中在耳蜗后腹核,为圆形或椭圆形双极神经元。下丘ＮＯＳ阳性反应神经元位于下丘中央核团,胞体形状和大小不一。内侧膝状体背侧核ＮＯＳ阳性神经元相对集中,多为双极神经元,部分神经元突起很长,散在阳性纤维,部分阳性纤维穿行于内侧膝状体背侧核与内侧膝状体之间。本研究提示,ＮＯ可能是听觉中枢的神经递质或调质,参与声信号传递的调节。     

Nitric oxide release from a single cell affects filopodial motility on growth cones of neighboring neurons

Nitric oxide (NO), a gaseous messenger, has been reported to be involved in a variety of functions in the nervous system, ranging from neuronal pathfinding to learning and memory. We have shown previously that the application of NO via NO donors to growth cones of identified Helisoma buccal neurons B5 in vitro induces an increase in filopodial length, a decrease in filopodial number, and a slowing in neurite advance. It is unclear, however, whether NO released from a physiological source would affect growth cone dynamics. Here we used cell bodies of identified neurons known to express the NO synthesizing enzyme nitric oxide synthase (NOS) as a source of constitutive NO production and tested their effect on growth cones of other cells in a sender-receiver paradigm. We showed that B5 cell bodies induced a rapid increase in filopodial length in NO-responsive growth cones, and that this effect was blocked by the NOS inhibitor 7-NI, suggesting that the effect was mediated by NO. Inhibition of soluble guanylyl cyclase (sGC) with ODQ blocked filopodial elongation induced by B5 somata, confirming that NO acted via sGC. We also demonstrate that the effect of NO was reversible and that a cell releasing NO can affect growth cones over a distance of at least 100 microm. Our results suggest that NO released from a physiological source can affect the motility of nearby growth cones and thus should be considered a signaling molecule with the potential to affect the outcome of neuronal pathfinding in vivo.     

Two enzymatic sources of nitric oxide in different organs of apple plant

Nitric oxide production, nitric oxide synthase (NOS) and mitochondrial nitrite-reducing activities in roots, leaves and stems of different developmental stages were investigated, using potted 3-year-old apple (Malus domestica Borkh.) trees. The arginine-dependent NOS activity is sensitive to NOS inhibitor L-NAME and aminoguanidine (AG), with L-NAME being more effective than AG. Endogenous NO production, NOS and mitochondrial nitrite-reducing activities are predominately presented in young leaves and especially in young white roots and young stems. Root and stem mitochondria can reduce nitrite to nitric oxide at the expense of NADH, however, this mitochondrial nitrite-reducing activity is absent in leaves.     

Preservation of neurological functions by nitric oxide synthase inhibitors in conscious rats following delayed hemorrhagic shock

Excessive production of nitric oxide (NO) as result of inducible nitric oxide synthase (iNOS) induction has been implicated in the pathophysiology of hemorrhagic shock. Our aim was to study the effects of NOS inhibitors, aminoguanidine (AG) and NG-nitro-L-arginine methyl ester (L-NAME), on survival rate, mean arterial blood pressure (MABP), temporal evolution of infarct volume, nitric oxide (NO) production and neurological deficit in a model of delayed hemorrhagic shock (DHS) in conscious rats. Our results showed that the NOS inhibitors significantly improved survival rate, MABP, and attenuated brain NO overproduction 24, 48 h and 72 h after DHS. AG reduced brain infarct volume and improved the neurological performance evaluated by the rotameric and grip strength tests while L-NAME did not show protective effect in rats following DHS. These findings suggest that NO formation via iNOS activation may contribute to organ damage and that the selective iNOS inhibitor, AG, may be of interest as a therapeutic agent for neurological recovery following DHS.     

The role of nitric oxide in regulation of the cardiovascular system in reptiles

The roles that nitric oxide (NO) plays in the cardiovascular system of reptiles are reviewed, with particular emphasis on its effects on central vascular blood flows in the systemic and pulmonary circulations. New data is presented that describes the effects on hemodynamic variables in varanid lizards of exogenously administered NO via the nitric oxide donor sodium nitroprusside (SNP) and inhibition of nitric oxide synthase (NOS) by l-nitroarginine methyl ester (l-NAME). Furthermore, preliminary data on the effects of SNP on hemodynamic variables in the tegu lizard are presented. The findings are compared with previously published data from our laboratory on three other species of reptiles: pythons (), rattlesnakes () and turtles (). These five species of reptiles possess different combinations of division of the heart and structural complexity of the lungs. Comparison of their responses to NO donors and NOS inhibitors may reveal whether the potential contribution of NO to vascular tone correlates with pulmonary complexity and/or with blood pressure. All existing studies on reptiles have clearly established a potential role for NO in regulating vascular tone in the systemic circulation and NO may be important for maintaining basal systemic vascular tone in varanid lizards, pythons and turtles, through a continuous release of NO. In contrast, the pulmonary circulation is less responsive to NO donors or NOS inhibitors, and it was only in pythons and varanid lizards that the lungs responded to SNP. Both species have a functionally separated heart, so it is possible that NO may exert a larger role in species with low pulmonary blood pressures, irrespective of lung complexity.     

一氧化氮的释放对海马脑片CA1区痫样放电的影响

用自制的一氧化氮（ＮＯ）敏感电极－Ｎａｆｉｏｎ－壳聚糖合镍修饰铂电极（Ｎａｆｉｏｎ－ＣＴＳ（Ｎｉ）－Ｐｔ）连续测定了青霉素致痫海马脑片ＣＡ１区锥体层神经元ＮＯ的释放,并同时观察了ＮＯ合酶抑制剂７－ｎｉｔｒｏ－ｉｎｄａｚｏｌｅ（７－ＮＩ）及Ｎ＾ω－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ（Ｌ－ＮＮＡ）对诱发痫波及ＮＯ释放量的影响。研究观察到：（１）在青霉素致痫脑片模型上,诱发的痫波随青霉素浓度的增加而增多,     

脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用

在乌拉坦麻醉的成年ＳＤ大鼠上,用玻璃微电极细胞外记录的方法,观察了脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用。结果发现：脑室内注射一氧化氮供体硝普钠对下丘脑室旁核中的加压素神经元产生剂量依赖性抑制作用;脑室内注射一氧化氮合酶抑制剂对加压素神经元也产生抑制作用。上述两种药物对催产素神经元均无作用。这些结果提示：一氧化氮可能在调节加压素和催产素神经元活动中起着不同的作用。     

Calcium-dependent release of NO from intracellular S-nitrosothiols

The paper describes a novel cellular mechanism for rapid calcium-dependent nitric oxide (NO) release. This release occurs due to NO liberation from S-nitrosothiols. We have analysed the changes of NO concentration in acutely isolated pancreatic acinar cells. Supramaximal acetylcholine (ACh) stimulation induced a Ca(2+)-dependent increase in the fluorescence in the majority of cells loaded with the NO probe DAF-FM via a patch pipette. The ACh-induced NO signals were insensitive to inhibitors of calmodulin and protein kinase C but were inhibited by calpain antagonists. The initial part of the NO signals induced by 10 muM ACh showed little sensitivity to inhibition of NO synthase (NOS); however, cell pretreatment with NO donors (increasing cellular S-nitrosothiol contents) substantially enhanced the initial component of NO responses. Pancreatic acinar cells were able to generate fast calcium-dependent NO responses when stimulated with physiological or supramaximal doses of secretagogues. Importantly, the source of this NO is the already available S-nitrosothiol store rather than de novo synthesis by NOS. A similar mechanism of NO release was found in dorsal root ganglia neurons.     

Attenuation of host NO production by MAMPs potentiates development of the host in the squid-vibrio symbiosis

Bacterial pathogens typically upregulate the host's production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents, a response that is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in attenuation of host NOS/NO, which occurs in high levels in hatchling light organs. Here, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we characterized levels of NOS with immunocytochemistry and NO with a NO-specific fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host's response to MAMPs depends on the context. These data also provide a mechanism by which symbiont MAMPs regulate host development.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

NO/cGMP signalling: <Emphasis Type="SmallCaps">L</Emphasis>-citrulline and cGMP immunostaining in the central complex of the desert locust <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>

Nitric oxide (NO) is a gaseous messenger molecule formed during conversion of L-arginine into L-citrulline by the enzyme NO synthase (NOS), which belongs to a group of NADPH diaphorases. Because of its gaseous diffusion properties, NO differs from classical neurotransmitters in that it is not restricted to synaptic terminals. In target cells, NO activates soluble guanylyl cyclase leading to an increase in cGMP levels. In insects, this NO/cGMP-signalling pathway is involved in development, memory formation and processing of visual, olfactory and mechanosensory information. We have analysed the distribution of putative NO donor and target cells in the central complex, a brain area involved in sky-compass orientation, of the locust Schistocerca gregaria by immunostaining for L-citrulline and cGMP. Six types of citrulline-immunostained neurons have been identified including a bilateral pair of hitherto undescribed neurons that connect the lateral accessory lobes with areas anterior to the medial lobes of the mushroom bodies. Three-dimensional reconstructions have revealed the connectivity pattern of a set of 18 immunostained pontine neurons of the central body. All these neurons appear to be a subset of previously mapped NADPH-diaphorase-positive neurons of the central complex. At least three types of central-complex neurons show cGMP immunostaining including a system of novel columnar neurons connecting the upper division of the central body and the lateral triangle of the lateral accessory lobe. Our results provide the morphological basis for further studies of the function of the labelled neurons and new insights into NO/cGMP signalling. This work was supported by DFG grant HO 950/16-2.     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.