A new species of cockroach, Periplaneta gajajimana sp. nov., collected in Gajajima, Kagoshima Prefecture, Japan.

We described a new species of cockroach, Periplaneta gajajimana sp. nov., which was collected in Gajajima, Kagoshima-gun Toshimamura, Kagoshima Prefecture, Japan, on November 2012. The new species is characterized by its reddish brown to blackish brown body, smooth surface pronotum, well developed compound eyes, dark brown head apex, dark reddish brown front face and small white ocelli connected to the antennal sockets. In male, the tegmen tip reach the abdomen end or are slightly shorter, while in the female, it does not reach the abdominal end and exposes the abdomen beyond the 7th abdominal plate. We confirmed the validity of this new species by breeding the specimens in our laboratory to demonstrate that the features of the progeny were maintained for several generations. For comparison and easy identification of this new species, the key to species identification of the genus Periplaneta that had been reported in Japan to date are also presented.

Antifungal activity of 3-acetylbenzamide produced by actinomycete WA23-4-4 from the intestinal tract of Periplaneta americana.

Actinomycetes are well-known for producing numerous bioactive secondary metabolites. In this study, primary screening by antifungal activity assay found one actinomycete strain WA23-4-4 isolated from the intestinal tract of Periplaneta americana that exhibited broad spectrum antifungal activity. 16S rDNA gene analysis of strain WA23-4-4 revealed close similarity to Streptomyces nogalater (AB045886) with 86.6% sequence similarity. Strain WA23-4-4 was considered as a novel Streptomyces and the 16s rDNA sequence has been submitted to GenBank (accession no. KX291006). The maximum antifungal activity of WA23-4-4 was achieved when culture conditions were optimized to pH 8.0, with 12% inoculum concentration and 210 ml ISP2 medium, which remained stable between the 5th and the 9th day. 3-Acetyl benzoyl amide was isolated by ethyl acetate extraction of WA23-4-4 fermentation broth, and its molecular formula was determined as C9H9NO2 based on MS, IR, 1H, and 13C NMR analyses. The compound showed significant antifungal activity against Candida albicans ATCC 10231 (MIC: 31.25 µg/ml) and Aspergillus niger ATCC 16404 (MIC: 31.25 µg/ml). However, the compound had higher MIC values against Trichophyton rubrum ATCC 60836 (MIC: 500 µg/ml) and Aspergillus fumigatus ATCC 96918 (MIC: 1,000 µg/ml). SEM analysis showed damage to the cell membrane of Candida albicans ATCC 10231 and to the mycelium of Aspergillus niger ATCC 16404 after being treatment with 3-acetyl benzoyl amide. In conclusion, this is the first time that 3-acetyl benzoyl amide has been identified from an actinomycete and this compound exhibited antifungal activity against Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.

Local tissue manipulation via a force- and pressure-controlled AFM micropipette for analysis of cellular processes.

Local manipulation of complex tissues at the single-cell level is challenging and requires excellent sealing between the specimen and the micromanipulation device. Here, biological applications for a recently developed loading technique for a force- and pressure-controlled fluidic force microscope micropipette are described. This technique allows for the exact positioning and precise spatiotemporal control of liquid delivery. The feasibility of a local loading technique for tissue applications was investigated using two fluorescent dyes, with which local loading behaviour could be optically visualised. Thus, homogeneous intracellular distribution of CellTracker Red and accumulation of SYTO 9 Green within nuclei was realised in single cells of a tissue preparation. Subsequently, physiological micromanipulation experiments were performed. Salivary gland tissue was pre-incubated with the Ca2+-sensitive dye OGB-1. An intracellular Ca2+ rise was then initiated at the single-cell level by applying dopamine via micropipette. When pre-incubating tissue with the nitric oxide (NO)-sensitive dye DAF-FM, NO release and intercellular NO diffusion was observed after local application of the NO donor SNP. Finally, local micromanipulation of a well-defined area along irregularly shaped cell surfaces of complex biosystems was shown for the first time for the fluidic force microscope micropipette. Thus, this technique is a promising tool for the investigation of the spatiotemporal effects of locally applied substances in complex tissues.

In-vivo biomagnetic characterisation of the American cockroach.

We present a quantitative method, utilising a highly sensitive quantum sensor, that extends applicability of magnetorelaxometry to biological samples at physiological temperature. The observed magnetic fields allow for non-invasive determination of physical properties of magnetic materials and their surrounding environment inside the specimen. The method is applied to American cockroaches and reveals magnetic deposits with strikingly different behaviour in alive and dead insects. We discuss consequences of this finding to cockroach magneto-reception. To our knowledge, this work represents the first characterisation of the magnetisation dynamics in live insects and helps to connect results from behavioural experiments on insects in magnetic fields with characterisation of magnetic materials in their corpses.

New insight into foregut functions of xenobiotic detoxification in the cockroach Periplaneta americana.

The physiological functions of insect foregut, especially in xenobiotic detoxification, are scarcely reported because of unimportance in appearance and insufficient molecular information. The cockroach Periplaneta americana, an entomological model organism, provides perfect material to study physiological functions of foregut tissue due to its architectural feature. Through Illumina sequencing of foregut tissue from P. americana individuals (control) or insects treated with cycloxaprid, as a novel neonicotinoid insecticide, 54 193 166 clean reads were obtained and further assembled into 53 853 unigenes with an average length of 366 bp. Furthermore, the number of unigenes involved in xenobiotic detoxification was analyzed, mainly including 70 cytochrome P450s, 12 glutathione S-transferases (GSTs), seven carboxylesterases (CarEs) and seven adenosine triphosphate-binding cassette (ABC) transporters. Compared to control, the expression of 22 xenobiotic detoxification unigenes was up-regulated after cycloxaprid application, mainly containing 18 P450s, one GST, two CarEs and one ABC adenosine triphosphate transporter, indicating that the oxidation-reduction was the major reactive process to cycloxaprid application. Through quantitative real-time polymerase chain reaction analysis, the expression of selected unigenes (six P450s, one GST and one CarE) was up-regulated at least two-fold following cycloxaprid treatment, and was generally in agreement with transcriptome data. Compared to the previous midgut transcriptome of P. americana, it looks like the expressive abundance of the xenobiotic detoxification unigenes might be important factors to the detoxifying functional differences between foregut and midgut. In conclusion, insect foregut would also play important roles in the physiological processes related to xenobiotic detoxification.

Not flying blind: a comparative study of photoreceptor function in flying and non-flying cockroaches.

Flying is often associated with superior visual performance, as good vision is crucial for detection and implementation of rapid visually guided aerial movements. To understand the evolution of insect visual systems it is therefore important to compare phylogenetically related species with different investments in flight capability. Here, we describe and compare morphological and electrophysiological properties of photoreceptors from the habitually flying green cockroach Panchlora nivea and the American cockroach Periplaneta americana, which flies only at high ambient temperatures. In contrast to Periplaneta, ommatidia in Panchlora were characterized by two-tiered rhabdom, which might facilitate detection of polarized light while flying in the dark. In patch-clamp experiments, we assessed the absolute sensitivity to light, elementary and macroscopic light-activated current and voltage responses, voltage-activated potassium (Kv) conductances, and information transfer. Both species are nocturnal, and their photoreceptors were similarly sensitive to light. However, a number of important differences were found, including the presence in Panchlora of a prominent transient Kv current and a generally low variability in photoreceptor properties. The maximal information rate in Panchlora was one-third higher than in Periplaneta, owing to a substantially higher gain and membrane corner frequency. The differences in performance could not be completely explained by dissimilarities in the light-activated or Kv conductances; instead, we suggest that the superior performance of Panchlora photoreceptors mainly originates from better synchronization of elementary responses. These findings raise the issue of whether the evolutionary tuning of photoreceptor properties to visual demands proceeded differently in Blattodea than in Diptera.

Fast and Powerful: Biomechanics and Bite Forces of the Mandibles in the American Cockroach Periplaneta americana.

Knowing the functionality and capabilities of masticatory apparatuses is essential for the ecological classification of jawed organisms. Nevertheless insects, especially with their outstanding high species number providing an overwhelming morphological diversity, are notoriously underexplored with respect to maximum bite forces and their dependency on the mandible opening angles. Aiming for a general understanding of insect biting, we examined the generalist feeding cockroach Periplaneta americana, characterized by its primitive chewing mouth parts. We measured active isometric bite forces and passive forces caused by joint resistance over the entire mandibular range with a custom-built 2D force transducer. The opening angle of the mandibles was quantified by using a video system. With respect to the effective mechanical advantage of the mandibles and the cross-section areas, we calculated the forces exerted by the mandible closer muscles and the corresponding muscle stress values. Comparisons with the scarce data available revealed close similarities of the cockroaches' mandible closer stress values (58 N/cm2) to that of smaller specialist carnivorous ground beetles, but strikingly higher values than in larger stag beetles. In contrast to available datasets our results imply the activity of faster and slower muscle fibres, with the latter becoming active only when the animals chew on tough material which requires repetitive, hard biting. Under such circumstances the coactivity of fast and slow fibres provides a force boost which is not available during short-term activities, since long latencies prevent a specific effective employment of the slow fibres in this case.

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.

Analyses of the mouthpart kinematics in Periplaneta americana (Blattodea, Blattidae) using synchrotron-based X-ray cineradiography.

The kinematics of the biting and chewing mouthparts of insects is a complex interaction of various components forming multiple jointed chains. The non-invasive technique of in vivo cineradiography by means of synchrotron radiation was employed to elucidate the motion cycles of the mouthparts in the cockroach Periplaneta americana. Digital X-ray footage sequences were used in order to calculate pre-defined angles and distances, each representing characteristic aspects of the movement pattern. We were able to analyze the interactions of the mouthpart components and to generate a functional model of maxillary movement by integrating kinematic results, morphological dissections and fluorescence microscopy. During the opening and closing cycles, which take about 450-500 ms on average, we found strong correlations between the measured maxillary and mandibular angles, indicating a strong neural coordination of these movements. This is manifested by strong antiphasic courses of the maxillae and the mandibles, antiphasic patterns of the rotation of the cardo about its basic articulation at the head and by the deflection between the cardo and stipes. In our functional model of the maxilla, its movement pattern is explained by the antagonistic activity of four adductor-promotor muscles and two abductor-remotor muscles. However, beyond the observed intersegmental and bilateral stereotypy, certain amounts of variation across subsequent cycles within a sequence were observed with respect to the degree of correlation between the various mouthparts, the maximum, minimum and time course of the angular movements. Although generally correlated with the movement pattern of the mandibles and the maxillary cardo-stipes complex, such plastic behaviour was especially observed in the maxillary palpi and the labium.

Serotonin-immunoreactive sensory neurons in the antenna of the cockroach Periplaneta americana.

The antennae of insects contain a vast array of sensory neurons that process olfactory, gustatory, mechanosensory, hygrosensory, and thermosensory information. Except those with multimodal functions, most sensory neurons use acetylcholine as a neurotransmitter. Using immunohistochemistry combined with retrograde staining of antennal sensory neurons in the cockroach Periplaneta americana, we found serotonin-immunoreactive sensory neurons in the antenna. These were selectively distributed in chaetic and scolopidial sensilla and in the scape, the pedicel, and first 15 segments of the flagellum. In a chaetic sensillum, A single serotonin-immunoreactive sensory neuron cohabited with up to four serotonin-negative sensory neurons. Based on their morphological features, serotonin-immunopositive and -negative sensory neurons might process mechanosensory and contact chemosensory modalities, respectively. Scolopidial sensilla constitute the chordotonal and Johnston's organs within the pedicel and process antennal vibrations. Immunoelectron microscopy clearly revealed that serotonin-immunoreactivities selectively localize to a specific type of mechanosensory neuron, called type 1 sensory neuron. In a chordotonal scolopidial sensillum, a serotonin-immunoreactive type 1 neuron always paired with a serotonin-negative type 1 neuron. Conversely, serotonin-immunopositive and -negative type 1 neurons were randomly distributed in Johnston's organ. In the deutocerebrum, serotonin-immunoreactive sensory neuron axons formed three different sensory tracts and those from distinct types of sensilla terminated in distinct brain regions. Our findings indicate that a biogenic amine, serotonin, may act as a neurotransmitter in peripheral mechanosensory neurons.

Locomotion- and mechanics-mediated tactile sensing: antenna reconfiguration simplifies control during high-speed navigation in cockroaches.

Animals can expend energy to acquire sensory information by emitting signals and/or moving sensory structures. We propose that the energy from locomotion itself could permit control of a sensor, whereby animals use the energy from movement to reconfigure a passive sensor. We investigated high-speed, antenna-mediated tactile navigation in the cockroach Periplaneta americana. We discovered that the passive antennal flagellum can assume two principal mechanical states, such that the tip is either projecting backward or forward. Using a combination of behavioral and robotic experiments, we demonstrate that a switch in the antenna's state is mediated via the passive interactions between the sensor and its environment, and this switch strongly influences wall-tracking control. When the tip of the antenna is projected backward, the animals maintain greater body-to-wall distance with fewer body collisions and less leg-wall contact than when the tip is projecting forward. We hypothesized that distally pointing mechanosensory hairs at the tip of the antenna mediate the switch in state by interlocking with asperities in the wall surface. To test this hypothesis, we performed laser ablation of chemo-mechanosensory hairs and added artificial hairs to a robotic antenna. In both the natural and artificial systems, the presence of hairs categorically increased an antenna's probability of switching state. Antennal hairs, once thought to only play a role in sensing, are sufficient for mechanically reconfiguring the state of the entire antenna when coupled with forward motion. We show that the synergy between antennal mechanics, locomotion and the environment simplifies tactile sensing.

Using DNA barcodes to confirm the presence of a new invasive cockroach pest in New York City.

Recently, specimens of a Periplaneta sp. were discovered in New York, NY, that did not match the typical morphology of Periplaneta americana L., the ubiquitous American cockroach. Here, we used DNA barcoding and morphological identification to confirm that this newly invasive pest species was indeed Periplaneta japonica Karny, 1908. We discuss this recent invasion in light of known life history traits of this species, with specific predictions for its impact in the urban northeastern United States.

Divergent and convergent projections to the two parallel olfactory centers from two neighboring, pheromone-receptive glomeruli in the male American cockroach.

Many animals utilize sex pheromone for detecting conspecific mates. Sex pheromone is usually a blend of two or more components with similar chemical compositions. The pheromone receivers are equipped with localized olfactory glomeruli in the first-order olfactory center for specifically processing these pheromone components. In the American cockroach, Periplaneta americana, either periplanone A or periplanone B emitted by virgin females evokes identical sexual behaviors in males. The antennal lobes of adult male cockroaches have enlarged, neighboring A- and B-glomeruli, which preferentially process periplanones A and B, respectively. By using intracellular recording and staining of neurons in the same preparations, we provide the first detailed projection maps of output neurons (projection neurons; PNs) from the A-glomerulus and the B-glomerulus. Although both PNs project to the mushroom body calyces and the lateral horn, their proximities in the two centers largely differ: in the calyces, the axon terminals of the A-PN were located more predominantly in the periphery compared with those of the B-PN, whereas axon terminals of both PNs were highly congruent in the anteromedial region of the lateral horn. These results suggest that pheromone component signals are dispersed in the mushroom body for specific odor discrimination but are integrated in the lateral horn for generating behaviors common to the pheromone components. Stimulation of the ipsilateral antenna with various odors showed that the odor specificity of A-PN is higher than that of B-PN. The different developmental lineages of A- and B-PNs suggested by these results are discussed.

Visual and olfactory input segregation in the mushroom body calyces in a basal neopteran, the American cockroach.

The cockroach Periplaneta americana is an evolutionary basal neopteran insect, equipped with one of the largest and most elaborate mushroom bodies among insects. Using intracellular recording and staining in the protocerebrum, we discovered two new types of neurons that receive direct input from the optic lobe in addition to the neuron previously reported. These neurons have dendritic processes in the optic lobe, projection sites in the optic tracts, and send axonal terminals almost exclusively to the innermost layer of the MB calyces (input site of MB). Their responses were excitatory to visual but inhibitory to olfactory stimuli, and weak excitation occurred in response to mechanosensory stimuli to cerci. In contrast, interneurons with dendrites mainly in the antennal lobe projection sites send axon terminals to the middle to outer layers of the calyces. These were excited by various olfactory stimuli and mechanosensory stimuli to the antenna. These results suggest that there is general modality-specific terminal segregation in the MB calyces and that this is an early event in insect evolution. Possible postsynaptic and presynaptic elements of these neurons are discussed.

Complete mapping of glomeruli based on sensory nerve branching pattern in the primary olfactory center of the cockroach Periplaneta americana.

Glomeruli are structural and functional units in the primary olfactory center in vertebrates and insects. In the cockroach Periplaneta americana, axons of different types of sensory neurons housed in sensilla on antennae form dorsal and ventral antennal nerves and then project to a number of glomeruli. In this study, we identified all antennal lobe (AL) glomeruli based on detailed innervation patterns of sensory tracts in addition to the shape, size, and locations in the cockroach. The number of glomeruli is approximately 205, and no sex-specific difference is observed. Anterograde dye injections into the antennal nerves revealed that axons supplying the AL are divided into 10 sensory tracts (T1-T10). Each of T1-T3 innervates small, oval glomeruli in the anteroventral region of the AL, with sensory afferents invading each glomerulus from multiple directions, whereas each of T4-T10 innervates large glomeruli with various shapes in the posterodorsal region, with a bundle of sensory afferents invading each glomerulus from one direction. The topographic branching patterns of all these tracts are conserved among individuals. Sensory afferents in a sub-tract of T10 had axon terminals in the dorsal margin of the AL and the protocerebrum, where they form numerous small glomerular structures. Sensory nerve branching pattern should reflect developmental processes to determine spatial arrangement of glomeruli, and thus the complete map of glomeruli based on sensory nerve branching pattern should provide a basis for studying the functional significance of spatial arrangement of glomeruli and its developmental basis.

Distinct electrophysiological properties in subtypes of nonspiking olfactory local interneurons correlate with their cell type-specific Ca2+ current profiles.

A diverse population of local interneurons (LNs) helps to process, structure, and spatially represent olfactory information in the insect antennal lobe. In Periplaneta americana, we identified two subtypes of nonspiking local interneurons (type II LNs) by their distinct morphological and intrinsic electrophysiological properties. As an important step toward a better understanding of the cellular mechanisms that mediate odor information processing, we present a detailed analysis of their distinct voltage-activated Ca(2+) currents, which clearly correlated with their distinct intrinsic electrophysiological properties. Both type II LNs did not posses voltage-activated Na(+) currents and apparently innervated all glomeruli including the macroglomerulus. Type IIa LNs had significant longer and thicker low-order neurites and innervated each glomerulus entirely and homogeneously, whereas type IIb LNs innervated only parts of each glomerulus. All type II LNs were broadly tuned and responded to odorants of many chemical classes with graded changes in the membrane potential. Type IIa LNs responded with odor-specific elaborate patterns of excitation that could also include "spikelets" riding on the depolarizations and periods of inhibition. In contrast, type IIb LNs responded mostly with sustained, relatively smooth depolarizations. Consistent with the strong active membrane properties of type IIa LNs versus type IIb LNs, the voltage-activated Ca(2+) current of type IIa LNs activated at more hyperpolarized membrane potentials and had a larger transient component.

Morphology and three-dimensional reconstruction of the digestive system of Periplaneta americana.

A three-dimensional (3-D) model of the digestive system of Periplaneta americana was built for the first time based on hematoxylin and eosin (H&E) staining, the study of multiple cross-sections of the larval cockroach, and 3-D reconstruction technology. The digestive system of P. americana includes the foregut, midgut, and hindgut and takes up most of the celom. The foregut comprises almost one half of the digestive system (43.57%). The midgut, the critical region for digestion and absorption, has the second highest volume ratio (35.21%). The hindgut, with the lowest volume ratio (21.22%), includes the ileum, colon, and rectum. After the ileal valve is the colon. The 3-D model presented in this paper provides a stereoscopic view for studying the adjacent relationship and arrangement of different gut sections of P. americana.

The pars intercerebralis as a modulator of locomotor rhythms and feeding in the American cockroach, Periplaneta americana.

It has been shown that in orthopteran insects each of the optic lobes (OLs) contains a circadian pacemaker controlling locomotor activity and that the pars intercerebralis (PI) modifies the activity level. However, the present study showed Period protein-like immunoreactivity (PER-ir) in the PI and dorsolateral protocerebrum (DL) as well as in the OLs in the American cockroach, Periplaneta americana, which raised the possibility that the PI or DL could be a clock element. Therefore, we removed the PI or DL surgically and observed the effects on locomotor rhythms and feeding behavior. In constant darkness (DD), cockroaches with an ablated PI (PIX-DD) showed arrhythmicity in locomotion and a massive increase in food consumption that led to increased body length and weight, while PIX cockroaches reared under LD 12:12 (PIX-LD) and the sham-treated cockroaches in DD (CNT-DD) showed rhythmicity and no increase in food consumption. Statistical analysis showed that arrhythmicity was not accompanied by hyperactivity, suggesting that the PI is involved in the regulation of locomotor activity and feeding in DD. The activities of alpha-amylase and proteases were found to be markedly elevated in the midgut of PIX-DD cockroaches but not in PIX-LD cockroaches. Taken together, these results indicate that the PI modulates locomotor rhythms and feeding behavior of cockroaches in a light-dependent manner. The PI and the OL may regulate circadian rhythms and feeding via distinct pathways.

Starvation induces apoptosis in the midgut nidi of Periplaneta americana: a histochemical and ultrastructural study.

The effects of starvation on cell death in the midgut of Periplaneta americana were studied histochemically and ultrastructurally. TUNEL assays showed that cell death began to increase in the columnar cells and nidi, the nests of stem cells and newborn cells from 2 weeks of starvation. A significant increase in cell death occurred in the nidi after 4 weeks of starvation. Cockroaches starved for 4 weeks showed active-caspase-3-like immuno-reactivity both in the columnar cells and nidi, whereas control cockroaches that were fed for 4 weeks showed this reactivity only in the apical cytoplasm of columnar cells. Electron microscopy revealed no chromatin condensation in the nucleus of columnar cells of cockroaches, whether fed or starved for 4 weeks. Starved cockroaches exhibited many small vacuoles in the cytoplasm of some columnar cells and "floating" organelles including nuclei in the lumen. A 4-week starvation induced the appearance of cytoplasmic fragmentation and secondary lysosomes in the nidi. Each fragment contained nuclear derivatives with condensed chromatin, i.e. apoptotic bodies. Mitotic cells were found in some, but not all nidi, even within the same starved sample. Fragmentation was not observed in the nidi of control cockroaches. Thus, starvation increases cell death not only in the columnar cells, but also in the nidi. The cell death in the nidi is presumably apoptosis executed by caspase 3.

Source, topography and excitatory effects of GABAergic innervation in cockroach salivary glands.

Cockroach salivary glands are innervated by dopaminergic and serotonergic neurons. Both transmitters elicit saliva secretion. We studied the distribution pattern of neurons containing gamma-aminobutyric acid (GABA) and their physiological role. Immunofluorescence revealed a GABA-immunoreactive axon that originates within the subesophageal ganglion at the salivary neuron 2 (SN2) and this extends within the salivary duct nerve towards the salivary gland. GABA-positive fibers form a network on most acinar lobules and a dense plexus in the interior of a minor fraction of acinar lobules. Co-staining with anti-synapsin revealed that some putative GABAergic terminals seem to make pre-synaptic contacts with GABA-negative release sites. Many putative GABAergic release sites are at some distance from other synapses and at distance from the acinar tissue. Intracellular recordings from isolated salivary glands have revealed that GABA does not affect the basolateral membrane potential of the acinar cells directly. When applied during salivary duct nerve stimulation, GABA enhances the electrical response of the acinar cells and increases the rates of fluid and protein secretion. The effect on electrical cell responses is mimicked by the GABA(B) receptor agonists baclofen and SKF97541, and blocked by the GABA(B) receptor antagonists CGP52432 and CGP54626. These findings indicate that GABA has a modulatory role in the control of salivation, acting presynaptically on serotonergic and/or dopaminergic neurotransmission.