Bacteriocytes and Blattabacterium Endosymbionts of the German Cockroach Blattella germanica, the Forest Cockroach Blattella nipponica, and Other Cockroach Species.

Cockroaches are commonly found in human residences and notorious as hygienic and nuisance pests. Notably, however, no more than 30 cockroach species are regarded as pests, while the majority of 4,500 cockroaches in the world are living in forest environments with little relevance to human life. Why some cockroaches have exceptionally adapted to anthropic environments and established pest status is of interest. Here we investigated the German cockroach Blattella germanica, which is a cosmopolitan pest species, and the forest cockroach Blattella nipponica, which is a wild species closely related to B. germanica. In contrast to easy rearing of B. germanica, laboratory rearing of B. nipponica was challenging-several trials enabled us to keep the insects for up to three months. We particularly focused on the distribution patterns of specialized cells, bacteriocytes, for harboring endosymbiotic Blattabacterium, which has been suggested to contribute to host's nitrogen metabolism and recycling, during the postembryonic development of the insects. The bacteriocytes were consistently localized to visceral fat bodies filling the abdominal body cavity, where a number of single bacteriocytes were scattered among the adipocytes, throughout the developmental stages in both females and males. The distribution patterns of the bacteriocytes were quite similar between B. germanica and B. nipponica, and also among other diverse cockroach species, plausibly reflecting the highly conserved cockroach-Blattabacterium symbiotic association over evolutionary time. Our study lays a foundation to experimentally investigate the origin and the processes of urban pest evolution, on account of possible involvement of microbial associates.

Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches.

Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA) from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba.

Morphology and ultrastructure of the tarsal adhesive organs of the Madagascar hissing cockroach Gromphadorhina portentosa.

The present transmission and scanning electron microscopic study of the ultramorphology of the pliable attachment pads (arolium, euplantulae) of the Madagascar hissing cockroach Gromphadorhina portentosa reveals structural evidence for their function in producing, storing, and secreting an adhesion-mediating secretion and releasing it to the exterior. The exocrine epidermal tissue of both the arolium and the euplantula is significantly enlarged by numerous invaginations stretching into the hemolymph cavity. Its cells show large nuclei, numerous mitochondria, Golgi complexes, and a prominent rough-surfaced endoplasmic reticulum integrated within an electron-dense cytoplasm that contains numerous vesicles of diverse electron density and size. Invaginations of the cell membrane provide evidence for strong membrane turnover. The glandular epithelium of both the arolium and the euplantula releases the adhesion-mediating secretion into a subcuticular void from which it has to permeate the thick cuticle of the adhesive pads. The subcuticular void is compartmentalized by cuticle bands through which the adhesion-mediating secretion permeates via small canals. The secretion subsequently enters a larger storage reservoir before being received by a prominent sponge-like cuticle. The structural differences between the arolium and the euplantula consist of the number and length of the interdigitations spanning the hemolymph cavity, of the subdivision of the subcuticular reservoir by cuticle bands, and of the thickness of the sponge-like cuticle. The structural results are discussed with respect to the production of a chemically complex (emulsion-like) adhesive, its controlled release to the exterior, and the micromechanical properties of the cuticle of the pliable pad.

The Notch pathway regulates both the proliferation and differentiation of follicular cells in the panoistic ovary of Blattella germanica.

The Notch pathway is an essential regulator of cell proliferation and differentiation during development. Its involvement in insect oogenesis has been examined in insect species with meroistic ovaries, and it is known to play a fundamental role in cell fate decisions and the induction of the mitosis-to-endocycle switch in follicular cells (FCs). This work reports the functions of the main components of the Notch pathway (Notch and its ligands Delta and Serrate) during oogenesis in Blattella germanica, a phylogenetically basal species with panoistic ovary. As is revealed by RNAi-based analyses, Notch and Delta were found to contribute towards maintaining the FCs in an immature, non-apoptotic state. This ancestral function of Notch appears in opposition to the induction of transition from mitosis to endocycle that Notch exerts in Drosophila melanogaster, a change in the Notch function that might be in agreement with the evolution of the insect ovary types. Notch was also shown to play an active role in inducing ovarian follicle elongation via the regulation of the cytoskeleton. In addition, Delta and Notch interactions were seen to determine the differentiation of the posterior population of FCs. Serrate levels were found to be Notch-dependent and are involved in the control of the FC programme, although they would appear to play no crucial role in panoistic ovary oogenesis.

ß/δ-PrIT1, a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria reidyi (F.O. Pickard-Cambridge, 1897).

A potent insecticidal toxin, ß/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. ß/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-ß/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes ß/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, ß/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, ß/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for ß/δ-PrIT1, leading to two different pharmacological responses. ß/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.

Humidity sensation, cockroaches, worms, and humans: are common sensory mechanisms for hygrosensation shared across species?

Although the ability to detect humidity (i.e., hygrosensation) represents an important sensory attribute in many animal species (including humans), the neurophysiological and molecular bases of such sensory ability remain largely unknown in many animals. Recently, Russell and colleagues (Russell J, Vidal-Gadea AG, Makay A, Lanam C, Pierce-Shimomura JT. Proc Natl Acad Sci USA 111: 8269-8274, 2014) provided for the first time neuromolecular evidence for the sensory integration of thermal and mechanical sensory cues which underpin the hygrosensation strategy of an animal (i.e., the free-living roundworm Caenorhabditis elegans) that lacks specific sensory organs for humidity detection (i.e., hygroreceptors). Due to the remarkable similarities in the hygrosensation transduction mechanisms used by hygroreceptor-provided (e.g., insects) and hygroreceptor-lacking species (e.g., roundworms and humans), the findings of Russell et al. highlight potentially universal mechanisms for humidity detection that could be shared across a wide range of species, including humans.

Allatostatin A-like immunoreactivity in the nervous system and gut of the larval midge Chironomus riparius: modulation of hindgut motility, rectal K+ transport and implications for exposure to salinity.

Evidence for the presence of allatostatin (AST) A-like neuropeptides in the larval midge Chironomus riparius is reported. Immunohistochemical studies on the nervous system and gut revealed the presence of AST A-like immunoreactive (AST-IR) cells and processes. The nerve cord contained AST-IR processes that originated from cells in the brain and travelled the length of nerve cord to the terminal ganglion. Within each ganglion, these processes gave rise to varicosities, suggesting that they formed synapses with neurons in the ganglia. Endocrine cells containing AST-IR were present in three regions of the midgut: near the attachment of the Malpighian tubules, between the anterior and posterior midgut, and in the vicinity of the gastric caecae. The terminal ganglion also contained four AST-IR cells that gave rise to axons that projected onto the hindgut and posterior midgut. Application of a cockroach AST to the semi-isolated hindgut of larval C. riparius led to dose-dependent inhibition of muscle contractions with an EC50 of ~10 nmol l(-1) and a decrease in rectal K(+) reabsorption resulting from reduced rectal Na(+)/K(+)-ATPase and vacuolar type H(+)-ATPase activities. The results suggest the presence of endogenous AST-like neuropeptides in larval C. riparius, where these factors play a role in the function of the gut. Furthermore, regulation of ion reabsorption by ASTs at the rectum could serve as an ideal mechanism of ion regulation in the face of abrupt and acute elevated salt levels.

Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells.

Calcium (Ca(2+)) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca(2+) concentration ([Ca(2+)]i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca(2+)]i is the use of fluorescent Ca(2+) indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca(2+) indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca(2+)-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca(2+)-free and Ca(2+)-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca(2+)-free and Ca(2+)-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca(2+) sensitivity is reduced. Nevertheless, quantitative [Ca(2+)]i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.

Calcium responses of circadian pacemaker neurons of the cockroach Rhyparobia maderae to acetylcholine and histamine.

The accessory medulla (aMe) is the pacemaker that controls circadian activity rhythms in the cockroach Rhyparobia maderae. Not much is known about the classical neurotransmitters of input pathways to the cockroach circadian system. The circadian pacemaker center receives photic input from the compound eye, via unknown excitatory and GABAergic inhibitory entrainment pathways. In addition, neuropeptidergic inputs couple both pacemaker centers. A histamine-immunoreactive centrifugal neuron connects the ventral aMe with projection areas in the lateral protocerebrum and may provide non-photic inputs. To identify neurotransmitters of input pathways to the circadian clock with Fura-2-dependent Ca(2+) imaging, primary cell cultures of the adult aMe were stimulated with acetylcholine (ACh), as the most prominent excitatory, and histamine, as common inhibitory neurotransmitter. In most of aMe neurons, ACh application caused dose-dependent increases in intracellular Ca(2+) levels via ionotropic nicotinic ACh receptors. These ACh-dependent rises in Ca(2+) were mediated by mibefradil-sensitive voltage-activated Ca(2+) channels. In contrast, histamine application decreased intracellular Ca(2+) levels in only a subpopulation of aMe cells via H2-type histamine receptor chloride channels. Thus, our data suggest that ACh is part of the light entrainment pathway while histamine is involved in a non-photic input pathway to the ventral circadian clock of the Madeira cockroach.

The evaporative function of cockroach hygroreceptors.

Insect hygroreceptors associate as antagonistic pairs of a moist cell and a dry cell together with a cold cell in small cuticular sensilla on the antennae. The mechanisms by which the atmospheric humidity stimulates the hygroreceptive cells remain elusive. Three models for humidity transduction have been proposed in which hygroreceptors operate either as mechanical hygrometers, evaporation detectors or psychrometers. Mechanical hygrometers are assumed to respond to the relative humidity, evaporation detectors to the saturation deficit and psychrometers to the temperature depression (the difference between wet-bulb and dry-bulb temperatures). The models refer to different ways of expressing humidity. This also means, however, that at different temperatures these different types of hygroreceptors indicate very different humidity conditions. The present study tested the adequacy of the three models on the cockroach's moist and dry cells by determining whether the specific predictions about the temperature-dependence of the humidity responses are indeed observed. While in previous studies stimulation consisted of rapid step-like humidity changes, here we changed humidity slowly and continuously up and down in a sinusoidal fashion. The low rates of change made it possible to measure instantaneous humidity values based on UV-absorption and to assign these values to the hygroreceptive sensillum. The moist cell fitted neither the mechanical hygrometer nor the evaporation detector model: the temperature dependence of its humidity responses could not be attributed to relative humidity or to saturation deficit, respectively. The psychrometer model, however, was verified by the close relationships of the moist cell's response with the wet-bulb temperature and the dry cell's response with the dry-bulb temperature. Thus, the hygroreceptors respond to evaporation and the resulting cooling due to the wetness or dryness of the air. The drier the ambient air (absolutely) and the higher the temperature, the greater the evaporative temperature depression and the power to desiccate.

The effects of huwentoxin-I on the voltage-gated sodium channels of rat hippocampal and cockroach dorsal unpaired median neurons.

Huwentoxin-I (HWTX-I) is a 33-residue peptide isolated from the venom of Ornithoctonus huwena and could inhibit TTX-sensitive voltage-gated sodium channels and N-type calcium channels in mammalian dorsal root ganglion (DRG) neurons. However, the effects of HWTX-I on mammalian central neuronal and insect sodium channel subtypes remain unknown. In this study, we found that HWTX-I potently inhibited sodium channels in rat hippocampal and cockroach dorsal unpaired median (DUM) neurons with the IC(50) values of 66.1±5.2 and 4.80±0.58nM, respectively. Taken together with our previous work on DRG neurons (IC(50)≈55nM), the order of sodium channel sensitivity to HWTX-I inhibition was insect central DUM≫mammalian peripheral>mammalian central neurons. HWTX-I exhibited no effect on the steady-state activation and inactivation of sodium channels in rat hippocampal and cockroach DUM neurons.

Sensillum-specific, topographic projection patterns of olfactory receptor neurons in the antennal lobe of the cockroach Periplaneta americana.

In vertebrates and many invertebrates, olfactory signals detected by peripheral olfactory receptor neurons (ORNs) are conveyed to a primary olfactory center with glomerular organization in which odor-specific activity patterns are generated. In the cockroach, Periplaneta americana, ORNs in antennal olfactory sensilla project to 205 unambiguously identifiable antennal lobe (AL) glomeruli that are classified into 10 glomerular clusters (T1-T10 glomeruli) innervated by distinct sensory tracts. In this study we employed single sensillum staining techniques and investigated the topographic projection patterns of individual ORNs to elucidate the relationship between sensillum types and glomerular organization in the AL. Axons of almost all ORNs projected to individual glomeruli. Axons of ORNs in perforated basiconic sensilla selectively innervated the anterodorsal T1-T4 glomeruli, whereas those in trichoid and grooved basiconic sensilla innervated the posteroventral T5-T9 glomeruli. About 90% of stained ORNs in trichoid sensilla sent axons to the T5 glomeruli and more than 90% of ORNs in grooved basiconic sensilla innervated the T6, T8, and T9 glomeruli. The T5 and T9 glomeruli exclusively receive sensory inputs from the trichoid and grooved basiconic sensilla, respectively. All investigated glomeruli received convergent input from a single type of sensillum except F11 glomerulus in the T6 glomeruli, which was innervated from both trichoid and grooved basiconic sensilla. These results suggest that ORNs in distinct sensillum types project to glomeruli in distinct glomerular clusters. Since ORNs in distinct sensillum types are each tuned to distinct subsets of odorant molecules, the AL is functionally compartmentalized into groups of glomeruli.

Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl- changes in cockroach salivary acinar cells.

The intracellular ion homeostasis in cockroach salivary acinar cells during salivation is not satisfactorily understood. This is mainly due to technical problems regarding strong tissue autofluorescence and ineffective ion concentration quantification. For minimizing these problems, we describe the successful application of two-photon (2P) microscopy partly in combination with fluorescence lifetime imaging microscopy (FLIM) to record intracellular Na(+) and Cl(-) concentrations ([Na(+)](i), [Cl(-)](i)) in cockroach salivary acinar cells. Quantitative 2P-FLIM Cl(-) measurements with the dye N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide indicate that the resting [Cl(-)](i) is 1.6 times above the Cl(-) electrochemical equilibrium but is not influenced by pharmacological inhibition of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and anion exchanger using bumetanide and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid disodium salt. In contrast, rapid Cl(-) reuptake after extracellular Cl(-) removal is almost totally NKCC mediated both in the absence and presence of dopamine. However, in physiological saline [Cl(-)](i) does not change during dopamine stimulation although dopamine stimulates fluid secretion in these glands. On the other hand, dopamine causes a decrease in the sodium-binding benzofuran isophthalate tetra-ammonium salt (SBFI) fluorescence and an increase in the Sodium Green fluorescence after 2P excitation. This opposite behavior of both dyes suggests a dopamine-induced [Na(+)](i) rise in the acinar cells, which is supported by the determined 2P-action cross sections of SBFI. The [Na(+)](i) rise is Cl(-) dependent and inhibited by bumetanide. The Ca(2+)-ionophore ionomycin also causes a bumetanide-sensitive [Na(+)](i) rise. We propose that a Ca(2+)-mediated NKCC activity in acinar peripheral cells attributable to dopamine stimulation serves for basolateral Na(+) uptake during saliva secretion and that the concomitantly transported Cl(-) is recycled back to the bath.

Involvement of the opioid system in the hypokinetic state induced in cockroaches by a parasitoid wasp.

The parasitoid wasp Ampulex compressa stings and injects venom into the cockroach brain to induce a long-lasting hypokinetic state. This state is characterized by decreased responsiveness to aversive stimuli, suggesting the manipulation of a neuromodulatory system in the cockroach's central nervous system. A likely candidate is the opioid system, which is known to affect responsiveness to stimuli in insects. To explore this possibility, we injected cockroaches with different opioid receptor agonists or antagonists before they were stung by a wasp and tested the escape behavior of these cockroaches to electric foot shocks. Antagonists significantly decreased the startle threshold in stung individuals, whereas agonists led to an increased startle threshold in controls. Yet, neither agonists nor antagonists had any effect on grooming. To further characterize the interaction between the venom and opioid receptors, we used an antenna-heart preparation. In un-stung individuals external application of crude venom completely inhibits antenna-heart contractions. In stung individuals the antenna-heart showed no contractions. Although acetylcholine restored contractions, the opioid receptor antagonist naloxone was unable to antagonize the venom inhibition. These results suggest that the venom of A. compressa might contribute to the manipulation of cockroach behavior by affecting the opioid system.

Insect hygroreceptor responses to continuous changes in humidity and air pressure.

The most favored model of humidity transduction views the cuticular wall of insect hygroreceptive sensilla as a hygromechanical transducer. Hygroscopic swelling or shrinking alters the geometry of the wall, deforming the dendritic membranes of the moist and dry cells. The small size the sensilla and their position surrounded by elevated structures creates technical difficulties to mechanically stimulate them by direct contact. The present study investigated hygroreceptors on the antennae of the cockroach and the stick insect. Accurately controlled, homogeneous mechanical input was delivered by modulating air pressure. Both the moist and dry cells responded not only to changes in air pressure but also in the opposite direction, as observed during changes in air humidity. The moist cell's excitatory response to increasing humidity and increasing air pressure implies that swelling of the hygroscopic cuticle compresses the dendrites, and the dry cell's excitatory response to decreasing humidity and decreasing air pressure implies that shrinking of the hygroscopic cuticle expands the dendrites. The moist and dry cells of the stick insect are more sensitive to pressure changes than those of the cockroach, but the responses to air pressure are generally weaker than to humidity. Therefore the hygroreceptive sensilla differ in their physical properties and constitutions. Furthermore, the mechanical parameters associated with homogeneous changes in air pressure on the sensillum surface can only partially account for the responses of the moist and dry cells of both species to humidity stimulation.

The fine structure of colleterial glands in two cockroaches and three termites, including a detailed study of Cryptocercus punctulatus (Blattaria, Cryptocercidae) and Mastotermes darwiniensis (Isoptera, Mastotermitidae).

The colleterial glands of insects are organs associated with the female genital apparatus. In cockroaches, these glands produce secretions that cover two parallel rows of eggs during oviposition, and in oviparous species, these secretions become the tanned, sculpted, rigid outer casing of the ootheca. The goal of this study was to compare the gross anatomy of the colleterial glands and the ultrastructure of their component tubules in the phylogenetically significant genera Cryptocercus (Blattaria) and Mastotermes (Isoptera). Recent studies indicate that cockroaches in the genus Cryptocercus are the sister group of termites, and Mastotermes is the only termite known to produce a cockroach-like ootheca. One additional oviparous cockroach, Therea, and two additional termites, Zootermopsis and Pseudacanthotermes, were also examined. As in other cockroaches, the colleterial glands of Cryptocercus and Therea are asymmetrical, with a well developed bipartite left gland and a smaller right gland. In the termites Mastotermes, Zootermopsis, and Pseudacanthotermes, the colleterial glands are composed of a well-developed, paired, anterior gland and a small posterior gland; histological staining and cytological evidence suggest that these are homologues of the left and the right colleterial glands of cockroaches, respectively. At the ultrastructural level, colleterial gland tubules are made of cells belonging to a modified class 1 type cell in the cockroaches, in Mastotermes, and in Zootermopsis; the latter lays its eggs singly, without a surrounding ootheca-like structure. In the advanced termite Pseudacanthotermes, the tubules are made of secretory units belonging to the class 3 cell type. This study demonstrates that the cytological characteristics of colleterial glands in basal termites are similar to those of cockroaches, whether the termite secretes an oothecal casing that covers two parallel rows of eggs, as in Mastotermes, or lays its eggs singly, as in Zootermopsis. The function of colleterial glands in non-mastotermitid termites is unknown.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons.

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Descending control of turning behavior in the cockroach, Blaberus discoidalis.

Legged locomotion has evolved as the most effective form of movement through unpredictable and tortuous environments. Upon encountering an obstacle, an animal must evaluate the object with its sense organs then use the information it acquires to direct appropriate transitional behaviors, such as turning. Previous studies using genetic and surgical lesions implicate the central body complex (CBC) in control of such transitional behaviors of various insects. In this study, lesions of the CBC and surrounding brain regions were used to examine the effects of damage on turning in free-moving and tethered cockroaches. Lesions were performed either as sagittal incisions or by inserting small pieces of foil into regions of the brain. Locomotor behaviors of intact and lesioned animals were compared using high speed video and kinematic analysis. The lesions locations were determined through histological methods. Sagittal lesions to the CBC often result in continuous or incorrect turns. Foil lesions in the CBC also increase the probability that individuals will show turning deficits. The location and degree of the lesion had a strong effect on the animal's ability to turn. These data strongly suggest that the CBC mediates the effects of head sense organs that produce changes in the direction of walking.

Chinese-scorpion (Buthus martensi Karsch) toxin BmK alphaIV, a novel modulator of sodium channels: from genomic organization to functional analysis.

In the present study, BmK alphaIV, a novel modulator of sodium channels, was cloned from venomous glands of the Chinese scorpion (Buthus martensi Karsch) and expressed successfully in Escherichia coli. The BmK alphaIV gene is composed of two exons separated by a 503 bp intron. The mature polypeptide contains 66 amino acids. BmK alphaIV has potent toxicity in mice and cockroaches. Surface-plasmon-resonance analysis found that BmK alphaIV could bind to both rat cerebrocortical synaptosomes and cockroach neuronal membranes, and shared similar binding sites on sodium channels with classical AaH II (alpha-mammal neurotoxin from the scorpion Androctonus australis Hector), BmK AS (beta-like neurotoxin), BmK IT2 (the depressant insect-selective neurotoxin) and BmK abT (transitional neurotoxin), but not with BmK I (alpha-like neurotoxin). Two-electrode voltage clamp recordings on rNav1.2 channels expressed in Xenopus laevis oocytes revealed that BmK alphaIV increased the peak amplitude and prolonged the inactivation phase of Na+ currents. The structural and pharmacological properties compared with those of other scorpion alpha-toxins suggests that BmK alphaIV represents a novel subgroup or functional hybrid of alpha-toxins and might be an evolutionary intermediate neurotoxin for alpha-toxins.

Nutrient-induced alpha-amylase and protease activity is regulated by crustacean cardioactive peptide (CCAP) in the cockroach midgut.

The midgut plays a major role in digestion and absorption of nutrients in insects, and contains endocrine cells throughout the epithelial layer that express neuropeptides, including crustacean cardioactive peptide (CCAP). In the present study, we demonstrate regulation of digestive enzyme activities by CCAP in response to nutrient ingestion in the cockroach, Periplaneta americana. The midgut of the cockroach exhibits maximal alpha-amylase and protease activities 3 h after intake of either starch or casein, but not of non-nutrients. Similar time-dependent responses of CCAP expression in midgut endocrine cells were observed after feeding starch and casein, but not after non-nutrients. We also show that incubation of the dissected midgut with CCAP leads to an increase in alpha-amylase and protease activity in a time-dependent manner, with the maximal activity at 2 h. Taken together, our data indicate the existence of an inducible mechanism where endocrine cells in the midgut are stimulated to synthesize and secrete CCAP by nutrients, and CCAP then up-regulates the activity of digestive enzymes.