A novel class of insecticidal alkylsulfones are potent inhibitors of vesicular acetylcholine transport.

Pyridine alkylsulfone derivatives typified by oxazosulfyl (Sumitomo Chemical Company Ltd.) and compound A2 (Syngenta) represent a new class of insecticides, with potent activity against several insect orders. Whilst the MOA of this class has been attributed to interaction with the voltage-gated sodium channel (VGSC), here we present strong evidence that their toxicity to insects is mediated primarily through inhibition of the vesicular acetylcholine transporter (VAChT). Alkylsulfone intoxication in insects is characterised by (i) a reduction in cholinergic synaptic transmission efficiency demonstrated by a depression of cercal afferent activity in giant-interneurone preparations of American cockroach (Periplaneta americana), (ii) selective block of cholinergic-transmission dependent post-synaptic potentials in the Drosophila giant-fibre pathway and (iii) abolition of miniature excitatory post-synaptic currents (mEPSCs) in an identified synapse in Drosophila larvae. Ligand-binding studies using a tritiated example compound ([3H]-A1) revealed a single saturable binding-site, with low nanomolar Kd value, in membrane fractions of green bottle fly (Lucilia sericata). Binding is inhibited by vesamicol and by several examples of a previously identified class of insecticidal compounds known to target VAChT, the spiroindolines. Displacement of this binding by analogues of the radioligand reveals a strong correlation with insecticidal potency. No specific binding was detected in untransformed PC12 cells but a PC12 line stably expressing Drosophila VAChT showed similar affinity for [3H]-A1 as that seen in fly head membrane preparations. Previously identified VAChT point mutations confer resistance to the spiroindoline class of insecticides in Drosophila by Gal-4/UAS directed expression in cholinergic neurones and by CRISPR gene-editing of VAChT, but none of these flies show detectable cross-resistance to this new chemical class. Oxazosulfyl was previously shown to stabilise voltage-gated sodium channels in their slow-inactivated conformation with an IC50 value of 12.3µM but inhibits binding of [3H]-A1 with approximately 5000 times greater potency. We believe this chemistry class represents a novel mode-of-action with high potential for invertebrate selectivity.

Insect Body Defence Reactions against Bee Venom: Do Adipokinetic Hormones Play a Role?

Bees originally developed their stinging apparatus and venom against members of their own species from other hives or against predatory insects. Nevertheless, the biological and biochemical response of arthropods to bee venom is not well studied. Thus, in this study, the physiological responses of a model insect species (American cockroach, Periplaneta americana) to honeybee venom were investigated. Bee venom toxins elicited severe stress (LD50 = 1.063 uL venom) resulting in a significant increase in adipokinetic hormones (AKHs) in the cockroach central nervous system and haemolymph. Venom treatment induced a large destruction of muscle cell ultrastructure, especially myofibrils and sarcomeres. Interestingly, co-application of venom with cockroach Peram-CAH-II AKH eliminated this effect. Envenomation modulated the levels of carbohydrates, lipids, and proteins in the haemolymph and the activity of digestive amylases, lipases, and proteases in the midgut. Bee venom significantly reduced vitellogenin levels in females. Dopamine and glutathione (GSH and GSSG) insignificantly increased after venom treatment. However, dopamine levels significantly increased after Peram-CAH-II application and after co-application with bee venom, while GSH and GSSG levels immediately increased after co-application. The results suggest a general reaction of the cockroach body to bee venom and at least a partial involvement of AKHs.

Azobenzene-diamides as Photopharmacological Ligands for Insect Ryanodine Receptor.

Photoresponsive ligands are powerful tool compounds for studying receptor function with spatiotemporal resolution. However, to the best of our knowledge, such a ligand is not available for the ryanodine receptor (RyR). Herein, we present a photochromic ligand (PCL) for insect RyR by decorating chlorantraniliprole (CHL) with photoswitchable azobenzene (AB). We demonstrated that one potent ligand, named ABCHL13, shows light-induced reversible trans-cis isomerization and 3.5-fold insecticidal activity decrease toward oriental armyworm (Mythimna separata) after UV-light irradiation, that is, trans-ABCH13 has higher activity than the cis-ABCH13. ABCHL13 enables optical control over intracellular Ca2+ release in dorsal unpaired median (DUM) neurons of M. separata and American cockroach (Periplaneta americana) and cardiac function of P. americana. Our results provide a first photopharmacological toolkit that is applicable to light-dependent regulation of RyR and heart beating.

Adipokinetic hormone promotes infection with entomopathogenic fungus Isaria fumosorosea in the cockroach Periplaneta americana.

The adipokinetic hormones (AKHs) are known to be involved in insect immunity, thus their role in the cockroach Periplaneta americana infected with the entomopathogenic fungus Isaria fumosorosea was examined in this study. The application of I. fumosorosea resulted in a significant increase in both Akh gene expression and AKH peptide levels. Further, co-application of I. fumosorosea with Peram-CAH-II significantly enhanced cockroach mortality compared with the application of I. fumosorosea alone. The mechanism of AKH action could involve metabolic stimulation, which was indicated by a significant increase in carbon dioxide production; this effect can increase the turnover and thus efficacy of toxins produced by I. fumosorosea in the cockroach's body. I. fumosorosea treatment resulted in a significant decrease in haemolymph nutrients (carbohydrates and lipids), but co-application with Peram-CAH-II restored control level of lipids or even further increased the level of carbohydrates. Such nutritional abundance could enhance the growth and development of I. fumosorosea. Further, both I. fumosorosea and Peram-CAH-II probably affected oxidative stress: I. fumosorosea alone curbed the activity of catalase in the cockroach's gut, but co-application with Peram-CAH-II stimulated it. Interestingly, the hormone alone had no effect on catalase activity. Taken together, the results of the present study demonstrate the interactions between the fungus and AKH activity; understanding this relationship could provide insight into AKH action and may have practical implications for insect pest control in the future.

Capsaicin-induced dysregulation of acid-base status in the American cockroach.

Members of TRP receptor family are involved in response to acidification. Here, we determined the effect of capsaicin, one of the TRP receptor activators, on hemolymph acid-base status in the American cockroach. Periplaneta americana adult individuals were injected with lactic acid (5% or 10%) and exposed to 100 µM capsaicin solution. Hemolymph pH was measured 15 min, 1, 4, 8 and 24 h after lactic acid and capsaicin application with a glass microelectrode. The results demonstrated that cockroaches recover from acidosis within 4 h from acid injection. Capsaicin impaired the buffering capacity of insects' hemolymph, resulting in significant drop of hemolymph pH observed even 24 h after application. Joint action of capsaicin and acidosis reveals new insight into possible mechanism of capsaicin action on TRP receptors in insects.

Effect of natural toxins and adipokinetic hormones on the activity of digestive enzymes in the midgut of the cockroach Periplaneta americana.

This study examined the effect of two natural toxins (a venom from the parasitic wasp Habrobracon hebetor and destruxin A from the entomopathogenic fungus Metarhizium anisopliae), and one pathogen (the entomopathogenic fungus Isaria fumosorosea) on the activity of basic digestive enzymes in the midgut of the cockroach Periplaneta americana. Simultaneously, the role of adipokinetic hormones (AKH) in the digestive processes was evaluated. The results showed that all tested toxins/pathogens elicited stress responses when applied into the cockroach body, as documented by an increase of AKH level in the central nervous system. The venom from H. hebetor showed no effect on digestive enzyme activities in the ceca and midgut in vitro. In addition, infection by I. fumosorosea caused a decrease in activity of all enzymes in the midgut and a variable decrease in activity in the ceca; application of AKHs did not reverse the inhibition. Destruxin A inhibited the activity of all enzymes in the midgut but none in the ceca in vitro; application of AKHs did reverse this inhibition, and no differences between both cockroach AKHs were found. Overall, the results demonstrated the variable effect of the tested toxins/pathogens on the digestive processes of cockroaches as well as the variable ability of AKH to counteract these effects.

Nanoencapsulated deltamethrin as synergistic agent potentiates insecticide effect of indoxacarb through an unusual neuronal calcium-dependent mechanism.

The use of neurotoxic chemical insecticides has led to consequences against the environment, insect resistances and side-effects on non-target organisms. In this context, we developed a novel strategy to optimize insecticide efficacy while reducing doses. It is based on nanoencapsulation of a pyrethroid insecticide, deltamethrin, used as synergistic agent, combined with a non-encapsulated oxadiazine (indoxacarb). In this case, the synergistic agent is used to increase insecticide efficacy by activation of calcium-dependant intracellular signaling pathways involved in the regulation of the membrane target of insecticides. In contrast to permethrin (pyrethroid type I), we report that deltamethrin (pyrethroid type II) produces an increase in intracellular calcium concentration in insect neurons through the reverse Na/Ca exchanger. The resulting intracellular calcium rise rendered voltage-gated sodium channels more sensitive to lower concentration of the indoxacarb metabolite DCJW. Based on these findings, in vivo studies were performed on the cockroach Periplaneta americana and mortality rates were measured at 24 h, 48 h and 72 h after treatments. Comparative studies of the toxicity between indoxacarb alone and indoxacarb combined with deltamethrin or nanoencapsulated deltamethrin (LNC-deltamethrin), indicated that LNC-deltamethrin potentiated the effect of indoxacarb. We also demonstrated that nanoencapsulation protected deltamethrin from esterase-induced enzymatic degradation and led to optimize indoxacarb efficacy while reducing doses. Moreover, our results clearly showed the benefit of using LNC-deltamethrin rather than piperonyl butoxide and deltamethrin in combination commonly used in formulation. This innovative strategy offers promise for increasing insecticide efficacy while reducing both doses and side effects on non-target organisms.

To Explore the Protective Mechanism of PTEN-Induced Kinase 1 (PINK1)/Parkin Mitophagy-Mediated Extract of Periplaneta Americana on Lipopolysaccharide-Induced Cardiomyocyte Injury.

BACKGROUND Sepsis is defined as a systemic inflammatory response syndrome caused by an infection (suspicious or confirmed). Its essence is inflammatory mediators and cytokines mediated by host immune response. The present study aimed to investigate the role of Periplaneta americana extracts (XML) on PTEN-induced kinase 1 (PINK1)/Parkin mediated mitophagy in cardiomyocyte injury by sepsis. MATERIAL AND METHODS H9C2 cells were cultured and transfected with Mdivi-1 and Atg7 siRNA. The cell viability and drug toxicity were detected using Cell Counting Kit-8 assay. ELISA (enzyme-linked immunosorbent assay) was used to assess cardiac injury factors and inflammatory factors. Fluorescence levels of LC3 were detected using immunofluorescence assay. Then, the protein and mRNA expression levels were analyzed using western blot and qRT-PCR. Intracellular adenosine triphosphate (ATP) levels were measured using an ATP kit. Finally, flow cytometry was used to detected apoptosis. RESULTS The result showed that XML significantly increase cell viability in H9C2 cells. Compared with XML+LPS (lipopolysaccharide) group, the level of cTNI, CK-MB, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α was significantly upregulation in LPS+XML+Mdivi-1 or LPS+XML+Atg7 siRNA group. In addition, the release of LC3 was significant decreased. The protein and mRNA expression of PINK1, Parkin, Nix, Beclin-1 was significantly increased, but decreased expression of Mitofusin1, Mitofusin2, Opa1, Drp1, and P62 in LPS+XML+Mdivi-1 or LPS+XML+Atg7 siRNA groups. More importantly, we found that cell apoptosis was induced by Mdivi-1 and Atg7 siRNA. CONCLUSIONS The study provided evidence that XML regulated the process of LPS-induced cardiomyocyte injury through mitophagy by the PINK1/Parkin pathway.

Synthesis, insecticidal evaluation and mode of action of novel anthranilic diamide derivatives containing sulfur moiety as potential ryanodine receptor activators.

Anthranilic diamide insecticide could control lepidopteran pests by selectively binding and activating insect ryanodine receptors (RyRs), and the unique mode of action is different from other conventional insecticides. In order to discover new anthranilic diamide insecticide as ryanodine receptors activators, a series of 11 novel anthranilic diamides derivatives (Ia-k) were synthesized and confirmed by melting point, 1H NMR, 13C NMR and elemental analyses. The preliminary bioactivity revealed that most title compounds showed moderate to remarkable activities against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella). Especially, compounds Ia and If, which exhibited 100% larvicidal activity against oriental armyworm at 1.0 mg L-1, and comparable to that of chlorantraniliprole (100% at 1 mg L-1). If displayed 60% insecticidal activity against diamondback moth at 0.01 mg L-1, better than chlorantraniliprole (45% at 0.01 mg L-1). The preliminary structure activity relationships were discussed. In addition, the calcium imaging experiment indicated that the insect ryanodine receptor is the potential target of If.

Altered heat nociception in cockroach Periplaneta americana L. exposed to capsaicin.

Some natural alkaloids, e.g. capsaicin and camphor, are known to induce a desensitization state, causing insensitivity to pain or noxious temperatures in mammals by acting on TRP receptors. Our research, for the first time, demonstrated that a phenomenon of pharmacological blockade of heat sensitivity may operate in American cockroach, Periplaneta americana (L.). We studied the escape reaction time from 50°C for American cockroaches exposed to multiple doses of different drugs affecting thermo-TRP. Capsaicin, capsazepine, and camphor induced significant changes in time spent at noxious ambient temperatures. Moreover, we showed that behavioral thermoregulation in normal temperature ranges (10-40°C) is altered in treated cockroaches, which displayed a preference for warmer regions compared to non-treated insects. We also measured the levels of malondialdehyde (MDA) and catalase activity to exclude the secondary effects of the drugs on these processes. Our results demonstrated that increase in time spent at 50°C (five versus one trial at a heat plate) induced oxidative stress, but only in control and vehicle-treated groups. In capsaicin, capsazepine, menthol, camphor and AITC-treated cockroaches the number of exposures to heat had no effect on the levels of MDA. Additionally, none of the tested compounds affected catalase activity. Our results demonstrate suppression of the heat sensitivity by repeated capsazepine, camphor and capsaicin administration in the American cockroach.

Biochemical efficacy, molecular docking and inhibitory effect of 2, 3-dimethylmaleic anhydride on insect acetylcholinesterase.

Evolution of resistance among insects to action of pesticides has led to the discovery of several insecticides (neonicotinoids and organophosphates) with new targets in insect nervous system. Present study evaluates the mode of inhibition of acetylchlonesterase (AChE), biochemical efficacy, and molecular docking of 2,3-dimethylmaleic anhydride, against Periplaneta americana and Sitophilus oryzae. The knockdown activity of 2,3-dimethylmaleic anhydride was associated with in vivo inhibition of AChE. At KD99 dosage, the 2,3-dimethylmaleic anhydride showed more than 90% inhibition of AChE activity in test insects. A significant impairment in antioxidant system was observed, characterized by alteration in superoxide dismutase and catalase activities along with increase in reduced glutathione levels. Computational docking programs provided insights in to the possible interaction between 2,3-dimethylmaleic anhydride and AChE of P. americana. Our study reveals that 2,3-dimethylmaeic anhydride elicits toxicity in S. oryzae and P. americana primarily by AChE inhibition along with oxidative stress.

Flupyrimin: A Novel Insecticide Acting at the Nicotinic Acetylcholine Receptors.

A novel chemotype insecticide flupyrimin (FLP) [N-[(E)-1-(6-chloro-3-pyridinylmethyl)pyridin-2(1H)-ylidene]-2,2,2-trifluoroacetamide], discovered by Meiji Seika Pharma, has unique biological properties, including outstanding potency to imidacloprid (IMI)-resistant rice pests together with superior safety toward pollinators. Intriguingly, FLP acts as a nicotinic antagonist in American cockroach neurons, and [3H]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [3H]FLP receptors is identical to the IMI receptor, and the alternative is IMI-insensitive subtype. Furthermore, FLP is favorably safe to rats as predicted by the very low affinity to the rat α4ß2 nAChR. Structure-activity relationships of FLP analogues in terms of receptor potency, featuring the pyridinylidene and trifluoroacetyl pharmacophores, were examined, thereby establishing the FLP molecular recognition at the Aplysia californica ACh-binding protein, a suitable structural surrogate of the insect nAChR. These FLP pharmacophores account for the excellent receptor affinity, accordingly revealing differences in its binding mechanism from IMI.

Adipokinetic hormones control amylase activity in the cockroach (Periplaneta americana) gut.

This study examined the biochemical characteristics of α-amylase and hormonal (adipokinetic hormone: AKH) stimulation of α-amylase activity in the cockroach (Periplaneta americana) midgut. We applied two AKHs in vivo and in vitro, then measured resultant amylase activity and gene expression, as well as the expression of AKH receptor (AKHR). The results revealed that optimal amylase activity is characterized by the following: pH: 5.7, temperature: 38.4 °C, Km (Michaelis-Menten constant): 2.54 mg starch/mL, and Vmax (maximum reaction velocity): 0.185 µmol maltose/mL/min. In vivo application of AKHs resulted in significant increase of amylase activity: by two-fold in the gastric caeca and 4-7 fold in the rest of the midgut. In vitro experiments supported results seen in vivo: a 24-h incubation with the hormones resulted in the increase of amylase activity by 1.4 times in the caeca and 4-9 times in the midgut. Further, gene expression analyses reveal that AKHR is expressed in both the caeca and the rest of the midgut, although expression levels in the former were 23 times higher than levels in the latter. A similar pattern was found for the amylase (AMY) gene. Hormonal treatment did not affect the expression of either gene. This study is the first to provide evidence indicating direct AKH stimulation of digestive enzyme activity in the insect midgut, supported by specific AKHR gene expression in this organ.

Mesoionic insecticides: a novel class of insecticides that modulate nicotinic acetylcholine receptors.

BACKGROUND: As the world population grows towards 9 billion by 2050, it is projected that food production will need to increase by 60%. A critical part of this growth includes the safe and effective use of insecticides to reduce the estimated 20-49% loss of global crop yields owing to pests. The development of new insecticides will help to sustain this protection and overcome insecticide resistance. RESULTS: A novel class of mesoionic compounds has been discovered, with exceptional insecticidal activity on a range of Hemiptera and Lepidoptera. These compounds bind to the orthosteric site of the nicotinic acetylcholine receptor and result in a highly potent inhibitory action at the receptor with minimal agonism. The synthesis, biological activity, optimization and mode of action will be discussed. CONCLUSION: Triflumezopyrim insect control will provide a powerful tool for control of hopper species in rice throughout Asia. Dicloromezotiaz can provide a useful control tool for lepidopteran pests, with an underexploited mode of action among these pests. © 2016 Society of Chemical Industry.

CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF ß1 SUBUNIT: INVOLVEMENT IN NICOTINE-INDUCED CURRENTS.

Nicotinic acetylcholine receptors are ligand-gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real-time polymerase chain reaction (PCR) experiments demonstrated that ß1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against ß1 subunit revealed that inhibition of ß1 expression strongly decreases nicotine-induced currents amplitudes. Moreover, co-application with 0.5 µM α-bungarotoxin completely inhibited nicotine currents whereas 10 µM d-tubocurarine had a partial effect demonstrating that ß1-containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α-bungarotoxin.

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami.

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.

Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor.

Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 µM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick ß2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid.

EVIDENCE THAT ECDYSIS IN THE LARVAL COCKROACH, Periplaneta americana L. IS TRIGGERED BY AN INCREASE IN THE CONCENTRATION OF HEMOLYMPH SUGAR.

Ecdysis in insects can be defined as shedding of the cuticle at the end of a larval stadium. This event can only occur after the peak titer of ecdysteroid in the hemolymph has returned to a low level. In the cockroach Periplaneta americana, ecdysis is strongly correlated with a rise in the concentration of trehalose and glucose in the hemolymph, leading to the idea that a causal relationship may exist between both events. The objective in this study was to determine if an increase in hemolymph sugar level would shorten the time to ecdysis in cockroach larvae with experimentally delayed ecdysis. The last larval stadium of P. americana averages 33.5 days but this increases significantly if the larva is injected with a small volume of saline. Injection of 10 µl of saline on day 20 and on four successive days lengthened the stadium by as much as 2 weeks. If, however, trehalose or glucose is incorporated into the saline, approximately 40% of the treated larvae undergo ecdysis at the same time as uninjected larvae. Injection of Peram-AKH, the hypertrehalosemic hormone, also decreases the time for ecdysis to occur. This suggests that peak levels of ecdysteroid trigger the release of Peram-AKH, which then leads to activation of trehalose synthesis. The results support the hypothesis that elevated hemolymph sugar is a contributing factor in the removal of ecdysteroid from the hemolymph.

Venom from the spider Araneus ventricosus is lethal to insects but inactive in vertebrates.

Araneus ventricosus spider venom, which was collected by electrical stimulation, is abundant in peptides and proteins with molecular weights ranging from 2 kDa to 70 kDa as determined by gel electrophoresis and mass spectrometry. Electrophysiological experiments showed that 50 µg/mL venom could block the voltage-gated sodium channels (VGSCs) currents of the dorsal unpaired median (DUM) neurons of Periplaneta americana cockroaches. However, 500 µg/mL venom could not block the VGSCs currents in rat dorsal root ganglion cells or the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm. Moreover, we also observed that injection of the venom in P. americana gave rise to obvious envenomation symptoms, with a LD50 value of 30.7 µg/g. Enzymatic analysis indicated that the venom possessed activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrate that A. ventricosus venom contains bioactive components targeting insects, which are the natural prey of these spiders. Furthermore, the venom was found to be not active in vertebrate. Thus, we suggest that A. ventricosus venom contains novel insect-selective compounds that might be helpful in developing new and safe insecticides.

Intraspecific Signals Inducing Aggregation in Periplaneta americana (Insecta: Dictyoptera).

Chemical communication is necessary to induce aggregation and to maintain the cohesion of aggregates in Periplaneta americana (L.) cockroaches. We aimed to identify the chemical message inducing aggregation in this species. Two types of bioassays were used-binary choice tests in Petri dishes and tests in Y-olfactometer. Papers conditioned by direct contact of conspecifics induce aggregation when proposed in binary choice tests and were attractive in a Y-olfactometer. The identification of the molecules present on these conditioned papers indicated that dichloromethane extracts contained mainly cuticular hydrocarbons whereas methanol extracts contained more volatile molecules. Only a mixture of extracts in both solvents induced aggregation. High concentrations of cuticular hydrocarbons are necessary to induce aggregation when presented alone. When presented with volatile molecules present in methanol extracts, low concentrations of cuticular hydrocarbons are sufficient to induce aggregation if they are presented in contact. Among volatile molecules collected on filter paper, a mixture of three compounds-hexadecanoic acid, pentadecanoic acid, and pentaethylene glycol-induced aggregation. Our results provide evidence that aggregation processes in P. americana relies on a dual mechanism: attraction over long distances by three volatile molecules and maintenance on site by contact with cuticular hydrocarbons.