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.

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.

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.

The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels.

One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large ß hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.

Three austin family compounds from Penicillium brasilianum exhibit selective blocking action on cockroach nicotinic acetylcholine receptors.

Austin (AT) and its derivatives (dehydroaustin (DAT) and acetoxydehydroaustin (ADAT)) produced by Penicillium brasilianum MG-11 exhibit toxicity to insects, yet their targets are unknown. Here, we used whole-cell patch-clamp electrophysiology to investigate the action of AT family compounds on cockroach acetylcholine (ACh), γ-aminobutyric acid (GABA) and l-glutamate receptors expressed in the American cockroach (Periplaneta americana) neuron. U-tube application of AT or its derivatives did not induce any current amplitudes, suggesting that they did not act as agonist of these three receptors. In the second step of experiments, they were bath-applied for 1min before co-application with the corresponding ligand. We found that AT and its derivatives had no effect on GABA and l-glutamate-induced currents, whereas they significantly reduced ACh- and epibatidine-induced currents, showing that these compounds acted as selective antagonists of nicotinic acetylcholine receptors (nAChRs) expressed in the cockroach neuron. Of the compounds, DAT showed the highest blocking potency for nAChRs, differentially attenuating the peak and slowly desensitizing current amplitude of ACh-induced responses with pIC(50) (=-logIC(50) (M)) values of 6.11 and 5.91, respectively. DAT reduced the maximum normalized response to ACh without a significant shift in EC(50), suggesting that the blocking action is not competitive with ACh.

[Effects of several exorphins and endorphins on the escape reaction of the cockroach Periplaneta americana under elevated temperature conditions].

The ability of several alimentary opioid peptides (exorphin C, rubiscolin-5, cytochrophi-4) and endorphins (met-enkephalin, dynotphin A(1-10), beta-neoendorphin) to change the escape reaction of the cockroaches Periplaneta americana at their placement into a hot chamber was studied. The ED50 values increasing twice the insect stay time in the hot chamber as well as duration and dynamics of the effects were determined. It has been shown that ED5 decreases statistically significantly with increase of the length of the peptide molecule and its affinity of duration of the effects and to an increase of their affinity to delta-receptors - to prolongation of the reaction (more than150 min). In the group of alimentary peptides (exorphins) the most active was a fragment of D-ribulose-1,5-biphosphate carboxylase/oxigenase rubiscolin-5 (ED5 = 386 nM per individual). This might indicate a specific ability of some plant proteins to regulate (decrease) the insect protective behavior.

Effects of phosphine on the neural regulation of gas exchange in Periplaneta americana.

Phosphine is used for fumigating stored commodities, however an understanding of the physiological response to phosphine in insects is limited. Here we show how the central pattern generator for ventilation in the central nervous system (CNS) responds to phosphine and influences normal resting gas exchange. Using the American cockroach, Periplaneta americana, that perform discontinuous gas exchange (DGE) at rest, we simultaneously measure ventilatory nervous output from the intact CNS, VCO(2) and water loss from live specimens. Exposure to 800 ppm phosphine at 25 degrees C for 2 h (n=13) during recording did not cause any mortality or obvious sub-lethal effects. Within 60 s of introducing phosphine into the air flow, all animals showed a distinct CNS response accompanied by a burst release of CO(2). The initial ventilatory response to phosphine displaced DGE and was typically followed by low, stable and continuous CO(2) output. CNS output was highest and most orderly under normoxic conditions during DGE. Phosphine caused a series of ventilatory CNS spikes preceding almost complete cessation of CNS output. Minimal CNS output was maintained during the 2 h normoxic recovery period and DGE was not reinstated. VCO(2) was slightly reduced and water loss significantly lower during the recovery period compared with those rates prior to phosphine exposure. A phosphine narcosis effect is rejected based on animals remaining alert at all times during exposure.

Comparison of effects of octopamine and insecticidal essential oils on activity in the nerve cord, foregut, and dorsal unpaired median neurons of cockroaches.

Essential oil constituents were tested for their neurophysiological effects in Periplaneta americana and Blaberus discoidalis. Eugenol depressed spontaneous and stimulus-evoked impulses recorded extracellularly in the abdominal nerve cord, with an almost complete block of spikes at 2 x 10(-3) M. Geraniol and citral had similar depressive effects but increased spontaneous firing at lower doses (threshold 2.5 x 10(-4) M). Similar effects occurred in dorsal unpaired median (DUM) neurons, recorded intracellularly in the isolated terminal abdominal ganglion of P. americana. Spontaneous firing was progressively reduced by increasing concentrations of eugenol, whereas geraniol and citral produced biphasic effects (excitation at 10(-4) M, depression at 2 x 10(-3) M). All three oils decreased excitability of silent DUM neurons that were depolarised by applied current, but eugenol (at 10(-3) M) also changed the firing pattern from single spikes to bursts driven by plateau potentials. All oils reduced spike undershoot. Low doses of citral and geraniol (threshold ca. 10(-4) M) reversibly increased the frequency of spontaneous foregut contractions and abolished them at 2 x 10(-3) M (together with response to electrical stimulation). Eugenol reversibly reduced spontaneous activity at 10(-4) M and above. Eugenol has been reported to exert its insecticidal properties via a low-dose activation of octopamine receptors. In our studies, however, octopamine was found to have opposing effects to eugenol on DUM neurons and foregut activity (excitatory in both). Furthermore, eugenol did not affect the response to octopamine in DUM neurons. These results suggest that reported effects of eugenol were on a different sub-type of octopamine receptor.

Molecular and pharmacological analysis of an octopamine receptor from American cockroach and fruit fly in response to plant essential oils.

Octopamine receptors from American cockroach, Periplaneta americana (Pa oa1), and fruit fly, Drosophila melanogaster (OAMB), were cloned and permanently expressed in HEK-293 cells, and found to activate adenylate cyclase activity and increase [Ca2+]i levels through G-protein coupled receptor signaling pathways. Sequencing information (GenBank accession number AY333178) and functional data of Pa oa1 were recently published. Saturation binding analysis with 3H-yohimbine was performed with Pa oa(1) and OAMB expressed in COS-7 cells. The K(d) values were determined to be 28.4 and 43.0 nM, respectively. B(max) was determined to be 11.8 and 8.04 pmol receptor/mg protein, respectively. Competitive binding data using cell membranes expressing either OAMB or Pa oa1 demonstrated significantly decreased binding activity in binding assays performed in the presence of plant essential oils, eugenol, cinnamic alcohol, and trans-anethole. Eugenol decreased cAMP level in HEK-293 cells expressing Pa oa1, but trans-anethole increased cAMP in HEK-293 cells expressing OAMB. All three chemicals increased [Ca2+]i level in both cell models. Toxicity data against fruit flies and American cockroaches demonstrated species differences in response to treatment with tested plant essential oils. The toxicity of tested chemicals against wild type and octopamine mutant (iav) fly strains suggested that an octopamine receptor mediates the toxicity of cinnamic alcohol, eugenol, trans-antehole, and 2-phenethyl propionate against fruit flies. Collectively, the data suggest a correlation between cellular changes induced by tested plant essential oils and their toxicity against fruit fly and American cockroach.

Wasp venom blocks central cholinergic synapses to induce transient paralysis in cockroach prey.

The parasitoid wasp Ampulex compressa induces a set of unique behavioral effects upon stinging its prey, the cockroach. It stings into the first thoracic segment inducing 2 to 3 min of transient flaccid paralysis of the front legs. This facilitates a second sting in the cockroach's head that induces 30 min of excessive grooming followed by a 2 to 5-week long lethargic state. In the present study, we examine the immediate effect of the first sting, which is a transient paralysis of the front legs. Using radiolabeled wasps, we demonstrate that the wasp injects its venom directly into the cockroach's first thoracic ganglion. The artificial injection of milked venom into a thoracic ganglion abolishes spontaneous and evoked responses of the motoneurons associated with leg movements. To investigate the physiological mechanism of action of the venom, we injected venom into the last abdominal ganglion of the cockroach, which houses a well-characterized cholinergic synapse. Injected venom abolishes both sensory-evoked and agonist-evoked postsynaptic potentials recorded in the postsynaptic neuron for 2 to 3 min without affecting action potential propagation. Thus, the venom blocking effect has a postsynaptic component that follows the same time course as the transient paralysis induced by the thoracic sting. Finally, injection of a nicotinic antagonist in the front thoracic ganglion induces paralysis of the front legs. We conclude that the transient paralytic effect of the thoracic sting can be mainly accounted for by the presence of a venom active component that induces a postsynaptic block of central cholinergic synaptic transmission.

Insecticidal activity of essential oils: octopaminergic sites of action.

UNLABELLED: A study was conducted to determine the insecticidal activity and mechanism of action of three essential oils (eugenol, alpha-terpineol and cinnamic alcohol) and an equal part mixture (3-blend) against American cockroaches (Periplaneta americana). To address species differences in response to treatment with the test oils, Carpenter ants (Camponotus pennsylvanicus De Geer), and German cockroaches (Blattella germanica) were included in this study. Exposed American cockroaches demonstrated hyperactivity followed by hyperextension of the legs and abdomen, then fast knockdown or quick immobilization followed by death. Ants and German cockroaches showed fast immobilization/knockdown followed by mortality. The 1:1:1 mixture (3-blend) was substantially effective against all test insects. One of the most remarkable observations was the increased frequency of heartbeats of American cockroaches in response to topical application of test oils. The changes in the pattern of cAMP level was biphasic. A significant increase in the cAMP level was found in response to 1 nmol/ml of eugenol, or 3-blend or 10 nmol/ml of alpha-terpineol. At higher concentrations a significant decrease in cAMP level was found. Blockage of octopamine receptors binding sites was also illustrated at lower concentrations of the test chemicals as judged by the decreased binding activity of [3H]octopamine to its receptors. IN CONCLUSION: (1) test oils are neuro-insecticides and their insecticidal activity is species-dependent; (2) a synergistic effect of the three oils was found when they were equally mixed (3-blend); and (3) the octpaminergic system mediates the insecticidal activity of eugenol, alpha-terpienol and the 3-blend.

Calcium-induced calcium release in neurosecretory insect neurons: fast and slow responses.

The dynamics of intracellular free Ca(2+)([Ca(2+)](i)) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca(2+) channels caused a steep increase in [Ca(2+)](i). Depolarizations lasting for < 100ms led to Ca(2+) release from intracellular stores as is indicated by the finding that the rise of [Ca(2+)](i) was greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca(2+)current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca(2+) influx enhanced in this way again caused a rise of [Ca(2+)](i) sensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca(2+)responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca(2+) responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca(2+) rises after having been treated with NHD. Thus, the variability of Ca(2+) signals might be caused by different filling states of Ca(2+) stores, and the resting Ca(2+) current seems to represent a source to fill empty Ca(2+) stores. In line with this notion, block of the endoplasmic Ca(2+) pump by thapsigargin (1 microM) produced either no or largely varying Ca(2+) responses. The Ca(2+) signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca(2+) release channels. Block of mitochondrial Ca(2+) uptake with carbonyl cyanide m -chlorophenylhydrazone (CCCP, 1 microM) completely abolished cADPR-induced Ca(2+) signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca(2+) uptake pathways and that some of these pathways involve mitochondria.

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

Structure-activity relationships for Periplaneta americana hypertrehalosemic hormone. I: The importance of side chains and termini.

Single amino acid replacement analogues for the native hypertrehalosemic hormone I of the American cockroach, Periplaneta americana (Pea-CAH-I: pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2), have been prepared by solid-phase peptide synthesis, and complete dose-response curves have been measured in P. americana monitoring the carbohydrate-mobilizing activity in vivo. All analogues that elicited hypertrehalosemia showed similar time-response courses, indicating that transport and degradation rates were comparable. Comparison of the potency and efficacy parameters of the analogues under study in the dose-response curves revealed four activity groups: 1) analogues that had the aromatic amino acids at positions 4 (phenylalanine) or 8 (tryptophan) replaced by alanine and glycine, respectively, had trace activity; 2) analogues with alanine at positions 1 or 2 had low potencies and an apparent biphasic dose-response relationship without much observable loss of efficacy; 3) analogues with glycine at positions 6 and 7 had potencies and efficacies most similar to Pea-CAH-I; and 4) analogues that had either an alanine instead of asparagine residue at position 3, or had a substitution of the carboxylamide function at the C-terminus by a carboxyl function reached apparent saturation, but only achieved 50-57% of the maximum activity of the native peptide. The potency profile for the analogue set is consistent with the importance of the N-terminal pentapeptide and the C-terminal tryptophan interacting with receptor(s) more closely than the side chains at positions 6 and 7, which are predicted to be the corner residues of a beta-turn. Finally, the biphasic dose-response curves observed for more than one analogue suggest the potential that receptors for Pea-CAH-I exist in more than one form.

Diflubenzuron affects gamma-thioGTP stimulated Ca2+ transport in vitro in intracellular vesicles from the integument of the newly molted American cockroach, Periplaneta americana L.

To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs (Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10(-8) M). Such an inhibitory effect on DFB on Ca2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na+, and Li+ did not affect the calcium uptake. Among ionophores, agents disrupting H+ gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and gamma-S-GTP stimulated Ca2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.

The presence of peptides related to the adipokinetic hormone/red pigment-concentrating hormone family in the nematode, Panagrellus redivivus.

Immunocytochemistry using polyclonal antisera raised to fragments or derivatives of locust adipokinetic hormone (AKH) I and IIs (Schooneveld et al., 1983, 1985, 1986) selectively stained cells in the nervous system of the free-living nematode, Panagrellus redivivus. Antiserum 528 (raised to the C-terminus of AKH IIs) stained the dorsal cephalic papillary cell bodies and the anterior nerve ring. Fibres in the lateral cords were stained with antiserum 241 that recognises the C-terminus of AKH I. Substances reacting to antisera 433 (raised to the N-terminal sequence of AKH I and IIs) 528 and 241 were present in the preanal ganglion and associated ventral nerve fibres. In males, all three antisera stained fibres leading to the base of the spicules. A peptide fraction from whole P. redivivus evoked an adipokinetic response in the locust, Schistocera gregaria which was dose dependent and was abolished by treatment with endopeptidase 24:11 but not by boiling or by incubation with leucine aminopeptidase. The adipokinetic activity was reduced by over 70% on incubation of the peptide fraction with pyroglutamyl aminopeptidase. The same fraction induced hyperglycaemia when injected into the cockroach, Periplaneta americana. These results are consistent with the existence in P. redivivus of peptides that are structurally related to the arthropod adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family.

Further studies on proctolin analogues modified in position 2 of the peptide chain and their influence on heart-beat frequency of insects.

Seven proctolin analogues (I-VII) modified in position 2 of the peptide chain by Phe (p-guanidino) (I), Phe (p-OEt) (II), Tyr (3'-NH2) (III), Tyr (3'-NO2) (IV), Afb (p-OH) (V) (Afb = 3-amino-4-phenyl-L-butyric acid), Afb (p-NH2) (VI), Afb (p-NO2) (VII), and the tetrapeptide Tyr (3'-NH2)-Leu-Pro-Thr (VIII) were synthesized by the classic liquid-phase method. The biological effects of the peptides were investigated in cardioexcitatory tests on two insect species, the cockroach Periplaneta americana L., and the yellow mealworm, Tenebrio molitor L. Within physiological concentrations (10(-9)-10(-7) M) peptides II, III, and IV stimulated the heart action of P. americana like proctolin itself. Under identical conditions, in the case of T. molitor, only peptide III showed cardiostimulatory properties, whereas other compounds (including II and IV) were inactive at concentrations up to 10(-7) M. Results reported here reflect, with reference to the analogues I-VII, selective recognition of receptors on myocardium of both insect species. The tetrapeptide VIII revealed a weak deacceleratory effect on P. americana and T. molitor heart action.

Effects of paraoxon on spike initiation and conduction block in the giant interneurons of the American cockroach, Periplaneta americana.

1. The effects of paraoxon were studied on spike initiation and conduction in the giant interneurons (GIs) of the American cockroach, using electrophysiological techniques. 2. Paraoxon treatment induced high-frequency bursts in GI axons. During these bursts, overshooting spikes recorded in the sixth abdominal ganglion were replaced, in phase, by small, decremental potentials. 3. These small potentials were not EPSPs since current injection could modulate their frequency. 4. An analysis of anteriorly conducted spikes indicates that the site of spike initiation is located near the dendritic region of the GI and is unchanged by paraoxon treatment.