Photosynthetic 1,8-cineole production using cyanobacteria.

Terpenoid is an important group of compounds not only as biocomponents but also as useful secondary metabolites. A volatile terpenoid 1,8-cineole, which is used as a food additive, flavoring agent, cosmetic, etc., is also attracting attention from a medical perspective due to its antiinflammation and antioxidation. The 1,8-cineole fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source supplement is necessary for a high-yield 1,8-cineole production. We constructed the 1,8-cineole-producing cyanobacteria toward a carbon-free and sustainable 1,8-cineole production. cnsA, the 1,8-cineole synthase gene in Streptomyces clavuligerus ATCC 27064, was introduced and overexpressed in the cyanobacterium Synechococcus elongatus PCC 7942. We succeeded in producing an average of 105.6 µg g-1 wet cell weight of 1,8-cineole in S. elongatus 7942 without supplementing any carbon source. Using the cyanobacteria expression system is an efficient approach to producing 1,8-cineole by photosynthesis.

Behavioral and Morphological Studies of the Membranous Tergal Structure of Male Blattella germanica (Blattodea: Ectobiidae) During Courtship.

The male German cockroach Blattella germanica (L.) exhibits a characteristic courtship display by raising its wings, turning around, and exposing its tergal glands on the seventh and eighth tergites to the female. The male secretes a courtship pheromone from the tergal glands which induces a strong feeding response in the female, facilitating copulation. Upon multiple, detailed microscopic observations of the courtship display, we found that the male markedly expanded the intersegmental area between the sixth and seventh tergites, but deflated this area as soon as it perceived a tactile stimulus on its back by the female, while continuing to raise its wings and exposing the tergal glands. The intersegmental area is composed of two parts: a membranous posterior part of the sixth tergite, and the regular intertergal membrane. The membranous posterior part was found to be crescentic in shape and clearly separated from the intertergal membrane by traverse tongue-shaped plates. Scanning electron microscopic observation revealed that there were many orifices or pores in the membranous crescentic zone, and its morphological structure was clearly different from that of the intertergal membrane. Our observations suggest that the crescentic zone is likely a secretory gland that may play an important role at the beginning of the male courtship display, different from the tergal glands.

Potency and Target Surface Interaction of Diazinoyl Nicotinic Insecticides.

Structure-activity relationships of diazinoyl nicotinic insecticides (diazinoyl isomers and 5- or 6-substituted pyrazin-2-oyl analogues) are considered in terms of affinity to the insect nicotinic acetylcholine receptor (nAChR) and insecticidal activity against the imidacloprid-resistant brown planthopper. Among the test compounds, 3-(6-chloropyridin-3-ylmethyl)-2-(pyrazinoyl)iminothiazoline shows the highest potency in nAChR affinity and insecticidal activity. Aplysia californica acetylcholine binding protein (AChBP) mutants (Y55W + Q57R and Y55W + Q57T) are utilized to compare molecular recognition of nicotinic insecticides with diverse pharmacophores. N-nitro- or N-cyanoimine imidacloprid or acetamiprid, respectively, exhibits a high affinity to these AChBP mutants at a similar potency level. Intriguingly, the pyrazin-2-oyl analogue has a higher affinity to AChBP Y55W + Q57R than that to Y55W + Q57T, thereby indicating that pyrazine nitrogen atoms contact Arg57 guanidinium and Trp55 indole NH. Furthermore, nicotine prefers AChBP Y55W + Q57T over Y55W + Q57R, conceivably suggesting that the protonated nicotine is repulsed by Arg57 guanidinium, consistent with its inferior potency to insect nAChR.

Emergent centrality in rank-based supplanting process.

We propose a stochastic process of interacting many agents, which is inspired by rank-based supplanting dynamics commonly observed in a group of Japanese macaques. In order to characterize the breaking of permutation symmetry with respect to agents' rank in the stochastic process, we introduce a rank-dependent quantity, overlap centrality, which quantifies how often a given agent overlaps with the other agents. We give a sufficient condition in a wide class of the models such that overlap centrality shows perfect correlation in terms of the agents' rank in the zero-supplanting limit. We also discuss a singularity of the correlation in the case of interaction induced by a Potts energy.

Noxious chemical discrimination by Tribolium castaneum TRPA1 channel in the HEK293 cell expression system.

Nociception is the sensory perception of noxious chemical stimuli. Repellent behavior to avoid noxious stimuli is indispensable for survival, and this mechanism has been evolutionarily conserved across a wide range of species, from mammals to insects. The transient receptor potential ankyrin 1 (TRPA1) channel is one of the most conserved noxious chemical sensors. Here, we describe the heterologous stable expression of Tribolium castaneum TRPA1 (TcTRPA1) in human embryonic kidney (HEK293) cells. The intracellular Ca2+ influx was measured when two compounds, citronellal and l-menthol, derived from plant essential oils, were applied in vitro using a fluorescence assay. The analysis revealed that citronellal evoked Ca2+ influx dose-dependently for TcTRPA1, whereas l-menthol did not. In combination with our present and previous results of the avoidance-behavioral assay at the organism level, we suggest that TcTRPA1 discriminates between these two toxic compounds, and diversification in the chemical nociception selectivity has occurred in TRPA1 channel among insect taxa.

Organophosphate agent action at the fatty acid amide hydrolase enhancing anandamide-induced apoptosis in NG108-15 cells.

Organophosphate (OP) agents are continuously utilized in large amount throughout the globe for crop protection and public health, thereby creating a potential concern on human health. OP agent as an anticholinesterase also acts on the endocannabinoid (EC)-hydrolases, i.e., fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), to reveal unexpected adverse effects including ADHD-like behaviors in adolescent male rats. The present investigation examines a hypothesis that OP compound inhibiting the EC-hydrolase(s) dysregulates the EC-signaling system, triggering apoptosis in neuronal cells. Ethyl octylphosphonofluoridate (EOPF), as an OP probe, preferably acts on FAAH over MAGL in intact NG108-15 cells. Anandamide (AEA), an endogenous FAAH substrate, is cytotoxic in a concentration-dependent manner, although 2-arachidonoylglycerol, an endogenous MAGL substrate, gives no effect in the concentrations examined here. EOPF pretreatment markedly enhances AEA-induced cytotoxicity. Interestingly, the cannabinoid receptor blocker AM251 diminishes AEA-induced cell death, whereas AM251 does not prevent the cell death in the presence of EOPF. The consistent results are displayed in apoptosis markers evaluation (caspases and mitochondrial membrane potential). Accordingly, FAAH inhibition by EOPF suppresses AEA-metabolism, and accumulated excess AEA overstimulates both the cannabinoid receptor- and mitochondria-mediated apoptotic pathways.

TRPA1-mediated repellency behavior in the red flour beetle Tribolium castaneum.

The sensory perception of irritant chemicals results in escape and repellency behavioral patterns in insects. Transient receptor potential channels are cation channels that function as sensor proteins for several types of signals, such as light, sound, temperature, taste, as well as chemical and physical stimuli; among these, the TRPA channel is widely conserved and activated by irritant chemicals. Certain plant-derived essential oils (EOs), produced by secondary metabolism, are mixtures of volatile compounds, which are used as repellents because they contain environmentally sustainable ingredients. Citronellal, which is present in citronella EO from Cymbopogon species, is a potentially viable insect repellent; however, the repellency capability against coleopteran beetles remains limited. We investigated the citronellal-derived repellency behavior for the red flour beetle Tribolium castaneum, in which TcTRPA1 and odorant receptor co-receptor (Orco) expressions were mediated by RNA interference. Area-preference tests showed dose-dependent repellency behavior for citronellal; additionally, both TcTRPA1 and TcOrco double-strand RNA (dsRNA) micro-injection showed clear TcTRPA1 and TcOrco transcript reductions, and only TcTRPA1 dsRNA treatment significantly impaired repellency behavior. The relative expression level of the TcTRPA1 transcripts, evaluated by quantitative reverse-transcription PCR (qRT-PCR), revealed dominant expression in the antennae, indicating the antennae-expressed TcTRPA1-mediated repellency behavior.

Antennal transcriptome analysis of chemosensory genes in the cowpea beetle, Callosobruchus maculatus (F.).

Olfaction, one of the most important sensory systems governing insect behavior, is a possible target for pest management. Therefore, in this study, we analyzed the antennal transcriptome of the cowpea beetle, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae: Bruchinae), which is a major pest of stored pulses and legumes. The de novo antennal RNA-seq assembly results identified 17 odorant, 2 gustatory, and 10 ionotropic receptors, 1 sensory neuron membrane protein, and 12 odorant-binding and 7 chemosensory proteins. Moreover, differential gene expression analysis of virgin male and female antennal samples followed by qRT-PCR revealed 1 upregulated and 4 downregulated odorant receptors in males. We also performed homology searches using the coding sequences built from previously proposed amino acid sequences derived from genomic data and identified additional chemosensory-related genes.

Contact repellency by l-menthol is mediated by TRPM channels in the red flour beetle Tribolium castaneum.

BACKGROUND: Among insects, beetles are one of the most destructive pests of agricultural and stored products. Researchers have been investigating alternatives to pesticides for more sustainable pest management. Here, we focused on insect transient receptor potential (TRP) channel-targeted repellency. Among transient receptor potential melastatin (TRPM) channels, mammalian TRPM8 is activated by menthol and its derivatives, but few previous studies have reported on whether the insect TRPM channel is activated by chemical compounds. Here, we investigated whether the TRPM channel (TcTRPM) of the red flour beetle Tribolium castaneum (Herbst), a major stored-products pest, mediated the repellent behavior of l-menthol and its derivatives. RESULTS: We initially investigated the repellent activity of l-menthol and menthoxypropanediol (MPD) against T. castaneum. The laboratory bioassay revealed that the repellent activities of l-menthol and MPD were dose dependent. RNA interference was used for transcriptional knockdown of TcTRPM and revealed that a reduced transcript level resulted in a significant decrease in l-menthol and MPD repellent activities. However, no significant decrease was observed for N,N-diethyl-3-methylbenzamide (DEET) repellency. The most abundant TcTRPM transcripts were observed in the antennae. However, antennae-plucked beetles maintained their repellent behavior with l-menthol. CONCLUSION: The repellent activities of l-menthol and MPD for T. castaneum are mediated by TcTRPM, and it was suggested that the olfactory response is not adequate for avoidance, but that contact repellency might be a more important repellant method. © 2020 Society of Chemical Industry.

Deciphering the Flupyrimin Binding Surface on the Insect Nicotinic Acetylcholine Receptor.

A novel insecticide flupyrimin (FLP) with a trifluoroacetyl pharmacophore acts as an antagonist at the insect nicotinic acetylcholine receptor (nAChR). This investigation examines a hypothesis that the FLP C(O)CF3 moiety is primarily recognized by the ß subunit-face in the ligand-binding pocket (interface between α and ß subunits) of the insect nAChR. Accordingly, we evaluate the atomic interaction between a fluorine atom of FLP and the partnering amino acid side chain on the ß subunit employing a recombinant hybrid nAChR consisting of aphid Mpα2 and rat Rß2 subunits (with a mutation at T77 on the Rß2). The H-donating T77R, T77K, T77N, or T77Q nAChR enhances the FLP binding potency relative to that of the wild-type receptor, whereas the affinity of neonicotinoid imidaclprid (IMI) with a nitroguanidine pharmacophore remains unchanged. These results facilitate the establishment of the unique FLP molecular recognition at the Mpα2/Mpß1 interface structural model, thereby underscoring a distinction in its binding mechanism from IMI.

Contact sex pheromone components of the seed beetle, Callosobruchus analis (F.).

Callosobruchus analis (Coleoptera: Chrysomelidae: Bruchinae), found throughout tropical Asia and Africa, is a pest of stored legumes. Previous work has shown that females of this species produce a contact sex pheromone that elicits copulatory behavior in males. Comparisons of copulatory activity between any two of four congeneric species suggest that the contact sex pheromones are species specific. In laboratory bioassays, male C. analis exhibited copulatory behavior to a female dummy to which a crude extract of virgin females was applied. The extract had been collected by a filter paper method and was purified by acid-base partition and chromatographic techniques. Gas chromatography-mass spectrometry (GC-MS) analyses of active fractions revealed that the active compounds were 2,6-dimethyloctane-1,8-dioic acid (1) and callosobruchusic acid, (E)-3,7-dimethyl-2-octene-1,8-dioic acid (2), previously identified as contact sex pheromones of Callosobruchus maculatus (F.) and C. chinensis (L.), respectively. The stereoisomeric and chemical compositions were determined by the 2D-HPLC-Ohrui-Akasaka method as (2S,6R)-1:(S)-2=1.8:1, which meant that both compounds in C. analis were stereochemically pure, unlike the case of C. maculatus and C. chinensis. An examination of the influence of synthetic pheromone compounds on male copulatory activity revealed that (2S,6R)-1 is the main component, and that (S)-2 has an additive effect. In the examination of the stereochemistry-activity relationship, no copulatory behavior was elicited by (2R,6S)-1, and, furthermore, the enantiomer significantly masked the pheromonal activity of (2S,6R)-1. Glass rod dummy assays also suggested the presence of synergists. These results could elucidate the specificity of mate recognition in C. analis.

2,3-Dihydrohomofarnesal: female sex attractant pheromone component of Callosobruchus rhodesianus (Pic).

Callosobruchus rhodesianus (Pic) (Coleoptera: Chrysomelidae: Bruchinae) is a pest of stored legumes through the Afro-tropical region. In laboratory bioassays, males of C. rhodesianus were attracted to volatiles collected from virgin females. Collections were purified by various chromatographic techniques, and the biologically active component isolated using gas chromatographic-electroantennographic detection analysis. Gas chromatography-mass spectrometry and NMR analyses suggested that the active compound was 2,3-dihydrohomofarnesal, i.e., 7-ethyl-3,11-dimethyl-6,10-dodecadienal. The structure was confirmed by non-stereoselective and enantioselective total synthesis. Using chiral gas chromatography, the absolute configuration of the natural compound was confirmed as (3S,6E)-7-ethyl-3,11-dimethyl-6,10-dodecadienal. Y-tube olfactomter assays showed that only the (S)-enantiomer attracted males of C. rhodesianus. The (R)-enantiomer and racemate did not attract males, suggesting that the (R)-enantiomer inhibits the activity of the natural compound. In combination with previous reports about sex attractant pheromones of congeners, we suggest that a saltational shift of the pheromone structure arose within the genus Callosobruchus.

Organophosphate Agent Induces ADHD-Like Behaviors via Inhibition of Brain Endocannabinoid-Hydrolyzing Enzyme(s) in Adolescent Male Rats.

Anticholinergic organophosphate (OP) agents act on the diverse serine hydrolases, thereby revealing unexpected biological effects. Epidemiological studies indicate a relationship between the OP exposure and development of attention-deficit/hyperactivity disorder (ADHD)-like symptoms, whereas no plausible mechanism for the OP-induced ADHD has been established. The present investigation employs ethyl octylphosphonofluoridate (EOPF) as an OP-probe, which is an extremely potent inhibitor of endocannabinoid (EC, anandamide and 2-arachidonoylglycerol)-hydrolyzing enzymes: that is, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). An ex vivo experiment shows that EOPF treatment decreases FAAH and MAGL activities and conversely increases EC levels in the rat brain. Subsequently, EOPF (treated intraperitoneally once at 0, 1, 2, or 3 mg/kg) clearly induces ADHD-like behaviors (in elevated plus-maze test) in both Wistar and spontaneously hypertensive rats. The EOPF-induced behaviors are reduced by a concomitant administration of cannabinoid receptor inverse agonist SLV-319. Accordingly, the EC system is a feasible target for OP-caused ADHD-like behaviors in adolescent rats.

Nonideal behavior of the intramolecular structure factor of dilute polymers in a theta solvent.

We study the configurational properties of single polymers in a theta solvent by Monte Carlo simulation of the bond fluctuation model. The intramolecular structure factor at the theta point is found to be distinctively different from that of the ideal chain. The structure factor shows a hump around q approximately 5/Rg and a dip around q approximately 10/Rg in the Kratky plot with Rg being the radius of gyration. This feature is apparently similar to that in a melt. The theoretical expression by the simple perturbation expansion to the first order in terms of the Mayer function can be fitted to the obtained structure factor quite well, but the second virial coefficient cannot be set to zero.

Homofarnesals: female sex attractant pheromone components of the southern cowpea weevil, Callosobruchus chinensis.

The southern cowpea weevil, Callosobruchus chinensis (Coleoptera: Bruchidae), is a major pest of stored legumes in warm temperate and tropical climates. The female sex attractant pheromone was extracted from filter-paper shelters taken from containers that housed virgin females. The extracts were purified by various chromatographic techniques, and the biologically active components in the fractions were screened by gas chromatographic-electroantennographic detection analysis with male antennae. Two compounds that elicited electrophysiological responses were isolated, and gas chromatography-mass spectrometry, nuclear magnetic resonance, and micro-chemical analyses suggested that the active compounds were homofarnesals, (2Z,6E)- and (2E,6E)-7-ethyl-3,11-dimethyl-2,6,10-dodecatrienals. Males of C. chinensis were significantly attracted to filter paper discs loaded with the synthetic compounds at 0.01-0.1 ng compared to solvent control in a Y-tube olfactometer assay. These pheromone components represent unique chemical structures within the genus Callosobruchus.

Contact sex pheromone components of the cowpea weevil, Callosobruchus maculatus.

The cowpea weevil, Callosobruchus maculatus, is a major pest of stored pulses. Females of this species produce a contact sex pheromone that elicits copulation behavior in males. Pheromone was extracted from filter-paper shelters taken from cages that housed females. Crude ether extract stimulated copulation in male C. maculatus. Initial fractionation showed behavioral activity in acidic and neutral fractions. Furthermore, bioassay-guided fractionation and gas chromatography-mass spectroscopy (GC-MS) analysis of active fractions revealed that the active components of the acidic fraction were 2,6-dimethyloctane-1,8-dioic acid and nonanedioic acid. These components along with the hydrocarbon fraction, a mixture of C(27)-C(35) straight chain and methyl branched hydrocarbons, had a synergistic effect on the behavior of males. Glass dummies treated with an authentic pheromone blend induced copulation behavior in males. The potential roles of the contact sex pheromone of C. maculatus are discussed.