Insect Receptors Research Articles

The neurotoxic mechanism of Jack Bean Urease in insects involves the interplay between octopaminergic and dopaminergic pathways

In the last decades, the entomotoxicity of JBU and its derived peptides became an object of study, due mainly to the ubiquitous interaction of these compounds with different species of insects and their potential as natural insecticides. In this work, we investigated the neurotoxic effects of JBU in Nauphoeta cinerea cockroaches by dissecting pharmacologically the monoaminergic pathways involved. Selective pharmacological modulators for monoaminergic pathways in in vivo and ex vivo experimental models were employed. Thus, the analysis of N. cinerea neurolocomotory behavior demonstrated that JBU (1.5 and 3 μg/g) induces a significant decrease in the exploratory activity. In these assays, pretreatment of animals with phentolamine, SCH23390 or reserpine, interfered significantly with the response of JBU. Using in vivo abductor metathoracic preparations JBU (1.5 μg/g) induced progressive neuromuscular blockade, in 120 min recordings. In this set of experiments, the previous treatment of the animals with phentolamine, SCH23390 or reserpine, completely inhibited JBU-induced neuromuscular blockade. The recordings of spontaneous compound neural action potentials in N. cinerea legs showed that JBU, only in the smallest dose, significantly decreased the number of potentials in 60 min recordings. When the animals were pretreated with phentolamine, SCH23390, or reserpine, but not with mianserin, there was a significant prevention of the JBU-inhibitory responses on the action potentials firing. Meanwhile, the treatment of the animals with mianserin did not affect JBU's inhibitory activity. The data presented in this work strongly suggest that the neurotoxic response of JBU in N. cinerea involves a cross talking between OCTOPAMIN-ergic and DOPAMIN-ergic nerve systems, but not the SEROTONIN-ergic neurotransmission. Further molecular biology studies with expression of insect receptors associated with voltage clamp techniques will help to discriminate the selectivity of JBU over the monoaminergic transmission.

(-)-Adaline from the Adalia Genus of Ladybirds Is a Potent Antagonist of Insect and Specific Mammalian Nicotinic Acetylcholine Receptors.

Ladybird beetles (Coleoptera: Coccinellidae) possess strong chemical defences that are secreted in response to stress and are also found on the coating of eggs, which are rich in alkaloids that are responsible for their toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists. Here, we have explored the actions of (−)-adaline, found in the 2-spot (Adalia bipunctata) and 10-spot (Adalia decempunctata) ladybirds, on both mammalian (α1β1γδ, α7, α4β2, α3β4) and insect nAChRs using patch-clamp of TE671 cells and locust brain neurons natively expressing nAChRs, as well as two-electrode voltage clamp of Xenopus laevis oocytes recombinantly expressing nAChRs. All nAChR subtypes were antagonised by (−)-adaline in a time-dependent, voltage-dependent and non-competitive manner with the lowest IC50s at rat α3β4 (0.10 μM) and locust neuron (1.28 μM) nAChRs, at a holding potential of −75 mV. The data imply that (−)-adaline acts as an open channel blocker of nAChRs.

A peptide Epitope − Synthetic hydrogel polymer conjugate that mimics insecticidal protein Receptors. Application in environmental and biological analysis

Monitoring transgenic crops (GMOs) is essential to track cross-breeding and ensure regulatory compliance but multiple protein variants now engineered into important crops challenges conventional antibody detection. Analysis of Bacillus thuringiensis (Bt) Cry protein − insect receptor complexes identified a peptide epitope, NITIHITDTNNK, with high sequence similarity to cadherin-like receptors across a family of Bt Cry proteins for broad-spectrum recognition. The peptide lacks the necessary physical, mechanical and chemical properties for practical applications. Conjugation of the peptide to a synthetic polymer creates a hybrid material that imparts robustness, adhesion, processability and polyvalence to the epitope. An epitope − hydrogel polymer conjugate library is created and screened for Bt Cry protein binding. A candidate with high adsorption capacity (∼995 mg g−1), fast adsorption kinetics (within 2 min), broad-spectrum specificity, and pH-responsive catch-and-release is identified as a selective sequestrant for extraction and isolation of Bt Cry variants from water and soil samples. An enrichment factor of 9.0 ∼ 12.5 and a linear range of 0.09 ∼ 0.45 µg g−1 was obtained for soil samples. The biotinylated epitope − polymer conjugate, a recognition element entirely different from an antibody, is integrated into an enzyme-linked immunosorbent assay (ELISA) for identification of cotton and rice crops transgenic with a variety of Bt Cry variants. The established ELISA assay exhibits wide linear range (0 ∼ 50 ng mL−1) and high accuracy (95.4 ∼ 108 %) for GMO cotton leaves. However, to distinguish GMO rice from non-GMO ones, background interference still needs to be overcome. We are now working for a more effective diagnostic for GMO rice.

Characterization and expression profiling of G protein-coupled receptors (GPCRs) in Spodoptera litura (Lepidoptera: Noctuidae)

Spodoptera litura is a highly destructive omnivorous pest, and they caused serious damage to various crops. G protein-coupled receptors (GPCRs) mediate dozens of physiological processes including reproduction, development, life span and behaviors, but the information of these receptors has been lacking in S. litura. Here, we methodically identified 122 GPCRs in S. litura and made an assay of their expression patterns in different tissues. Comparing the identified GPCRs with homologous genes of other insects, it is obvious that the subfamily A2 (biogenic amine receptors) and the subfamily A3 (neuropeptide and protein hormone receptors) of S. litura have expanded to a certain extent, which may be related to the omnivorous nature and drought environment resistance of S. litura. Besides, the large Methuselah (Mth)/Methuselah-like (Mthl) subfamily of S. litura may be involved in many physiological functions such as longevity and stress response. Apart from duplicate receptors, the loss of parathyroid hormone receptor (PTHR) and the bride of sevenless (Boss) receptor in the lepidopteran insects may imply a new pattern of wing formation and energy metabolism in lepidopteran insects. In addition, the high expression level of GPCRs in different tissues reflects the functional diversity of GPCRs regulating. Systemic identification and initial characterization of GPCRs in S. litura provide a basis for further studies to reveal the functions of these receptors in regulating physiology and behavior.

Different efficiency of auxiliary/chaperone proteins to promote the functional reconstitution of honeybee glutamate and acetylcholine receptors in Xenopus laevis oocytes.

Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co‐expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8β1, α7, α2α8β1 and α2α7α8β1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR‐1) and potential chaperone proteins (e.g., SOL‐1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR‐1 and α1α8β1 when co‐expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N‐terminal sequence. We recorded small ACh‐gated currents in few oocytes when the α7 subunit was co‐expressed with Caenorhabditis elegans RIC‐3, but none of the chaperone proteins allowed efficient expression of α2α8β1 or α2α7α8β1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species.

Effects of nicotinic acetylcholine receptor subunit deletion mutants on insecticide susceptibility and fitness in Drosophila melanogaster.

Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in insects and also the target site for many insecticides. Unfortunately, the effectiveness of these insecticides is diminishing as a consequence of the evolution of insecticide resistance. Further exploration of insecticide targets is important to sustainable pest management. In order to validate the role of nAChR subunits in insecticide susceptibility and test whether the subunit's absence imposes the fitness cost on insects, we determined the susceptibility of eight nAChR subunit deletion mutants of Drosophila melanogaster to nine insecticides. These findings highlighted the specific resistance of the Dα6 deletion mutant to spinosyns. Although triflumezopyrim, dinotefuran and imidacloprid are competitive modulators of nAChRs, differences in susceptibility of the insect with different deletion mutants suggested that the target sites of these three insecticides do not overlap completely. Mutants showed decreased susceptibility to insecticides, accompanied by a reduction in fitness. The number of eggs produced by Dα1attP , Dα2attP , Dβ2attP and Dβ3attP females was significantly lesser than that of the vas-Cas9 strain as the control. In addition, adults of Dα2attP , Dα3attP and Dα7attP strains showed lower climbing performance. Meanwhile, males of Dα3attP , Dα5attP , Dβ2attP and Dβ3attP , and females of Dβ2attP showed significantly shorter longevity than those of the vas-Cas9 strain. This study provides new insights into the interactions of different insecticides with different nAChRs subunit in D.melanogaster as a research model, it could help better understand such interaction in agricultural pests whose genetic manipulations for toxicological research are often challenging. © 2022 Society of Chemical Industry.

Secretion and Detection of Defensive Compounds by the Red Flour Beetle Tribolium castaneum Interacting with the Insect Pathogenic Fungus Beauveria bassiana.

Entomopathogenic fungi such as Beauveria bassiana are extensively used for the control of insect pests worldwide. They infect mostly by adhesion to the insect surface and penetration through the cuticle. However, some insects, such as the red flour beetle Tribolium castaneum (Herbst), have evolved resistance by embedding their cuticle with antifungal compounds. Thus, they avoid fungal germination on the cuticle, which result in low susceptibility to entomopathogenic fungi. In adult T. castaneum, these antifungals are the well-known defensive compounds methyl-1,4- and ethyl-1,4-benzoquinone. In this study, we added B. bassiana conidia on the diet of adult beetles to study the effect of the entomopathogen on the secretion and detection of the beetle volatile blend containing both benzoquinones. The compounds were analyzed by solid phase microextraction coupled to gas chromatography–flame ionization detection, and were detected by electroantennography. In addition, we measured the expression level of four genes encoding for two odorant-binding proteins (OBP), one chemosensory protein (CSP), and one odorant receptor (OR) in both healthy and fungus-treated insects. Significant alterations in the secretion of both benzoquinones, as well as in the perception of methyl-1,4-benzoquinone, were found in fungus-treated insects. TcOBP7D, TcOBP0A and TcCSP3A genes were down-regulated in insects fed conidia for 12 and 48 h, and the latter gene was up-regulated in 72 h samples. TcOR1 expression was not altered at the feeding times studied. We conclude that fungus-treated insects alter both secretion and perception of benzoquinones, but additional functional and genetic studies are needed to fully understand the effects of fungal infection on the insect chemical ecology.

Abiotic Synthetic Antibodies to Target a Specific Protein Domain and Inhibit Its Function.

The Bacillus thuringiensis (Bt) Cry proteins are widely used in insect pest control. Despite their economic benefits, remaining concerns over potential ecological and health risks warrant their ongoing surveillance. Affinity reagents, most often antibodies, protein scaffolds, and aptamers, are the traditional tools used for protein binding and detection. We report a synthetic antibody (SA) alternative to traditional biological affinity reagents for binding Bt Cry proteins. Analysis of hotspots of the Bt Cry protein-insect midgut cadherin-like receptor complexes was used for the design of the SA. The SA was selected from a small focused library of hydrogel copolymers containing functional monomers complementary to key exposed hotspots of Bt Cry proteins. A directed chemical evolution identified a SA, APhe-NP23, with affinity and selectivity for Bt Cry1Ab/Ac proteins. The putative intermolecular polymer-protein interfaces were identified by the SA's uptake of Bt Cry1Ac pepsin hydrolysates, binding epitope mutation studies, and protein-protein inhibition studies of the toxin binding to its native insect receptor binding domains. The SA inhibitor binds to the same protein domains as the insect's cadherin-like receptors, Bt-R1 and SeCad1b. The SA binds rapidly to Bt Cry1Ab/Ac with high capacity, is pH-responsive, and is synthesized reproducibly. We believe that a hotspot-directed approach is general for creation of abiotic protein affinity reagents that target functional protein domains. Affinity ligands are typically high-information content biologicals. Their structure and function are determined from their amino acid or oligo sequence. In contract, the SA described in this work is a statistical copolymer that lacks sequence specificity. These results are an important contribution to the concept that randomness and biospecificity are not mutually exclusive.

Xenopus Oocytes: A Tool to Decipher Molecular Specificity of Insecticides towards Mammalian and Insect GABA-A Receptors.

The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel pharmacological studies on the sensitivity of these different insect GABAr to various agonists or antagonists. The resulting analysis of the molecular basis of the species-specific GABAr responses to insecticides is necessary not only to depict and understand species toxicity, but also to help at the early identification of unacceptable toxicity of insecticides toward beneficial insects such as Apis mellifera (honeybees). Using heterologous expression in Xenopus laevis oocytes, and two-electrode voltage-clamp recording to assess the properties of the GABAr, we performed a comparative analysis of the pharmacological sensitivity of RDL subunits from A. pisum, A. mellifera and Varroa destructor GABAr to three pesticides (fipronil, picrotoxin and dieldrin). These data were compared to similar characterizations performed on two Homo sapiens GABA-A receptors (α2β2γ2 and α2β2γ2). Our results underline a global conservation of the pharmacological profiles of these receptors, with some interesting species specificities, nonetheless, and suggest that this approach can be useful for the early identification of poorly specific molecules.

The ecdysone receptor regulates several key physiological factors in Anopheles funestus

BackgroundMalaria is a devastating disease, transmitted by female Anopheles mosquitoes infected with Plasmodium parasites. Current insecticide-based strategies exist to control the spread of malaria by targeting vectors. However, the increase in insecticide resistance in vector populations hinder the efficacy of these methods. It is, therefore, essential to develop novel vector control methods that efficiently target transmission reducing factors such as vector density and competence. A possible vector control candidate gene, the ecdysone receptor, regulates longevity, reproduction, immunity and other physiological processes in several insects, including malaria vectors. Anopheles funestus is a prominent vector in sub-Saharan Africa, however, the function of the ecdysone receptor in this mosquito has not previously been studied. This study aimed to determine if the ecdysone receptor depletion impacts An. funestus longevity, reproduction and susceptibility to Plasmodium falciparum infection.MethodsRNA interference was used to reduce ecdysone receptor expression levels in An. funestus females and investigate how the above-mentioned phenotypes are influenced. Additionally, the expression levels of the ecdysone receptor, and reproduction genes lipophorin and vitellogenin receptor as well as the immune gene, leucine rich immune molecule 9 were determined in ecdysone receptor-depleted mosquitoes using quantitative polymerase chain reaction.ResultsEcdysone receptor-depleted mosquitoes had a shorter lifespan, impaired oogenesis, were less fertile, and had reduced P. falciparum infection intensity.ConclusionsOverall, this study provides the first experimental evidence that supports ecdysone receptor as a potential target in the development of vector control measures targeting An. funestus.

Sex Pheromone Receptors of Lepidopteran Insects

The sex pheromone receptors (SPRs) of Lepidopteran insects play important roles in chemical communication. In the sex pheromone detection processes, sex pheromone molecule (SPM), SPR, co-receptor (Orco), pheromone binding protein (PBP), sensory neuron membrane protein (SNMP), and pheromone degradation enzyme (PDE) play individual and cooperative roles. Commonly known as butterfly and moth, the Lepidopteran insects are widely distributed throughout the world, most of which are pests. Comprehensive knowledge of the SPRs of Lepidopteran insects would help the development of sex lure technology and the sex communication pathway research. In this review, we summarized SPR/Orco information from 10 families of Lepidopteran insects from corresponding studies. According to the research progress in the literature, we speculated the evolution of SPRs/Orcos and phylogenetically analyzed the Lepidopteran SPRs and Orcos with the neighbor-joining tree and further concluded the relationship between the cluster of SPRs and their ligands; we analyzed the predicted structural features of SPRs and gave our prediction results of SPRs and Orcos with Consensus Constrained TOPology Prediction (CCTOP) and SwissModel; we summarized the functional characterization of Lepidopteran SPRs and SPR-ligand interaction and then described the progress in the sex pheromone signaling pathways and metabotropic ion channel. Further studies are needed to work out the cryo-electron microscopy (EM) structure of SPR and the SPR-ligand docking pattern in a biophysical perspective, which will directly facilitate the understanding of sex pheromone signal transduction pathways and provide guidance in the sex lure technology in field pest control.

昆虫の嗅覚・嗅覚受容体とその応用

昆虫の嗅覚・嗅覚受容体とその応用

Insect RDL Receptor Models for Virtual Screening: Impact of the Template Conformational State in Pentameric Ligand-Gated Ion Channels.

The RDL receptor is one of the most relevant protein targets for insecticide molecules. It belongs to the pentameric ligand-gated ion channel (pLGIC) family. Given that the experimental structures of pLGICs are difficult to obtain, homology modeling has been extensively used for these proteins, particularly for the RDL receptor. However, no detailed assessments of the usefulness of homology models for virtual screening (VS) have been carried out for pLGICs. The aim of this study was to evaluate which are the determinant factors for a good VS performance using RDL homology models, specially analyzing the impact of the template conformational state. Fifteen RDL homology models were obtained based on different pLGIC templates representing the closed, open, and desensitized states. A retrospective VS process was performed on each model, and their performance in the prioritization of active ligands was assessed. In addition, the three best-performing models among each of the conformations were subjected to molecular dynamics simulations (MDS) in complex with a representative active ligand. The models showed variations in their VS performance parameters that were related to the structural properties of the binding site. VS performance tended to improve in more constricted binding cavities. The best performance was obtained with a model based on a template in the closed conformation. MDS confirmed that the closed model was the one that best represented the interactions with an active ligand. These results imply that different templates should be evaluated and the structural variations between their channel conformational states should be specially examined, providing guidelines for the application of homology modeling for VS in other proteins of the pLGIC family.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Fatal Neonicotinoid poisoning : A case Series

Poisoning due to pesticides has long been seen as a severe public health problem. A task force of the World Health Organization in early 1990 estimated that about one million unintentional pesticide poisonings occur annually, leading to approximately 20,000 deaths. Even after thirty years, there is no clear and accurate picture of global pesticide poisoning despite an increase in pesticide use. Organo phosphates are the major cause of self-poisoning in southern and central India and aluminium phosphide in northern India. Neonicotinoids are a newer class of insecticides with increasing usage in recent decades because of their favourable toxicological profile. Neonicotinoids have a very high margin of safety because of the specificity of the insecticides for nicotinic acetylcholine receptors in insects than mammals combined with rapid metabolism and poor penetration to the blood-brain barrier. The present study was carried out on cases brought for autopsy which revealed Neonicotinoid poisoning. Four cases were studied with the purpose to highlight the toxic effects of a new class of insecticide i.e., neonicotinoids which impose health hazards to humans and animals including death.

UV-C radiation-induced changes in the legs and trichoid sensilla of the red flour beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae)

Since various kind of receptors in insects are mainly located on their legs. We estimate the influence of ultraviolet-C on the leg morphology and trichoid sensilla of Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), zero-day-old pupae were irradiated with UV-C at a distance of 35 cm for 1, 2, 4, 8, 16, 32, and 64 min. Changes in leg morphologies and trichoid sensilla were determined using light and scanning electron microscopes. UV-C radiation caused the abnormalities of an adult’s legs that were classified into three levels; mild, moderate, and strong. Legs of the resulting adults were developed into malformed structures that lacked clear segmental identity. The severity of leg abnormalities was recorded in a dose-dependent manner. UV-C significantly decreased the length and width of the femur, tibia, and tarsus. In the strongly affected level, the femur was reduced in size while the tibia and tarsus were completely deleted, and there was no evidence of claws. Scanning electron microscopy showed the wrinkled cuticle on the leg segment, and the sensilla of the resulting adults appeared less in number. After UV-C radiation, the trichoid sensilla on the femur became longer and wider, but the trichoid sensilla on the tibia were shorter and narrower. Taken together, UV-C irradiation during pupal stage interfered development of legs and brought about specific malformation features.

California court orders end to sulfoxaflor use

The pesticide sulfoxaflor can no longer be used on crops in California, a state superior court ruled Dec. 3 . The decision is a win for beekeepers and environmental groups, which challenged California’s approval of agricultural uses for sulfoxaflor in 2020. The groups claimed that the state’s Department of Pesticide Regulation (DPR) violated state environmental law by failing to consider the chemical’s adverse impacts on bees. The court agreed with the petitioners, represented by the environmental law group Earthjustice, that the DPR violated state law when it approved sulfoxaflor pesticides, which are produced by Corteva Agriscience, formerly Dow AgroSciences. Sulfoxaflor targets the same nicotinic acetylcholine receptor in insects as do neonicotinoid pesticides, which have been under scrutiny for their ability to harm bees. Both sulfoxaflor and neonicotinoids are systemic insecticides, meaning they are taken up by plants and distributed throughout plant tissues. Both are harmful to bees at low doses.

Enhancing the resilience of transgenic cotton for insect resistance.

The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.

Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides.

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

A new subfamily of ionotropic glutamate receptors unique to the echinoderms with putative sensory role.

Chemosensation is a critical signalling process in animals and especially important in sea cucumbers, a group of ecologically and economically important marine echinoderms (class Holothuroidea), which lack audio and visual organs and rely on chemical sensing for survival, feeding and reproduction. The ionotropic receptors are a recently identified family of chemosensory receptors in insects and other protostomes, related to the ionotropic glutamate receptor family (iGluR), a large family of membrane receptors in metazoan. Here we characterize the echinoderm iGluR subunits and consider their possible role in chemical communication in sea cucumbers. Sequence similarity searches revealed that sea cucumbers have in general a higher number of iGluR subunits when compared to other echinoderms. Phylogenetic analysis and sequence comparisons revealed GluH as a specific iGluR subfamily present in all echinoderms. Homologues of the vertebrate GluA (aka α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA), GluK (aka kainate) and GluD (aka delta) were also identified. The GluN (aka N-methyl-d-aspartate, NMDA) as well as the invertebrate deuterostome subfamily GluF (aka phi) are absent in echinoderms. The echinoderm GluH subfamily shares conserved structural protein organization with vertebrate iGluRs and the ligand binding domain (LBD) is the most conserved region; genome analysis indicates evolution via lineage and species-specific tandem gene duplications. GluH genes (named Grih) are the most highly expressed iGluRs subunit genes in tissues in the sea cucumber Holothuria arguinesis, with Griha1, Griha2 and Griha5 exclusively expressed in tentacles, making them candidates to have a chemosensory role in this species. The multiple GluH subunits may provide alternative receptor assembly combinations, thus expanding the functional possibilities and widening the range of compounds detected during aggregation and spawning in echinoderms.