Insect Biochemistry and Molecular Biology最新文献

The diverse cytochrome P450 enzymes of insects play essential physiological roles and also play important roles in the metabolism of environmental chemicals such as insecticides. We manually curated the complement of P450 (CYP) genes, or CYPome, of the black fungus gnat, Bradysia (Sciara) coprophila (Diptera, Sciaroidea), a species with a variable number of chromosomes. This CYPome carries two types of “alien” P450 genes. The first type of alien P450s was found among the 163 CYP genes of the core genome (autosomes and X). They consist of 28 sequences resulting from horizontal gene transfer, with closest sequences not found in insects, but in other arthropods, often Collembola. These genes are not contaminants, because they are expressed genes with introns, found in synteny with regular dipteran genes, also found in B. odoriphaga and B. hygida. Two such “alien” genes are representatives of CYP clans not otherwise found in insects, a CYP53 sequence related to fungal CYP53 genes, and a CYP19-like sequence similar to some collembolan sequences but of unclear origin. The second type of alien P450s are represented by 99 sequences from germline-restricted chromosomes (GRC). While most are P450 pseudogenes, 33 are apparently intact, with half being more closely related to P450s from Cecidomyiidae than from Sciaridae, thus supporting the hypothesis of a cross-family hybridization origin of the GRC.

Sexual dimorphisms of the brain play essential roles in successful reproduction. Silkmoth Bombyx mori exhibits extensive sexual differences in sexual behavior, as well as their morphology. Although the neural circuits that transmit information about sex pheromone in the male brain are extensively analyzed, the molecular mechanisms that regulate their development are still elusive. In the present study, we focused on the silkmoth ortholog of fruitless (fru) as a candidate gene that regulates sexual dimorphisms of the brain. fru transcripts were expressed from multiple promoters in various tissues, and brain-specific transcripts were sex-specifically spliced, in a manner similar to Drosophila. Interestingly, fru was highly expressed in the adult female brain and the male larval testis. Analysis of CRISPR/Cas9-mediated fru knockout strains revealed that fru plays important roles in survival during late larval and pupal stages, testis development, and adult sexual behavior. fru mutant males exhibited highly reduced levels of courtship and low copulation rate, indicating that fru plays significant roles in the sexual behavior of silkmoths, although it is not absolutely necessary for copulation. In the fru mutant males, sexually dimorphic pattern of the odorant receptor expression was impaired, possibly causing the defects in courtship behavior. These results provide important clues to elucidate the development of sexual dimorphisms of silkmoth brains, as well as the evolution of fruitless gene in insects.

Mosquitoes rely mainly on the sense of smell to decipher their environment and locate suitable food sources, hosts for blood feeding and oviposition sites. The molecular bases of olfaction involve multigenic families of olfactory proteins that have evolved to interact with a narrow set of odorants that are critical for survival. Understanding the complex interplay between diversified repertoires of olfactory proteins and ecologically-relevant odorant signals, which elicit important behaviors, is fundamental for the design of novel control strategies targeting the sense of smell of disease vector mosquitoes. Previously, large multigene families of odorant receptor and ionotropic receptor proteins, as well as a subset of odorant-binding proteins have been shown to mediate the selectivity and sensitivity of the mosquito olfactory system. In this study, we identify a mosquito-specific antennal protein (MSAP) gene as a novel molecular actor of odorant reception. MSAP is highly conserved across mosquito species and is transcribed at an extremely high level in female antennae. In order to understand its role in the mosquito olfactory system, we generated knockout mutant lines in Anopheles gambiae, and performed comparative analysis of behavioral and physiological responses to human-associated odorants. We found that MSAP promotes female mosquito attraction to human odor and enhances the sensitivity of the antennae to a variety of odorants. These findings suggest that MSAP is an important component of the mosquito olfactory system, which until now has gone completely unnoticed.

Excessive use of azole fungicides in agriculture poses a potential threat to honeybees and other pollinator insects; however, the detailed effects of these molecules remain largely unclear. Hence, in the present study it was aimed to investigate the acute sublethal effects of tebuconazole on the redox homeostasis and fatty acid composition in the brain of honeybees. Our findings demonstrate that tebuconazole decreased total antioxidant capacity, the ratio of reduced to oxidized glutathione and disturbed the function of key antioxidant defense enzymes along with the induction of lipid peroxidation indicated by increased malondialdehyde levels, while it also altered the fatty acid profile of the brain. The present study highlights the negative impact of tebuconazole on honeybees and contributes to the understanding of potential consequences related to azole exposure on pollinator insects’ health, such as the occurrence of colony collapse disorder.

Trypanosoma rangeli is a protozoan parasite that infects triatomines and mammals in the Americas, producing mixed infections with Trypanosoma cruzi, the etiological agent of Chagas disease. The former parasite is not pathogenic to humans, but has different levels of pathogenicity, as well as causing physiological and behavioral alterations, to its invertebrate hosts. In this study, we measured locomotory activity, and the glyceride accumulation profile in the hemolymph and fat body, as well as the expression of key genes related to triglyceride metabolism, of Rhodnius prolixus nymphs infected with T. rangeli. We found that the locomotory activity of the insects was correlated with the amount of triglycerides in the fat body. Infected nymphs had increased activity when starved, and also had an accumulation of glycerides in the fat body and hemolymph. These alterations were also associated with a higher expression of the diacylglycerol acyltransferase, lipophorin and lipophorin receptor genes in the fat body. We infer that T. rangeli is able to alter the energetic processes of its invertebrate host, in order to increase the availability of lipids to the parasite, which, in turn modifies the activity levels of the insect. These alterations are discussed with regard to their potential to increase the transmission rate of the parasite.

The Arachnida subclass of Acari comprises many harmful pests that threaten agriculture as well as animal health, including herbivorous spider mites, the bee parasite Varroa, the poultry mite Dermanyssus and several species of ticks. Especially in agriculture, acaricides are often used intensively to minimize the damage they inflict, promoting the development of resistance. Beneficial predatory mites used in biological control are also subjected to acaricide selection in the field. The development and use of new genetic and genomic tools such as genome and transcriptome sequencing, bulked segregant analysis (QTL mapping), and reverse genetics via RNAi or CRISPR/Cas9, have greatly increased our understanding of the molecular genetic mechanisms of resistance in Acari, especially in the spider mite Tetranychus urticae which emerged as a model species. These new techniques allowed to uncover and validate new resistance mutations in a larger range of species. In addition, they provided an impetus to start elucidating more challenging questions on mechanisms of gene regulation of detoxification associated with resistance.

Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary glands, we demonstrated that saliva-producing type II and III acini are innervated by neuropeptidergic axons which release different classes of neuropeptides via their terminals (Šimo et al., 2009b, 2013). Among these, the neuropeptide SIFamide—along with its cognate receptor—were postulated to control the basally located acinar valve via basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionally characterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genome and proved that it senses a low nanomolar level of its corresponding ligand. Insect SIFamide paralogs, SMYamides, also activated the receptor but less effectively compared to SIFamide. Bioinformatic and molecular dynamic analyses suggested that I. scapularis SIFamide receptors are class A GPCRs where the peptide amidated carboxy-terminus is oriented within the receptor binding cavity. The receptor was found to be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, and ovaries, but not in Malpighian tubules. Investigation of the temporal expression patterns suggests that the receptor transcript is highly expressed in unfed I. ricinus female salivary glands and then decreases during feeding. In synganglia, a significant transcript increase was detected in replete ticks. In salivary gland acini, an antibody targeting the SIFa_R2 recognized basal epithelial cells, myoepithelial cells, and basal granular cells in close proximity to the SIFamide-releasing axon terminals. Immunoreactivity was also detected in specific neurons distributed throughout various I. ricinus synganglion locations. The current findings, alongside previous reports from our group, indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in I. ricinus salivary glands. Our study investigates the peptidergic regulation of the I. ricinus salivary gland in detail, emphasizing the complexity of this system.

ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase α and β family, including the α and β subunits of ATP synthase (RpATPSynα and RpATPSynβ), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organsn highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynβ knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynβ increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynα knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.

Personal protection measures against the mosquitoes like the use of repellents constitute valuable tools in the effort to prevent the transmission of vector-borne diseases. Therefore, the discovery of novel repellent molecules which will be effective at lower concentrations and provide a longer duration of protection remains an urgent need.

Mosquito Odorant-Binding Proteins (OBPs) involved in the initial steps of the olfactory signal transduction cascade have been recognized not only as passive carriers of odors and pheromones but also as the first molecular filter to discriminate semiochemicals, hence serving as molecular targets for the design of novel pest control agents. Among the three-dimensional structures of mosquito OBPs solved in the last decades, the OBP1 complexes with known repellents have been widely used as reference structures in docking analysis and molecular dynamics simulation studies for the structure-based discovery of new molecules with repellent activity.

Herein, ten compounds known to be active against mosquitoes and/or displaying a binding affinity for Anopheles gambiae AgamOBP1 were used as queries in an in silico screening of over 96 million chemical samples in order to detect molecules with structural similarity. Further filtering of the acquired hits on the basis of toxicity, vapor pressure, and commercial availability resulted in 120 unique molecules that were subjected to molecular docking studies against OBP1. For seventeen potential OBP1-binders, the free energy of binding (FEB) and mode of interaction with the protein were further estimated by molecular docking simulations leading to the selection of eight molecules exhibiting the highest similarity with their parental compounds and favorable energy values. The in vitro determination of their binding affinity to AgamOBP1 and the evaluation of their repellent activity against female Aedes albopictus mosquitoes revealed that our combined ligand similarity screening and OBP1 structure-based molecular docking successfully detected three molecules with enhanced repellent properties. A novel DEET-like repellent with lower volatility (8.55 × 10⁻⁴ mmHg) but a higher binding affinity for OBP1 than DEET (1.35 × 10⁻³ mmHg). A highly active repellent molecule that is predicted to bind to the secondary Icaridin (sIC)-binding site of OBP1 with higher affinity than to the DEET-site and, therefore, represents a new scaffold to be exploited for the discovery of binders targeting multiple OBP sites. Finally, a third potent repellent exhibiting a high degree of volatility was found to be a strong DEET-site binder of OBP1 that could be used in slow-release formulations.

Brochosomes derived from the specialized glandular segments of the Malpighian tubules (MTs) form superhydrophobic coatings for insects of Membracoidea, and have multiple hypothetical functions. However, the constituents, biosynthesis and evolutionary origin of brochosomes remain poorly understood. We investigated general chemical and physical characteristics of the integumental brochosomes (IBs) of the leafhopper Psammotettix striatus, determined the constituents of IBs, identified the unigenes involved in brochosomal protein synthesis, and investigated the potential associations among brochosomal protein synthesis, amino acid composition of food source, and the possible roles of endosymbionts in brochosome production. The results show that IBs are mainly composed of glycine- and tyrosine-rich proteins and some metal elements, which contain both essential and non-essential amino acids (EAAs and NEAAs) for insects, including EAAs deficient in the sole food source. All 12 unigenes involved in synthesizing the 12 brochosomal proteins (BPs) with high confidence are exclusively highly expressed in the glandular segment of MTs, confirming that brochosomes are synthesized by this segment. The synthesis of BPs is one of the key synapomorphies of Membracoidea but may be lost secondarily in a few lineages. The synthesis of BPs might be related to the symbiosis of leafhoppers/treehoppers with endosymbionts that provide these insects with EAAs, including those are deficient in the sole diet (i.e., plant sap) and could only be made available by the symbionts. We hypothesize that the functional modification of MTs have combined with the application of BPs enabling Membracoidea to colonize and adapt to novel ecological niches, and evolve to the dramatic diversification of this hemipteran group (in particular the family Cicadellidae). This study highlights the importance of evolutionary plasticity and multiple functions of MTs in driving the adaptations and evolution of sap-sucking insects of Hemiptera.