BackgroundThe functional and evolutionary diversification of insect cytochrome P450s (CYPs) shaped the success of insects. CYPs constitute one of the largest and oldest gene superfamilies that are found in virtually all aerobic organisms. Because of the availability of whole genome sequence and well functioning RNA interference (RNAi), the red flour beetle, Tribolium castaneum serves as an ideal insect model for conducting functional genomics studies. Although several T. castaneum CYPs had been functionally investigated in our previous studies, the roles of the majority of CYPs remain largely unknown. Here, we comprehensively analyzed the phylogenetic relationship of all T. castaneum CYPs with genes in other insect species, investigated the CYP6BQ gene cluster organization, function and evolution, as well as examined the mitochondrial CYPs gene expression patterns and intron-exon organization.ResultsA total 143 CYPs were identified and classified into 26 families and 59 subfamilies. The phylogenetic trees of CYPs among insects across taxa provided evolutionary insight for the genetic distance and function. The percentage of singleton (33.3%) in T. castaneum CYPs is much less than those in Drosophila melanogaster (52.5%) and Bombyx mori (51.2%). Most members in the largest CYP6BQ gene cluster may make contribution to deltamethrin resistance in QTC279 strain. T. castaneum genome encodes nine mitochondrial CYPs, among them CYP12H1 is only expressed in the final instar larval stage. The intron-exon organizations of these mitochondrial CYPs are highly diverse.ConclusionOur studies provide a platform to understand the evolution and functions of T. castaneum CYP gene superfamily which will help reveal the strategies employed by insects to cope with their environment.

The adaptation of herbivorous insects to their host plants is hypothesized to be intimately associated with their ubiquitous development of resistance to synthetic pesticides. However, not much is known about the mechanisms underlying the relationship between detoxification of plant toxins and synthetic pesticides. To address this knowledge gap, we used specialist pest Colorado potato beetle (CPB) and its host plant, potato, as a model system. Next-generation sequencing (454 pyrosequencing) was performed to reveal the CPB transcriptome. Differential expression patterns of cytochrome P450 complement (CYPome) were analyzed between the susceptible (S) and imidacloprid resistant (R) beetles. We also evaluated the global transcriptome repertoire of CPB CYPome in response to the challenge by potato leaf allelochemicals and imidacloprid. The results showed that more than half (51.2%) of the CBP cytochrome P450 monooxygenases (P450s) that are up-regulated in the R strain are also induced by both host plant toxins and pesticide in a tissue-specific manner. These data suggest that xenobiotic adaptation in this specialist herbivore is through up-regulation of multiple P450s that are potentially involved in detoxifying both pesticide and plant allelochemicals.

As a superfamily of multifunctional enzymes that is mainly associated with xenobiotic adaptation, glutathione S-transferases (GSTs) facilitate insects' survival under chemical stresses in their environment. GSTs confer xenobiotic adaptation through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. In this article, a comprehensive overview of current understanding on the versatile functions of insect GSTs in detoxifying chemical compounds is presented. The diverse structures of different classes of insect GSTs, specifically the spatial localization and composition of their amino acid residues constituted in their active sites are also summarized. Recent availability of whole genome sequences of numerous insect species, accompanied by RNA interference, X-ray crystallography, enzyme kinetics and site-directed mutagenesis techniques have significantly enhanced our understanding of functional and structural diversity of insect GSTs.

Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the P-X-P-X motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.

The two-spotted spider mite, Tetranychus urticae Koch, is a constant threat to sustainable production of numerous economically important crops globally. Management of T. urticae is heavily reliant on the application of synthetic acaricides. However, T. urticae has rapidly developed resistance to most of the acaricides available for its control due to its very broad host plant range, extremely short lifecycle, high fecundity, arrhenotokous parthenogenesis, and overwintering strategy. Despite the recent progress in identifying genetic changes/markers associated with resistance toward some commonly used acaricides for T. urticae, there is still limited studies that select and apply these markers in field populations to guide sustainable pest management strategy design. Temporal and spatial characterization of acaricide-resistant phenotypes and their underlying mechanisms are crucial for the design and implementation of successful and sustainable integrated mite management programs. This review highlights the current acaricide resistance status of field-collected T. urticae populations and the underlying molecular mechanisms of resistance. Our review found that some genetic mutations in target sites and/or overexpression of metabolic genes confer resistance in geographically exclusive populations, while some resistance markers appear to be specific to populations at biogeographical areas. Thus, there is a need for locally based coordinated efforts to understand the mechanisms of resistance present in endemic T. urticae populations. Moreover, we discuss a prospective template for designing an effective acaricide resistance management program within various agroecosystems.

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Insect G protein coupled receptors (GPCRs) have been identified as a highly attractive target for new generation insecticides discovery due to their critical physiological functions. However, few insect GPCRs have been functionally characterized. Here, we cloned the full length of a methuselah-like GPCR gene (Ldmthl1) from the Asian gypsy moth, Lymantria dispar. We then characterized the secondary and tertiary structures of Ldmthl1. We also predicted the global structure of this insect GPCR protein which is composed of three major domains. RNA interference of Ldmthl1 resulted in a reduction of gypsy moths' resistance to deltamethrin and suppressed expression of downstream stress-associated genes, such as P450s, glutathione S transferases, and heat shock proteins. The function of Ldmthl1 was further investigated using transgenic lines of Drosophila melanogaster. Drosophila with overexpression of Ldmthl1 showed significantly longer lifespan than control flies. Taken together, our studies revealed that the physiological functions of Ldmthl1 in L. dispar are associated with longevity and resistance to insecticide stresses. Potentially, Ldmthl1 can be used as a target for new insecticide discovery in order to manage this notorious forest pest.

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