Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) resistance in the RDDT R strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT R strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2b position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.Keywords: cytochrome P450; mutation; insecticide; resistance; structure.Many cytochrome P450 enzymes are known to be essential for the protection of organisms against xenobiotics. In insects, the involvement of cytochrome P450 enzymes in plant toxin or insecticide resistance has already been suggested or demonstrated [1][2][3][4][5][6][7], although high resistance levels to insecticides still remain unexplained. To date, only three of the cytochrome P450 enzymes linked to resistance have been shown to be able to metabolize insecticides. Two were cloned from the house fly: CYP6A1 metabolizes aldrin, heptachlor [8], terpenoids [9] and diazinon [10] and CYP12A1 metabolizes aldrin, heptachlor, diazinon and azinphosmethyl [11]. The third is CYP6A2 from Drosophila melanogaster. Baculovirus-directed production of wild-type CYP6A2 showed metabolism of cyclodiene and organophosphorous insecticides, but 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) metabolism could not be detected [12]. In addition, sequence polymorphism of CYP6A1 and CYP6D1 has been documented in the house fly, but there is no link between these instances of polymorphism and insecticide resistance [7,13,14]. These results are in contrast with known instances of cytochrome P450 polymorphisms in humans, which are well known to affect the metabolism of drugs [15,16] and even pesticides [17]. In fact, only two examples of pesticide...

BackgroundEndoparasitoid wasps are important natural enemies of the widely distributed aphid pests and are mainly used as biological control agents. However, despite the increased interest on aphid interaction networks, only sparse information is available on the factors used by parasitoids to modulate the aphid physiology. Our aim was here to identify the major protein components of the venom injected at oviposition by Aphidius ervi to ensure successful development in its aphid host, Acyrthosiphon pisum.ResultsA combined large-scale transcriptomic and proteomic approach allowed us to identify 16 putative venom proteins among which three γ-glutamyl transpeptidases (γ-GTs) were by far the most abundant. Two of the γ-GTs most likely correspond to alleles of the same gene, with one of these alleles previously described as involved in host castration. The third γ-GT was only distantly related to the others and may not be functional owing to the presence of mutations in the active site. Among the other abundant proteins in the venom, several were unique to A. ervi such as the molecular chaperone endoplasmin possibly involved in protecting proteins during their secretion and transport in the host. Abundant transcripts encoding three secreted cystein-rich toxin-like peptides whose function remains to be explored were also identified.ConclusionsOur data further support the role of γ-GTs as key players in A. ervi success on aphid hosts. However, they also evidence that this wasp venom is a complex fluid that contains diverse, more or less specific, protein components. Their characterization will undoubtedly help deciphering parasitoid-aphid and parasitoid-aphid-symbiont interactions. Finally, this study also shed light on the quick evolution of venom components through processes such as duplication and convergent recruitment of virulence factors between unrelated organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-342) contains supplementary material, which is available to authorized users.

The aphid Acyrthosiphon pisum population is composed of different morphs, such as winged and wingless parthenogens, males, and sexual females. The combined effect of reduced photoperiodicity and cold in fall triggers the apparition of sexual morphs. In contrast they reproduce asexually in spring and summer. In our current study, we provide evidence that clonal individuals display phenotypic variability within asexual morph categories. We describe that clones sharing the same morphological features, which arose from the same founder mother, constitute a repertoire of variants with distinct behavioral and physiological traits. Our results suggest that the prevailing environmental conditions influence the recruitment of adaptive phenotypes from a cohort of clonal individuals exhibiting considerable molecular diversity. However, we observed that the variability might be reduced or enhanced by external factors, but is never abolished in accordance with a model of stochastically produced phenotypes. This overall mechanism allows the renewal of colonies from a few adapted individuals that survive drastic episodic changes in a fluctuating environment.[Supplemental material is available online at http://www.genome.org.]Aphids exhibit a complex mode of reproduction, combining parthenogenesis (spring/summer) and sexual activity (fall/winter) in species such as Acyrthociphon pisum (Dixon 1973;Blackman and Eastop 1984). Aphids thus constitute an excellent model system to investigate how this double reproductive system generates polyphenism, a generic concept used to describe the emergence of distinct morphs, such as winged, wingless, sexual female, and male (Blackman and Eastop 1984;Blackman 1987;Muller et al. 2001). Aphid morph distribution, particularly wing dimorphism, is influenced by environmental conditions, such as population density (crowding effects) (Sutherland 1969) and/or host plant vitality, as well as physical parameters including humidity, temperature, and photoperiodicity (Walters and Dixon 1982;Dixon 1998). This raises fascinating questions regarding the outcomes of alternative developmental mechanisms that cause morph switching in a predictable way (Stearns 1989;Nijhout 1999).Some aphid species are ''sexual'' lineages committed exclusively to sexual reproduction, others are ''facultative asexual'' lineages, which alternate between sexual and parthenogenetic modes depending on the season, while some others are obligate parthenogens (Delmotte et al. 2002;Le Trionnaire et al. 2008). This combined double system of reproduction is shared with many other species like Daphnia ( Despite some divergent reports, most authors seem to agree that sexual populations in aphids present a high allelic polymorphism of many genes and predominance of homozygous loci within individuals. In contrast, asexual populations seem to present less allelic polymorphism, but strong heterozygosity at most loci (Delmotte et al. 2002;Kanbe and Akimoto, 2009). It is largely assumed that organisms reproducing asexually should maintain lower g...

Background: Parasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biological control. Their success depends on adapting to develop inside aphids and overcoming both host aphid defenses and their protective endosymbionts. Results: We present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp) and the most AT-rich reported thus far for any arthropod (GC content: 25.8 and 23.8%). This nucleotide bias is accompanied by skewed codon usage and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and energy efficiency. We identify missing desaturase genes, whose absence may underlie mimicry in the cuticular hydrocarbon profile of L. fabarum. We highlight key gene groups including those underlying venom composition, chemosensory perception, and sex determination, as well as potential losses in immune pathway genes. Conclusions: These findings are of fundamental interest for insect evolution and biological control applications. They provide a strong foundation for further functional studies into coevolution between parasitoids and their hosts. Both genomes are available at https://bipaa.genouest.org.