The insulin signaling pathway in Drosophila melanogaster: A nexus revealing an "Achilles' heel" in DDT resistance.

Insecticide resistance is an ongoing challenge in agriculture and disease vector control. Here, we demonstrate a novel strategy to attenuate resistance. We used genomics tools to target fundamental energy-associated pathways and identified a potential "Achilles' heel" for resistance, a resistance-associated protein that, upon inhibition, results in a substantial loss in the resistance phenotype. Specifically, we compared the gene expression profiles and structural variations of the insulin/insulin-like growth factor signaling (IIS) pathway genes in DDT-susceptible (91-C) and -resistant (91-R) Drosophila melanogaster (Drosophila) strains. A total of eight and seven IIS transcripts were up- and down-regulated, respectively, in 91-R compared to 91-C. A total of 114 nonsynonymous mutations were observed between 91-C and 91-R, of which 51.8% were fixed. Among the differentially expressed transcripts, phosphoenolpyruvate carboxykinase (PEPCK), down-regulated in 91-R, encoded the greatest number of amino acid changes, prompting us to perform PEPCK inhibitor-pesticide exposure bioassays. The inhibitor of PEPCK, hydrazine sulfate, resulted in a 161- to 218-fold decrease in the DDT resistance phenotype (91-R) and more than a 4- to 5-fold increase in susceptibility in 91-C. A second target protein, Glycogen synthase kinase 3ß (GSK3ß-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3ß-PO inhibitor, lithium chloride, likewise reduced the resistance but to a lesser extent than did hydrazine sulfate for PEPCK. We demonstrate the potential role of IIS genes in DDT resistance and the potential discovery of an "Achilles' heel" against pesticide resistance in this pathway.

Identification of transcriptional responsive genes to acetic acid, ethanol, and 2-phenylethanol exposure in Drosophila melanogaster.

The fruit fly, Drosophila melanogaster, is predominantly found in overripe, rotten, fermenting, or decaying fruits and is constantly exposed to chemical stressors such as acetic acid, ethanol, and 2-phenylethanol. D. melanogaster has been employed as a model system for studying the molecular bases of various types of chemical-induced tolerance. Expression profiling using Illumina sequencing has been performed for identifying changes in gene expression that may be associated with evolutionary adaptation to exposure of acetic acid, ethanol, and 2-phenylethanol. We identified a total of 457 differentially expressed genes that may affect sensitivity or tolerance to three chemicals in the chemical treatment group as opposed to the control group. Gene-set enrichment analysis revealed that the genes involved in metabolism, multicellular organism reproduction, olfaction, regulation of signal transduction, and stress tolerance were over-represented in response to chemical exposure. Furthermore, we also detected a coordinated upregulation of genes in the Toll- and Imd-signaling pathways after the chemical exposure. Quantitative reverse transcription PCR analysis revealed that the expression levels of nine genes within the set of genes identified by RNA sequencing were up- or downregulated owing to chemical exposure. Taken together, our data suggest that such differentially expressed genes are coordinately affected by chemical exposure. Transcriptional analyses after exposure of D. melanogaster with three chemicals provide unique insights into subsequent functional studies on the mechanisms underlying the evolutionary adaptation of insect species to environmental chemical stressors.

A review of DDT resistance as it pertains to the 91-C and 91-R strains in Drosophila melanogaster.

While insecticide resistance presents a challenge for those intent on controlling insect populations, these challenges have also generated a set of tools that can be used to ask fundamental biological questions about that resistance. Numerous species of insects have evolved resistance to multiple classes of insecticides. Each one of these species and their respective resistant populations represent a potential tool for understanding the molecular basis of the evolution of resistance. However, in-laboratory maintenance of resistant insect populations (and their comparative susceptible populations) suitable for asking the needed set of questions around the molecular consequences of long-term pesticide exposure requires a significant, in places prohibitive, level of resources. Drosophila melanogaster (hereafter referred to as Drosophila) is a model insect system with populations easily selected with pesticides and readily maintainable over decades. Even within Drosophila, however, few populations exist where long-term pesticide selection has occurred along with contrasting non-selected population. As such, the Drosophila 91-C and 91-R populations, which exhibit insecticide resistance to DDT (91-R), compared to a non-selection population (91-C), represent a unique resource for the study of high level DDT resistance. Moreover, with the availability of "omics" technologies over the past several decades, this paired population has emerged as a useful tool for understanding both the molecular basis of pesticide resistance and the molecular consequences of long-term pesticide exposure. In this review, we summarize the studies with these aforementioned populations over the past several decades, addressing what has been learned from these efforts.

Cytochrome P450s Cyp4p1 and Cyp4p2 associated with the DDT tolerance in the Drosophila melanogaster strain 91-R.

Cytochrome P450s are part of a super-gene family that has undergone gene duplication, divergence, over-expression and, in some cases, loss of function. One such case is the 91-R and 91-C strains of common origin, in Drosophila melanogaster, whereby 91-R (DDT resistant strain) overexpresses Cyp4p1 and Cyp4p2 and both genes are lost in 91-C (DDT susceptible strain). In this study, we used a comparative approach to demonstrate that transcription of Cyp4p1 and Cyp4p2 were constitutively up-regulated in the Drosophila melanogaster strain 91-R as compared to another DDT susceptible strain Canton-S which does not have a loss of function of these genes. Furthermore, significantly increased expression of Cyp4p1 and Cyp4p2 was induced in 91-R in response to sublethal DDT exposure, however, such induction did not occur in the DDT treated Canton-S. Additionally, fixed nucleotide variation within putative transcription factor binding sites of Cyp4p1 and Cyp4p2 promoters were observed between 91-R and Canton-S, however, their impact on transcription remains to be determined. Two GAL4/UAS transgenic strains with integrated heat shock-inducible Cyp4p1- or Cyp4p2-RNAi constructs within wild-type genetic backgrounds were developed. Following heat shock induction of Cyp4p1 and Cyp4p2 knockdown, these transgenic lines showed increased DDT mortality as compared to their corresponding non-heat shock controls. These results provide a functional link of Cyp4p1 and Cyp4p2 in conferring tolerance to DDT exposure.

Phylogenomics from Whole Genome Sequences Using aTRAM.

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.].

Lethal and sub-lethal effects of select macrocyclic lactones insecticides on forager worker honey bees under laboratory experimental conditions.

Selective insecticide application is one important strategy for more precisely targeting harmful insects while avoiding or mitigating collateral damage to beneficial insects like honey bees. Recently, macrocyclic lactone-class insecticides have been introduced into the market place as selective bio-insecticides for controlling many arthropod pests, but how to target this selectivity only to harmful insects has yet to be achieved. In this study, the authors investigated the acute toxicity of fourmacrocyclic lactone insecticides (commercialized as abamectin, emamectin benzoate, spinetoram, and spinosad) both topically and through feeding studies with adult forager honey bees. Results indicated emamectin benzoate as topically 133.3, 750.0, and 38.3-fold and orally 3.3, 7.6, and 31.7-fold more toxic, respectively than abamectin, spinetoram and spinosad. Using Hazard Quotients for estimates of field toxicity, abamectin was measured as the safest insecticide both topically and orally for honey bees. Moreover, a significant reduction of sugar solution consumption by treatment group honey bees for orally applied emamectin benzoate and spinetoram suggests that these insecticides may have repellent properties.

Identification and interaction of multiple genes resulting in DDT resistance in the 91-R strain of Drosophila melanogaster by RNAi approaches.

4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will likely increase in terms of level and extent. Drosophila melanogaster is a model dipteran with a well annotated genome allowing both forward and reverse genetic manipulation, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold) and recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms. Their interactions, however, remain unclear. Use of Gal4/UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has identified the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in decreased sensitivity to DDT. These above findings have been further validated in 91-R flies using a nanoparticle-enhanced RNAi strategy, directly implication these genes in DDT resistance in 91-R flies.

Variation in Mitochondria-Derived Transcript Levels Associated With DDT Resistance in the 91-R Strain of Drosophila melanogaster (Diptera: Drosophilidae).

The organochloride insecticide dichlorodiphenyltrichloroethane (DDT) and its metabolites can increase cellular levels of reactive oxygen species (ROS), cause mitochondrial dysfunction, and induce apoptosis. The highly DDT-resistant Drosophila melanogaster Meigen 1830 (Drosophila) strain, 91-R, and its susceptible control, 91-C, were used to investigate functional and structural changes among mitochondrial-derived pathways. Resequencing of mitochondrial genomes (mitogenomes) detected no structural differences between 91-R and 91-C, whereas RNA-seq suggested the differential expression of 221 mitochondrial-associated genes. Reverse transcriptase-quantitative PCR validation of 33 candidates confirmed that transcripts for six genes (Cyp12d1-p, Cyp12a4, cyt-c-d, COX5BL, COX7AL, CG17140) were significantly upregulated and two genes (Dif, Rel) were significantly downregulated in 91-R. Among the upregulated genes, four genes are duplicated within the reference genome (cyt-c-d, CG17140, COX5BL, and COX7AL). The predicted functions of the differentially expressed genes, or known functions of closely related genes, suggest that 91-R utilizes existing ROS regulation pathways of the mitochondria to combat increased ROS levels from exposure to DDT. This study represents, to our knowledge, the initial investigation of mitochondrial genome sequence variants and functional adaptations in responses to intense DDT selection and provides insights into potential adaptations of ROS management associated with DDT selection in Drosophila.

Socially responsive effects of brain oxidative metabolism on aggression.

Despite ongoing high energetic demands, brains do not always use glucose and oxygen in a ratio that produces maximal ATP through oxidative phosphorylation. In some cases glucose consumption exceeds oxygen use despite adequate oxygen availability, a phenomenon known as aerobic glycolysis. Although metabolic plasticity seems essential for normal cognition, studying its functional significance has been challenging because few experimental systems link brain metabolic patterns to distinct behavioral states. Our recent transcriptomic analysis established a correlation between aggression and decreased whole-brain oxidative phosphorylation activity in the honey bee (Apis mellifera), suggesting that brain metabolic plasticity may modulate this naturally occurring behavior. Here we demonstrate that the relationship between brain metabolism and aggression is causal, conserved over evolutionary time, cell type-specific, and modulated by the social environment. Pharmacologically treating honey bees to inhibit complexes I or V in the oxidative phosphorylation pathway resulted in increased aggression. In addition, transgenic RNAi lines and genetic manipulation to knock down gene expression in complex I in fruit fly (Drosophila melanogaster) neurons resulted in increased aggression, but knockdown in glia had no effect. Finally, honey bee colony-level social manipulations that decrease individual aggression attenuated the effects of oxidative phosphorylation inhibition on aggression, demonstrating a specific effect of the social environment on brain function. Because decreased neuronal oxidative phosphorylation is usually associated with brain disease, these findings provide a powerful context for understanding brain metabolic plasticity and naturally occurring behavioral plasticity.

Alternative Splice in Alternative Lice.

Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation.

RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance.

Utilization of the human louse genome to study insecticide resistance and innate immune response.

Since sequencing the human body louse genome, substantial advances have occurred in the utilization of the information gathered from louse genomes and transcriptomes. Comparatively, the body louse genome contains far fewer genes involved in environmental response, such as xenobiotic detoxification and innate immune response. Additionally, the body louse maintains a primary bacterial endosymbiont, Candidatus Riesia pediculicola, and a number of bacterial pathogens that it vectors, which have genomes that are also reduced in size. Thus, human louse genomes offer unique information and tools for use in advancing our understanding of coevolution among vectors, endosymbionts and pathogens. In this review, we summarize the current literature on the extent of pediculicide resistance, the availability of new pediculicides and information establishing this organism as an efficient model to study how xenobiotic metabolism, which is involved in insecticide resistance, is induced and how insects modify their innate immune response upon bacterial challenge resulting in enhanced vector competence.

Rates of genomic divergence in humans, chimpanzees and their lice.

The rate of DNA mutation and divergence is highly variable across the tree of life. However, the reasons underlying this variation are not well understood. Comparing the rates of genetic changes between hosts and parasite lineages that diverged at the same time is one way to begin to understand differences in genetic mutation and substitution rates. Such studies have indicated that the rate of genetic divergence in parasites is often faster than that of their hosts when comparing single genes. However, the variation in this relative rate of molecular evolution across different genes in the genome is unknown. We compared the rate of DNA sequence divergence between humans, chimpanzees and their ectoparasitic lice for 1534 protein-coding genes across their genomes. The rate of DNA substitution in these orthologous genes was on average 14 times faster for lice than for humans and chimpanzees. In addition, these rates were positively correlated across genes. Because this correlation only occurred for substitutions that changed the amino acid, this pattern is probably produced by similar functional constraints across the same genes in humans, chimpanzees and their ectoparasites.

Differential effects of RNAi treatments on field populations of the western corn rootworm.

RNA interference (RNAi) mediated crop protection against insect pests is a technology that is greatly anticipated by the academic and industrial pest control communities. Prior to commercialization, factors influencing the potential for evolution of insect resistance to RNAi should be evaluated. While mutations in genes encoding the RNAi machinery or the sequences targeted for interference may serve as a prominent mechanism of resistance evolution, differential effects of RNAi on target pests may also facilitate such evolution. However, to date, little is known about how variation of field insect populations could influence the effectiveness of RNAi treatments. To approach this question, we evaluated the effects of RNAi treatments on adults of three western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) populations exhibiting different levels of gut cysteine protease activity, tolerance of soybean herbivory, and immune gene expression; two populations were collected from crop rotation-resistant (RR) problem areas and one from a location where RR was not observed (wild type; WT). Our results demonstrated that RNAi targeting DvRS5 (a highly expressed cysteine protease gene) reduced gut cysteine protease activity in all three WCR populations. However, the proportion of the cysteine protease activity that was inhibited varied across populations. When WCR adults were treated with double-stranded RNA of an immune gene att1, different changes in survival among WT and RR populations on soybean diets occurred. Notably, for both genes, the sequences targeted for RNAi were the same across all populations examined. These findings indicate that the effectiveness of RNAi treatments could vary among field populations depending on their physiological and genetic backgrounds and that the consistency of an RNAi trait's effectiveness on phenotypically different populations should be considered or tested prior to wide deployment. Also, genes that are potentially subjected to differential selection in the field should be avoided for RNAi-based pest control.

Characterization of the inheritance of field-evolved resistance to diamides in the fall armyworm (Spodoptera frugiperda) (Lepidoptera: Noctuidae) population from Puerto Rico.

The fall armyworm (Spodoptera frugiperda) is one of the most destructive pests of corn. New infestations have been reported in the East Hemisphere, reaching India, China, Malaysia, and Australia, causing severe destruction to corn and other crops. In Puerto Rico, practical resistance to different mode of action compounds has been reported in cornfields. In this study, we characterized the inheritance of resistance to chlorantraniliprole and flubendiamide and identified the possible cross-resistance to cyantraniliprole and cyclaniliprole. The Puerto Rican (PR) strain showed high levels of resistance to flubendiamide (RR50 = 2,762-fold) and chlorantraniliprole (RR50 = 96-fold). The inheritance of resistance showed an autosomal inheritance for chlorantraniliprole and an X-linked inheritance for flubendiamide. The trend of the dominance of resistance demonstrated an incompletely recessive trait for H1 (♂ SUS × â™€ PR) × and an incompletely dominant trait for H2 (♀ SUS × â™‚ PR) × for flubendiamide and chlorantraniliprole. The PR strain showed no significant presence of detoxification enzymes (using synergists: PBO, DEF, DEM, and VER) to chlorantraniliprole; however, for flubendiamide the SR = 2.7 (DEM), SR = 3.2 (DEF) and SR = 7.6 (VER) indicated the role of esterases, glutathione S- transferases and ABC transporters in the metabolism of flubendiamide. The PR strain showed high and low cross-resistance to cyantraniliprole (74-fold) and cyclaniliprole (11-fold), respectively. Incomplete recessiveness might lead to the survival of heterozygous individuals when the decay of diamide residue occurs in plant tissues. These results highlight the importance of adopting diverse pest management strategies, including insecticide rotating to manage FAW populations in Puerto Rico and other continents.

miRNA Dynamics for Pest Management: Implications in Insecticide Resistance.

Utilizing chemical agents in pest management in modern agricultural practices has been the predominant approach since the advent of synthetic insecticides. However, insecticide resistance is an emerging issue, as pest populations evolve to survive exposure to chemicals that were once effective in controlling them, underlining the need for advanced and innovative approaches to managing pests. In insects, microRNAs (miRNAs) serve as key regulators of a wide range of biological functions, characterized by their dynamic expression patterns and the ability to target genes. Recent studies are increasingly attributed to the significance of miRNAs in contributing to the evolution of insecticide resistance in numerous insect species. Abundant miRNAs have been discovered in insects using RNA sequencing and transcriptome analysis and are known to play vital roles in regulation at both the transcriptional and post-transcriptional levels. Globally, there is growing research interest in the characterization and application of miRNAs, especially for their potential role in managing insecticide resistance. This review focuses on how miRNAs contribute to regulating insecticide resistance across various insect species. Furthermore, we discuss the gain and loss of functions of miRNAs and the techniques for delivering miRNAs into the insect system. The review emphasizes the application of miRNA-based strategies to studying their role in diminishing insecticide resistance, offering a more efficient and lasting approach to insect management.

Identification of differentially expressed miRNAs associated with diamide detoxification pathways in Spodoptera frugiperda.

The fall armyworm (FAW) Spodoptera frugiperda is a severe economic pest of multiple crops globally. Control of this pest is often achieved using insecticides; however, over time, S. frugiperda has developed resistance to new mode of action compounds, including diamides. Previous studies have indicated diamide resistance is a complex developmental process involving multiple detoxification genes. Still, the mechanism underlying the possible involvement of microRNAs in post-transcriptional regulation of resistance has not yet been elucidated. In this study, a global screen of microRNAs (miRNAs) revealed 109 known and 63 novel miRNAs. Nine miRNAs (four known and five novel) were differentially expressed between insecticide-resistant and -susceptible strains. Gene Ontology analysis predicted putative target transcripts of the differentially expressed miRNAs encoding significant genes belonging to detoxification pathways. Additionally, miRNAs are involved in response to diamide exposure, indicating they are probably associated with the detoxification pathway. Thus, this study provides comprehensive evidence for the link between repressed miRNA expression and induced target transcripts that possibly mediate diamide resistance through post-transcriptional regulation. These findings highlight important clues for further research to unravel the roles and mechanisms of miRNAs in conferring diamide resistance.

Insect resistance management for stored product pests: a case study of cowpea weevil (Coleoptera: Bruchidae).

The cowpea weevil, Callosobruchus maculatus F. (Coleoptera: Bruchidae), can cause up to 100% yield loss of stored cowpea seeds in a few months in West Africa. Genes expressing toxins delaying insect maturation (MDTs) are available for genetic engineering. A simulation model was used to investigate the possible use of MDTs for managing C. maculatus. Specifically, we studied the effect of transgenic cowpea expressing an MDT, an insecticide, or both, on the evolution of resistance by C. maculatus at constant temperature. Transgenic cowpea expressing only a nonlethal MDT causing 50-100% maturation delay did not control C. maculatus well. Mortality caused by a maturation delay improved the efficacy of transgenic cowpea expressing only a lethal MDT, but significantly reduced the durability of transgenic cowpea Transgenic cowpea expressing only a lethal MDT causing 50% maturation delay and 90% mortality controlled C. maculatus better than one expressing only a nonlethal MDT, but its durability was only 2 yr. We concluded that transgenic cowpea expressing only an MDT has little value for managing C. maculatus. The resistance by C. maculatus to transgenic cowpea expressing only an insecticide rapidly evolved. Stacking a gene expressing a nonlethal MDT and a gene expressing an insecticide in transgenic cowpea did not significantly improve the durability of an insecticide, but stacking a gene expressing a lethal MDT and a gene expressing an insecticide in transgenic cowpea significantly improved the durability of an insecticide and an MDT. We also discussed this approach within the idea of using transgenic RNAi in pest control strategies.

Evolutionary toxicogenomics: diversification of the Cyp12d1 and Cyp12d3 genes in Drosophila species.

Gene duplication and divergence are overwhelmingly considered to be the primary mechanisms by which cytochrome P450 monooxygenases (P450s) have radiated into a large and diverse gene superfamily. To address how environmental stress drives the fixation and diversification of gene duplications, we have analyzed Cyp12d1 and Cyp12d3, a pair of duplicated genes found in the sequenced Drosophila genomes of the melanogaster group. The paralog Cyp12d3, which is not found in Drosophila melanogaster, is basal to the melanogaster group, after it split from the obscura group (ca. 50 mya), and has a significant signature of positive selection in two species (D. sechellia and D. ananassae). Examination of the Cyp12d1 region in D. melanogaster wildtype and isoline populations revealed variation both in copy number and sequence, including splice-site variations, which certainly alter gene function. Further investigations of several strains have identified three cases in which differences in the Cyp12d1 gene region are associated with the differences in transcript abundance and transcriptional responses to the environmental stresses that have not been seen for other detoxificative loci. Together, these data highlight the value of using both macro- and microevolutionary approaches in studying the duplication and divergence events associated with detoxification genes and lay important groundwork for future studies in the field of evolutionary toxicogenomics, which uses the principles of phylogenetic analysis to predict possible enzymatic functions.

Geographic distribution of phylogenetically-distinct legume pod borer, Maruca vitrata (Lepidoptera: Pyraloidea: Crambidae).

Maruca vitrata Fabricius is a pantropical lepidopteran pest of legumes. Phylogenetic analysis of a mitochondrial cytochrome c oxidase-I gene (cox1) fragment indicates that three Maruca sp. mitochondrial lineages have unique geographic distributions [lineages 1 and 2: Australia, Taiwan, and West Africa (Niger, Nigeria, and Burkina Faso), and lineage 3: Puerto Rico]. The haplotype (T30, T114) is specific to lineages 1&2 and was assayed by NsiI and SacI polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) within population samples; it was not observed in the Puerto Rican samples, but was nearly fixed among samples from West Africa, Australia and Taiwan (85.5-100%). Re-sequencing and phylogenetic analyses of PCR-RFLP defined cox1 haplotypes indicate that nucleotide diversity is highest among samples from West Africa. Phylogenetic reconstruction based upon ribosomal DNA (rDNA) internal transcribed spacer-2 (ITS-2) sequences provided additional evidence for three Maruca sp. clades. These data suggest that multiple unique Maruca species or subspecies are present worldwide, which has implications for the management of this pest species-complex.