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.

Physiological and molecular correlates of the screwworm fly attraction to wound and animal odors.

The screwworm fly, Cochliomyia hominivorax (Coquerel), was successfully eradicated from the United States by the sterile insect technique (SIT). However, recent detection of these flies in the Florida Keys, and increased risk of introductions to the other areas warrant novel tools for management of the flies. Surveillance, a key component of screwworm control programs, utilizes traps baited with rotting liver or a blend of synthetic chemicals such as swormlure-4. In this work, we evaluated the olfactory physiology of the screwworm fly and compared it with the non-obligate ectoparasitic secondary screwworm flies, C. macellaria, that invade necrotic wound and feed on dead tissue. These two species occur in geographically overlapping regions. C. macellaria, along with other blowflies such as the exotic C. megacephala, greatly outnumber C. hominivorax in the existing monitoring traps. Olfactory responses to swormlure-4 constituents between sex and mating status (mated vs unmated) in both species were recorded and compared. Overall, responses measured by the antennograms offered insights into the comparative olfactory physiology of the two fly species. We also present detailed analyses of the antennal transcriptome by RNA-Sequencing that reveal significant differences between male and female screwworm flies. The differential expression patterns were confirmed by quantitative PCR. Taken together, this integrated study provides insights into the physiological and molecular correlates of the screwworm's attraction to wounds, and identifies molecular targets that will aid in the development of odorant-based fly management strategies.

Dietary antioxidants impact DDT resistance in Drosophila melanogaster.

Insects experience a diversity of subtoxic and/or toxic xenobiotics through exposure to pesticides and, in the case of herbivorous insects, through plant defensive compounds in their diets. Many insects are also concurrently exposed to antioxidants in their diets. The impact of dietary antioxidants on the toxicity of xenobiotics in insects is not well understood, in part due to the challenge of developing appropriate systems in which doses and exposure times (of both the antioxidants and the xenobiotics) can be controlled and outcomes can be easily measured. However, in Drosophila melanogaster, a well-established insect model system, both dietary factors and pesticide exposure can be easily controlled. Additionally, the mode of action and xenobiotic metabolism of dichlorodiphenyltrichloroethane (DDT), a highly persistent neurotoxic organochlorine insecticide that is detected widely in the environment, have been well studied in DDT-susceptible and -resistant strains. Using a glass-vial bioassay system with blue diet as the food source, seven compounds with known antioxidant effects (ascorbic acid, ß-carotene, glutathione, α-lipoic acid, melatonin, minocycline, and serotonin) were orally tested for their impact on DDT toxicity across three strains of D. melanogaster: one highly susceptible to DDT (Canton-S), one mildly susceptible (91-C), and one highly resistant (91-R). Three of the antioxidants (serotonin, ascorbic acid, and ß-carotene) significantly impacted the toxicity of DDT in one or more strains. Fly strain and gender, antioxidant type, and antioxidant dose all affected the relative toxicity of DDT. Our work demonstrates that dietary antioxidants can potentially alter the toxicity of a xenobiotic in an insect population.

Dietary antioxidant vitamin C influences the evolutionary path of insecticide resistance in Drosophila melanogaster.

Herbivorous insects encounter a variety of toxic environmental substances ranging from ingested plant defensive compounds to human-introduced insecticidal agents. Dietary antioxidants are known to reduce the negative physiological impacts of toxins in mammalian systems through amelioration of reactive oxygen-related cellular damage. The analogous impacts to insects caused by multigenerational exposure to pesticides and the effects on adaptive responses within insect populations, however, are currently unknown. To address these research gaps, we used Drosophila as a model system to explore adaptive phenotypic responses to acute dichlorodiphenyltrichloroethane (DDT) exposure in the presence of the dietary antioxidant vitamin C and to examine the structural genomic consequences of this exposure. DDT resistance increased significantly among four replicates exposed to a low concentration of DDT for 10 generations. In contrast, dietary intake of vitamin C significantly reduced DDT resistance after mutigenerational exposure to the same concentration of DDT. As to the genomic consequences, no significant differences were predicted in overall nucleotide substitution rates across the genome between any of the treatments. Despite this, replicates exposed to a low concentration of DDT without vitamin C showed the highest number of synonymous and non-synonymous variants (3196 in total), followed by the DDT plus vitamin C (1174 in total), and vitamin C alone (728 in total) treatments. This study demonstrates the potential role of diet (specifically, antioxidant intake) on adaptive genome responses, and thus on the evolution of pesticide resistance within insect populations.

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.

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.

The complete mitogenome of the Emerald Ash Borer (EAB), Agrilus planipennis (Insecta: Coleoptera: Buprestidae).

The complete mitogenome of the Emerald Ash Borer (EAB, Agrilus planipennis) was obtained by gleaning mitochondrial sequences from whole-genome Illumina sequencing data. The circular genome has 15,942 base pairs and contains 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and an A-T-rich region. All PCGs begin with ATN codons. The nucleotide composition is highly asymmetric (31.65% A, 40.25% T, 17.39% G, 10.71% C), with an overall A-T content of 71.9%. Phylogenetic analysis based on insect mitogenomes indicated that EAB is closely related to other Buprestoidea species, clustering most closely with Chrysochroa fulgidissima.

Transcriptome Analysis of the Emerald Ash Borer (EAB), Agrilus planipennis: De Novo Assembly, Functional Annotation and Comparative Analysis.

BACKGROUND: The Emerald ash borer (EAB), Agrilus planipennis, is an invasive phloem-feeding insect pest of ash trees. Since its initial discovery near the Detroit, US- Windsor, Canada area in 2002, the spread of EAB has had strong negative economic, social and environmental impacts in both countries. Several transcriptomes from specific tissues including midgut, fat body and antenna have recently been generated. However, the relatively low sequence depth, gene coverage and completeness limited the usefulness of these EAB databases. METHODOLOGY AND PRINCIPAL FINDINGS: High-throughput deep RNA-Sequencing (RNA-Seq) was used to obtain 473.9 million pairs of 100 bp length paired-end reads from various life stages and tissues. These reads were assembled into 88,907 contigs using the Trinity strategy and integrated into 38,160 unigenes after redundant sequences were removed. We annotated 11,229 unigenes by searching against the public nr, Swiss-Prot and COG. The EAB transcriptome assembly was compared with 13 other sequenced insect species, resulting in the prediction of 536 unigenes that are Coleoptera-specific. Differential gene expression revealed that 290 unigenes are expressed during larval molting and 3,911 unigenes during metamorphosis from larvae to pupae, respectively (FDR< 0.01 and log2 FC>2). In addition, 1,167 differentially expressed unigenes were identified from larval and adult midguts, 435 unigenes were up-regulated in larval midgut and 732 unigenes were up-regulated in adult midgut. Most of the genes involved in RNA interference (RNAi) pathways were identified, which implies the existence of a system RNAi in EAB. CONCLUSIONS AND SIGNIFICANCE: This study provides one of the most fundamental and comprehensive transcriptome resources available for EAB to date. Identification of the tissue- stage- or species- specific unigenes will benefit the further study of gene functions during growth and metamorphosis processes in EAB and other pest insects.

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis).

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a ß-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis.

Evidence for trade-offs in detoxification and chemosensation gene signatures in Plutella xylostella.

BACKGROUND: Detoxification genes have been associated with insecticide adaptation in the diamondback moth, Plutella xylostella. The link between chemosensation genes and adaptation, however, remains unexplored. To gain a better understanding of the involvement of these genes in insecticide adaptation, the authors exposed lines of P. xylostella to either high uniform (HU) or low heterogeneous (LH) concentrations of permethrin, expecting primarily physiological or behavioral selection respectively. Initially, 454 pyrosequencing was applied, followed by an examination of expression profiles of candidate genes that responded to selection [cytochrome P450 (CYP), glutathione S-transferase (GST), carboxylesterase (CarE), chemosensory protein (CSP) and odorant-binding protein (OBP)] by quantitative PCR in the larvae. Toxicity and behavioral assays were also conducted to document the effects of the two forms of exposure. RESULTS: Pyrosequencing of the P. xylostella transcriptome from adult heads and third instars produced 198,753 reads with 52,752,486 bases. Quantitative PCR revealed overexpression of CYP4M14, CYP305B1 and CSP8 in HU larvae. OBP13, however, was highest in LH. Larvae from LH and HU lines had up to five- and 752-fold resistance levels respectively, which could be due to overexpression of P450s. However, the behavioral responses of all lines to a series of permethrin concentrations did not vary significantly in any of the generations examined, in spite of the observed upregulation of CSP8 and OBP13. CONCLUSION: Expression patterns from the target genes provide insights into behavioral and physiological responses to permethrin and suggest a new avenue of research on the role of chemosensation genes in insect adaptation to toxins.

RNA-Seq reveals a xenobiotic stress response in the soybean aphid, Aphis glycines, when fed aphid-resistant soybean.

BACKGROUND: While much recent research has expanded our understanding of the molecular interactions between aphids and their host plants, it is lacking for the soybean aphid, Aphis glycines. Since its North American invasion, A. glycines has become one of the most damaging insect pests on this important crop. Five soybean genes for host plant resistance to A. glycines have been identified, but populations of A. glycines have already adapted to overcome these resistance genes. Understanding the molecular interactions between resistant soybean and A. glycines can provide clues to its adaptation mechanisms. Here, we used RNA-Sequencing to compare and contrast A. glycines gene expression when fed resistant (Rag1) and susceptible soybean. RESULTS: Combining results from a previous A. glycines transcriptome, we generated 64,860 high quality transcripts, totaling 41,151,086 bases. Statistical analysis revealed 914 genes with significant differential expression. Most genes with higher expression in A. glycines on resistant plants (N = 352) were related to stress and detoxification such as cytochrome P450s, glutathione-S-transferases, carboxyesterases, and ABC transporters. A total of 562 genes showed lower transcript abundance in A. glycines on resistant plants. From our extensive transcriptome data, we also identified genes encoding for putative salivary effector proteins (N = 73). Among these, 6 effector genes have lower transcript abundance in A. glycines feeding on resistant soybean. CONCLUSIONS: Overall, A. glycines exhibited a pattern typical of xenobiotic challenge, thereby validating antibiosis in Rag1, presumably mediated through toxic secondary metabolites. Additionally, this study identified many A. glycines genes and gene families at the forefront of its molecular interaction with soybean. Further investigation of these genes in other biotypes may reveal adaptation mechanisms to resistant plants.

Molecular characterization and expression analysis of soluble trehalase gene in Aphis glycines, a migratory pest of soybean.

In insects, the enzyme trehalase plays a crucial role in energy metabolism, chitin synthesis and possibly during plant-insect interactions. We have characterized a soluble trehalase gene (Tre-1) from cDNA of Aphis glycines, a serious migratory pest of soybean. The full-length cDNA of Tre-1 in A. glycines (AyTre-1) was 2550 bp long with an open reading frame of 1770 bp that encoded for a 589 amino acid residues protein. Sequence assessment and phylogenetic analysis of the putative protein suggested that the selected cDNA belongs to soluble trehalase group. Quantitative PCR (qPCR) analysis in different tissues and developmental stages revealed peak mRNA levels of AyTre-1 in the gut (compared with other tissues assayed) and highest expression in the second instar compared with the other developmental stages assayed. Interestingly, a significantly increased expression of AyTre-1 (1.9-fold, P < 0.05) was observed in the alate morphs compared with that in apterate morphs. However, there was no significant difference in AyTre-1 expression in A. glycines-nymphs fed with resistant and susceptible plants. Expression patterns identified in this study provide a platform to investigate the role of AyTre-1 in physiological activities such as flight and feeding in A. glycines. The characterization of soluble trehalase gene may help to develop novel strategies to manage A. glycines using trehalase inhibitors and using RNA interference for knock-down of AyTre-1 expression.

Glutathione-S-transferase profiles in the emerald ash borer, Agrilus planipennis.

The emerald ash borer, Agrilus planipennis Fairmaire is a recently discovered invasive insect pest of ash, Fraxinus spp. in North America. Glutathione-S-transferases (GST) are a multifunctional superfamily of enzymes which function in conjugating toxic compounds to less toxic and excretable forms. In this study, we report the molecular characterization and expression patterns of different classes of GST genes in different tissues and developmental stages plus their specific activity. Multiple sequence alignment of all six A. planipennis GSTs (ApGST-E1, ApGST-E2, ApGST-E3, ApGST-O1, ApGST-S1 and ApGST-µ1) revealed conserved features of insect GSTs and a phylogenetic analysis grouped the GSTs within the epsilon, sigma, omega and microsomal classes of GSTs. Real time quantitative PCR was used to study field collected samples. In larval tissues high mRNA levels for ApGST-E1, ApGST-E3 and ApGST-O1 were obtained in the midgut and Malpighian tubules. On the other hand, ApGST-E2 and ApGST-S1 showed high mRNA levels in fat body and ApGST-µ1 showed constitutive levels in all the tissues assayed. During development, mRNA levels for ApGST-E2 were observed to be the highest in feeding instars, ApGST-S1 in prepupal instars; while the others showed constitutive patterns in all the developmental stages examined. At the enzyme level, total GST activity was similar in all the tissues and developmental stages assayed. Results obtained suggest that A. planipennis is potentially primed with GST-driven detoxification to metabolize ash allelochemicals. To our knowledge this study represents the first report of GSTs in A. planipennis and also in the family of wood boring beetles.

Identification of odor-processing genes in the emerald ash borer, Agrilus planipennis.

BACKGROUND: Insects rely on olfaction to locate food, mates, and suitable oviposition sites for successful completion of their life cycle. Agrilus planipennis Fairmaire (emerald ash borer) is a serious invasive insect pest that has killed tens of millions of North American ash (Fraxinus spp) trees and threatens the very existence of the genus Fraxinus. Adult A. planipennis are attracted to host volatiles and conspecifics; however, to date no molecular knowledge exists on olfaction in A. planipennis. Hence, we undertook an antennae-specific transcriptomic study to identify the repertoire of odor processing genes involved in A. planipennis olfaction. METHODOLOGY AND PRINCIPAL FINDINGS: We acquired 139,085 Roche/454 GS FLX transcriptomic reads that were assembled into 30,615 high quality expressed sequence tags (ESTs), including 3,249 isotigs and 27,366 non-isotigs (contigs and singletons). Intriguingly, the majority of the A. planipennis antennal transcripts (59.72%) did not show similarity with sequences deposited in the non-redundant database of GenBank, potentially representing novel genes. Functional annotation and KEGG analysis revealed pathways associated with signaling and detoxification. Several odor processing genes (9 odorant binding proteins, 2 odorant receptors, 1 sensory neuron membrane protein and 134 odorant/xenobiotic degradation enzymes, including cytochrome P450s, glutathione-S-transferases; esterases, etc.) putatively involved in olfaction processes were identified. Quantitative PCR of candidate genes in male and female A. planipennis in different developmental stages revealed developmental- and sex-biased expression patterns. CONCLUSIONS AND SIGNIFICANCE: The antennal ESTs derived from A. planipennis constitute a rich molecular resource for the identification of genes potentially involved in the olfaction process of A. planipennis. These findings should help in understanding the processing of antennally-active compounds (e.g. 7-epi-sesquithujene) previously identified in this serious invasive pest.

Molecular characterization of genes encoding inward rectifier potassium (Kir) channels in the bed bug (Cimex lectularius).

The molecular genetics of inward-rectifier potassium (Kir) channels in insects is poorly understood. To date, Kir channel genes have been characterized only from a few representative dipterans (i.e., fruit flies and mosquitoes). The goal of the present study was to characterize Kir channel cDNAs in a hemipteran, the bed bug (Cimex lectularius). Using our previously reported bed bug transcriptome (RNA-seq), we identified two cDNAs that encode putative Kir channels. One was a full-length cDNA that encodes a protein belonging to the insect 'Kir3' clade, which we designate as 'ClKir3'. The other was a partial cDNA that encodes a protein with similarity to both the insect 'Kir1' and 'Kir2' clades, which we designate as 'ClKir1/2'. Quantitative real-time PCR analysis revealed that ClKir1/2 and ClKir3 exhibited peak expression levels in late-instar nymphs and early-instar nymphs, respectively. Furthermore, ClKir3, but not ClKir1/2, showed tissue-specific expression in Malpighian tubules of adult bed bugs. Lastly, using an improved procedure for delivering double-stranded RNA (dsRNA) to male and female bed bugs (via the cervical membrane) we demonstrate rapid and systemic knockdown of ClKir3 transcripts. In conclusion, we demonstrate that the bed bug possesses at least two genes encoding Kir channels, and that RNAi is possible for at least Kir3, thereby offering a potential approach for elucidating the roles of Kir channel genes in bed bug physiology.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2012 - 30 September 2012.

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

Characterization of a chitin synthase encoding gene and effect of diflubenzuron in soybean aphid, Aphis glycines.

Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS) in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS) was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The quantitative real-time PCR (qPCR) analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument); however, it had the highest expression in integument (~3.5 fold compared to gut). Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ®), a chitin-synthesis inhibitor, on A. glycines' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold) of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression.

Transcriptome analysis of the salivary glands of potato leafhopper, Empoasca fabae.

The potato leafhopper, Empoasca fabae, is a pest of economic crops in the United States and Canada, where it causes damage known as hopperburn. Saliva, along with mechanical injury, leads to decreases in gas exchange rates, stunting and chlorosis. Although E. fabae saliva is known to induce plant responses, little knowledge exists of saliva composition at the molecular level. We subjected the salivary glands of E. fabae to Roche 454-pyrosequencing which resulted significant number (30,893) of expressed sequence tags including 2805 contigs and 28,088 singletons. A high number of sequences (78%) showed similarity to other insect species in GenBank, including Triboliumcastaneum, Drosophilamelanogaster and Acrythosiphonpisum. KEGG analysis predicted the presence of pathways for purine and thiamine metabolic, biosynthesis of secondary metabolites, drug metabolism, and lysine degradation. Pfam analysis showed a high number of cellulase and carboxylesterase protein domains. Expression analysis of candidate genes (alpha amylase, lipase, pectin lyase, etc.) among different tissues revealed tissue-specific expression of digestive enzymes in E. fabae. This is the first study to characterize the sialotranscriptome of E. fabae and the first for any species in the family of Cicadellidae. Due to the status of these insects as economic pests, knowledge of which genes are active in the salivary glands is important for understanding their impact on host plants.

Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae).

Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs. We evaluated seven potential HKGs (SDFS, succinate dehydrogenase flavoprotein subunit; EF1a, elongation factor-la; HEL, helicase; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; RPS9, ribosomal protein S9; TBP, TATA-box binding protein; and UBQ, ubiquitin-conjugating protein) to determine the most efficient HKGs that have stable expression among tissues, developmental stages, and aphids fed on susceptible and host plant-resistant soybean. HKG stability was determined using GeNorm and NormFinder. Results from three different experimental conditions revealed high stability of TBP compared with the other HKGs profiled across the samples assayed. RPS9 showed stable expression among aphids on susceptible and resistant plants, whereas EF1a showed stable expression in tissues and developmental stages. Therefore, we recommend the TBP as a suitable HKG for efficient normalization among treatments, tissues, and developmental stages of A. glycines. In addition, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate and reliable normalization of qRT-PCR data in A. glycines.

RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug.

BACKGROUND: Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. RESULTS: We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. CONCLUSIONS: We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.