Search

The emerald ash borer ( Agrilus planipennis) is an important invasive insect pest of Fraxinus spp. that feeds on host tissues containing high levels of sucrose. However, little is known about how it digests sucrose. Here, using larval midgut transcriptome data and preliminary genome sequence efforts, two β-fructofuranosidase-encoding ScrB genes, Apla Scr B-1 and Apla Scr B-2, were identified, and proved to reside within the A. planipennis genome. Homology and phylogenetic analysis revealed that they were acquired by A. planipennis via horizontal gene transfer ( HGT) from bacteria, possibly an event independent from that reported in bark beetles (eg ScrB genes). Microsynteny between A. planipennis DNA scaffold #2042940, which hosts Apla Scr B-1, and a region in the Tribolium castaneum chromosome LG4 suggested that A. planipennis gained this gene after the separation of Buprestidae and Tenebrionidae. Although both of the putative Apla Scr B proteins have conserved β-fructofuranosidase motifs, only Apla Scr B-2 was predicted to be a secretory protein. Expression of Apla Scr B-1 seemed constitutive during development and in all tissues examined, whereas Apla Scr B-2 showed a peak expression in adults and in the midgut. We propose that acquisition of these genes by A. planipennis from bacteria is adaptive, and specifically Apla Scr B-2 is involved in breaking down dietary sucrose to obtain energy for development. [ABSTRACT FROM AUTHOR]

The soybean aphid ( Aphis glycines Matsumura) is one of the most important insect pests of soybean [ Glycine max (L.) Merr.] in North America, and three biotypes of the aphid have been confirmed. Genetic studies of the soybean aphid are needed to determine genetic diversity, movement pattern, biotype distribution and mapping of virulence genes for efficient control of the pest. Simple sequence repeats (SSR) markers are useful for population and classical genetic studies, but few are currently available for the soybean aphid. In this study, we designed primers for 342 genic-SSR markers from a dataset of more than 102 024 transcript reads generated by 454 GS FLX sequencing of a cDNA library of the soybean aphid. Two hundred forty-six markers generated PCR products of expected size and 26 were polymorphic among four pooled aphid DNA samples. An additional five markers that were fixed for two alleles among the pooled samples were found to be polymorphic when tested on 96 individual aphids. Sequencing of the PCR products generated by two polymorphic SSR markers revealed that the polymorphisms were strictly because of variations in the SSR repeats among the aphids tested. The genetic diversity among 96 soybean aphids, 24 each from two field collections (South Dakota and Michigan, biotype unknown) and two laboratory colonies [biotype 1 (B1) and biotype 2 (B2)], was assessed with 29 polymorphic SSR markers. These markers discriminated laboratory colonies from field collections and field collections from different states. The genic-SSR markers developed and validated in this study will be a significant addition to the limited number of SSR markers, mostly genomic-SSR, currently available for the soybean aphid. These markers will be useful for genetic studies, including population genetics, genetic and QTL mapping, migration and biotype differentiation of the soybean aphid. [ABSTRACT FROM AUTHOR]

Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is currently the most important insect pest of soybean (Glycine max (L.) Merr.) in the United States and causes significant economic damage worldwide, but little is known about the aphid at the molecular level. Mariner-like transposable elements (MLEs) are ubiquitous within the genomes of arthropods and various other invertebrates. In this study, we report the cloning of MLEs from the soybean aphid genome using degenerate PCR primers designed to amplify conserved regions of mariner transposases. Two of the ten sequenced clones (designated as Agmar1 and Agmar2) contained partial but continuous open reading frames, which shared high levels of homology at the protein level with other mariner transposases from insects and other taxa. Phylogenetic analysis revealed Agmar1 to group within the irritans subfamily of MLEs and Agmar2 within the mellifera subfamily. Southern blot analysis and quantitative PCR analysis indicated a low copy number for Agmar1-like elements within the soybean aphid genome. These results suggest the presence of at least two different putative mariner-like transposases encoded by the soybean aphid genome. Both Agmar1 and Agmar2 could play influential roles in the architecture of the soybean aphid genome. Transposable elements are also thought to potentially mediate resistance in insects through changes in gene amplification and mutations in coding sequences. Finally, Agmar1 and Agmar2 may represent useful genetic tools and provide insights on A. glycines adaptation. [ABSTRACT FROM PUBLISHER]

Oligoarray analysis was used to determine the number and nature of genes expressed in third instar Drosophila melanogaster larval midguts. The majority of transcripts were associated with protein synthesis and metabolism. Serine proteases were the main proteolytic enzymes detected. Some 40% of the cytochrome P450 genes and 74% of the glutathione S transferases (GSTs) in the genome of D. melanogaster were observed to be expressed in the midgut by oligoarray analysis. We also identified potential transcription factor binding motifs (TFBMs) of P450s, GSTs and carboxylesterases. Many of the midgut-expressed GST genes contained candidate TFBMs homologous to TFBMs in mammals that have been associated with responses to oxidative stress. We also investigated the response of GSTs in the midgut to dietary H2O2, which showed a dosage-based differential response. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Entomologist is the property of Cambridge University Press and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Full-length cDNA and genomic sequences for two genes (designatedmdesprot-Iandmdesprot-II) encoding digestive serine proteases in Hessian fly,Mayetiola destructor, have been cloned and characterized. The deduced amino acid sequences revealed similarity with trypsin-like digestive serine proteases from other Dipterans. Bothmdesprot-Iandmdesprot-IIencoded proteins with secretion signal peptides at the N-terminals, indicating the proteins are secreted proteases that should function as midgut digestive proteases. A cytological analysis with fluorescentin situhybridization revealed the cytological localization ofmdesprot-Iandmdesprot-IIon the long arm of Autosome 2. Results are discussed in the context of the efficacy of potential protease inhibitors to develop Hessian fly resistant wheat through genetic engineering approaches. [ABSTRACT FROM AUTHOR]

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 3β (GSK3β-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3β-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., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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, β-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 β-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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 ) 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 ., Competing Interests: The authors report no conflicts of interest. The authors are responsible for the content and writing of the paper. This work was funded by a Genomics Research and Development Initiative (GRDI) grant from NRCan to DD, GQ and MC., (© 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.

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.

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., (© 2014 Society of Chemical Industry.)

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 β-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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (© 2012 Blackwell Publishing Ltd.)

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.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

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.

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.

Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

Phytophagous insects frequently encounter reactive oxygen species (ROS) from exogenous and endogenous sources. To overcome the effect of ROS, insects have evolved a suite of antioxidant defense genes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX). The emerald ash borer (Agrilus planipennis Fairmaire), an exotic invasive insect pest from Asia has killed millions of ash trees and continues to invade North America at a rapid pace. From an on-going expressed sequence tag (EST) project of A. planipennis larval tissues, we identified ESTs coding for a Cu-Zn SOD (ApSOD1), a CAT (ApCAT1) and a GPX (ApGPX1). A multiple sequence alignment of the derived A. planipennis sequences revealed high homology with other insect sequences at the amino acid level. Phylogenetic analysis of ApSOD1 grouped it with Cu-Zn SODs of other insect taxa. Quantitative real time PCR (qRT-PCR) analysis in different larval tissues (midgut, fat body, Malpighian tubule and cuticle) revealed high mRNA levels of ApCAT1 in the midgut. Interestingly, high mRNA levels for both ApSOD1 and ApGPX1 were observed in the Malpighian tubules. Assay of mRNA levels in developmental stages (larva, prepupa and adults) by qRT-PCR indicated high transcript levels of ApCAT1 and ApGPX1 in larval and prepupal stages with a decline in adults. On the other hand, the transcript levels of ApSOD1 were observed to be constitutive in all the developmental stages assayed. Results obtained reflect a plausible role of these A. planipennis antioxidant genes in quenching ROS from both diet (ash allelochemicals) as well as endogenous sources. These studies further help in understanding the adaptation/invasiveness of A. planipennis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Blood-feeding insects have evolved resistance to various insecticides (organochlorines, pyrethroids, carbamates, etc.) through gene mutations and increased metabolism. Bed bugs (Cimex lectularius) are hematophagous ectoparasites that are poised to become one of the major pests in households throughout the United States. Currently, C. lectularius has attained a high global impact status due to its sudden and rampant resurgence. Resistance to pesticides is one factor implicated in this phenomenon. Although much emphasis has been placed on target sensitivity, little to no knowledge is available on the role of key metabolic players (e.g., cytochrome P450s and glutathione S-transferases) towards pesticide resistance in C. lectularius. In this review, we discuss different modes of resistance (target sensitivity, penetration resistance, behavioral resistance, and metabolic resistance) with more emphasis on metabolic resistance.

Background: Ash (Fraxinus spp.) is a dominant tree species throughout urban and forested landscapes of North America (NA). The rapid invasion of NA by emerald ash borer (Agrilus planipennis), a wood-boring beetle endemic to Eastern Asia, has resulted in the death of millions of ash trees and threatens billions more. Larvae feed primarily on phloem tissue, which girdles and kills the tree. While NA ash species including black (F. nigra), green (F. pennsylvannica) and white (F. americana) are highly susceptible, the Asian species Manchurian ash (F. mandshurica) is resistant to A. planipennis perhaps due to their co-evolutionary history. Little is known about the molecular genetics of ash. Hence, we undertook a functional genomics approach to identify the repertoire of genes expressed in ash phloem., Methodology and Principal Findings: Using 454 pyrosequencing we obtained 58,673 high quality ash sequences from pooled phloem samples of green, white, black, blue and Manchurian ash. Intriguingly, 45% of the deduced proteins were not significantly similar to any sequences in the GenBank non-redundant database. KEGG analysis of the ash sequences revealed a high occurrence of defense related genes. Expression analysis of early regulators potentially involved in plant defense (i.e. transcription factors, calcium dependent protein kinases and a lipoxygenase 3) revealed higher mRNA levels in resistant ash compared to susceptible ash species. Lastly, we predicted a total of 1,272 single nucleotide polymorphisms and 980 microsatellite loci, among which seven microsatellite loci showed polymorphism between different ash species., Conclusions and Significance: The current transcriptomic data provide an invaluable resource for understanding the genetic make-up of ash phloem, the target tissue of A. planipennis. These data along with future functional studies could lead to the identification/characterization of defense genes involved in resistance of ash to A. planipennis, and in future ash breeding programs for marker development.

Background: Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance., Methodology and Principal Findings: Using 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database., Conclusions: To our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies.

Background: The insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level., Methodology and Principal Findings: Newer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level., Conclusions and Significance: To our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis.

The Hessian fly, Mayetiola destructor, is a serious pest of wheat and an experimental organism for the study of gall midge-plant interactions. In addition to food digestion and detoxification, the gut of Hessian fly larvae is also an important interface for insect-host interactions. Analysis of the genes expressed in the Hessian fly larval gut will enhance our understanding of the overall gut physiology and may also lead to the identification of critical molecules for Hessian fly-host plant interactions. Over 10,000 Expressed Sequence Tags (ESTs) were generated and assembled into 2007 clusters. The most striking feature of the Hessian fly larval transcriptome is the existence of a large number of transcripts coding for so-called small secretory proteins (SSP) with amino acids less than 250. Eleven of the 30 largest clusters were SSP transcripts with the largest cluster containing 11.3% of total ESTs. Transcripts coding for diverse digestive enzymes and detoxification proteins were also identified. Putative digestive enzymes included trypsins, chymotrypsins, cysteine proteases, aspartic protease, endo-oligopeptidase, aminopeptidases, carboxypeptidases, and alpha-amylases. Putative detoxification proteins included cytochrome P450s, glutathione S-transferases, peroxidases, ferritins, a catalase, peroxiredoxins, and others. This study represents the first global analysis of gut transcripts from a gall midge. The identification of a large number of transcripts coding for SSPs, digestive enzymes, detoxification proteins in the Hessian fly larval gut provides a foundation for future studies on the functions of these genes.

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

Background: Aphids are one of the most important insect taxa in terms of ecology, evolutionary biology, genetics and genomics, and interactions with endosymbionts. Additionally, many aphids are serious pest species of agricultural and horticultural plants. Recent genetic and genomic research has expanded molecular resources for many aphid species, including the whole genome sequencing of the pea aphid, Acrythosiphon pisum. However, the invasive soybean aphid, Aphis glycines, lacks in any significant molecular resources., Methodology/principal Findings: Two next-generation sequencing technologies (Roche-454 and Illumina GA-II) were used in a combined approach to develop both transcriptomic and genomic resources, including expressed genes and molecular markers. Over 278 million bp were sequenced among the two methods, resulting in 19,293 transcripts and 56,688 genomic sequences. From this data set, 635 SNPs and 1,382 microsatellite markers were identified. For each sequencing method, different soybean aphid biotypes were used which revealed potential biotype specific markers. In addition, we uncovered 39,822 bp of sequence that were related to the obligatory endosymbiont, Buchnera aphidicola, as well as sequences that suggest the presence of Hamiltonella defensa, a facultative endosymbiont., Conclusions and Significance: Molecular resources for an invasive, non-model aphid species were generated. Additionally, the power of next-generation sequencing to uncover endosymbionts was demonstrated. The resources presented here will complement ongoing molecular studies within the Aphididae, including the pea aphid whole genome, lead to better understanding of aphid adaptation and evolution, and help provide novel targets for soybean aphid control.

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1(st)-, 2(nd)-, 3(rd)-, 4(th)-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.