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.

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.

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.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle.

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.

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.

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.

Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.

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.

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.

Characterization and expression analysis of a gene encoding a secreted lipase-like protein expressed in the salivary glands of the larval Hessian fly, Mayetiola destructor (Say).

In a salivary gland transcriptomics study we identified a cDNA with a full-length open reading frame for a gene (MdesL1) encoding a lipase-like protein expressed in the salivary glands of the larval Hessian fly, Mayetiola destructor (Say). Fluorescent in situ hybridization on salivary polytenes positioned MdesL1 on the long arm of Autosome 1. BLASTp and conserved domain searches revealed the deduced amino acid sequence contained a lipase superfamily domain with similarity to lipases and phospholipases from other insects. A secretion signal peptide was identified at the amino terminus of the deduced amino acid sequence. Analysis of the transcript of MdesL1 in larval Hessian fly tissues by quantitative real-time PCR (qPCR) revealed the greatest abundance was in salivary glands. Analysis of transcript levels during development showed the greatest level was detected in feeding 1st-instar and early 2nd-instar larvae. Transcript levels increased dramatically over time in larvae feeding on susceptible wheat but were detected at low levels in larvae feeding on resistant wheat. These data suggest the protein encoded by MdesL1 is likely secreted into host-plant cells during larval feeding and could be involved in extra-oral digestion and changes in host-cell permeability or in generating a second messenger in a host-cell-signaling cascade.

Molecular characterization and responsive expression of a defender against apoptotic cell death homologue from the Hessian fly, Mayetiola destructor.

Apoptosis or programmed cell death is an active process occurring in multicellular organisms to maintain growth and development. The Hessian fly, Mayetiola destructor, is rapidly emerging as a model insect species to study insect-plant interactions and to decipher some exceptional physiological phenomena. In this study, we report the characterization and expression profiles of a putative Hessian fly defender against apoptotic cell death (DAD1) homologue designated MdesDAD1. The deduced amino acid sequence of MdesDAD1 revealed significant similarity (75% identity, 9e-42) to other insect and non-insect DAD1 sequences. Phylogenetic analysis grouped MdesDAD1 within a sub-clade consisting of other insect DAD1 homologues. Quantitative analysis indicated constitutive levels of MdesDAD1 mRNA in all the tissues examined but an altered expression pattern during development, wherein the highest mRNA levels observed were prior to pupation. Most interestingly, MdesDAD1 transcript was found to be up-regulated during incompatible (larvae reared on resistant wheat) Hessian fly/wheat interactions compared to compatible (larvae reared on susceptible wheat) interactions. These results suggest MdesDAD1 to have a putative role in the inhibition of unwanted apoptosis triggered during development and in incompatible Hessian fly/wheat interactions. The results obtained provide clues to plausible insect and host-plant factors that could be responsible for the induction of MdesDAD1.

Tissue and life stage specificity of glutathione S-transferase expression in the Hessian fly, Mayetiola destructor: implications for resistance to host allelochemicals.

Two new Delta and Sigma glutathione S-transferases (GSTs) in the Hessian fly, Mayetiola destructor (Diptera: Cecidomyiidae), were characterized and transcription profiles described. The deduced amino acid sequences for the two M. destructor Delta GSTs (MdesGST-1 and MdesGST-3) showed high similarity with other insect Delta GSTs including the conserved catalytic serine residue. The deduced amino acid sequence for the M. destructor Sigma GST (MdesGST-2) showed high similarity with other insect Sigma GSTs including the conserved glutathione and substrate binding sites. Quantitative tissue expression analysis showed that mRNA levels for MdesGST-1 were predominant in fat body, whereas for MdesGST-2 and MdesGST-3 expression was predominant in the midgut. Temporal expression during development showed peak mRNA levels for MdesGST-1 during larval development, but in the pupal stage for MdesGST-2. MdesGST-3 showed a constitutive expression pattern throughout development. M. destructor feeds on wheat, and expression analysis after feeding indicated that mRNA levels for MdesGST-1 were significantly higher in incompatible interactions in which larvae fed on resistant wheat, while MdesGST-3 was significantly higher in compatible interactions when larvae fed on susceptible wheat. MdesGST-2 showed an equivalent expression pattern during both interactions. These results suggest that the M. destructor Delta GSTs are important in detoxifying wheat allelochemicals during feeding, while Sigma GST participates in metabolism of endogenous substrates.

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.

Gene-for-gene defense of wheat against the Hessian fly lacks a classical oxidative burst.

Genetic similarities between plant interactions with microbial pathogens and wheat interactions with Hessian fly larvae prompted us to investigate defense and counterdefense mechanisms. Plant oxidative burst, a rapid increase in the levels of active oxygen species (AOS) within the initial 24 h of an interaction with pathogens, commonly is associated with defenses that are triggered by gene-for-gene recognition events similar to those involving wheat and Hessian fly larvae. RNAs encoded by Hessian fly superoxide dismutase (SOD) and catalase (CAT) genes, involved in detoxification of AOS, increased in first-instar larvae during both compatible and incompatible interactions. However, mRNA levels of a wheat NADPH oxidase (NOX) gene that generates superoxide (O2-) did not increase. In addition, inhibiting wheat NOX enzyme with diphenyleneiodonium did not result in increased survival of avirulent larvae. However, nitro blue tetrazolium staining indicated that basal levels of O2- are present in both uninfested and infested wheat tissue. mRNA encoded by wheat genes involved in detoxification of the cellular environment, SOD, CAT, and glutathione-S-transferase did not increase in abundance. Histochemical staining with 3,3-diaminobenzidine revealed no increases in wheat hydrogen peroxide (H2O2) during infestation that were correlated with the changes in larval SOD and CAT mRNA. However, treatment with 2',7'-dichlorofluorescin demonstrated the presence of basal levels of H2O2 in the elongation zone of both infested and uninfested plants. The accumulation of a wheat flavanone 3-hydroxylase mRNA did show some parallels with larval gene mRNA profiles. These results suggested that larvae encounter stresses imposed by mechanisms other than an oxidative burst in wheat seedlings.

Characterization of a serine carboxypeptidase in the salivary glands and fat body of the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae).

A full-length cDNA encoding a serine carboxypeptidase (designated SmSCP-1) was recovered from an ongoing salivary gland EST project of the wheat midge. The deduced 461-amino acid sequence had a putative signal sequence at the amino terminus, indicating it was a secreted protein. The protein shared homology with serine carboxypeptidases from other insects, mammals, plants, and yeasts. SmSCP-1 mRNA was expressed in all stages of development and detected in salivary gland and fat body tissues but not in midgut tissue. Expression analysis and quantitative real-time PCR assays in male and female wheat midges and the fat body tissue of adult midges revealed that SmSCP-1 was up-regulated nearly four-fold in the female midges compared to males and nearly two-fold in female fat body compared to male fat body. The wheat midge serine carboxypeptidase (SmSCP-1) most likely has a dual function. As a secreted digestive enzyme, it could play a role in mobilizing host-plant seed reserves for feeding larvae and as expressed in fat body could function as an exopeptidase in degradation of vitellogenin and/or in post-translational processing of other enzymes.

Expression patterns of antibacterial genes in the Hessian fly.

We report on the transcriptional patterns of three antibacterial genes, a defensin (MdesDEF-1), a diptericin (MdesDIP-1) and a lysozyme (MdesLYS-1), during development in Hessian fly, Mayetiola destructor. Quantitative analysis by real-time PCR of mRNA levels in different tissues revealed a predominance of the transcripts for all three genes in the midgut, while analysis during development revealed greatest abundance in mRNA during the 3rd-instar. An evaluation of the midgut lumen revealed the presence of a diverse bacterial flora in larvae maintained on susceptible wheat. Further, the titer of bacteria in the midgut increased approximately 250-fold from the 1st-instar through the 2nd-instar. However, no detectable titer of bacteria was observed from the midgut lumen of larvae maintained on resistant plants. PCR amplicons produced using primers designed to conserved regions of the Pseudomonas 16S rRNA gene supported taxonomic identification for some of the bacteria comprising the midgut flora as belonging to the genus Pseudomonas. Analysis of mRNA for the Hessian fly antibacterial genes in larvae feeding on susceptible and resistant plants revealed an increase in the transcript level for MdesDEF-1 in 1st-instar larvae on susceptible plants, while the transcript levels for MdesDIP-1 and MdesLYS-1 were constant. Results suggest the transcriptional patterns of the Hessian fly antibacterial genes observed could be associated with the developing midgut bacterial flora present in larvae feeding on susceptible wheat as well as microbial challenge encountered at other stages in development.

Differential expression of two cytochrome P450 genes in compatible and incompatible Hessian fly/wheat interactions.

We have recovered two Hessian fly cytochrome P450 cDNAs from an ongoing midgut EST project. CYP6AZ1 and CYP6BA1 represent two new subfamilies within the CYP6 family. The deduced amino acid sequences for CYP6AZ1 and CYP6BA1 show conserved structural and functional domains of insect P450s. Expression analysis with reverse transcription-polymerase chain reaction (RT-PCR) indicated that CYP6AZ1 is midgut specific and induced during active larval feeding, whereas CYP6BA1 was expressed in all tissues and developmental stages examined. Further expression analysis of CYP6AZ1 with RT-PCR in compatible and incompatible Hessian fly/wheat interactions suggested that CYP6AZ1 may be required for larval feeding in compatible interactions. These results should lead to a better understanding of the Hessian fly/wheat interaction with emphasis on the larval midgut as a critical interface with its host plant.