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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.

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.

Herbivorous insect species are constantly challenged with reactive oxygen species (ROS) generated from endogenous and exogenous sources. ROS produced within insects because of stress and prooxidant allelochemicals produced by host plants in response to herbivory require a complex mode of antioxidant defense during insect/plant interactions. Some insect herbivores have a midgut-based defense against the suite of ROS encountered. Because the Hessian fly (Mayetiola destructor) is the major insect pest of wheat worldwide, and an emerging model for all gall midges, we investigated its antioxidant responses during interaction with its host plant. Quantitative data for two phospholipid glutathione peroxidases (MdesPHGPX-1 and MdesPHGPX-2), two catalases (MdesCAT-1 and MdesCAT-2), and two superoxide dismutases (MdesSOD-1 and MdesSOD-2) revealed high levels of all of the mRNAs in the midgut of larvae on susceptible wheat (compatible interaction). During development of the Hessian fly on susceptible wheat, a differential expression pattern was observed for all six genes. Analysis of larvae on resistant wheat (incompatible interaction) compared with larvae on susceptible wheat showed increased levels of mRNAs in larvae on resistant wheat for all of the antioxidant genes except MdesSOD-1 and MdesSOD-2. We postulate that the increased mRNA levels of MdesPHGPX-1, MdesPHGPX-2, MdesCAT-1, and MdesCAT-2 reflect responses to ROS encountered by larvae while feeding on resistant wheat seedlings and/or ROS generated endogenously in larvae because of stress/starvation. These results provide an opportunity to understand the cooperative antioxidant defense responses in the Hessian fly/wheat interaction and may be applicable to other insect/plant interactions.

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.

One of the well-studied components of the insect gut is the peritrophic matrix (PM). This semipermeable structure primarily functions in digestion, and protection against invasive microorganisms and mechanical damage. We report the cDNA cloning and transcription profiles of a peritrophin-A like gene (designated MdesPERI-A1) in the Hessian fly Mayetiola destructor. The predicted amino acid sequence of MdesPERI-A1 revealed a putative secretion signal peptide at its amino terminus, similarity to peritrophins from other insects including dipterans, and the presence of two chitin binding domains each containing six cysteine residues. Quantitative expression analysis of MdesPERI-A1 mRNA in different larval tissues revealed the transcript to be predominantly present in the midgut (597.9-fold) compared to other tissues assayed including salivary glands and fat bodies. Spatial expression patterns during development showed a peak expression of MdesPERI-A1 in the feeding second-instars (146-fold) and a decline in expression in the pupal and adult stages. Transcription profiling of MdesPERI-A1 during compatible (larvae on susceptible plants) and incompatible (larvae on resistant plants) interactions with wheat revealed a greater level (1.7-fold) of MdesPERI-A1 transcript in larvae on resistant plants in the initial time point examined. However, MdesPERI-A1 expression declined in larvae on resistant plants at the later time points.

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.

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.

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.

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.