Transcriptome profiling reveals differential gene expression of detoxification enzymes in a hemimetabolous tobacco pest after feeding on jasmonate-silenced Nicotiana attenuata plants.

BACKGROUND: The evolutionary arms race between plants and insects has driven the co-evolution of sophisticated defense mechanisms used by plants to deter herbivores and equally sophisticated strategies that enable phytophagous insects to rapidly detoxify the plant's defense metabolites. In this study, we identify the genetic determinants that enable the mirid, Tupiocoris notatus, to feed on its well-defended host plant, Nicotiana attenuata, an outstanding model for plant-insect interaction studies. RESULTS: We used an RNAseq approach to evaluate the global gene expression of T. notatus after feeding on a transgenic N. attenuata line which does not accumulate jasmonic acid (JA) after herbivory, and consequently accumulates very low levels of defense metabolites. Using Illumina sequencing, we generated a de novo assembled transcriptome which resulted in 63,062 contigs (putative transcript isoforms) contained in 42,610 isotigs (putative identified genes). Differential expression analysis based on RSEM-estimated transcript abundances identified 82 differentially expressed (DE) transcripts between T. notatus fed on wild-type and the defenseless plants. The same analysis conducted with Corset-estimated transcript abundances identified 59 DE clusters containing 85 transcripts. In both analyses, a larger number of DE transcripts were found down-regulated in mirids feeding on JA-silenced plants (around 70%). Among these down-regulated transcripts we identified seven transcripts possibly involved in the detoxification of N. attenuata defense metabolite, specifically, one glutathione-S-transferase (GST), one UDP-glucosyltransferase (UGT), five cytochrome P450 (P450s), and six serine proteases. Real-time quantitative PCR confirmed the down-regulation for six transcripts (encoding GST, UGT and four P450s) and revealed that their expression was only slightly decreased in mirids feeding on another N. attenuata transgenic line specifically silenced in the accumulation of diterpene glycosides, one of the many classes of JA-mediated defenses in N. attenuata. CONCLUSIONS: The results provide a transcriptional overview of the changes in a specialist hemimetabolous insect associated with feeding on host plants depleted in chemical defenses. Overall, the analysis reveals that T. notatus responses to host plant defenses are narrow and engages P450 detoxification pathways. It further identifies candidate genes which can be tested in future experiments to understand their role in shaping the T. notatus-N. attenuata interaction.

Insight into the Sialome of the Bed Bug, Cimex lectularius.

The evolution of insects to a blood diet leads to the development of a saliva that antagonizes their hosts' hemostasis and inflammation. Hemostasis and inflammation are redundant processes, and thus a complex salivary potion composed of dozens or near 100 different polypeptides is commonly found by transcriptome or proteome analysis of these organisms. Several insect orders or families evolved independently to hematophagy, creating unique salivary potions in the form of novel pharmacological use of endogenous substances and in the form of unique proteins not matching other known proteins, these probably arriving by fast evolution of salivary proteins as they evade their hosts' immune response. In this work we present a preliminary description of the sialome (from the Greek Sialo = saliva) of the common bed bug Cimex lectularius, the first such work from a member of the Cimicidae family. This manuscript is a guide for the supplemental database files http://exon.niaid.nih.gov/transcriptome/C_lectularius/S1/Cimex-S1.zip and http://exon.niaid.nih.gov/transcriptome/C_lectularius/S2/Cimex-S2.xls.

Biochemical and molecular analysis of deltamethrin resistance in the common bed bug (Hemiptera: Cimicidae).

This study establishes deltamethrin resistance in a common bed bug, Cimex lectularius L., population collected from New York City (NY-BB). The NY-BB population was 264-fold more resistant to 1% deltamethrin in contact bioassay compared with an insecticide-susceptible population collected in Florida (FL-BB). General esterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase activities of NY-BB were not statistically different from those of FL-BB. cDNA fragments that encoded the open reading frame of voltage-sensitive sodium channel alpha-subunit genes from the FL-BB and NY-BB populations, respectively, were obtained by homology probing polymerase chain reaction (PCR) and sequenced. Sequence alignment of the internal and 5' and 3' rapid amplification of cDNA ends (RACE) fragments generated a 6500-bp cDNA sequence contig, which was composed of a 6084-bp open reading frame (ORF) encoding 2027 amino acid residues and 186-bp 5' and 230-bp 3' untranslated regions (5' and 3' UTRs, respectively). Sequence comparisons of the open reading frames of the alpha-subunit genes identified two point mutations (V419L and L925I) that were presented only in the NY-BB population. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly (Bemisia tabaci). V419L, located in the IS6 transmembrane segment, is a novel mutation. A Val to Met mutation at the corresponding position of the bed bug V419, however, has been identified in the tobacco budworm as a kdr-type mutation. This evidence suggests that the two mutations are likely the major resistance-causing mutations in the deltamethrin-resistant NY-BB through a knockdown-type nerve insensitivity mechanism.

The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases.

Apyrases are enzymes that hydrolyze nucleotide di- and triphosphates to orthophosphate and mononucleotides. At least two families of enzymes, belonging to the 5'-nucleotidase and to the actin/heat shock 70/sugar kinase superfamily, have evolved independently to serve the apyrase reaction. Both families require either Ca(2+) or Mg(2+) for their action. A novel apyrase enzyme sequence, with no homology to any other known protein sequence, was found recently in the salivary glands of the hematophagous bed bug Cimex lectularius. This enzyme functions exclusively with Ca(2+). Here, we report the finding of a cDNA similar to that of the C. lectularius salivary apyrase isolated from a salivary gland cDNA library of Phlebotomus papatasi. Transfection of insect cells with the P. papatasi salivary gland apyrase cDNA resulted in the secretion of a Ca(2+)-dependent apyrase whose activity was indistinguishable from that in salivary homogenates of P. papatasi. Homologous sequences were found in humans, in another sand fly (Lutzomyia longipalpis), in the fruit fly Drosophila melanogaster, in the nematode Caenorhabditis elegans and in the protozoan Cryptosporidium parvum, indicating that this family of enzymes is widespread among animal species.