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Abstract Background The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs). The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. Results Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH) families: GH11 (xylanases), GH28 (polygalacturonases or pectinases), and GH45 (β-1,4-glucanases or cellulases). Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs) families as well as polygalacturonase-inhibiting proteins (PGIPs) were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome analyses could be partially explained by differences in transcriptional level. Conclusions Combining proteome and transcriptome sequencing analyses proved to be a powerful tool for the discovery of active PCWDEs in a non-model species. Our data represent the starting point of an in-depth functional and evolutionary characterization of PCWDE gene families in phytophagous beetles and their contribution to the adaptation of these highly successful herbivores to their host plants.

Abstract Background Hormesis is a biphasic biological response characterized by the stimulatory effect at relatively low amounts of chemical compounds which are otherwise detrimental at higher concentrations. A hormetic response in larval growth rates has been observed in cotton-feeding insects in response to increasing concentrations of gossypol, a toxic metabolite found in the pigment glands of some plants in the family Malvaceae. We investigated the developmental effect of gossypol in the cotton bollworm, Helicoverpa armigera, an important heliothine pest species, by exposing larvae to different doses of this metabolite in their diet. In addition, we sought to determine the underlying transcriptional responses to different gossypol doses. Results Larval weight gain, pupal weight and larval development time were measured in feeding experiments and a hormetic response was seen for the first two characters. On the basis of net larval weight gain responses to gossypol, three concentrations (0%, 0.016% and 0.16%) were selected for transcript profiling in the gut and the rest of the body in a two-color double reference design microarray experiment. Hormesis could be observed at the transcript level, since at the low gossypol dose, genes involved in energy acquisition such as β-fructofuranosidases were up-regulated in the gut, and genes involved in cell adhesion were down-regulated in the body. Genes with products predicted to be integral to the membrane or associated with the proteasome core complex were significantly affected by the detrimental dose treatment in the body. Oxidoreductase activity-related genes were observed to be significantly altered in both tissues at the highest gossypol dose. Conclusions This study represents the first transcriptional profiling approach investigating the effects of different concentrations of gossypol in a lepidopteran species. H. armigera's transcriptional response to gossypol feeding is tissue- and dose-dependent and involves diverse detoxifying mechanisms not only to alleviate direct effects of gossypol but also indirect damage such as pH disturbance and oxygen radical formation. Genes discovered through this transcriptional approach may be additional candidates for understanding gossypol detoxification and coping with gossypol-induced stress. In a generalist herbivore that has evolved transcriptionally-regulated responses to a variety of different plant compounds, hormesis may be due to a lower induction threshold of growth-promoting, stress-coping responses and a higher induction threshold of detoxification pathways that are costly and cause collateral damage to the cell.

Abstract Background The cell suicide pathway of apoptosis is a necessary event in the life of multicellular organisms. It is involved in many biological processes ranging from development to the immune response. Evolutionarily conserved proteases, called caspases, play a central role in regulating apoptosis. Reception of death stimuli triggers the activation of initiator caspases, which in turn activate the effector caspases. In Lepidoptera, apoptosis is crucial in processes such as metamorphosis or defending against baculovirus infection. The discovery of p35, a baculovirus protein inhibiting caspase activity, has led to the characterization of the first lepidopteran caspase, Sf-Caspase-1. Studies on Sf-Caspase-1 mode of activation suggested that apoptosis in Lepidoptera requires a cascade of caspase activation, as demonstrated in many other species. Results In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived EST datasets. We identified 66 sequences distributed among 27 species encoding putative caspases. Phylogenetic analyses showed that Lepidoptera possess at least 5 caspases, for which we propose a unified nomenclature. According to homology to their Drosophila counterparts and their primary structure, we determined that Lep-Caspase-1, -2 and -3 are putative effector caspases, whereas Lep-Caspase-5 and -6 are putative initiators. The likely function of Lep-Caspase-4 remains unclear. Lep-Caspase-2 is absent from the silkworm genome and appears to be noctuid-specific, and to have arisen from a tandem duplication of the Caspase-1 gene. In the tobacco hawkmoth, 3 distinct transcripts encoding putative Caspase-4 were identified, suggesting at least 2 duplication events in this species. Conclusions The basic repertoire of five major types of caspases shared among Lepidoptera seems to be smaller than for most other groups studied to date, but gene duplication still plays a role in lineage-specific increases in diversity, just as in Diptera and mammals.

Abstract Background The chemical components of sex pheromones have been determined for more than a thousand moth species, but so far only a handful of genes encoding enzymes responsible for the biosynthesis of these compounds have been identified. For understanding the evolution of moth sexual communication, it is essential to know which genes are involved in the production of specific pheromone components and what controls the variation in their relative frequencies in the pheromone blend. We used a transcriptomic approach to characterize the pheromone gland of the Noctuid moth Heliothis virescens, an important agricultural pest, in order to obtain substantial general sequence information and to identify a range of candidate genes involved in the pheromone biosynthetic pathway. Results To facilitate identifying sets of genes involved in a broad range of processes and to capture rare transcripts, we developed our majority of ESTs from a normalized cDNA library of Heliothis virescens pheromone glands (PG). Combining these with a non-normalized library yielded a total of 17,233 ESTs, which assembled into 2,082 contigs and 6,228 singletons. Using BLAST searches of the NR and Swissprot databases we were able to identify a large number of putative unique gene elements (unigenes), which we compared to those derived from previous transcriptomic surveys of the larval stage of Heliothis virescens. The distribution of unigenes among GO Biological Process functional groups shows an overall similarity between PG and larval transcriptomes, but with distinct enrichment of specific pathways in the PG. In addition, we identified a large number of candidate genes in the pheromone biosynthetic pathways. Conclusion These data constitute one of the first large-scale EST-projects for Noctuidae, a much-needed resource for exploring these pest species. Our analysis shows a surprisingly complex transcriptome and we identified a large number of potential pheromone biosynthetic pathway and immune-related genes that can be applied to population and systematic studies of Heliothis virescens and other Noctuidae.