Expressed sequence tags from the midgut of Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae).

The midgut is a key tissue in insect science. Physiological roles include digestion and peritrophic membrane function, as well as being an important target for insecticides. We used an expressed sequence tag (EST) approach to identify candidate genes and gene families involved in these processes in the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). Two cDNA libraries were constructed from dissected midgut of third to fifth instar larvae. Clustering analysis of 6416 expressed sequence tags produced 1178 tentative unique genes comprising 725 tentative contigs and 453 singletons. The sequences show similar codon usage to sequences from other lepidopterans, a Kozak consensus sequence similar to Drosophila and single nucleotide polymorphisms (SNPs) were detected at a frequency of 1.35/kb. The identity of the most common Interpro families correlates well with major known functions of the midgut. Phylogenetic analysis was conducted on representative sequences from selected multigene families. Gene families include a broad range of digestive proteases, lipases and carbohydrases that appear to have degradative capacity against the major food components found in leaves, the diet of these larvae; and carboxylesterases, glutathione-S-transferases and cytochrome P450 monooxygenases, potentially involved in xenobiotic degradation. Two of the larger multigene families, serine proteases and lipases, expressed a high proportion of genes that are likely to be catalytically inactive.

Molecular-based approach to the differentiation of mealybug (Hemiptera: Pseudococcidae) species.

The rDNA internal transcribed spacer (ITS) region of 4 mealybug species, Pseudococcus viburni (Signoret), P. longispinus (Targiono-Tozzetti), P. calceolariae (Maskell), and P. similans (Lidgett), was isolated by polymerase chain reaction (PCR) amplification, cloned, and sequenced. In this region of the genome there were numerous differences, including nucleotide substitutions, insertions, or deletions between P. viburni, P. longispinus, and P. calceolariae, whereas P. calceolariae and P. similans were very similar. Based on sequence differences between the ITS regions, we designed PCR primers that were able to differentiate the 4 mealybug species and that correlated with morphological differences found between adult females of these species. The PCR amplification by using the species-specific primers enabled the differentiation of not only adult females but also eggs, juveniles, and adult males, which was not previously possible by using conventional identification methods.

Evolution of the proteinase inhibitor I family and apparent lack of hypervariability in the proteinase contact loop.

A protein phylogenetic tree was constructed from 24 homologous proteinase inhibitor I sequences identified in the EMBL/Genbank and Swiss-Prot databases and from translated amino acid data from four constitutive cDNA clones of proteinase inhibitor I characterized from potato tuber mRNA. The tree suggests that divergence of at least four paralogous proteins with functional specialization occurred at different times during the evolutionary history of the proteinase inhibitor I family. Five distinct regions in the primary structure, earlier identified by structural studies, were used to analyze the inhibitor family for hypervariability (Creighton and Darby, Trends Biochem Sci 14:319-324, 1989). Mutations did not occur with higher-than-random frequency within the proteinase binding region. When isoinhibitor, orthologous, or paralogous data subsets were subsequently analyzed the same results were obtained. Comparison of the amino acid sequences for all the known potato proteinase isoinhibitor I proteins identified ten highly variable sites. These also were distributed randomly. Thus hypervariability, which has been observed in all other serine proteinase inhibitor families to date, appears to be lacking in the proteinase inhibitor I family.