Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.     

Cloning and expression of a novel juvenile hormone-metabolizing epoxide hydrolase during larval–pupal metamorphosis of the cabbage looper, Trichoplusia ni

A full-length cDNA encoding for a microsomal juvenile hormone (JH)-metabolizing epoxide hydrolase (TmEH-1) was isolated from a cDNA library constructed from fat body of last stadium (wandering) cabbage loopers, Trichoplusia ni, at the exact developmental time of maximum JH epoxide hydrolase activity. TmEH-1 was 1887 base pairs in lenght with a 1389 base pair open reading frame encoding 463 amino acids. Amino acid sequence analysis showed that TmEH-1 was most similar to and contained the exact catalytic triad (Asp-226, Glu-403 and His-430) found in microsomal epoxide hydrolases. TmEH-1-specific message was present along with JH III epoxide hydrolase activity in fat body in feeding (days 1 and 2) and wandering (day 3) larvae with the peak in message level preceding the peak in JH epoxide hydrolase activity by 1 day. When TmEH-1 was expressed in baculovirus-infected Spodoptera frugiperda cells, a 46,000 molecular weight protein appeared on SDS-PAGE which corresponded to the predicted size coded by the TmEH-1 message and which was positively correlated with increases in JH III epoxide hydrolase activity above that of wild-type controls. In subcellular distribution studies, 58% of the juvenile hormone III epoxide hydrolase activity was in the insoluble fractions. Baculovirus expressed TmEH-1 demonstrated a higher specific activity for JH III as compared to the general EH substrates, cis- and trans-stibene oxide. Southern blot analyses suggested that multiple epoxide hydrolase genes are present in T. ni.     

Microarray-based survey of a subset of putative olfactory genes in the mosquito Anopheles gambiae

Female Anopheles gambiae mosquitoes respond to odours emitted from humans in order to find a blood meal, while males are nectar feeders. This complex behaviour is controlled at several levels, but is probably initiated by the interaction of various molecules in the antennal sensilla. Important molecules in the early odour recognition events include odourant binding proteins (OBPs), which may be involved in odour molecule transport, odourant receptors (ORs) that are expressed in the chemosensory neurones and odour degrading enzymes (ODEs). To obtain a better understanding of the expression patterns of genes that may be involved in host odour reception in females, we generated a custom microarray to study their steady state mRNA levels in chemosensory tissues, antennae and palps. These results were supported by quantitative RT PCR. Our study detected several OBPs that are expressed at significantly higher levels in antennae and palps of females vs. males, while others showed the opposite expression pattern. Most OBPs are slightly down-regulated 24 h after blood feeding, but some, especially those with higher expression levels in males, are up-regulated in blood-fed females, suggesting a shift in blood-fed females from human host seeking to nectar feeding.     

Functional analysis of AeSCP-2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti

The effect of gene expression knockdown was used to study the function of the sterol carrier protein-2 (AeSCP-2) in the yellow fever mosquito, Aedes aegypti. Injection of small double stranded AeSCP-2 RNAs into mosquito larvae resulted in the knockdown of gene products. The lack of AeSCP-2 in larvae coincided with a reduction in accumulated cholesterol in pupae, supporting the hypothesis that AeSCP-2 may be involved in cholesterol uptake in mosquito larvae. Knockdown of AeSCP-2 caused a high mortality rate in developing adult and reduced egg viability. Results from this study indicate that AeSCP-2 is important for adult development and for the viability of the eggs.     

Molecular characterization of a gene encoding juvenile hormone esterase in the red flour beetle,Tribolium castaneum

Juvenile hormone esterases (JHEs) are required for the degradation of juvenile hormones (JHs) in insects. Here, we report the cloning and analysis of the jhe gene in the red flour beetle, Tribolium castaneum, a model insect of Coleoptera. The Tcjhe gene was strongly expressed at the final instar larva, as would be expected if it functioned to decrease the JH titer at this stage. A recombinant TcJHE protein efficiently degraded JH III, suggesting that the enzyme functions in vivo as a JH‐specific degradation enzyme. This is the first report describing the developmental expression profile of the jhe gene whose enzymatic activity was shown in Coleoptera, and the new data reported here will aid elucidation of the mechanism of JH titer regulation in insects.     

A novel cadherin-like gene from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), larval midgut tissue

A cadherin-like gene associated with larval midgut tissues was cloned from western corn rootworm (Diabrotica virgifera virgifera: Coleoptera), an economically important agricultural pest in North America and Europe and the primary target pest species for corn hybrids expressing Cry3 toxins from Bacillus thuringiensis (Bt). The full-length cDNA (5371 bp in length) encodes an open reading frame for a 1688 amino acid polypeptide. The putative protein has similar architecture to cadherin-like proteins isolated from lepidopteran midguts that have been shown to bind to Cry1 Bt toxins and have been implicated in Bt resistance. The D. v. virgifera cadherin-like gene is expressed primarily in the larval midgut and regulated during development, with high levels of expression observed in all instars and adults but not pupae. The corresponding genomic sequence spans more than 90 kb and is interspersed with 30 large introns. The genomic organization of the cadherin-like gene for this coleopteran species bears strong resemblance to lepidopteran cadherins suggesting a common molecular basis for susceptibility to Cry3 toxins in Coleoptera.