Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti.

BACKGROUND: Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production. CONCLUSIONS/SIGNIFICANCE: Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.

Phenotypic analysis of EcR-A mutants suggests that EcR isoforms have unique functions during Drosophila development.

The steroid hormone ecdysone triggers transitions between developmental stages in Drosophila by acting through a heterodimer consisting of the EcR and USP nuclear receptors. The EcR gene encodes three protein isoforms (EcR-A, EcR-B1, and EcR-B2) that have unique amino termini but that contain a common carboxy-terminal region including DNA-binding and ligand-binding domains. EcR-A and EcR-B1 are expressed in a spatially complementary pattern at the onset of metamorphosis, suggesting that specific responses to ecdysone involve distinct EcR isoforms. Here, we describe phenotypes of EcR-A specific deletion mutants isolated using transposon mutagenesis. Western blot analysis shows that each of these mutants completely lacks EcR-A protein, while the EcR-B1 protein is still present. The EcR(112) strain has a deletion of EcR-A specific non-coding and regulatory sequences but retains the coding exons, while the EcR(139) strain has a deletion of EcR-A specific protein coding exons but retains the regulatory region. In these mutants, the developmental progression of most internal tissues that normally express EcR-B1 is unaffected by the lack of EcR-A. Surprisingly, however, we found that one larval tissue, the salivary gland, fails to degenerate even though EcR-B1 is the predominant isoform. This result may indicate that the low levels of EcR-A in this tissue are in fact required. We identified yet another type of mutation, the EcR(94) deletion, that removes the EcR-A specific protein coding exons as well as the introns between the EcR-A and EcR-B transcription start sites. This deletion places the EcR-A regulatory region adjacent to the EcR-B transcription start site. While EcR(112) and EcR(139) mutant animals die during mid and late pupal development, respectively, EcR(94) mutants arrest prior to pupariation. EcR-A mutant phenotypes and lethal phases differ from those of EcR-B mutants, suggesting that the EcR isoforms have distinct developmental functions.

Piv site-specific invertase requires a DEDD motif analogous to the catalytic center of the RuvC Holliday junction resolvases.

Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.