A targeted approach to the identification of candidate genes determining susceptibility to Plasmodium gallinaceum in Aedes aegypti

Abstract

The malaria parasite, Plasmodium, has evolved an intricate life cycle that includes stages specific to a mosquito vector and to the vertebrate host. The mosquito midgut represents the first barrier Plasmodium parasites encounter following their ingestion with a blood meal from an infected vertebrate. Elucidation of the molecular interaction between the parasite and the mosquito could help identify novel approaches to preventing parasite development and subsequent transmission to vertebrates. We have used an integrated Bulked Segregant Analysis-Differential Display (BSA-DD) approach to target genes expressed that are in the midgut and located within two genome regions involved in determining susceptibility to P. gallinaceum in the mosquito Aedes aegypti. A total of twenty-two genes were identified and characterized, including five genes with no homologues in public sequence databases. Eight of these genes were mapped genetically to intervals on chromosome 2 that contain two quantitative trait loci (QTLs) that determine susceptibility to infection by P. gallinaceum. Expression analysis revealed several expression patterns, and ten genes were specifically or preferentially expressed in the midgut of adult females. Real-time PCR quantification of expression with respect to the time of blood meal ingestion and infection status in mosquito strains permissive and refractory for malaria revealed a differential expression pattern for seven genes. These represent candidate genes that may influence the ability of the mosquito vector to support the development of Plasmodium parasites. Here we describe their isolation and discuss their putative roles in parasite-mosquito interactions and their use as potential targets in strategies designed to block transmission of malaria.

Molecular characterization of genes encoding trypsin-like enzymes from Aedes aegypti larvae and identification of digestive enzymes

“…Several Ae. aegypti digestive enzymes have been identified, including trypsins (Barillas-Mury and Wells, 1993), chymotrypsins (Jiang et al, 1997), aminopeptidases (Morlais et al, 2003) and carboxypeptidases (Isoe et al, 2009). Trypsin expression persists throughout developmental instars of Ae.…”

Aedes Aegypti Vectorial Capacity Is Determined by the Infecting Genotype of Dengue Virus

“…aegypti housekeeping gene to quantify tissue and normalize samples. The ribosomal protein S17 (RpS17) RNA has been used as a gene expression standard in other experiments; 22,23 we have adapted this for use as a qRT-PCR standard, which requires a shorter amplicon. Primer and probe sequences were identified from the LF272 EST sequence (Genbank accession number BM005484) using PrimerExpress (Applied Biosystems, Foster City, CA).…”

Biosynthesis of isoprenoids – studies on the mechanism of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase

“…The development of that compound as an antibiotic drug was aborted in the 1980s, but recent work has shown activity against various Plasmodium spp., including Plasmodium falciparum, a major human pathogen [17,[20][21][22]. These studies have validated IspC as a target for the development of novel antimalarial agents, which are urgently needed in light of the enormous death toll of malaria [23] and the rapid dissemination of variants with resistance against currently available drugs [24]. Moreover, IspC and the consecutive enzymes of the pathway are believed to be potential targets for the chemotherapy of infections by a variety of eubacterial pathogens, most notably Mycobacterium tuberculosis [14,[25][26][27].…”