Mosquito transcriptome changes and filarial worm susceptibility in Armigeres subalbatus

Armigeres subalbatus is a natural vector of the filarial worm Brugia pahangi, but it kills Brugia malayi microfilariae (mf) by melanotic encapsulation. Because B. malayi and B. pahangi are morphologically and biologically similar, this mosquito-parasite system serves as a valuable model for studying resistance mechanisms in mosquito vectors. Comparing Ar. subalbatus-B. pahangi susceptibility and Ar. subalbatus-B. malayi refractoriness could provide significant insight into recognition mechanisms required to mount an effective anti-filarial worm immune response in the mosquito, as well as provide considerable detail into the molecular components involved in vector competence. Accordingly, we initiated transcriptome profiling studies of Ar. subalbatus in relation to filarial worm infection to provide information on the molecular components involved in B. pahangi susceptibility for comparison with our earlier studies on B. malayi refractoriness (Aliota et al., 2007). In addition, these studies also provide information on the infection response of a natural vector, i.e., the overall transcriptional and physiological change that occurs in the mosquito as a result of parasite infection, for comparison with our previous studies that employed a highly susceptible laboratory model, Aedes aegypti (Erickson et al., 2009). The time course chosen facilitated an examination of key events in the development of the parasite, beginning with the very start of filarial worm infection and spanning to well after infective-stage parasites had completed development in the mosquito. Herein, we demonstrate that filarial worm susceptibility in Ar. subalbatus is a highly complex process during the first 24 hours of infection. It is a process that involves many factors of both known and unknown function which are most likely associated with filarial worm penetration through the midgut lumen, invasion into thoracic muscle cells, and maintenance of homeostasis in the hemolymph environment. The data show that there are distinct and separate transcriptional patterns associated with filarial worm susceptibility as compared to refractoriness, and that an infection response in Ar. subalbatus, a natural vector, can differ significantly from that observed in Ae. aegypti, a common laboratory model. Finally, the data presented herein provide us with a cadre of information to design wet lab experiments and select candidates for further study to more fully dissect the nature of the anti-filarial worm immune response in this mosquito-parasite system. Nine sample groups were created from thirteen timepoints to study mosquito transcript changes in response to B. pahangi development. In each sample group, comparisons were made between mosquitoes exposed to an infective bloodmeal containing B. pahangi mf and those exposed to a bloodmeal without parasites. In addition, a separate set of microarray analyses directly compared transcriptome profiles between mosquitoes in which filarial worms develop to infective stage larvae (B. pahangi) and mosquitoes in which an anti-filarial worm immune response had been initiated (B. malayi). This direct comparison contained groups of mosquitoes exposed to an infective bloodmeal containing B. pahangi mf or exposed to a bloodmeal containing B. malayi mf. Samples were collected 1, 6, 12, and 24 h PI and 2-3 d PI. Three biological replicates, each with two technical replicates done as dye-swapped pairs (Cy5 experimental vs. Cy3 control), were performed for each experimental set in an effort to eliminate bias in the dye coupling. Each biological replicate consisted of mosquitoes from distinct generations to take into account stochastic variations. RNA integrity was verified via gel electrophoresis or via Bioanalyzer (Agilent, Santa Clara, CA) and only quality intact RNA was used for cDNA synthesis.