Elucidating the processes mediating mosquito basement membrane repair and the implications on within-mosquito virus dynamics

Abstract/Project Summary: Recent work has demonstrated that mosquito feeding biology, namely multiple blood feeding, can greatly impact within-mosquito virus dissemination dynamics as well as time to transmission. When mosquitoes feed, their midgut expands dramatically, the surrounding midgut basement membrane (BM) becomes disordered, and constituent BM collagen IV triple helices become unfolded. If mosquitoes feed a second time following an infectious blood meal, this transient damage is correlated with earlier virus escape from the midgut and a weakening of the midgut escape barrier. Although some studies have addressed BM development and repair in Drosophila, little is known from adult mosquitoes. Initial RT-qPCR studies suggest that expression of the two forms of collagen IV, viking (vkg) and collagen at 25C (Cg25C), increase in the midgut as BM damage decreases. Expression of secreted protein acidic and rich in cysteine (SPARC), a gene important for BM collagen IV integration in Drosophila, was evident in the mosquito carcass (containing the fat body). Other tissues and genes may also be important as earlier studies have noted expression of important BM genes during development in the insect fat body, midguts, and hemocytes. In this application, the spatiotemporal expression of BM genes and proteins following blood feeding will be investigated and knockdown of genes of interest such as vkg, cg25C, and SPARC will reveal which genes are critical for BM repair. Further, transgenic mosquito lines will be created with tissue-specific inducible expression of fluorescently tagged genes involved in BM repair to identify the tissue of origin for proteins incorporated into the repairing BM. From this, the relative importance of the different tissues that may be involved in BM repair processes will be determined. Transgenic lines with constitutive expression of vkg and cg25C will be created to determine if overexpression of collagen IV can affect BM repair and the timing of virus dissemination. This investigation of mosquito midgut BM repair and the tissues involved will expand our knowledge of mosquito biology, allow for the manipulation of these processes, and provide new targets for mosquito or pathogen control measures.