The molecular and immunochemical expression of innexins in the yellow fever mosquito, Aedes aegypti: Insights into putative life stage- and tissue-specific functions of gap junctions

Abstract

Gap junctions (GJ) mediate direct intercellular communication by forming channels through which certain small molecules and/or ions can pass. Connexins, the proteins that form vertebrate GJ, are well studied and known to contribute to neuronal, muscular and epithelial physiology. Innexins, the GJ proteins of insects, have only recently received much investigative attention and many of their physiological roles remain to be determined. Here we characterize the molecular expression of six innexin (Inx) genes in the yellow fever mosquito Aedes aegypti (AeInx1, AeInx2, AeInx3, AeInx4, AeInx7, and AeInx8) and the immunochemical expression of one innexin protein, AeInx3, in the alimentary canal. We detected the expression of no less than four innexin genes in each mosquito life stage (larva, pupa, adult) and tissue/body region from adult males and females (midgut, Malpighian tubules, hindgut, head, carcass, gonads), suggesting a remarkable potential molecular diversity of GJ in mosquitoes. Moreover, the expression patterns of some innexins were life stage and/or tissue specific, suggestive of potential functional specializations. Cloning of the four full-length cDNAs expressed in the Malpighian tubules of adult females (AeInx1, AeInx2, AeInx3, and AeInx7) revealed evidence for 1) alternative splicing of AeInx1 and AeInx3 transcripts, and 2) putative N-glycosylation of AeInx3 and AeInx7. Finally, immunohistochemistry of AeInx3 in the alimentary canal of larval and adult female mosquitoes confirmed localization of this innexin to the intercellular regions of Malpighian tubule and hindgut epithelial cells, suggesting that it is an important component of GJ in these tissues.

Physiological characterization and regulation of the contractile properties of the mosquito ventral diverticulum (crop)

“…RNA isolation and qPCR were performed as described in Calkins et al (2015). In brief, RNA was isolated using the method of Chomczynski and Sacchi (1987).…”

“…cDNA was synthesized using 1 µg of total RNA in the GoScript® Reverse Transcriptase kit (Promega, Madison, WI). Primers for qPCR to measure innexin and reference gene (RPS7) mRNA expression were from Calkins and Piermarini (2015). For a given sample, each reaction consisted of 10 µl: 5 µl of GoTaq Master Mix, 40 ng of cDNA (0.2 µl of 200 ng/µl cDNA), 1 µl of 4 µM primers, and 3.8 µl of nuclease free water.…”

Gap junction gene and protein families: Connexins, innexins, and pannexins

“…Some innexins may also be glycosylated; for example, two yellow fever mosquito ( Aedes aegypti ) innexins (AeInx3 and AeInx7) contain predicted N-glycosylation sites, and immunoblots of AeInx3 show multiple immunoreactive bands. This similarity to the pannexins suggests that some innexins might have other functional roles besides intercellular communication [15]. …”

Localization and role of inward rectifier K+ channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti

“…Intact, isolated Malpighian tubules were fixed with 4% paraformaldehyde, washed with a phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 (Thermo Scientific), and blocked with 10% normal goat serum (Thermo Scientific) supplemented with casein (Vector Laboratories, Burlingame, CA), exactly as described previously (Calkins et al, 2015). The PBS was diluted from a 10Â commercially-supplied concentrate (Thermo Scientific) and consisted of 9.5 mM phosphate, 2.7 mM KCl, and 137 mM NaCl (pH 7.4).…”