The response of claudin-like transmembrane septate junction proteins to altered environmental ion levels in the larval mosquito Aedes aegypti

Abstract

Septate junctions (SJs) occlude the paracellular pathway and function as paracellular diffusion barriers within invertebrate epithelia. However, integral components of SJs and their contribution to barrier properties have received considerably less attention than those of vertebrate occluding junctions. In arthropods, SJ proteins have only been identified in Drosophila and among these are three integral claudin-like proteins, Megatrachea (Mega), Sinuous (Sinu) and Kune-kune (Kune), as well as a receptor-like transmembrane SJ protein known as Neurexin IV (Nrx IV). In this study, mega, sinu, kune and nrx IV are identified and characterized in aquatic larvae of the mosquito Aedes aegypti and a role for these proteins in ionoregulatory homeostasis is considered. Transcripts encoding Mega, Sinu, Kune and Nrx IV were found in iono/osmoregulatory tissues such as the midgut, Malpighian tubules, hindgut and anal papillae, but abundance was greater in the hindgut and anal papillae. Using immunohistochemical and western blot analysis it was found that Kune localized to the regions of intercellular contact between epithelial cells of the rectum and posterior midgut and in the apical membrane domain of the syncytial epithelium of anal papillae. To investigate a potential role for integral SJ proteins in larval A. aegypti iono/osmoregulation, abundance was examined in animals reared in freshwater or brackish water (30 % seawater). In iono/osmoregulatory epithelia, larvae exhibited tissue-specific alterations in mega mRNA and Kune protein abundance, but not sinu or nrx IV mRNA. These studies provide a first look at the potential contribution of integral SJ components to iono/osmoregulatory homeostasis in an aquatic invertebrate.

Identification of the septate junction protein gliotactin in the mosquito, Aedes aegypti: evidence for a role in increased paracellular permeability in larvae

“…In a second study, a salinity-induced increase in the protein abundance of the integral SJ protein kunekune (Kune) was observed in the posterior midgut as well as anal papillae of A. aegypti larva while other SJ proteins were unaltered (Jonusaite et al, 2016b). Taken together, these observations suggest that select SJ proteins contribute to osmoregulatory homeostasis in larval A. aegypti (Jonusaite et al, 2016b(Jonusaite et al, , 2017.…”

“…The changes in PEG-400 flux across the midgut and Malpighian tubules occurred in association with increased transcript abundance of the sSJ proteins Ssk and mesh in these epithelia of BW-reared larvae when compared with FW animals (Jonusaite et al, 2017). In a second study, a salinity-induced increase in the protein abundance of the integral SJ protein kunekune (Kune) was observed in the posterior midgut as well as anal papillae of A. aegypti larva while other SJ proteins were unaltered (Jonusaite et al, 2016b). Taken together, these observations suggest that select SJ proteins contribute to osmoregulatory homeostasis in larval A. aegypti (Jonusaite et al, 2016b(Jonusaite et al, , 2017.…”

“…Larvae of Aedes aegypti (Linnaeus in Hasselquist 1762) were obtained from a colony maintained in the Department of Biology at York University as previously described (Jonusaite et al, 2016b Total RNA was extracted from larval A. aegypti tissues (midgut with gastric caecae, Malpighian tubules, hindgut and anal papillae) using TRIzol ® reagent (Invitrogen, Burlington, ON, Canada) according to the manufacturer's instructions. Tissues from 50 larvae were pooled per one biological sample.…”

“…Expression of mRNA encoding gli in the whole body and osmoregulatory tissues of A. aegypti larvae was examined by routine reverse transcriptase PCR (RT-PCR). 18S rRNA mRNA abundance was used as a loading control and was amplified using primers previously described (Jonusaite et al, 2016b). Resulting RT-PCR amplicons were resolved by agarose gel electrophoresis and sequence identities were confirmed after sequencing at the York University Core Molecular Facility (Department of Biology, York University, ON, Canada).…”

“…For qPCR analyses, gli mRNA abundance was normalized to either 18S rRNA or rp49 transcript abundance. Aedes aegypti 18S rRNA and rp49 mRNA were amplified using primers previously described (Jonusaite et al, 2016b).…”