Date of Award

Summer 5-2021

Document Type

Thesis

Degree Name

Bachelor of Arts

Department

Biochemistry & Molecular Biology

First Advisor

Christopher M. Gillen

Abstract

Aedes aegypti expresses three putative Na+-dependent cation-chloride cotransporters (CCCs). Whereas AeNKCC1 is orthologous to the Drosophila Na+-K+-2Cl- cotransporter Ncc69, aeCCC2 and aeCCC3 separate into an insect-specific clade with Drosophila Ncc83. In previous work, aeCCC2-expressing Xenopus oocytes demonstrated increased Li+ (Na+ tracer) but equal Rb+ (K+ tracer) uptake compared to H2O-injected controls (Kalsi et al., 2019). To further explore the transport properties of aeCCC2, we exposed Sf9 insect cells transiently transfected with aeCCC2, Ncc83, Ncc83-pHluorin, and Ncc69 to 20 mM [Li+ ] and 5 mM [Rb+ ] saline and measured uptake via cation chromatography. In Ncc83-pHluorin transfected cells, we confirmed membrane expression via fluorescent microscopy. Baseline Na+:K+ ratios of aeCCC2 transfected cells were 1.8 to 2.0 fold greater than controls and Ncc69 transfected cells. Following hypotonic pre-incubation, Ncc69 transfected cells had 2 to 3 fold greater Rb+ uptake compared to controls and cells transfected with aeCCC2. Additionally, aeCCC2 transfected cells had 1.8 and 3 fold greater Li+:Rb+ uptake ratio compared to controls and Ncc69 transfected cells, respectively. To evaluate the role of aeCCC2 in epithelia, we performed whole-mount immunohistochemistry on larval and adult Ae. aegypti gut tissues. Antibodies against aeCCC2 stained the midgut, ileum, and Malpighian tubules (MTs) in adults and larvae. Staining of MT was on the basolateral membrane of principal cells. Notably, whereas ileum and MTs were equally stained in larvae, staining was more pronounced in ileum compared to MTs in adult females. Our findings suggest aeCCC2 contributes to non-canonical K+-independent Na+ transport in various epithelia involved in osmoregulation.

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