Term of Award

Summer 2019

Degree Name

Master of Science in Biology (M.S.)

Document Type and Release Option

Thesis (open access)

Copyright Statement / License for Reuse

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

Department

Department of Biology

Committee Chair

Christopher Cutler

Committee Member 1

Johanne Lewis

Committee Member 2

Vinoth Sittaramane

Abstract

Osmoregulation is facilitated by using an assortment of ion and water channels to assist acclimation to changing conditions and to maintain cellular homeostasis. Euryhaline fish can be found in both seawater and freshwater (SW and FW) environments. Expression of ion and water channels have been experimentally demonstrated to change as a fish acclimates to different environmental conditions. Relatively recently, a new group of water channels has been discovered that are primarily intracellular and includes aquaporin 11 (AQP11). Typically, AQPs are located on the cell plasma membrane to allow water to flow in and out of the cell by osmosis to assist in cellular homeostasis. Since AQP11 has been primarily located in the membrane of the endoplasmic reticulum, it was expected that the expression of AQP11 would not be affected by the acclimation of fish to SW. For this study, Anguilla rostrata (the American eel) was selected partly because it has two paralogs of AQP11 (AQP11a and AQP11b). Eels were subjected to a SW-acclimation experiment where they were acclimated to SW for predetermined times, while concurrent FW control samples were also kept. Initially, genomic sequences for AQP11a and AQP11b genes were available for the closely related Anguilla japonica (the Japanese eel), thus cloning and sequencing of the AQP11a and 11b paralog cDNA sequences from the American eel were performed. The obtained sequences were used to generate primers and antibodies for further studies. Quantification of mRNA/cDNA was performed using quantitative PCR (qPCR). The analysis of the qPCR data determined that both AQP11a and AQP11b had significant differences between FW and SW in the expression of mRNA/cDNA in multiple osmoregulatory tissues (i.e. kidney, gill, and gastrointestinal tract). Quantification of AQP11 proteins was facilitated using western blotting. The antibody generated for AQP11a was found to be non-specific and unusable for quantification potentially due to a possible PDZ domain located at the C-terminal end where polypeptides for the antibody were made. The antibody made against AQP11b produced quantifiable western blots. Significant differences between AQP11b bands were found in all intestinal tissues. Additionally, localization of AQP11 paralogs was carried out using immunohistochemistry in multiple intestinal tissues as well as kidney and liver in both FW and SW-acclimated fish. In SW tissues AQP11a and AQP11b were seen to be present in smooth muscle in intestinal tissues as well as on the luminal side of epithelial cells and expression of AQP11a and AQP11b seems to be lower in corresponding FW tissues. In kidney tissue AQP11b was occasionally present in nephron tubules in SW tissue but not FW tissue, and AQP11b was also found in the liver surrounding the liver ducts. The within-group and between-group quantitative analysis of the FW and SW-acclimated AQP11 paralogs transcript and protein expression suggests that AQP11 paralogs are affected by SW-acclimation even though they are intracellular. These results may also point to the endoplasmic reticulum being affected by SW-acclimation due to subsequent related changes in AQP11 expression.

OCLC Number

1112110078

Research Data and Supplementary Material

No

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