Anderson, David

Gillen, Christopher M.

English

Kenyon College: Digital Kenyon - Research, Scholarship, and Creative Exchange

Digital Kenyon: Research, Scholarship, and Creative ExchangeKenyon Summer Science Scholars Program Summer Student Research ScholarshipSummer 2017Expression and activity of cation cotransporters inyellow fever mosquito (Aedes aegypti) in differentsalinitiesDavid AndersonKenyon College, andersondj@kenyon.eduChristopher M. GillenKenyon College, gillenc@kenyon.eduFollow this and additional works at: https://digital.kenyon.edu/summerscienceprogramPart of the Ecology and Evolutionary Biology CommonsThis Poster is brought to you for free and open access by the Summer Student Research Scholarship at Digital Kenyon: Research, Scholarship, andCreative Exchange. It has been accepted for inclusion in Kenyon Summer Science Scholars Program by an authorized administrator of Digital Kenyon:Research, Scholarship, and Creative Exchange. For more information, please contact noltj@kenyon.edu.Recommended CitationAnderson, David and Gillen, Christopher M., "Expression and activity of cation cotransporters in yellow fever mosquito (Aedesaegypti) in different salinities" (2017). Kenyon Summer Science Scholars Program. Paper 17.https://digital.kenyon.edu/summerscienceprogram/17Gene expression:.• Aedes aegypti larvae depend on anal papillae (AP) to absorb ions and MalpigianTubules (MT) to secrete ions.• By regulating ion flow between the hemolymph and outside environment,mosquitoes are able to completely adapt to changes in saline within as little as 6hours.• In larvae reared in 30% SW, Na+ and Cl- uptake is significantly decreasedcompared larvae reared in FW [6].• We are interested in Na+-dependent cation-chloride cotransporters (CCCs)which, are proposed to contribute to homeostasis of Na+, K+ and Cl- ions withina mosquitoes’ hemolymph [7].• aeCCC3 is suspected to contribute in ion absorption while aeCCC1 and aeCCC2genes are responsible for the secretion of ions.Efflux:• Began to characterize the various roles of the NKCC transport pathway bypharmaceutically inhibiting larvae ion efflux.• Previously bumetanide has been used to attempt to inhibit the function of NKCCbut there has been no effect on the Cl-/H+ transporter mechanism [5].• Bumetanide uptake is dependent on Na+, K+, and Cl- ion uptake through NKCCtransport.• In order to have bumetanide taken into the larvae’s body, there must besufficient ions in the water to allow for the NKCC transport to take place anduptake the inhibitor.Throughout its complex life cycle, the Yellow Fever mosquito, Aedes aegypti, endures varyingextremes of salinity stress. Once hatched, larvae maintain ion balances while living in a primarilyfreshwater environment. Adults readily lose water via evaporation and must limit water loss andfeed to replace the lost water. Adult females feed on blood to provide necessary nutrients fordevelopment of their eggs, and take in salt and water as a result. Larvae and adults both usecation chloride cotransporters (CCC) to osmoregulate. Larvae can osmoregulate in environmentsranging from freshwater to 30% saltwater. Larvae reared in freshwater undergo greaterosmoregulatory stress than larvae reared in saltwater and thus we predicted that larvae rearedin deionized water would show an increase in aeCCC3 gene expression. Quantitative PCR analysisshowed no significant difference between the expression of aeCCC1, aeCCC2 and aeCCC3 inlarvae reared in deionized water and larvae reared in freshwater, but showed aeCCC3 tendingtowards statistical significance. In order to further understand the roles of the cation chloridecotransporters we exposed larvae to varying concentrations of bumetanide, a known inhibitor ofmosquito efflux of sodium, calcium, potassium and ammonium. We found bumetanide increasedthe level of sodium ion efflux with the largest increase in larvae exposed to 2 μM. Our resultssuggest bumetanide is possibly inhibiting a transporter besides aeCCC3 and perhaps larvaeexposed to extreme ion-deficient environments more readily use aeCCC3 than those undernormal conditions.AbstractIntroductionExpression and activity of cation cotransporters in yellowfever mosquito (Aedes aegypti) in different salinitiesDavid ANDERSON 1, and Christopher M.GILLEN11Kenyon College, Gambier OHIon Efflux RateThank you to the Kenyon College Biology department for their assistance and training, and especially Professor Christopher Gillen for his help every step of the way. This research was funded by Kenyon College Summer Science and the NSF.AcknowledgementsaeCCC1, aeCCC2, and aeCCC3 Gene ExpressionFigure 2: Gene expression of aeCCC1, aeCCC2 and aeCCC3 in larval Aedes aegypti reared inFreshwater or Deionized water. Lower ∆CT indicates higher expression levels. Fold differencesare calculated by the following: RQ = 2-∆∆CT. Error bars are SEM. For aeCCC1: ANOVA,F=0.0053, p=0.9428, RQ=0.98, n=11(DI), 15(FW). For aeCCC2: ANOVA, F=2.2172, p=0.1495,RQ=0.62, n=11(DI), 15(FW). For aeCCC3: ANOVA, F=5.6528, p=0.02574, RQ=0.62, n=11(DI),15(FW).Time 50: Na+ EffluxMethodsCation Chromatography:• A 200 mM stock solution of bumetanide dissolved inethanol was used to create 0.2 µM, 2 µM, 20 µM, and 200µM treatment solutions in deionized water.• A pair of larvae were placed into the one of the fourtreatment group solutions for a 20-minute incubationperiod within a 24 well plate.• 300 µL samples were taken from each experimentalgroup at time 10, 30 and 50 minutes.• Samples were analyzed using a 500-DX-chromatographysystem to measure concentrations of Na+, Ca2+, NH4+,and K+ ions.Statistics: ANOVA with Tukey comparisons via RStudiowere used to compare ion concentrations and relative geneexpression. Rstudio was used to calculate the rates of ionefflux for each treatment group.Figure 3: Example cationchromatography graph showingdistribution of ions present.Figure 5: Cation efflux of a) Na, b) K, c) NH4, d) Ca under varyingconcentrations of Bumetanide treated deionized water. For sodium:ANOVA, F=1.2146, p=.3206. For potassium: ANOVA, F=0.5692,p=0.6866. For ammonium: ANOVA, F=0.2014, p=0.936. For calcium:ANOVA, F = 0.9251, p = 0.4596.ReferencesFigure 1: Typical Aedes aegypti larvae. Photograph by Michele Cutwa-Francis, University of Florida. Figure 6: Larval Aedes aegypti sodium efflux at minute 50. All 50 minute values were compared to minute 10 values to produce deltaNa. Efflux of sodium cations increased as the concentration of bumetanide increased. ANOVA, F =1.5588 , p = 0.2051.Mosquito Rearing:• Eggs were hatched while submerged in freshwater within a vacuum chamber and rearedat 28 ˚C and 80% humidity on a 12/12 hour light/dark cycle.• Larvae used in qPCR gene analysis were split into Deionized or freshwater 3 days afterhatching.• Water was replaced everyday after day 3. On day 7, whole body 4th instar larvae werecollected in 1 mL TRIzol reagent for RNA isolation.Quantititative PCR:• Whole body larvae were homogenized in 150 µl Trizol®, ground up vigoriously with apestle, and 850 µl more of Trizol® was be added.• Sample RNA was run through clean and concentrating steps with a RNA Clean &Concentrator™-5 Kit (Zymo Research).• RNA samples were converted into cDNA with an OneStep RT-PCR kit (QIAGEN©).Controls did not contain reverse transcriptase enzyme.• Four primers; aeCCC1-2, aeCCC2-2, aeCCC3-2 and aeRps5 wereused to detect therelative quantification of all three proteins in comparison to aeRps5.• Gene expression will be quantified using a comparative Ct method described bySchmittgen and Livak (2008). A threshold cycle (Ct) is the first detectable level of cDNAamplified. Expression levels of each gene were compared to a housekeeping gene, Rps5.Figure 4: Diagram of varying Bumetanide  treatment groups used in each experiment for ion efflux.0 µM0 µMA BC D1. Albers, M.A and Bradley, T.J. (2011). On the Evolution of Saline Tolerance in the Larvae of Mosquitoes in the Genus Ochlerotatus. Physiological and Biochemical Zoology 84(3), 258-267. 2. Beyenbach, K.W. (2003). Review: Transport Mechanisms of Diuresis in Malpighian Tubules of Insects 206, 3845-3856. 3. Beyenbach, K.W.; Skaer, H.; and Dow, J.A.T. (2010). The Developmental , Molecular, and Transport Biology of Malpighian Tubules. Annual Review of Entomology 55: 351-374. 4. Bradley, T.J. (1987). Physiology of Osmoregulation in Mosquitoes. Annual Review of Entomology 32, 439-362. 5. Del Duca, O.; Nasirian, A.; Galperin, V.; Donini, A.  (2011). Pharmacological characterization of apical Na+ and Cl- transport mechanisms of the anal papillae in the larval mosquito Aedes aegypti. Journal of Experimental Biology 214: 3992-3999. 6. Donini, Andrew; Gaidhu P. Mandeep; Strasberg R. Dana; O’Donnell J. Michael. 2007. Changing Salinity Induces Alterations in Hemolymph Ioon Concetrations and Na+ and Cl- Transport Kinetics of the Anal Papillae in the Larval Mosquito, Aedes aegypti. Journal of Experimental Biology 210: 983-992.7. Filippova V.; Aimanoa K.; and Gill S.S. 2003. Expression of an Aedes aegpyti Cation-Chloride Cotransporter and its DosophilaHomologues. Insect Molecular Biology 12(4), 319-331. 8. Piermarini, Peter M. and Gillen, Christopher M. 2015. “Chapter 13: Non-Traditional Models: The Molecular Physiology of Sodium and Water Transport in Mosquito Malpighian Tubules.” Sodium and Water Homeostasis. Ed. By Hyndman, K.A. and Pannabecker, T.L. 200 µM 200 µM200 µM20 µM 20 µM 20 µM2 µM 2 µM2 µM0.2 µM 0.2 µM0.2 µM0 µMHypotheses• Larvae raised in deionized water will have greater expression of aeCCC3 compared to larvae raised in freshwater.• As bumetanide concentrations increase, levels of efflux will decrease.Conclusions• Mosquito larvae reared in deionized water have greater expression of aeCCC3 compared to larvae reared in freshwater.• Unlike Del Duca (2011), there is a difference in efflux between larvae exposed to bumetanide and those who were not.• Sodium efflux increased as bumetanide concentration increased.

Expression and activity of cation cotransporters in yellow fever mosquito (Aedes aegypti) in different salinities

https://digital.kenyon.edu/cgi/viewcontent.cgi?article=1016&amp;context=summerscienceprogram

Expression and activity of cation cotransporters in yellow fever mosquito (Aedes aegypti) in different salinities

Abstract

Similar works

Full text

Available Versions

Kenyon College: Digital Kenyon - Research, Scholarship, and Creative Exchange