Development of methods to identify the sex chromosomal genotype (XX, XY, YY) in pejerrey and its application in a field survey of sex-reversals and super-males

YAMAMOTO Y; TASHIRO S; HATTORI RS; ZHANG Y; MIRANDA LA; COLAUTTI DC; BERASAIN E; STRUSSMANN CA

Simposio; 8 ISFE; 2016

8 ISFE

Developmentof methods to identify the sex chromosomalgenotype (XX, XY, YY) in pejerrey and its application in a fieldsurvey of sex-reversals and super-malesYoji Yamamoto1, Seiya Tashiro1, Ricardo S Hattori2, Yan Zhang1, Leandro AMiranda3, Dario C Colautti4, Gustavo E Berasain5, Carlos A Strüssmann1Tokyo University of Marine Science and Technology, Japan, 2APTA/SAA,Brazil, 3IIB-INTECH, Argentina, 4UNLP, Argentina, 5MAAGBA, Argentina.yoji@m.kaiyodai.ac.jpThe pejerrey Odontesthes bonariensis is known for its strongtemperaturedependent sex determination (TSD). However, we have also shown that this species possess a major, if not master, testis determining gene, theY-chromosome-linked anti-Müllerian hormone (amhy). This finding makespossible to monitor wild pejerrey populations for sex-reversals and may prove instrumental for field studies addressing the effects of abnormal temperatures on reproduction. It is also possible that super-male YY individuals occur in the wild, for example, by mating of sex-reversed (XY) females to normal XY males, but current assayscannot distinguish XY from YY individuals. Because of the possible impact thatsex-reversed and YY individuals might have on the sex ratios of wildpopulations,we artificially produced YY individuals to test their survival and fertilityand developed a molecular method to discriminate fish with single (XY) anddouble (YY)copies of amhy. Finally, we carried out a field study to assess thepresence of sexreversals and YY individuals in wild populations. First, captive-rearedbroodstockwere genetically screened for amhy and a sex-reversed XY female wasmated toan XY male. Their progeny was reared until sexual maturity and screened bygenomic DNA PCR analysis using amhy-specific primers. In this screening,66%(53 out of 80) of the progeny was amhy-positive, which suggested thepresence ofYY fish assuming Mendelian segregation (XX 1:3 XY+YY). We then performed aqPCR analysis on genomic DNA which detected 16 individuals, presumably YYindividuals, with amhy values twice higher than the other 37 amhy-positivefishes.Progeny tests with amhy-negative (XX) females showed that the presumedYYindividuals were in fact YY as their offspring was 100% amhy-positive(XY). Wethen used these molecular tools to screen wild pejerrey (n=158) collected inLakeChascomus, Argentina, in 2014. The analysis revealed the presence of 35XXfemales, 4 XY-females, 12 XX-males, 105 XY-males, and 2 YY-males. The resultsof this study reveal that YY pejerrey are both viable and fertile and confirmtheoccurrence of sex reversal in both directions, probably as a result of TSD, andeven YY individuals in a wild pejerrey population. The molecular toolsdevelopedin this study may be useful for surveying the effects of temperature and otherfactors on sex determination of wild populations.