Crowding stress during the period of sex determination causes masculinization in pejerrey Odontesthes bonariensis, a fish with temperature-dependent sex determination.

The pejerrey is an atherinopsid species from South America that presents a combination of genotypic and environmental (temperature-dependent) sex determination whereby low and high temperatures induce feminization and masculinization, respectively. Masculinization involves a heat-induced stress response leading to increased circulating cortisol and androgens. We tested whether crowding would elicit a similar response as high temperature and affect the sex ratios of pejerrey. Larvae with XX and XY genotypes were reared at 15, 62 and 250 larvae/L in 0.4, 1.6, and 6.4 L containers during a period considered critical for sex determination at 25 °C, a mixed-sex promoting temperature. Fish were analysed at 3-7 weeks for whole-body cortisol and 11-ketotestosterone (11-KT) titer and hydroxy-steroid dehydrogenase (hsd11b2) mRNA transcript abundance, and after completion of gonadal sex differentiation (10-14 weeks) for determination of phenotypic and genotypic sex mismatches. Crowding was associated with depressed growth, higher cortisol and 11-KT titers, increased hsd11b2 transcription, and increased frequency of masculinization compared to intermediate and/or low rearing densities. Perceived crowding (by rearing in containers with mirror-finish, reflecting walls) also caused masculinization. These results suggest the possibility that other environmental factors besides temperature can also affect sex determination in pejerrey and that a stress response leading to increased cortisol and androgen levels, which is potentially perceived by the brain, may be a common feature among different forms of environmental sex determination in this species.