Sex differentiation in grayling (Salmonidae) goes through an all-male stage and is delayed in genetic males who instead grow faster

Abstract

Fish populations can be threatened by distorted sex ratios that arise during sex differentiation. Here we describe sex differentiation in a wild grayling (Thymallus thymallus) population that suffers from distorted sex ratios. We verified that sex determination is linked to the sex determining locus (sdY) of salmonids. This allowed us to study sex-specific gene expression and gonadal development. Sex-specific gene expression could be observed during embryogenesis and was strong around hatching. About half of the fish showed immature testes around eleven weeks after fertilization. This phenotype was mostly replaced by the “testis-to-ovary” or “ovaries” phenotypes during development. The gonads of the remaining fish stayed undifferentiated until six months after fertilization. Genetic sexing revealed that fish with undifferentiated gonads were all males, who grew larger than the genetic females during the observational period. Only 12% of the genetic males showed testicular tissue six months after fertilization. We conclude that sex differentiation starts before hatching, goes through an all-male stage for both sexes (which represents a rare case of “undifferentiated” gonochoristic species that usually go through an all-female stage), and is delayed in males. During these juvenile stages males grow faster than females instead of developing their gonads.

Sex-specific changes in gene expression and delayed sex differentiation in response to estrogen pollution in grayling (Salmonidae)

“…until several days after hatching, when a subset of each treatment group was distributed to two 200 L tanks each filled with lake water (pumped from Lake Geneva at 40 m depth), fed with live Artemia and copepods and later dry food, and sampled in 5 monthly intervals for histological examination of the gonads (see Maitre et al . [27] for a description of sex differentiation). For the EE2-treated groups, 200 ng EE2 were dissolved in 200 L tanks each to reach a starting concentration of 1 ng/L.…”

“…Because the RNA extraction protocol did not include a DNase treatment, DNA traces inside the RNA samples were amplified to determine the sdY genotype [26] of each individual, using the 18S gene as PCR internal control, either in a multiplex reaction used for samples with high amount of DNA, or after a second PCR amplification in single reactions with half the amounts of the respective primers each for samples with low DNA content (see Maitre et al . [27] for a more detailed protocol). Based on the sdY genotype, one female and one male per family and treatment group was haphazardly chosen for further analyses (in 2 of in total 30 combinations of family x treatment x time point, two females or two males, respectively, were used because only one sex could be found in the sample).…”

“…The RNA-seq analysis steps are described in Maitre et al . [27]. To summarize, reads were quality trimmed or filtered, and PCR duplicates were removed, resulting in a set of 60 RNA libraries with, on average, 2*40 millions of 80 bp reads each (standard deviation of six million reads).…”

“…Yano et al [26] established sex-linked genetic markers that could be used to determine the genetic sex of many salmonids, including their sample of grayling taken from a fish farm in France. These markers could be successfully verified in over 100 phenotypically sexed adult grayling sampled from our study population [27]. We therefore use them here to separate effects of EE2 on gene expression in genetic males and females.…”

“…Maitre et al . [27] found large effects of genetic sex on gene expression around the time of hatching from eggs, while gene expression did not seem to differ significantly at late embryogenesis. Their findings thus suggest that the physiological cascade of sex determination starts during embryogenesis and before hatching.…”