Zebrafish change their sex in warm water

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There are some species of fish where their sex is not clearly fixed Photo: Dr ShThere are some species of fish where their sex is not clearly fixed Photo: Dr Shahrbanou Hosseini

Research team led by Göttingen University identify DNA -hotspots- that tell zebrafish to change sex in warmer waters. Environment leaves its mark on genome through DNA methylation.

In many species, such as zebrafish, sex is partly or completely determined by the environment. Genes can predispose to a particular sex but may be -overruled- by the influence of the environment, for example temperature or population density. However, the mechanisms are not fully understood. Now, a research team led by the University of Göttingen has identified the DNA -hotspots- that tell zebrafish to change sex in warmer waters. These ’epigenetic’ changes can then reroute genetic pathways, so that the embryos change sex. This is relevant for conservation since an influence of temperature on sex determination could be recipe for disaster for species living through rapid climate change. The results were published in Frontiers in Cell and Developmental Biology.

For each of 17 families of zebrafish, the researchers raised a single generation from fertilization to adulthood, approximately 3.5 months later. Each family consisted of approximately 200 full siblings. The water temperature was 28 °C, except that half of the embryos within each family were transiently exposed to 35 °C when five to 24 hours old - a critical time window. The sex ratio of the adults differed strongly between families, but in many families the percentage of males became between 15% and 20% higher after brief exposure to 35 °C. Effectively, the influence of higher temperature had ’overruled’ the genetic predisposition of some individuals, causing them to change sex into males. But which adult males were ’born’ male and which reversed sex can’t be determined at present.

The researchers then selected a single female-biased family and a single male-biased family for experiments. They dissected out the ovaries and testes of zebrafish from each of the two families, separately for the two treatments. They performed a technique, bisulfite sequencing (BS-Seq) on the DNA in 72 of these gonad samples, to determine the proportion of methylated cytosines across the genome. They focused on 174 selected genes whose involvement in sex determination was suspected from earlier studies. They also looked at promoter regions of genes in general, where DNA methylation is known to be important for regulating gene expression.

In zebrafish, embryos hatch with rudimentary ovary-like gonads. But depending on the ’crosstalk’ between the many sex-determining genes (scattered across the chromosomes) and the environment, individuals may resorb these proto-ovaries and develop testes, thus becoming males. The researchers found that in the zebrafish, a model species, the answers appear to be DNA methylation. They demonstrate that high temperature prompts methyl groups to be added to many of the C nucleobases (cytosines) in regulatory stretches of DNA. There, methylation typically dials down the expression of downstream genes.

The researchers were able to identify 54 genes that are ’hotspots’ for the crosstalk between environment and genes predisposing to either sex. Here, DNA methylation prompted by higher temperatures during embryonic development can change the sex ratio, yielding male-biased broods. Many of the genes here identified have been shown elsewhere to be important for reproduction or regulating DNA methylation.

The study’s first author, Dr Shahrbanou Hosseini, a postdoctoral researcher at the Molecular Livestock Science and Diagnostics Group of the Department of Animal Sciences at Göttingen University, said: "Here we show that epigenetic modifications influence the variation in sex ratio between zebrafish families. This implies that the interaction between genotype and environment in determining sex is mediated by epigenetics." Co-author Prof Bertram Brenig, the director of the Institute of Veterinary Medicine at Göttingen University, concluded: "Our results show that epigenetic regulatory mechanisms, such as DNA methylation, are important for shaping sex in species with genotype-by-environment sex determination, like zebrafish."

The researchers suspect that their results can be generalized to other species with a ’genes-by-environment’ sex determination system, for example lizards, crocodilians, and fish like Nile tilapia, Japanese flounder, European sea bass, and African catfish. Brenig warned: "Because their sex-determining system is sensitive to temperature, such species run a greater risk of becoming extinct when they produce maladaptive, strongly skewed sex ratios under the predicted global warming scenario."

Original publication: Hossein et al, -Epigenetic Regulation of Phenotypic Sexual Plasticity Inducing Skewed Sex Ratio in Zebrafish-, Frontiers in Cell and Developmental Biology.