The Flathead grey mullet ( Mugil cephalus ) is a cosmopolitan marine fish. Most of its production comes from capture fisheries, yet, the aquaculture of this species, where fries are captured in estuaries and acclimated to grow out in fresh- to brackish-water ponds is becoming more and more significant, with Egypt being the major producer. Mullet females grow faster than males and ovaries of mature females are desired in the roe industry. Hence, producing all-female progeny populations is a major advantage for mullet hatcheries and producers. Only recently, steady production of mullet fry in hatcheries has become a reality. Now, it is the appropriate time to integrate genetically improved broodstock for production of all-female fingerlings. Developing such genetically improved broodstock requires understanding the sex-determination system in mullet, developing sex-reversed broodfish and developing assays to determine the genetic sex of individuals to identify sex-reversed fish. Here we report on our advancements towards developing sex-reversed males for mullet.
In mullet males and females are hard to distinguish until ready to spawn. We sampled mature fish and identified their sex based on gonad type. These fish were also genotyped by several thousands of SNP markers. We constructed a genetic map and mapped a single sex-determination genomic region. Using comparative genomics, a few candidate sex-determining gene located to this genomic region were identified. Our results corroborate two other, recently published studies, identifying the same sex-determining genomic region for several mullet populations from the Mediterranean Sea. All these studies pointed to a XX/XY sex-determination system in most populations. Thus, sex-reversed males (males with an XX rather than XY genotype) are needed as broodfish to cross with regular XX females to produce all-female (XX) progeny populations.
Developing a protocol for sex-reversal in the mullet takes time since fish mature after about two years, only then allowing to test if the natural 1:1 sex ratio was biased. Fish at early stages were fed with androgen (Methyl-testosterone) treated food. In one experiment, the control group showed a 1:1 sex ratio, whereas the treated group had 63% males, significantly more than expected, suggesting some of the males in this treated group are sex-reversed. From this group, we have fish grown under broodstock conditions.
Finally, based on the SNP markers, we developed molecular sexing assays, allowing to determine the sex of individuals based on their DNA only. By applying the molecular assays to control-group fish, we determined which genotypes are of females and which are of males. By applying these assays to fish from the sex-reversal group, some fish with testes but a female genotype were identified, in accordance to the biased sex ratio. Therefore, indeed this group included some sex-reversed males. We then, tested live fish held as broodfish by stripping and identified males. By testing their DNA, some were indeed sex-reversed. These will be soon tested as broodfish to produce all-female progeny population.
Taken together, all elements of the required methodology were developed to a stage when production of all-female groups can be tested. Further experiments are needed to tune-up the different parts of the methodology in order to allow full-scale and efficient production of all-female hatchery fingerlings, a technology that will significantly contribute to production of mullet in aquaculture and protection of the natural populations in seas.