Introduction
The grey mullet, Mugil cephalus, are fished and farmed world-wide. To avoid the continuous pressure on the wild populations, we developed at the IOLR-NCM breeding and larval rearing protocols for captive mullets, along with developing formulated grow-out feeds for their rearing to market size. Following a success in closing the species’ life cycle in captivity, improved economics of production is expected by culturing all-female mullet populations, largely due to their highly prized roe used for preparing a seafood delicacy called “Karasumi”. To achieve this goal, the current study has adopted the indirect feminization strategy, involving the masculinisation of genotypic females, and crosses of the produced neomales with normal females, to produce a female monosex mullet population. Along these lines, our specific objectives were to: (i) optimize broodstock management and improve spawning success, (ii) establish functional neomales (=sex reversed females) (iii) develop sex-specific molecular markers for identification of the neomales, (iv) produce an all-female grey mullet genetic line.
Materials and Methods
Induced gonadal development and spawning – To stimulate gonadal development several hormone-based treatments were tested. In females, the effects of twined combinations of Metoclopramide (an antagonist of dopamine D2 receptors; DA) with either rFSH (yeast produced recombinant FSH) or GnRH-EVAc (implants for sustained release of the GnRH analogue) were tested. Experimentation with males evaluated the effect of r-FSH as a sole therapeutic agent vs. r-FSH use to prime the fish prior to the administration of MT-EVAc (implants for sustained release of methyltestosterone). To induce spawning, fully mature grey mullet females and males were treated with two consecutive injections consisting of GnRHa +DA given 22.5-h apart.
Induced masculinization – A masculinized phenotype was obtained by exposing for 4 months, hatchery produced mullet fry, at 3 age categories (3, 6 and 9- month old), to food supplemented with methyltestosterone (MT; 10 or 15 mg MT/Kg of food). The sex phenotype was monitored by histological secession of the gonads and/or gonadal biopsies.
Sex-specific molecular markers – Analysis of genomic alleles was based on whole- genome sequencing of a male and female. Based on sequence variation along the assembled target gene and read-pair information, sex-specific alleles were manually separated using Gap5 viewer. Variants that fit the XY system model were tested for association with sex by Sanger sequencing of sample of individuals from two families. Variants that passed this test were further examined on the population level by Sanger sequencing. The sex specific markers were validated and used to identify mullet neomales.
Results and conclusions
The optimal treatment consisting of DA+r-FSH, resulted with 91% post-vitellogenic females within the treatment-group. The r-FSH was a potent steroidogenic hormone during the onset of spermatogenesis. All males that were primed with r-FSH and then subjected to MT-EVAc implantation produced sperm. The spawning induction trials gave rise to high quality eggs and larvae and later on to large numbers of robust juveniles during natural (September-November) and shifted (January-February) reproductive seasons.
The results of the masculinizing studies highlight the period of 6 to 9 month of age, as a labile phase when the differentiating gonads are most susceptible to androgens. Regardless of the dose, administration of MT to the 6-month old fish, produced 100 % males upon the completion of sexual differentiation. After sexual maturity was achieved, spermiating mullet neomales were crossed with normal females giving rise to, the first all-female mullet stock, ever produced. Furthermore, body weight measurements revealed enhanced growth of the all-female mullet juveniles relative to mixed population of the same age class.
Altogether, our results corroborate the notion that the culturing of all-female mullet populations would improve production significantly due to the faster growth of females in comparison to sibling males and could provide mullet roe for Karasumi production if fish are reared to sexual maturity. Both of these advantages would improve the economics of mullet culture and justify rearing them in monoculture.
