Induction of gynogenesis in the turbot (Scophthalmus maximus):

Abstract

Fish in which gynogenesis has been induced have all their chromosomes inherited from the mother and, if females are the homogametic sex, they usually are all females. Because turbot females grow faster than males, the production of all-female populations is highly desirable. The sperm of turbot is of poor quality and its larvae are small and fragile. These circumstances represent a challenge for the induction of gynogenesis in the turbot. As a first step towards this goal, effective conditions for the induction of gynogenesis through UV irradiation of sperm followed by a cold shock were established. When diluted 1:10 with Ringer-200 saline solution and placed in a thin layer (∼0.3 mm), a dose-dependent effect of UV light on sperm motility was found. The dose at which both the amount of motile sperm and the duration of sperm motility was reduced to 50% of the original value (ID₅₀) was ∼28,000 erg mm⁻². A typical Hertwig effect was elicited with a dose of 30,000 erg mm⁻². The resulting embryos exhibited the typical “haploid syndrome” and died shortly after hatching. Application of a cold shock (−1 to 0°C for 25 min starting at 6.5 min after fertilization) to activated eggs with UV-irradiated (30,000 erg mm⁻²) and diluted (1:10) sperms restored diploidy and resulted in the production of gynogenetic diploids (2n=44 chromosomes). These conditions were used in a pilot-scale experiment and found effective in inducing gynogenesis in ∼47,000 eggs. The rate of gynogenesis induction was 100% as verified by an analysis with microsatellite DNA markers. Survival of the gynogenetics was approximately 10% of diploids at 6 months of age, although growth was similar during this period. If this species turns out to have female homogamety, as is the case in most pleuronectiformes examined so far, the method presented here is the first necessary step for the production of all-female populations of this economically important species.

Introduction

Turbot (Scophthalmus maximus) is a species of growing importance for European aquaculture. Its production has steadily increased from 2966 mT in 1995 (FAO, 1997) to 5320 mT in 2002 (FEAP, 2003). Growth of turbot is affected by both sex and maturation. Males start to grow less than females as early as 8 months from hatch (Imsland et al., 1997), and this differential growth rate is maintained throughout the remainder of the production cycle including sexual maturation. Maturing females can reach 1.8 kg in 20 months whereas weight of males reaches only around 1 kg. As is practiced with other cultured species, it has been suggested that methods should be developed for the production of all-female populations of turbot (Imsland et al., 1997).

All-female populations of fish can be produced by direct hormonal treatment with estrogens to feminize sexually undifferentiated fish (see Piferrer, 2001 for review). However, despite that steroids are permitted for sex control during early development of fish in the legislation of many countries, this practice provokes consumer rejection and is not advisable. An indirect method based on the production of neomales (genetic females/phenotypic males) can be applied to obtain all-female progenies when the female is the homogametic sex (Piferrer, 2001). To the best of our knowledge, for turbot, there are no available data on hormonal methods, either direct or indirect, to produce all-female populations.

Alternatively, a short-cut approach to obtain all-female populations in fish is through the induction of gynogenesis. Gynogenesis is a chromosome set manipulation technique consisting of the generation of progenies whose chromosomes are exclusively inherited from the mother Chourrout, 1982, Thorgaard, 1983. The induction of gynogenesis involves DNA sperm inactivation while maintaining its capacity for triggering of embryonic development. The resulting embryos are haploid and nonviable posthatch, unless diploidy is restored by retaining the second polar body or by inhibiting the first mitotic division after shock treatment (Thorgaard, 1983). The induction of gynogenesis results in a low percentage of viable fish because of the manipulations involved and because of high inbreeding, but the resulting fish should be all females when sex determination involves female homogamety (Devlin and Nagahama, 2002). In practice, even in these cases, gynogenesis does not always ensure 100% females, although the offsprings are highly skewed in that direction (Felip et al., 2001, for review). These deviations can be explained by the influence of the environment or the role of secondary sex determination mechanisms Komen et al., 1992, Devlin and Nagahama, 2002. Because of the low viability of inbred gynogenetics, a practical approach is to sex-reverse gynogenetics for obtaining neomales for monosex milt production and to produce all-female progenies Piferrer et al., 1994, Donaldson, 1996, Felip et al., 2001.

Gynogenesis has other important applications for aquaculture and specifically to that of turbot. First, analysis of sex ratios in gynogenetic progenies can provide valuable data for assessing the sex determination mechanism Hunter and Donaldson, 1983, Nanda et al., 1992 which is not yet known in the turbot. Although inbreeding decreases viability, highly inbred lines could be crossed to exploit the dominant component of genetic variance Purdom, 1993, Tave, 1993. Finally, the use of haploid and diploid gynogenetics is broadly recognized as a useful tool for constructing genetic maps (Danzmann and Gharbi, 2001), which now are being implemented in turbot (L. Sánchez, personal communication).

A critical point of gynogenesis induction is the application of the appropriate UV dose to achieve the complete DNA sperm inactivation while maintaining the capacity to trigger embryonic development (Felip et al., 1999). Turbot exhibit poor sperm quality, with considerable variation in concentration among different males (Suquet et al., 1994), and lower larval survival (Devauchelle et al., 1988) when compared to other teleosts. On the other hand, of relevance for this study are the knowledge of turbot sperm physiological features (Suquet et al., 1994), the initiation of movement and swimming characteristics (Chauvaud et al., 1995), and the determination of the optimal sperm-to-egg ratio for fertilization Suquet et al., 1995, Chereguini et al., 1999. Furthermore, an optimized cold shock procedure to retain the second polar body is available Piferrer et al., 2000, Piferrer et al., 2003. The induction of gynogenesis has been reported for other flatfishes including the hirame, Paralichthys olivaceus Tabata, 1991, Kim et al., 1993, Yamamoto, 1999 and the common sole, Solea solea (Howell et al., 1995). Currently, gynogenesis is used in the practical aquaculture of rainbow (Oncorhynchus mykiss) and brown trout (Salmo trutta) in France, common carp (Cyprinus carpio) in China and Japan and of hirame in Japan (Hulata, 2001).

The objectives of the present study were: (1) to investigate the effects of UV light on turbot sperm in regard to its ability to fertilize (activate) eggs and trigger embryonic development, (2) to determine the optimal conditions to induce gynogenesis in the turbot, and (3) to study early development and viability of gynogenetic progeny.