Induced reproduction in Pātiki (Rhombosolea leporina), a novel endemic aquaculture candidate

Abstract

The rapid global population growth combined with the decline of wild-capture fisheries has resulted in the global expansion of aquaculture. New Zealand’s aquaculture industry remains largely underdeveloped and as such, industry growth would benefit from species diversification. The endemic yellowbelly flounder (Rhombosolea leporina) is a promising candidate species for New Zealand aquaculture due to its high market value, fast growth rate and the global demand for flatfishes. At present, very limited knowledge exists regarding the reproductive biology of this species, and notably the control of reproduction in captivity. Successful finfish aquaculture requires a robust understanding of reproduction for effective broodstock management. This thesis investigates induced ovulation in yellowbelly flounder using a gonadotropin releasing hormone analogue (GnRHa). The study pairs physiology and genetics to advance the current knowledge of reproduction in this species. GnRHa was administrated to induce ovulation in yellowbelly flounder, over a 28-day period. One of three treatments were assigned to 27 female yellowbelly flounder (n=9): 50 µg/kg GnRHa, 100 µg/kg GnRHa, or a sham treatment of 50 µg/kg Ringer’s solution. Histological and fresh oocyte analyses were carried out and batch fecundity, fertilization and ovulation rates were assessed. Reproductive dysfunction experienced by yellowbelly flounder in this study was through incomplete germinal vesicle migration and a failure to enter hydration. Ovulation was only observed in GnRHa treated fish and the ovulatory periodicity appeared to be approximately 72 hours in fish that ovulated more than once. This study confirms that yellowbelly flounder are multiple group synchronous batch spawners. Although fewer fish than expected ovulated (four), significantly more GnRHa treated fish completed final oocyte maturation and entered hydration compared to the controls. Furthermore, fecundity, fertilization rates and egg viability were high, with fertilization >80% achieved in some initial ovulations. In contrast, eggs obtained from fish that had ovulated for a second time, were characterised by low fecundity and fertilization rates. Nonetheless, this data indicates GnRHa can successfully induce ovulation at dosages of at least 50 µg/kg without adversely affecting egg quality in at least the first batch. Further refinement is essential to optimize a protocol for the reliable induction of reproduction in this species. Low incidences of ovulation may be a factor of dosage or inappropriate ovarian maturity at the time of hormone administration. This highlights the need to confirm oocyte maturity immediately prior to GnRHa treatment. To aid future work, a classification index for the identification of ovarian developmental stage based on fresh oocyte morphology was developed for yellowbelly flounder. To further advance future research into yellowbelly flounder reproduction, brain, pituitary and gonad transcriptomic libraries were generated for female yellowbelly flounder using RNA-seq. Protein sequences of eight reproduction related genes were successfully identified and characterised, which were StAR, Gpr54, Lhβ, Fshβ, Fshr, Cyp19a1a, Cyp19a1b and Hsd17b1. Conserved functional regions, phylogenetic analysis and homology with sequences from other Pleuronectiformes confirmed the identities of these genes and indicated that their functional roles are likely conserved. Characterisation of these genes provides highly valuable information for future quantitative assessment of gene expression throughout the yellowbelly flounder reproductive axis. The results presented in this thesis will help to inform future work to control and optimize reproduction in captive yellowbelly flounder. In addition, the genetic data will enable molecular tools to be developed, including quantitative PCR primers as well as the potential for recombinant gonadotropins to develop enzyme-linked immunosorbent assays. These tools will be highly valuable to accurately assess the effects of GnRHa treatment on reproduction and gene expression under differing culture conditions. This study indicates that successful ovulation and good quality eggs may be achieved in yellowbelly flounder using GnRHa treatment.