The influence of environmental factors, sex steroids and tributyltin on growth, survival and reproductive parameters in the European flat oyster (Ostrea edulis Linnaeus, 1758)

Abstract

The flat oyster Ostrea edulis (Linnaeus, 1758), a protandrous sequential hermaphrodite, is commercially valuable species in Europe but its populations have been collapsing since the mid1800s until recent years. In the Solent, Southern England, one such population of oysters has seen a significant reduction in the number of brooding female-phase oysters and skewed sex ratio towards male-phase oysters in recent years. Due to their ecological and economic importance, and numerous reintroduction schemes coming forward, understanding the many uncertainties still existing regarding their biology and reproduction are of critical importance. The aim of this thesis was to investigate some of the environmental factors (abiotic and biotic factors, and environmental pollutants) affecting physiological, biochemical and reproductive attributes in O. edulis. In order to understand the effect of temperature on sex determination and the timing of gametogenesis, two experiments were carried out. In the first experiment animals (n=49 per treatment) were treated at different temperatures (10, 14 and 18°C) under laboratory conditions for four months. To understand the changes occurring during different seasons of the year and the interaction with other environmental factors such as food availability, a second experiment was completed using 120 oysters kept under semi-enclosed conditions at the National Oceanography Centre Southampton from May 2016 to June 2017. For these two experiments the variables response included biometric parameters, gametogenic stage and sex determination. Gonadal tissue from these oysters was analysed by Enzyme Linked Immunosorbent Assay (ELISA) to determine sex steroid concentrations. The results suggested a significant influence of temperature on reproductive parameters during both experiments. High temperatures were related to accelerated gametogenesis in all the experiments. Sex ratios changed throughout both experiments with lower temperatures causing a female-biased sex ratio whereas more males were found with the increase of temperature. Food availability also showed a direct relationship with gonadal maturation, suggesting that this factor could play an important role in terms of energy allocation for sexual maturation. This study found a lack of relation between sex steroids in gonadal tissue and reproductive parameters, suggesting a different molecular pathway involved in reproduction in O. edulis and independent of vertebrate-related sex steroids.

In order to understand the effect and mechanism of action of exogenous sex steroids O. edulis was exposed to different concentrations of testosterone (20 ng/L (n=20) and 200 ng/L (n=20)), estradiol (5 ng/L (n=20) and 50 ng/L (n=20)), and a negative control (n=30) for 10 weeks. At the end of the exposure, gametogenic stage, hormones concentrations, energy reserves content and metabolomic profiling analysis were conducted to elucidate the metabolic alterations that occur in individuals exposed to those compounds. Results showed that oysters exposed to sex steroids presented an increase in mortality. It was also evidenced that O. edulis can uptake and accumulate estradiol and testosterone from the water. Although testosterone and estradiol did not cause masculinising or feminising effects other processes such as glycogenolysis and the synthesis of proteins and lipids were affected. In the same manner a reduction in metabolism evidenced by a lower content of energy reserves, down-regulation of molecules involved in the Tricarboxylic Acid (TCA) cycle and energy molecules was observed indicating an effect on homeostasis in O. edulis exposed to sex steroids.

Additionally, an experiment was carried out to evaluate the effect of a well-known endocrine disrupting environmental pollutant -tributyl tin (TBT)- to establish effects on sex determination, the gametogenic cycle, biochemical composition and the metabolome of O. edulis. Oysters were exposed to TBT chloride (20 ng/L (n=30), 200 ng/L (n=30) and 2000 ng/L (n=30) for 9 weeks. At the end of the exposure, gametogenic stage, hormones concentrations, energy reserves content and metabolomic profiling analysis were conducted to elucidate the metabolic alterations that occur in individuals exposed to those compounds. Oysters exposed to TBTCl presented an increase in mortality and changes in the gametogenic cycle with arrest in stages G0 and G1. Sex determination was affected by TBT causing a masculinization effect in O. edulis treated with low and environmentally relevant concentrations and an increase of inactive stages in oysters treated with high concentrations of this pollutant. The analysis of metabolites showed significant changes in the global biochemistry of oysters exposed to exogenous TBTCl by affecting ions involved in different and important molecular pathwayssuch as TCA cycle and creating an imbalance between molecules involved in oxidative stress and the antioxidant system. A TBT mechanism of actions is proposed based on the results obtained in this study and the literature review.

Overall this study showed that a rise in sea temperatures and food quality through the year could influence reproductive parameters in O. edulis, potentially affecting the long term health of populations and contributing to recent declines. The presence and persistence of environmental pollutants, such as sex steroids and TBT, could cause an additional threat to the declining O. edulis populations and related taxa around the world, by increasing mortality, changing reproductive maturation and skewing sex-ratios in natural populations. Furthermore, the presence of some intermediate molecules in the steroidogenesis pathway was detected in this study but their role in reproduction in invertebrates needs confirmation.

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Identifiers

ORCID for Christopher Hauton: orcid.org/0000-0002-2313-4226

ORCID for Ian Williams: orcid.org/0000-0002-0121-1219

ORCID for Antony Jensen: orcid.org/0000-0002-8924-1198

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