Systematic, molecular, and (eco)toxicology investigations of endocrine mechanisms in molluscs

Abstract

Molluscs, integral to ecosystems as habitat engineers and food sources, are extremely sensitive to environmental pollution. While these animals can serve as early indicators for endocrine-disrupting chemicals (EDCs), current testing guidelines lack mechanistic underpinning due to our poor understanding of molluscan endocrinology. Previous molecular investigations have demonstrated that molluscan genomes lack essential enzymes and nuclear steroid receptors to induce vertebrate steroidogenesis. However, some steroidogenic enzymes do appear to exist in molluscs, but their function remains unknown. These include the enzymes 5-alpha-reductases (5αR1, 5αR2) that metabolise testosterone to dihydrotestosterone in vertebrates. Previous work has shown that developmental exposure of Biomphalaria glabrata embryos to pharmaceutical 5αR disruptors (dutasteride or finasteride) causes a highly reproducible and dose-dependent disruption to development, resulting in altered shell morphology. However, the impact of dutasteride on adult B. glabrata remains unexplored. This doctoral thesis aimed to fill significant knowledge gaps relating to endocrinological pathways in molluscs, investigate the expression of 5αR genes in the embryonic B. glabrata and evaluate the effects of dutasteride disruption on B. glabrata adults. Using the systematic review guidelines PRISMA, data on the occurrence of hormones, hormone receptors and hormone-metabolising enzymes in Mollusca was identified from 145 eligible studies (published between 2012-2021) and was systematically collected, evaluated and visualised in an openly accessible interactive database. By developing a RT-qPCR assay and validating stable reference genes across embryonic stages of B. glabrata, the transcript expression of genes encoding 5αR1 and 5αR2 was assessed. Lastly, the effects of dutasteride on B. glabrata adults were evaluated using a 21-day flow-through exposure and an OECD 243 static-renewal test, both with nominal exposure concentrations of 0, solvent control, 1 μg/L, 3.2 μg/L, 10 μg/L, 32 μg/L and 100 μg/L dutasteride. The systematic investigations revealed that most studies assessed were found to be heavily skewed towards vertebrate-type sex steroidogenesis, with over 62% measuring 17β-estradiol in mollusc tissues, despite unconvincing evidence that molluscs can biosynthesise vertebrate-type steroids. However, a limited number of studies are now looking beyond vertebrate-type sex steroids and diverting their focus towards the role of thyroid hormones, phytosterols (plant sterols) and ecdysteroids (insect steroids) in molluscs. The results of the RT-qPCR experiments suggest that 5αR1 and 5αR2 genes are not differentially expressed across day 2 - day 4 post-oviposition embryonic stages. Whereas the minimal variation of the five candidate reference genes UBI, TUB, EF1a, ACTIN-1, and H2A across day 2 - day 5 post-oviposition stages, makes them reliable models for normalising gene expression in embryonic B. glabrata. Importantly, under static-renewal conditions, dutasteride was observed to cause unexpected mortalities and significant declines in reproductive output of adults at concentrations above 3.2 μg/L and 32 μg/L, respectively. Together, the findings of this thesis provide novel insights into underexplored endocrinological pathways in molluscs that could be targets of endocrine disruption. Moreover, novel findings on the expression of 5αR in embryonic B. glabrata and data on the reproductive toxicity of dutasteride may help enhance our understanding of the role of 5αR in this species.