Crustacean intersexuality is feminisation without de-masculinisation: implications

19 20 The dysfunction associated with intersexuality in vertebrates and molluscs is often a 21 serious threat to ecosystems. Although poorly understood, crustacean intersexuality is 22 associated with contamination and includes forms linked to increased sex-ratio distorting 23 parasites at polluted sites. Despite the importance of crustaceans for monitoring 24 vulnerable aquatic habitats, little is known about the molecular basis of this abnormal 25 sexual differentiation and any associated sexual dysfunction. To increase the relevance 26 of crustaceans to environmental toxicologists, we comprehensively analysed gene 27 expression in amphipods presenting parasite and non-parasite associated intersexuality. 28 Our findings reveal existing vertebrate biomarkers of feminisation should not be applied 29 to crustaceans, as orthologous genes are not induced in feminised amphipods. 30 Furthermore, in contrast to vertebrates, where feminisation and intersexuality is often 31 associated with deleterious de-masculinisation, we find males maintain masculinity even 32 when unambiguously feminised. This reveals a considerable regulatory separation of the 33 gene pathways responsible for male and female characteristics and demonstrates that 34 evidence of feminisation (even if detected with appropriate biomarkers) is not a proxy for 35 de-masculinisation in crustaceans. This study has also produced a comprehensive 36 spectrum of potential molecular biomarkers that, when combined with our new molecular 37 understanding, will greatly facilitate the use of crustaceans to monitor aquatic habitats. 38