Pacific oysters, the most widely cultivated oyster species globally, contribute to over $89 million in sales in the United States alone. Oysters are intriguing sequential hermaphrodites, undergoing sex changes at various points during their lifespan. Nevertheless, the absence of secondary sex characteristics and the lack of sex chromosomes in this species remain a challenge for studying basic mechanisms of sex determination and also for the non-lethal sexing of animals. We set out to answer two questions to address cryptic sex states among oysters: 1. Are there specific early and/or later gene expression patterns that reliably predict final sex state of oysters at spawning time? and 2. Can these gene expression signals be detected from sampling hemolymph, which serves as the invertebrate equivalent of blood?
Hemolymph samples were repeatedly and non-lethally collected four times from the same group of marked individual oysters during the period prior to conditioning and throughout gonad maturation (Figure 1). Sex of each oyster were determined at the end of gonad maturation by gonadal biopsy. RNA-seq was used as a comprehensive discovery method to detect temporal differential expression patterns in male and female hemolymph samples. Genes involved in muscle growth, such as paramyosin (Ensembl Gene ID: G24796), were upregulated in females compared to males at the first sampled time point (26 days) (Figure 2). Genes involved in nutrient transport were upregulated in females compared to males at 36, 51 and 79 days, consistent with the observation of enlargement of eggs and vitellogenesis. Resolving markers and mechanisms of oyster sex determination will enhance the efficiency of spawning and crossing practices, potentially enabling controlled production of specific sex ratios and/or sterile oysters.