Characterizing transcriptome in female scallop Chlamys farreri provides new insights into the molecular mechanisms of reproductive regulation during ovarian development and spawn

Highlights

• The study conducted a multi-stage ovarian transcriptome analysis in Chlamys farreri.

• The study identified some key signaling pathways and 14 candidate genes.

• This study will help reveal the reproductive molecular mechanisms behind bivalves.

Abstract

Bivalve mollusks are descendants of an early-Cambrian lineage and have successfully evolved unique strategies for reproduction. Nonetheless, the molecular mechanisms underlying reproductive regulation in mollusks remain to be elucidated. In this study, transcriptomes of ovary at four reproductive stages in female Chlamys farreri were characterized by RNA-Seq. Regarding signaling pathways, ECM-receptor interaction pathway, mTOR signaling pathway, Fanconi anemia pathway, FoxO signaling pathway, Wnt signaling pathway and Hedgehog signaling pathway were enriched during ovarian development processes. In addition, pathways related to energy metabolism such as Nitrogen metabolism and Arachidonic acid metabolism were enriched at spawn stage. Interestingly, Neuroactive ligand-receptor interaction was significantly enriched involved in ovarian development and spawn, and indicated the potential functions of nervous system on reproductive regulation in C. farreri. What’s more, this study identified and characterized fourteen genes involved in “sex hormones synthesis and regulation”, “ovarian development and spawn” and “maternal immunity” during the four reproductive stages in C. farreri. We determined that CYP17 uniquely affected gamete release by influencing the physiological balance among the steroid hormones and showed that receptors of the 5-HT and GABA neurotransmitters were tightly associated with ovarian maturation. Furthermore, to the best of our knowledge, this is the first study to report the maternal effect gene Zar1 in bivalve mollusks, likewise the maternal immunity genes displayed coordinated and cooperative expression during reproductive periods, which strengthened the environmental adaptation mechanisms of bivalves. Taken together, this study provides the first dynamic transcriptomic analysis of C. farreri at four key reproductive stages, which will assist in revealing the molecular mechanisms underlying bivalves on reproductive regulation in ovarian development and spawn.

Introduction

Reproductive success of individual animals is crucial to the survival of any species. Through the process animals maintain a diversity of characteristics, acclimatize to labile environments, and propagate for continuation of species (Morishita et al., 2010). Mollusks as one of the largest phyla of existing invertebrates appeared in 530 million years ago, has strong-adaptation to environment, little-alteration in morphology and habitual nature, and mollusks have successfully evolved unique strategies for reproduction (Feng et al., 2014, Vinther et al., 2017). For example, there are diverse reproduction modes possessed by gastropod and bivalve mollusks, mainly including dioecious and simultaneous or sequential hermaphrodites, besides some species are capable of changing sex under genotype and/or environment stress (Collin, 2013, Piferrer, 2013). Bivalves (e.g., Pacific oysters and pearl oysters) are known to adopt opportunistic reproductive tactics, adjusting their internal energy to meet the growth demand and using their surplus energy for multiple spawn throughout the year under certain conditions, such as low phytoplankton (Pouvreau et al., 2000, Royer et al., 2008). Therefore, studies on molluscan reproduction will not only strengthen our knowledge of the molecular mechanisms involved but may also be of value in understanding reproductive regulation in mollusks.

Normal ovarian growth contributes substantially to fertilization and improvement of zygote quality, and manipulating the development, maturation and release of oocytes are of great significance to ensure molluscan reproductive success (Chung, 2008, Erkan, 2009, Doucet-Beaupré et al., 2010). Additionally, oocytes contribute to zygote quality by the transfer of maternal immunity to offspring (Wang et al., 2015, Allam and Espinosa, 2016). However, few studies have conducted the systematic analysis on the development of gonads and regulation of gametogenesis over the reproductive processes in mollusks. Research has illustrated that molluscan reproduction is a genetically controlled response to the outside environment, regulated by endocrine system and nervous system, which accompanied with various biochemicals exquisitely orchestrated, including endogenous hormones, neurotransmitters and neuropeptides (Croll and Wang, 2007, Morishita et al., 2010). Although the effects of the above factors on ovarian growth and spawn have been investigated, the molecular mechanisms controlling their biosynthesis and mediating their physiological functions are still largely unknown (Li et al., 2016). Given that only limited studies have investigated the regulatory mechanisms of reproductive processes in mollusks, it remains challenging to adopt effective methods that can elucidate the underlying molecular mechanisms.

Next generation sequencing (NGS) technology is a powerful tool with outstanding advantages of high throughput, low cost, and high information output, which has created opportunities for exploring the transcriptome of non-model species at unprecedented sensitivity and depth (Meng et al., 2015). RNA-Seq technology studies on gonadal tissues have been conducted in a relatively large number of aquatic species, both in vertebrates (e.g., the rainbow darter, Etheostoma caeruleum; European seabass, Dicentrarchus labrax; ungeo, Coilia nasus) and invertebrates (e.g., crab Portunus trituberculatus, shrimp Procambarus clarkia, oyster Pinctada margaritifera), and many reproduction-related genes and pathways have been identified (Bahamonde et al., 2016, Ribas et al., 2019, Duan et al., 2015, Meng et al., 2015, Jiang et al., 2014, Teaniniuraitemoana et al., 2014). However, gonadal transcriptome studies of mollusks have mainly focused on screening and identifying genes that are related to sex differentiation and determination and comparing differences between the two genders (Teaniniuraitemoana et al., 2014, Yue et al., 2018, Ghiselli et al., 2012, Li et al., 2016). Studies which exploring the molecular mechanisms of gonadal development and gametogenesis regulated by reproductive endocrinology are relatively scarce, especially those investigating the specific stage of gamete release.

Bivalves are widely distributed with approximately 30,000 extant species and making up the second largest group of mollusks. Due to their diverse modes of reproduction, bivalves are used as an excellent model to study reproductive regulation (Appeltans et al., 2012, Yue et al., 2018). Chlamys farreri, naturally distributed around the seacoasts of North China, Korea, and Japan, is one of the most commercially important cultured bivalves with substantial ecological and economic value (Zhang et al., 2011). In the present study, we performed a comprehensive multi-stage (proliferative, growth, mature, and spawn stage) analysis of C. farreri ovarian transcriptome with the aim of elucidating the potential molecular mechanisms underlying reproductive regulation in ovarian development and spawn, which are not only of great significance to strengthen the biological cognition, but also make enormous contributions to accelerate the genetic improvement of economic species.