Research paperCharacterizing transcriptome in female scallop Chlamys farreri provides new insights into the molecular mechanisms of reproductive regulation during 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.
Section snippets
Sample collection and preparation
Scallops used in this study were maricultured in strict accordance with national and institutional (the Ocean University of China) guidelines for the protection of human subjects and animal welfare. Experimental C. farreri scallops, mostly at the proliferative stage, were purchased from Shazikou (Yellow Sea, Qingdao, China). Female scallops were further selected based on the color of gonad, the female was pale-orange, while the male was pale-white (Chung, 2008) and maintained in 60 L tanks (1 L
Analysis of sequenced data quality
The 12 ovarian cDNA libraries constructed for Illumina sequencing and the data processing results are summarized in Table 2, showing the clean reads used for further analyses after removing low quality data. A total of 631,853,466 clean reads were filtered from 645,811,106 raw reads of 150 bp pair-end generated by Illumina sequencing. The values of Q20 (%) and Q30 (%) ranged from 96.7 to 97.7% and 91.05 to 93.46%, respectively. Additionally, the average size of the clean data generated from
Discussion
Recently, the genome of C. farreri has been sequenced and published (Li et al., 2017), indicating that the studies on C. farreri have entered the post-genomic era. Nonetheless, the regulatory molecular mechanisms underlying reproduction in mollusks has not been elucidated to date, and the clarification of the underpinning signaling pathways, as well as the reproductive candidate genes with gonadal development and gamete release remains challenging. In this study, we examined four defined stages
Conclusion
The current study profiled ovarian transcriptomic variations in the course of four key reproductive stages in C. farreri, and explored the underpinning signaling pathways that regulate the reproductive process. Additionally, this study identified and characterized 14 candidate genes involved in “Sex Hormones Synthesis and Regulation”, “Ovarian Development and Spawn” and “Maternal Immunity”, a number of which, such as Zar1 and Wnt10a, are the first to be investigated in bivalve mollusks. The
CRediT authorship contribution statement
Ruiyi Xu: Conceptualization, Investigation, Data curation, Writing - original draft, Writing - review & editing, Funding acquisition. Luqing Pan: Conceptualization, Supervision, Project administration, Funding acquisition. Yingying Yang: Data curation. Yueyao Zhou: Data curation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by The Key Research Project of Shandong (2018) (2018GHY115007) and Fundamental Research Funds for the Central Universities (201861030). We thank the staff at the Laboratory of Physiology for the help with sampling and taking care of the scallops. The authors would like to thank the editors and the anonymous reviewers for their valuable comments and suggestions on this paper.
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