Understanding gene functions in marine invertebrates has been limited, largely due to the lack of suitable assay systems. Such a system requires investigative methods that are reproducible and can be quantitatively evaluated, such as a cell line, and a strong promoter that can drive high expression of a transgene. In this study, we established primary cell culture from a marine bivalve mollusc,Mizuhopecten yessoensis. Using scallop primary cells, we optimized electroporation conditions for transfection and carried out a luciferase-based promoter activity assay to identify strong promoter sequences that can drive expression of a gene of interest. We evaluated potential promoter sequences from genes of endogenous and exogenous origin and discovered a strong viral promoter derived from a bivalve-infectious virus, ostreid herpesvirus-1 (OsHV-1). This promoter, we termed OsHV-1 promoter, showed 24.7-fold and 16.1-fold higher activity than the cytomegalovirus immediate early (CMV IE) promoter and the endogenous EF1α promoter, the two most commonly used promoters in bivalves so far. Our GFP assays showed that the OsHV-1 promoter is active not only in scallop cells but also in HEK293 cells and zebrafish embryos. The OsHV-1 promoter practically enables functional analysis of marine molluscan genes, which can contribute to unveiling gene-regulatory networks underlying astonishing regeneration, adaptation, reproduction, and aging in marine invertebrates.

The objective of the present study was to analyze the phenotypic stability of sex after sex differentiation in the Yesso scallop, which is a gonochoristic species that has been described as protandrous. So far, no study has investigated in detail the sexual fate of the scallop after completion of sex differentiation, although bivalve species often show annual sex change. In the present study, we performed a tracking experiment to analyze the phenotypic stability of sex in scallops between one and two years of age. We also conducted molecular marker analyses to describe sex differentiation and gonad development. The results of the tracking experiment revealed that all scallops maintained their initial sex phenotype, as identified in the last reproductive period. Using molecular analyses, we characterized my-dmrt2 and my-foxl2 as sex identification markers for the testis and ovary, respectively. We conclude by proposing that the Yesso scallop is a sex-stable bivalve after its initial sex differentiation and that it maintains a sex-stable maturation system throughout its life. The sex-specific molecular markers identified in this study are useful tools to assess the reproductive status of the Yesso scallop.

It has been observed that 5-HT excites the heart nerves in hard clam and regulates contraction and relaxation of the anterior byssus retractor muscle in the blue mussel. It is now known that 5-HT regulates several neurobehavioral systems such as mood, appetite, sleep, learning, and memory. It also plays critical roles in the physiological functions of peripheral organs involved in stress, growth, and reproduction in the animal kingdom. The present study reviews conserved 5-HT biosynthesis and its localization in the nervous system, and its physiological contribution to regulate reproduction in bivalves. In the cytosol of neurons, tryptophan hydroxylase catalyzes hydroxylation of l-tryptophan to 5-hydroxytryptophan, which is converted to 5-HT by aromatic l-amino acid decarboxylase. A 5-HT transporter and a monoamine oxidase reuptakes and metabolizes 5-HT to control the amount of released 5-HT in the nervous system and peripheral organs. Perikarya and fibers of 5-HT neurons are mostly located in the cortices and neuropil of ganglia, respectively, and innervate the gonad. However, distribution and 5-HT content differ among species and sexes and undergo seasonal variations associated with gonadal development. The present review pays a special attention to future research perspectives to better understand 5-HT regulation of reproduction in bivalves.