Background: Pearl production by transplantation in Akoya pearl oyster (Pinctada fucata) is a biotechnology developed in Japan that skillfully utilizes the pearl-forming ability of oysters. In this method, cultured pearls are formed from a pearl nucleus and a small piece of mantle transplanted into the gonads of recipient pearl oysters. In this study, we hypothesized that the sex of the recipient pearl oyster might affect the quality of pearl produced. While some previous studies have examined the sex of Akoya pearl oyster, detailed information is lacking. Results: To investigate sex in Akoya pearl oyster, we collected small gonadal fragments from 1-year-old pearl oysters by biopsy. Using the collected gonad fragment, the sex of the oysters was determined by microscopic observation, and the remaining samples were stored for gene expression analyses. All oysters were labeled to distinguish each individual for serial samplings every four months over the 2-year study period. At the start of experiment, nearly all of the pearl oysters were male, but the male:female ratio ofmale decreased over the course of the experiment. Interestingly, the number of males increased after spring, during the breeding season. This suggests that, in pearl oyster, sex is affected by season. Expression analysis of sex-related genes (Dmrt2, Vtg, Zp) indicated that all genes were expressed in all individuals and all periods. Conclusions: These results suggest that Akoya pearl oysters are hermaphroditic, and that females appear as necessary, such as during the breeding season. These findings could contribute to higher efficiency and quality of pearl cultivation. Keywords: Gene expression, Germ cells, Gonadal stage, Hermaphrodite, Male ratio Background pearls has been underestimated. In transplantation, the Cultivation of Japanese Akoya pearl oyster (Pinctada pearl nucleus and a small piece of mantle are trans- fucata) for pearl production is an important traditional planted into the gonads of the recipient pearl oyster, marine industry in Japan, with a history of more than thus the gonadal condition of the recipient pearl oyster 100 years. The characteristics of cultured pearls are af- should affect the efficiency of pearl culture . Since fected by two kinds of pearl oysters: the donor, which germ cells, such as sperm and egg, in the gonads are dir- provides the small piece of mantle to be transplanted, ectly linked to the quality and efficiency of cultured and the recipient, in which the pearl nucleus and a small pearls, most pearl farmers have focused on managing piece of mantle are transplanted to produce a pearl. the condition of the gonads in pearl oysters, which is a Generally, the brightness, luster, and color of pearls are costly and labor-intensive process. affected by the donor oyster, while the thickness of the Although the sex of pearl oysters has been studied in nacre is affected by the recipient. In pearl culture, only the past, detailed information on the sex characteristics the recipient oyster needs to have a rapid growth rate of this specied is lacking. Understanding of the sex of and disease-resistance; hence, the quality of produced the pearl oyster is very important for their breeding, as well as for pearl culture. In a previous study , we * Correspondence: email@example.com found that the sex of the recipient pearl oyster greatly Laboratory of Fish Reproductive Physiology, Graduate School of Agriculture, affected the quality of cultured pearls. Male recipient Ehime University, Matsuyama, Ehime, Japan oysters produced commercially valuable pearls at higher Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mulyana et al. Zoological Letters (2018) 4:11 Page 2 of 7 rates than female recipient oysters. The average percent- Histological analysis of gonad age of low-quality pearls from male recipient oysters was Gonads of Akoya pearl oyster were collected in the final lower than that of the female recipient oyster. Differ- sampling period in August 2016 (40 months after hatch- ences in pearl formation ability between male and female ing) and were fixed in Davidson solution. Fixed gonads oyster in pearl formation can be explained by nacre were embedded in paraffin, cut into 5 μm sections, and growth. Nacre grew evenly every month in males, while stained with Delafield’s hematoxylin–eosin. These gonad there was a nacre growth stagnation phase between July sections were observed under a microscope to confirm and August and between September and October in fe- the sex and gonadal stage. male oysters . Male Akoya pearl oyster is more favor- able for pearl culture because it produces high-quality RNA extraction and cDNA synthesis pearls on average. However, Akoya pearl oyster’s sexual Total RNA extraction from gonad tissue was performed cycle is complex. There are three sexes in Akoya pearl using RNeasy® Plus Universal Mini Kit (QIAGEN, oyster: male, female, and hermaphrodite . Interest- Germany) following the manufacturer’s protocol. The ingly, several studies have reported that oyster species cDNA was synthesized using QuantiTect® Reverse have the ability to change sex [4–9], which adds further Transcription Kit (QIAGEN, Germany) following the complexity. Achieving a better understanding of manufacturer’s protocol. Synthesized cDNAs were then sex-changing patterns is important for Akoya pearl oys- diluted 10 times for gene expression analysis using spe- ter culture, due to the differences in pearl formation cific primers for genes involved in sex determination. ability in male and female oysters. The aim of the present study is to describe sex characteristics in Akoya Sex-related genes expression analysis pearl oyster using histology and gene expression RNA expression of three different sex-related genes, analysis. Dmrt2 (Double-sex and mab-3-related transcription fac- tor 2), Vtg (Vitellogenin), and Zp (Zona pellucida)in Methods gonad of Akoya pearl oyster were analyzed using reverse Biopsy of gonad fragment of Akoya pearl oysters transcription polymerase chain reaction (RT-PCR). Two μl In this study, we used 256 Akoya pearl oysters (Pinctada of the diluted cDNA solution were used for the PCR reac- fucata) produced from a single pearl oyster hatchery (Pearl tion (total volume: 25 μl). Amplification procedure of these Oyster Research Laboratory, Shimonada Fisheries genes followed protocol of GoTaq® G2 Hot Start Green Cooperative, Ehime Prefecture, Japan) in February and Master Mix 2X (Promega, USA). The specific primers for March 2013. All oysters were marked to distinguish each in- each gene were designed using a Blast Search of the Pinc- dividual for serial sampling. A fragment of gonad of Akoya tada fucata Genome (http://marinegenomics.oist.jp/pearl/ pearloysterwas takenbybiopsymethodonceinevery four blast/search?project_id=36)(Table 1). Amplification was months from April 2014 until August 2016, for a total of performed with programmable thermal cycler (Biometra seven sampling periods. Biopsy method was performed by Tprofessional BASIC 96 Gradient) consisting of needle aspiration through the gonad after the oysters were pre-denaturation at 95 °C for 2 min, followed by 40 cycles anesthetized with 0.35 M MgCl . Approximately of denaturation at 95 °C for 15 s, annealing at 55 °C for 100 mg gonad fragment was taken from each individual. 15 s, extension at 72 °C for 15 s, and an additional exten- Each sample was then divided into two parts. One part was sion step at 72 °C for 5 min. Amplicons were separated on fixed in Davidson solution and used for smeared-slide glass 2% agarose gel (100 V; 15 min) and visualized by staining preparation, while the other was subsequently treated with with ethidium bromide under UV light. RNAlater (Thermo Fisher Scientific, MA) or flash frozen in liquid nitrogen for gene expression analysis. Table 1 Nucleotide sequence of primers used in RT-PCR analysis Smeared-slide glass and Giemsa staining of biopsy Primer name Nucleotide sequence (5′-3′) sample Dmrt2 Forward primer ctc cat ttc caa cat tca tac aat a After fixation, a maximum of 20 mg of fixed gonad frag- Reverse primer tga tga agt tgc aga ctt tgg t ment was smeared on a slide glass with 12 rounded Vtg Forward primer gtt atg gag tca gaa ccg ttg a wells. Each well contained one sample. These slide Reverse primer gaa tga agc ggc att tcc glasses were stained by Giemsa staining solution for 30 min at room temperature. Giemsa-stained samples Zp Forward primer tga agg ttg cca tgg aga gt on slide glasses were then observed under an optical Reverse primer gat ttg ccc tct aag ttt gat cgt microscope (Olympus CX21). Sex of Akoya pearl oyster GAPDH Forward primer acc act gtc cac gcc att was determined by the presence of sperm or oocytes on Reverse primer act ctg gta taa ctt tgc cta cgg the smeared-slide samples. Mulyana et al. Zoological Letters (2018) 4:11 Page 3 of 7 Results (Fig. 3b), female-to-male sex change (FM) (Fig. 3c), and Sex ratio of Akoya pearl oyster male-to-female sex change (MF2) (Fig. 3d). Sperms were Male ratio based on biopsy results of gonad are shown observed in all seven sampling times of all-period male in Fig. 1. The graph shows fluctuating trends in the male oysters, while oocytes were observed throughout the ex- ratio in both groups throughout the study period. As perimental period in all-period female oysters based on much as 95% of total individuals were males on the first biopsy results. sex check 12 months after hatching. Male ratio always increased from spring to summer, which is the breeding Sex-related genes expression season in Akoya pearl oyster, while it always decreased RT-PCR analysis showed that Dmrt2 and Vtg were from summer to winter. Male ratio decreased from the expressed in four representative individuals, which ex- first winter to spring, and then increased in the next hibited different sex-changing patterns in all periods. such period (Fig. 1). These two genes were expressed in all periods, regard- Of individuals in the total sample, 35% remained male, less of the sex. Zp was also expressed in both male and while only 2% remained female throughout the experi- female, although not in all periods (Fig. 4). These results mental period, as determined from biopsy results. indicated that Akoya pearl oyster displays hermaphrodit- ism during its life cycle. Gonadal stage of Akoya pearl oyster Gonadal stage was determined by histological observa- Discussion tion (Fig. 2). Two types of spermatogenesis stage were Nearly all one-year old Akoya pearl oysters were males observed in male oysters. The mature stage was indi- during the first determination of sex in this experiment. cated by the dense volume of ripe spermatozoa, while in Initial functional sex of Akoya pearl oyster is typically the resting or spent stage of spermatogenesis residual male because male germ cells generally mature before spermatozoa filled the acini. All female oysters had female germ cells in . The increase in the number of already entered the resting stage of oogenesis, as residual males during spring to summer is equal to the decrease oocytes or atretic oocytes were observed in the gonads. of the number of females. One of the spawning seasons of Akoya pearl oyster is around June, when the water Determination of sex by biopsy temperature is warm. Spawning in closely related species There were several typical sex-changing patterns of of Akoya pearl oyster, such as Pteria sterna, P. imbricata Akoya pearl oysters found in this experiment, including and P. radiata, is initially triggered by rising water all-period male (AM) (Fig. 3a), all-period female (AF) temperature [8, 12, 13]. The oocytes are released outside Fig. 1 Male ratio of Akoya pearl oyster (P. fucata) at seven sampling times. The sex of individual Akoya pearl oysters in two different hatching groups were determined every four months by biopsy. Male ratio was calculated based on the sex obtained from biopsy check Mulyana et al. Zoological Letters (2018) 4:11 Page 4 of 7 Fig. 2 Photomicrographs of transversal section of representative Akoya pearl oyster (P. fucata) gonads showing stages in gametogenesis (a) male gonad in resting stage with residual spermatozoa, (b) male gonad in mature stage with ripe spermatozoa in dense volume, (c) female gonad in resting stage with residual or atretic oocytes. Res, residual spermatozoa; Rs, ripe spermatozoa; Ato, atretic oocyte. Scale bar, 200 μm to the water during spawning, so there are only a few or The presence of sperm and oocyte in the gonad of even no oocytes left in the gonad. It is predicted that Akoya pearl oyster is important because it directly af- Akoya pearl oyster always has two kinds of germ cells in fects the quality and efficiency of cultured pearls . a given individual at the same time. Some sperm cells Dmrt2 (double-sex and mab-3-related transcription remained in the female gonads after spawning, as factor 2) is an important gene in the maturation of spawning is generally incomplete . The sex was de- sperm because it plays critical role in spermatocytes termined to be male if only spermatogonia were ob- and spermatids differentiation into sperms . Vtg served when the gonad was checked by biopsy after (vitellogenin) expression suggests the occurrence of the spawning season had passed. Other reasons are vitellogenesis in the form of yolk protein autosynth- that gametogenesis always occurs actively and rapidly esis in ovary of marine bivalves . Zona pellucida , and spermatogonia proliferate more rapidly than (ZP) domain proteins are components of the egg en- oogonia . velope in invertebrates . Gonadal stage was observed using transverse sections Determination of sex by biopsy revealed that some of Akoya pearl oyster gonads. Whole gonad sampling Akoya pearl oysters possessed oocytes at certain times, time was done in August, when the spawning season and possessed sperm cells without any oocytes at a sub- had already elapsed two months before sampling time. sequent biopsy. The same pattern was also observed for The time of final sampling was the reason why the ma- individuals with sperm cells at certain sex determination jority of pearl oysters were in the resting or spent stage tests, but oocytes were observed in the gonad instead of of gametogenesis. Other than resting stage of gameto- sperm at the next biopsy. These results indicate that genesis as the majority of gonadal stage, the mature Akoya pearl oyster exhibit phenotypic bidirectional stage was also observed in several individuals. The differ- sex-change. However, RT-PCR result of three sex-related ent gonadal stages observed in P. fucata used in this genes, i.e. Dmrt2, Vtg and Zp showed that these genes study indicate that this species is a continuous spawner were expressed in all male and female pearl oysters dur- and could be due to continuously active and rapid gam- ing experimental period. This result was supported by etogenesis . Moreover, the reproductive cycle in P. similar gene expression trends of those male- and fucata is less distinct in tropical temperatures and female-related genes in all types of sex-changing pattern. spawning occurs continuously . The concerted expression of three sex-related genes Mulyana et al. Zoological Letters (2018) 4:11 Page 5 of 7 Fig. 3 Four typical sex-changing patterns of Akoya pearl oyster (P. fucata)(a) all-period male, (b) all-period female, (c) female changed to male, (d) changing between male and female. Sex-changing pattern was based from the sex of Akoya checked by biopsy at seven sampling times. Each symbol indicates: Sp, sperms; Ooc, oocyte. Scale bar, 200 μm indicates that sperm and oocyte are present at the same Several previous research studies reported similar re- time inside the gonad of Akoya pearl oyster. Our results sults supporting our results in the present study. In suggest that the Akoya pearl oyster is a simultaneous addition to being a transitional hermaphrodite, 0.2% of hermaphrodite species rather than a sex-changing P. margaritifera population exhibited simultaneous species. hermaphroditism . As much as 1.1% of P. radiata Fig. 4 RT-PCR analysis of Akoya pearl oyster (P. fucata) Dmrt2, Vtg and ZPB at seven sampling times. (a) AM: all-period male; (b) AF: all-period female; (c) FM: female changed to male; (d) MF2: changing between male and female. 1st-7th: sequence of sampling time Mulyana et al. Zoological Letters (2018) 4:11 Page 6 of 7 were simultaneous hermaphroditic pearl oysters, as they Funding This study was supported by a Grant-in-Aid from the Project of the NARO had both sperm and oocyte in the gonad concurrently Bio-oriented Technology Research Advancement Institution (the special . A few simultaneous hermaphrodites in P. imbricata scheme project on regional developing strategy) (c232). were also observed, although at very low levels . The Authors’ contributions occurrence of both male and female germ cells in JSM, TI, and MT performed the experiments. TI, CM, and TM contributed to the same gonad was also recorded in P. albina  conception and design of the experiments. AF and YW contributed to and P. fucata . supervision of the work and critical review of the manuscript. JSM and TI analyzed the data. JSM, TI, and TM wrote the manuscript. All authors read In contrast with the findings in this study, P. margari- and approved the final manuscript. tifera develops as male first then progressively changes to female after two years [6, 9]. Another member of Ethics approval and consent to participate genus Pinctada, P. maxima matures first as male during Not applicable. year one, indicating protandrous hermaphroditism . Competing interests Similar with P. margaritifera and P. maxima, Pteria The authors declare that they have no competing interests. sterna was also reported as a potential transitional hermaphrodite . Publisher’sNote The sex of Akoya pearl oyster was expected to be Springer Nature remains neutral with regard to jurisdictional claims in strongly affected by environmental factors, as females published maps and institutional affiliations. appear at breeding season; however, few individuals Author details with no sex change have been found. These results Department of Biology, Bogor Agricultural University, Bogor, West Jawa, indicated that sex of Akoya pearl oyster is influenced Indonesia. Laboratory of Fish Reproductive Physiology, Graduate School of Agriculture, Ehime University, Matsuyama, Ehime, Japan. Pearl Oyster by both environmental and genetic factors. Studies Research Laboratory, Shimonada Fisheries Cooperative, Uwajima, Ehime, involving oysters, namely Crassostrea gigas and Japan. Department of Aquatic Resources Management, Bogor Agricultural P. margaritifera , supported the theory that University, Bogor, West Jawa, Indonesia. Department of Global Environment Studies, Faculty of Environmental Studies, Hiroshima Institute of Technology, environmental factors combined with genetic mechan- Hiroshima, Hiroshima, Japan. ism control sex determination of adult oysters. 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Published: Jun 5, 2018