Reunov et al. Helgol Mar Res (2018) 72:8 https://doi.org/10.1186/s10152-018-0510-x Helgoland Marine Research ORIGINAL ARTICLE Open Access Variation of sperm morphology in Pacifc oyster precludes its use as a species marker but enables intraspecifc geo‑authentifcation and aquatic monitoring Arkadiy Reunov1,2*, Evgenia Vekhova2, Evgeny Zakharov3, Yulia Reunova2, Yana Alexandrova2, Svetlana Sharina2,4 and Andrey Adrianov2,4 Abstract According to recent reports, shell morphology is unreliable for the identifcation of oysters because of the high phe- notypic plasticity of these bivalves. Using COI DNA barcoding and sperm morphology, we reinvestigated the species validity of wild Pacifc oyster Crassostrea gigas habituating the Peter the Great Bay (Sea of Japan). DNA barcoding con- frmed the species validity of samples collected. Application of the single sperm pattern was not possible for species identifcation due to pronounced sperm plasticity being found. Six sperm morphs were discovered in the testes of each oyster collected. The amount of abundant sperm morphs and the type of the most dominant sperm pattern are particular to geographical localities that are individual depending on the environmental factors. Ecological monitor- ing of marine areas and commercially assigned intraspecifc geo-authentifcation of the Pacifc oyster seems possible based on the analysis of this species’ heterogenic sperm. Further work will be needed to test if sperm heterogeneity exists in other Ostreidae species and if heterogenic sperms could be used for interspecifc analysis. Keywords: Pacifc Ocean, Sea of Japan, Crassostrea gigas, DNA barcoding, COI, Sperm Introduction both genetic data and morphological feature. Tus, a Te Pacifc shore in the far eastern region of Russia (Pri- reliable morphological trait is needed. Considering that morye) is a habitat for the wild Pacifc oyster that is not in some cases the sperm structure is used for bivalve an object of commercial fshing and represents a natural mollusc identifcation [7], it seems reasonable to inves- reserve of this commercially valuable bivalve mollusc. tigate if the morphology of spermatozoa could be used Tis oyster has been identifed as Crassostrea gigas based as a feature for the identifcation of the Pacifc oysters in on shell morphology [1–3]. However, shell appearance is Primorye. not a reliable feature for oyster identifcation because of During this work, we aimed to test if sperm morphol- high phenotypic plasticity in these bivalves [4, 5]. Taking ogy is specifc enough to be used as a taxonomically into account that the validity of C. gigas has never been signifcant feature for Pacifc oyster identifcation in Pri- genetically confrmed in Primorye, the genetic analysis morye. Using COI DNA barcoding that is assigned as of specimens seems necessary. According to Will et al. most convenient for species validity identifcation [8], [6], the most valid identifcation of species must be con- and proven for successful discrimination of other oyster ducted by integrative taxonomy using a combination of species [9], we also aimed to determine if the samples collected belong to C. gigas. *Correspondence: [email protected] 1 Electron Microscopy Laboratory, University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, ON K1Y 4W7, Canada Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/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. Reunov et al. Helgol Mar Res (2018) 72:8 Page 2 of 11 Materials and methods Total DNA was extracted from pieces of mantle Specimen collection (3–5 mm3) according to a standard phenol/chlorophorm Living specimens of the Pacifc oyster Crassostrea gigas extraction method [10]. Partial sequences of mitochon- (Tunberg, 1793) were collected in 2015. Te end of July drial cytochrome c oxidase subunit I gene (COI) were was chosen for collection because it is a period of natural amplifed and sequenced using the universal invertebrate spawning of oysters in Primorye. Four geographic locali- primer pairs: LCO1490—5′-GGT CAA CAA ATC ATA ties were chosen for sampling (Fig. 1). 1—location in the AAG ATA TTG G-3′ and HCO2198—5′-TAA ACT TCA Amursky Bay (the Peter the Great Bay, Sea of Japan, Rus- GGG TGA CCA AAA AAT CA-3′ [11]. sia, 43°14′27.69″N, 131°89′72.60″E). 2—location in the PCR amplifcation was performed in a 25 µl reaction Eastern Bosphorus Strait (the Peter the Great Bay, Sea of volume containing 17.4 µl of water; 2.5 µl of 10 × bufer Japan, Russia, 43°07′32.14″N, 131°84′34.27″E); 3—loca- (Evrogen); 2.0 µl of dNTP (concentration 2.5 mM of each, tion in the Ussuriisky Bay (the Peter the Great Bay, Sea Evrogen); 1 µl of each forward and reverse PCR primer of Japan, Russia, 43°18′83.65″N, 132°11′23.44″E); 4— (5 pmol/µl); 0.1 µl of Taq DNA polymerase (5 units/µl, location in the Vostok Bay (the Peter the Great Bay, Sea Evrogen); and 1 µl of template DNA solution. Te cycling of Japan, Russia, 42°89′32.45″N, 132°73′47.65″E). Tree parameters were an initial denaturation step at 94 °C males from each location were selected for both the (3 min), followed by 30 cycles of denaturation for 45 s at genetic analysis and study of male gametes. 94 °C, annealing for 90 s at 42 °C, and extension for 120 s at 72 °C; and a fnal extension phase at 72 °C for 5 min. Genetic analysis Amplifcation products were applied as templates for Specimens were deposited at the Canadian Centre for sequencing, using the same primers as for PCR and “Big DNA Barcoding, Biodiversity Institute of Ontario (Uni- Dye Terminator Cycle Sequencing Kit” v. 3.1 (Applied versity of Guelph, Canada) with the following catalogue Biosystems, USA) following the manufacturer’s protocol. numbers: YARRA063-12, YARRA064-12, YARRA065- Te cycling parameters were an initial denaturation step 12 (Amursky Bay, Sea of Japan, Russia); YARRA066- at 96 °C (1 min), followed by 25 cycles of 10 s at 96 °C, 12, YARRA067-12, YARRA068-12 (Eastern Bosporus 10 s at 42–49 °C and 4 min at 60 °C. Sequencing reaction Strait, Sea of Japan, Russia); YARRA069-12, YARRA070- products were purifed by ethanol precipitation and ana- 12, YARRA (Ussuriisky Bay, Sea of Japan, Russia); lysed on an ABI-3130 Genetic Analyzer (Applied Biosys- YARRA060-12, YARRA061-12, YARRA062-12 (Vostok tems, ABI, USA). Sequences were verifed by forward and Bay, Sea of Japan, Russia). reverse comparisons. Fig. 1 Pacifc oyster Crassostrea gigas. The map showing the geographic locations of specimens collected in Primorye. 1—location in the Amursky Bay (the Peter the Great Bay, Sea of Japan, Russia, 43°14′27.69″N, 131°89′72.60″E). 2—location in the Eastern Bosphorus Strait (the Peter the Great Bay, Sea of Japan, Russia, 43°07′32.14″N, 131°84′34.27″E); 3—location in the Ussuriisky Bay (the Peter the Great Bay, Sea of Japan, Russia, 43°18′83.65″N, 132°11′23.44″E); 4—location in the Vostok Bay (the Peter the Great Bay, Sea of Japan, Russia, 42°89′32.45″N, 132°73′47.65″E) Reunov et al. Helgol Mar Res (2018) 72:8 Page 3 of 11 Te contigs were obtained and edited using Chro- and critical-point dried in CO2. Dried materials were masPro v. 1.7.6 (http://www.techn elysi um.com.au/ mounted onto aluminum stubs, coated with gold, and chrom as.html). Eleven sequences, 658 bp in length, were examined with a scanning electron microscope LEO-430. obtained. All sequences and their metadata determined in this study have been deposited in the BOLD database Quantitative analysis (Barcode of Life Data System) (http://bolds ystem s.prg) under accession numbers YARRA060-12|RRYA-60– Te quantifcation of sperm morphology patterns was YARRA070|RRYA-70. Our dataset was compared done for the oysters with catalogue numbers YARRA063- with the sequences of C. gigas from GenBank (NCBI, 12, YARRA064-12, YARRA065-12 (Amursky Bay, http://www.ncbi.nlm.nih.gov/) and BOLD. Sequences Sea of Japan, Russia); YARRA066-12, YARRA067- of two species from the genus Crassostrea (C. virginica 12, YARRA068-12 (Eastern Bosporus Strait, Sea of (EU007485) and C. sikamea (AY632568)) were selected Japan, Russia), YARRA069-12, YARRA070-12, YARRA as the outgroup. (Ussuriisky Bay, Sea of Japan, Russia); YARRA060-12, Sequences were aligned using MUSCLE [12] imple- YARRA061-12, YARRA062-12 (Vostok Bay, Sea of Japan, mented in the program MEGA 5 [13]. MEGA 5 was also Russia). For each sample, 100 sperm cells were consid- used to calculate pairwise genetic distances (p distances) ered, thus 300 cells were studied for each location, and and to build trees. Kimura’s two-parameter model (K2P) 1200 sperms were investigated altogether. Sperm pat- [14] was used for estimation of intraspecifc and inter- terns were identifed by scanning electron microscopy specifc distances and constructed neighbor-joining (NJ) and the frequency of each pattern was calculated. All trees. All estimations of sequences and distances were values are expressed as means with standard error of the performed excluding the outgroup. Bootstrap analysis mean (SEM). Diferences between groups were calcu- with 1000 replications was used to assess the stability of lated using Student’s t test. P < 0.05 was considered statis- each node.