Sympatric Two-species Infestation by Rhizocephalan Barnacle Parasites in the Spider Crab Pugettia aff. ferox Ohtsuchi & Kawamura, 2019 from Peter the Great Bay (Northwestern Sea of Japan)

© 2021 Academia Sinica, Taiwan Open Access Sympatric Two-species Infestation by Rhizocephalan Barnacle Parasites in the Spider Crab Pugettia aff. ferox Ohtsuchi & Kawamura, 2019 from Peter the Great Bay (Northwestern Sea of Japan) Darya D. Golubinskaya1,*, Olga M. Korn1, Svetlana N. Sharina1, and Nikolai I. Selin1 1A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690041, Russia. Correspondence: E-mail: [email protected] (Golubinskaya). E-mail: [email protected] (Korn); [email protected] (Sharina); [email protected] (Selin) Received 12 January 2021 / Accepted 23 June 2021 / Published 1 October 2021 Communicated by Benny K.K. Chan Sympatric two-species infestation by rhizocephalan parasites in the spider crab Pugettia aff. ferox Ohtsuchi & Kawamura, 2019 (Brachyura: Epialtidae) was investigated in the Vostok Bay (Peter the Great Bay, northwestern Sea of Japan). Morphological and molecular analyses showed that this crab was infested simultaneously by Sacculina pugettiae Shiino, 1943 and Parasacculina pilosella (Van Kampen et Boschma, 1925) (Cirripedia: Rhizocephala). Sacculina pugettiae was found in the northwestern Sea of Japan for the first time. The two rhizocephalan species are clearly distinguishable by the morphology of their external cuticles, the shape and position of their receptacles, and the structure of their colleteric glands. Retinacula are present in the mature externae of both species. Molecular analysis showed that these rhizocephalans are unrelated, although both species parasitize Pugettia aff. ferox and are sympatric. Sacculina pilosella should be placed in the genus Parasacculina Høeg & Glenner, 2019, belonging to the family Polyascidae Høeg & Glenner, 2019. The intensity of infestation reached two externae in P. pilosella and three externa in S. pugettiae per host. A simultaneous settlement of two rhizocephalans on the same crab specimen was shown for the first time. The intensity of the two-species multiple infestations reached four externae per host. Externae with developing embryos occurred from June to September in P. pilosella and July to September in S. pugettiae, at water temperatures of 15–24°C, indicating that the reproductive periods of these species are confined to the summer months in the investigated locality. Key words: Rhizocephala, Parasacculina pilosella, Sacculina pugettiae, Morphology, Multiple infestation. Citation: Golubinskaya DD, Korn OM, Sharina SN, Selin NI. 2021. Sympatric two-species infestation by rhizocephalan barnacle parasites in the spider crab Pugettia aff. ferox Ohtsuchi & Kawamura, 2019 from Peter the Great Bay (northwestern Sea of Japan). Zool Stud 60:54. doi:10.6620/ ZS.2021.60-54. BACKGROUND The rhizocephalan barnacles (Crustacea: Cirripedia) are extremely simplified parasites that infest mostly decapods and some other crustaceans. The rhizocephalan female consists of two functional parts: an external reproductive body (the externa) connected through a stalk to an internal system of trophic rootlets (the interna). The externa contains an ovary, receptacles with dwarf males reduced to the spermatogenic cells, and a mantle cavity with developing embryos (Høeg et al. 2014). Since the number of morphological characters of the externa is very limited, molecular analysis is required to correctly identify rhizocephalan species. Zoological Studies 60:54 (2021) doi:10.6620/ZS.2021.60-54 1 © 2021 Academia Sinica, Taiwan Parasacculina pilosella (Rhizocephala: Polyascidae) was first described by Van Kampen and Boschma (1925) in Sumatra (Indonesia) on Quadrella coronata Dana, 1852 (Brachyura: Trapeziidae) and in Java on Ozius tuberculosus H. Milne Edwards, 1834 (Brachyura: Oziidae) and Eriphia sebana (Shaw & Nodder 1803) (Brachyura: Eriphiidae). Later, this species was also found in Seto (Honshu, Japan) on Q. coronata, Menaethius monoceros (Latreille, 1825) and Pugettia quadridens (De Haan, 1839) (Brachyura: Epialtidae) (Shiino 1943). Sacculina pugettiae (Rhizocephala: Sacculinidae) was described by Shiino (1943) in Seto (Honshu, Japan) on P. quadridens. Later, it was found in Samani (Hokkaido, Japan); peculiarities of this species in northern Japan were described by Boschma (1960). Shiino (1943) noted that these two parasites are easily distinguished from each other by the morphology of their external cuticle and the position of the receptacles, which are within the visceral mass in S. pugettiae and outside in P. pilosella. Thus, short morphological descriptions of both species are available, but the present study is the first to conduct any molecular analysis on either species. In Russian waters, P. pilosella was found on P. quadridens in 1997. The larvae of this rhizocephalan were reared under laboratory conditions. It was shown that the development of P. pilosella comprises five naupliar stages, as in most rhizocephalans (Korn and Rybakov 2001). Later, the muscular system in the interna of P. pilosella was visualized (Miroliubov et al. 2019) and specialized rootlets used to interact with the host’s nervous system were described (Lianguzova et al. 2021). In 2019, we found a second rhizocephalan parasite of P. quadridens, tentatively identified as S. pugettiae. Until now, it was believed that the spider crab recorded frequently in northeast Asian waters – including Japan, Korea, northern China, Hong Kong, and far-eastern Russia – was P. quadridens (De Haan, 1839) (Vinogradov 1950; Sakai 1976; Fuseya and Watanabe 1993). However, a detailed morphological investigation of Pacific Pugettia species showed that all specimens of P. quadridens from northeastern Japan – as well as from the Korean Peninsula, northern China and Russian waters – were most probably actually Pugettia ferox Ohtsuchi & Kawamura, 2019. Ohtsuchi and Kawamura (2019) did not present the molecular data on this new species from its type locality. Our material from Russia was identified by Dr. Ohtsuchi as P. ferox based on morphological characters (personal communication). Comparative molecular investigation of Pugettia from Peter the Great Bay using partial sequences of two mitochondrial loci (16S rDNA and COI) showed that these specimens differ from the typical Japanese P. quadridens. However, until a molecular analysis of P. ferox in the type locality is made, we identified them as Pugettia aff. ferox. The aim of this investigation was to identify both rhizocephalans from Russian waters using morphological and molecular methods and obtain preliminary data on spider crab infestations in the investigated locality. MATERIALS AND METHODS Sampling Specimens of Pugettia aff. ferox infested by Parasacculina pilosella and Sacculina pugettiae (Fig. 1) were collected by SCUBA diving at a depth of 1.5–3 m in Vostok Bay (Peter the Great Bay, Sea of Japan). Crabs were sampled once a month from May to September 2019. All material was fixed in 95% ethanol. One male and one female of Pugettia aff. ferox with the rhizocephalan externae undetached were deposited into the Museum of the A. V. Zhirmunsky National Scientific Center of Marine Biology, Vladivostok, Russia (MIMB, catalogue numbers 40810 and 40811). Morphological investigation of the externa The species identification of rhizocephalans was carried out by the shape and position of receptacles. This character can be seen on the living or fixed adults; the virginal stage was investigated using SEM. In both species, we measured the width of each detached parasite externa (the greatest dorsoventral distance), then recorded their developmental stage and the position on the abdominal segments. The following stages were identified: virginal externa (white, without spermatogenic cells in the receptacles), immature externa (yellow, without larvae in the mantle cavity), mature 1 (yellow, embryos in the mantle cavity without eyes), mature 2 (light brown, embryos in the mantle cavity with eyes). The carapace width (including lateral spines) of the host crabs (males and females) was also measured. The mantle cuticles from numerous externae of both species were fixed in 70% ethanol, dehydrated in an ethanol series and acetone, critically point dried in CO2, and sputtered with chromium. The SEM micrographs were taken with a Zeiss Sigma 300 VP microscope. Three externae of each species were detached from the host crabs and fixed in Bouin solution, dehydrated through a gradient ethanol-xylene series and embedded in paraffin. Transverse and longitudinal sections, 6 μm page 2 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan thick, were stained with Ehrlich hematoxylin, examined with a Carl Zeiss Axio Imager Z.2 light microscope furnished with a digital camera. The data on the dynamics of water temperature were obtained from a hydrometeorological station at the Vostok Marine Biological Station (A.V. Zhirmunsky National Scientific Center of Marine Biology FEB RAS). Fig. 1. The host crab, Pugettia aff. ferox, infested by Parasacculina pilosella (A, C), Sacculina pugettiae (B, D, E) and both rhizocephalans (F). 1, P. pilosella, 2, S. pugettiae, mo, mantle opening. page 3 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan Molecular investigation of the externa Live externae of both rhizocephalans were fixed in 95% ethanol. Voucher specimens were deposited into the Museum of the A.V. Zhirmunsky National Scientific Center of Marine Biology FEB RAS (MIMB, catalogue numbers 40795–40809). Total DNA was extracted from a piece of ovarian tissue using a CTAB extraction method (Dawson et al. 1998). Fragments of the mitochondrial largesubunit ribosomal RNA (16S rRNA) and cytochrome c oxidase subunit I (COI) genes were amplified and sequenced using the universal invertebrate primer pairs: 16SL3-Ven (5'-GCAAYGAGAGTTGTRCTAAGGT AGC-3') (Kappner and Bieler 2006) and 16SRHTB (5'-ACGCCGGTTTGAACTCAGATC-3') (Kocher et al. 1989) for 16S rDNA; LCO1490(F) (5'-GGTCAA CAAATCATAAAGATATTGG-3') and HCO2198(R) (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer at al. 1994) for COI. We also used: 18S5' (F) (5'-CTGGTTGATYCTGCCAGT-3') and 5R (5'-CTTGGCAAATGCTTTCGC-3') (Giribet et al. 1996) for fragments of nuclear markers 18S rDNA; LSU5 (F) (5'-TAGGTCGACCCGCTGAAYTTAAG CA-3') and LSU3 (5'-TCC TGA GGG AAA CTT CGG3') for 28S rDNA. PCR amplification was performed with a ScreenMix kit (Evrogen) and cycling parameters according the manufacturer’s protocol. The annealing temperatures were 42°C for COI, 52°C for 16S rDNA, and 60°C for 18S and 28S rDNA. Amplification products were applied as templates for sequencing, using the same primers as for PCR and BrilliantDye™ Terminator Cycle Sequencing kit v3.1 (NimaGen) according to the manufacturer’s protocol. Sequencing reaction products were purified by ethanol precipitation and analyzed on an ABI-3500 Genetic Analyzer (Applied Biosystems). Sequences were verified by forward and reverse comparisons. The contigs were obtained and edited using ChromasPro v. 1.7.6 (http://www.technelysium. com.au/chromas.html). A BLAST search (https:// blast.ncbi.nlm.nih.gov/Blast.cgi) was used to check new sequences against the database for possible contamination and sequence artifacts. All sequences determined in the present study were deposited deposited into GenBank (NCBI, http://www.ncbi.nlm. nih.gov/) under the accession numbers MW418446– MW418458 (16S rDNA), MW418428–MW418436 (18S rDNA), MW418439–MW418444 (28S rDNA), and MW401796, MW401797 (COI). Sequences of Peltogaster paguri (Peltogastridae) were selected as the outgroup. Sequences were aligned using MUSCLE (Edgar 2004) implemented in the MEGA X program (Kumar et al. 2018). The quality of alignment was checked visually. The models of nucleotide substitution for trees were selected using jModelTest v. 2.1.4 (Software Foundation, Inc., Boston, MA) (Darriba et al. 2012). The ТN+I+G model were selected for all genes separately under the Akaike information criterion. To construct BI-trees, MrBayes 3.2.6 was used (Huelsenbeck and Ronquist 2001), implemented in CIPRES Science Gateway (http://www.phylo. org/) (Miller et al. 2010), for the Bayesian analysis of 10,000,000 generations, with four parallel chains and sample frequencies set to 500, in two separate runs. Based on the convergence of likelihood scores, 25% of the sampled trees were discarded as burn-in. The uncorrected pairwise genetic distances (p-distances) for these species were calculated using MEGA X. The manuscript provides the tables and phylogenetic trees for only 16S and 18S rRNA genes. The rest of the data are available as supplemental materials. RESULTS Morphological identification of two rhizocephalan species Superorder Rhizocephala Müller, 1862 Family Sacculinidae Lilljeborg, 1861, amended by Høeg et al. (2020) Genus Sacculina Thompson, 1836 Sacculina pugettiae Shiino, 1943: 23–24, fig. 16. Sacculina pugettiae – Boschma 1960: 19–24, figs. 1–5. Family Polyascidae Høeg & Glenner, 2019 in Høeg et al. (2020) Genus Parasacculina Høeg & Glenner, 2019 in Høeg et al. (2020) Parasacculina pilosella (Van Kampen et Boschma, 1925) comb. nov.: 24–27, figs. 14, 15. Sacculina pilosella – Shiino 1943: 11–12, figs. 1E, 7; Korn and Rybakov 2001: 177–179; Miroliubov et al. 2019: 48–56, fig. 3; Lianguzova et al. 2021: 101009. Host: Carapace width of the males of Pugettia aff. ferox infested by rhizocephalans ranged from 14.0 to 31.0 mm, females – from 9.9 to 25.0 mm. Bathymetrical range: In Vostok Bay (Peter the Great Bay, Sea of Japan), crabs infested by both rhizocephalans were found at a depth of 1.5–3 m. Location on the host: The position of the externae of both rhizocephalans was not connected with specific abdominal segment of the host. Parasites were found on page 4 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan 1, 2, 3, 4, 5, 6 segments and also on the borders between 2 and 3, 3 and 4, 4 and 5 segments. Most crabs had only one rhizocephalan externa. External morphology: Parasacculina pilosella and Sacculina pugettiae were externally very similar (Fig. 1C, D). The width of the externae varied from 1.1 to 13.5 mm in P. pilosella and from 1.0 to 13.7 mm in S. pugettiae. The virginal externae of both species were white (Fig. 1E), immature externae – yellow (Fig. 1C, D), mature externae – yellow (embryos without eyes) or light brown (embryos with eyes) (Fig. 1F). The mature externae of both species had prominent dorsoventral wrinkles. The external cuticle varied from 22 to 40 µm thick. In P. pilosella, the external cuticle was covered by numerous hyaline spines 28–38 µm length united in groups with a common base. This character is visible on the SEM photos (Fig. 2A, D) as well as on the histological sections of the externa (Fig. 3A). In S. pugettiae, we found two types of the external surface. The external cuticle of about 2 thirds of the investigated specimens was smooth, without spines and excrescences, but often covered with the epibionts (Fig. 2B), the cuticle of about 1 third of specimens was divided into small star-shaped areas with a diameter of 5–8 µm (Fig. 2C). The cuticle of three found specimens was two-layered: smooth cuticle was folded back, revealing a star-shaped surface (Fig. 2E). The receptacles of the mature externae in both rhizocephalans were easily detached. In P. pilosella, isolated receptacles were globular with the cavity (lumen) inside (Fig. 4A). Their diameter was of 300–800 µm. The spermatogenic cells were placed in the central part of the receptacle. Each receptacle was connected to a folded receptacle duct by a short probably chitinous tubule (Fig. 4C, E). In S. pugettiae, the receptacles presented the elongate tubes, directed dorsoventrally, of 1200–1700 µm length and with a diameter of 200–600 µm (Fig. 4B). They were placed closely together but always clearly separated. The spermatogenic cells were found in the narrower dorsal part of the receptacle (Fig. 4D, F). The receptacle ducts were slightly flattened, of 200–300 µm width. In P. pilosella, the receptacles were located outside of the visceral mass in the basal region of the stalk (Fig. 4C, E), whereas in S. pugettiae, they were placed within the visceral mass (Fig. 4D, F). In P. pilosella, the colleteric glands were weakly branched from the atrium (central part of the gland) attaining 16 tubes (canals), arranged in one layer (Fig. 3C, E). In S. pugettiae, they were highly branched exceeding 33 tubes, arranged in several layers (Fig. 3D, F). The maximum number of tubes was located in the central part of the externa. Their diameter was 40–90 µm in P. pilosella and 30–80 µm in S. pugettiae. Retinacula: In both rhizocephalans, the internal cuticle had a wrinkled surface. In the mature externae of P. pilosella, the internal cuticle was covered with ridges spirally twisted and ended with short finger-like processes (Fig. 2F). We have not found the retinacula in the virginal externa of P. pilosella (4.4 mm width). In three virginal externae of Sacculina pugettiae (3–3.5 mm width), the retinacula were also not found. The internal cuticle of the fourth virginal specimen (2.3 mm width) was covered with numerous undeveloped flattened retinacula of 5 µm in diameter (Fig. 2G). In the mature externa of P. pilosella (10.5 mm width), rare solitary barbed spindles (9 µm length) placed in the shallow depressions were noted in the region of the stalk (Fig. 2H, I). In the mature externae of S. pugettiae (6.5–9.3 mm width), numerous retinacula presented the groups of 4–5 barbed spindles (6–8 µm length) at a common base placed in the shallow depressions (Fig. 2J, K). The retinacula of both species were covered with a layer of secretion and with numerous bacteria. Molecular identification of two rhizocephalan species The investigated samples of rhizocephalans were relegated into two clades – Sacculinidae for Sacculina pugettiae and Polyascidae for Parasacculina pilosella (pp = 1 for all markers) – confirming their status as different and not closely related species. Molecular data showed that all rhizocephalans implemented into the analysis form two monophyletic clades with high posterior probability (pp = 1 for all markers). These clades correspond to the families Sacculinidae and Polyascidae (Figs. 5, 6). However, for the family Sacculinidae, branch topologies within this clade are not identical for each gene. The specimens in the family Polyascidae form three groups of sequences (pp = 1). The first consists of Polyascus species and the second consists of Parasacculina species. The third group contains P. shiinoi (for 16S rDNA) and P. shiinoi + P. bicuspidata (for 18S rDNA), which are basal to other Polyascidae on the trees presented (Figs. 5, 6). The comparison of pairwise genetic distances indicated stronger differences between species of the families Sacculinidae and Polyascidae (Tables 1, 2). Preliminary investigation of the multiple infestation of Pugettia aff. ferox by rhizocephalans The preliminary data on the infestation of the spider crab Pugettia aff. ferox in Vostok Bay showed page 5 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan Fig. 2. SEM showing external cuticle (A–E), internal cuticle (F, G) and retinacula (H–K) of Parasacculina pilosella (A, D, F, H, I) and Sacculina pugettiae (B, C, E, G, J, K). hs, hyaline spines of external cuticle; r, retinacula. page 6 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan Fig. 3. Mantle (A, B) and colleteric glands (C–F) of Parasacculina pilosella (A, C, E) and Sacculina pugettiae (B, D, F). C, D, transverse sections; E, F, longitudinal sections. a, atrium of colleteric gland; em, embryos; exc, external cuticle; hs, hyaline spines, mc, mantle cavity; ov, ovary; tu, tubes of colleteric gland. page 7 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan that Sacculina pugettiae occurred more often than Parasacculina pilosella (Table 3). Over seven months, we found 86 specimens of Pugettia aff. ferox infested by rhizocephalans. Among them, 56 specimens (65.1%) possessed the externae of S. pugettiae, 14 (16.3%) – the externae of P. pilosella, and 16 specimens (18.6%) were infested by both rhizocephalans simultaneously. 80.3% of crabs with S. pugettiae had one externa, 12.5% – two externae and 7.2% – three externae of the parasite. All crabs with P. pilosella had only one externa. Moreover, each of 10 crabs possessed two externae of different species, five crabs – three externae, but one crab – four externae (two of S. pugettiae and two of P. pilosella). Thus, the intensity of infestation reached two externae Fig. 4. Receptacles of Parasacculina pilosella (A, C, E) and Sacculina pugettiae (B, D, F). A, B, light microscopy; C, D, longitudinal sections; E, F, transverse sections. l, lumen; ov, ovary; rd, receptacle duct; sc, spermatogenic cells. page 8 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan per host in P. pilosella and three externae per host in S. pugettiae. The intensity of two-species multiple infestations reached four externae per host. Pugettia females were infested more often than males. The virginal externae were found on host crabs from May to August in P. pilosella, and from May to September in S. pugettiae, gradually decreasing in number (Fig. 7). The externae of P. pilosella with developing embryos appeared in June, at a temperature of 14.6 ± 2.0°C; ovigerous externae of S. pugettiae appeared in July, at a temperature of 18.2 ± 1.7°C. Both ovigerous parasites occurred until to September. The immature externae were noted from May to September. In spring and early summer, the “old” immature Fig. 5. Bayesian inference analysis of 16S rDNA sequences for the Sacculinidae and Polyascidae. Numerals above or below the branches are Bayesian posterior probabilities. page 9 of 16Zoological Studies 60:54 (2021) © 2021 Academia Sinica, Taiwan Komai, 2000 (Decapoda: Anomura: Paguridae) in the northern part of the species range. J Mar Biol Assoc U K 99(1):101–109. doi:10.1017/S0025315417001679. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. doi:10.1093/molbev/ msw054. Lianguzova AD, Ilyutkin SA, Korn OM, Miroliubov AA. 2021. Specialised rootlets of Sacculina pilosella (Rhizocephala: Sacculinidae) used for interactions with its host’s nervous system. Arthropod Struct Dev 60:101009. doi:10.1016/ j.asd.2020.101009. Lützen J, Yamaguchi T. 1999. 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Crabs of Japan and the adjacent seas. Vol. 1. Kodansha Ltd., Tokyo. Shiino SM. 1943. Rhizocephala of Japan. J Sigenkagaku Kenkyusyo 1:1–36. Tsuchida K, Lützen J, Nishida M. 2006. Sympatric three-species infection by Sacculina parasites (Cirripedia: Rhizocephala: Sacculinidae) of an intertidal grapsoid crab. J Crustac Biol 26(4):474–479. doi:10.1651/S-2682.1. Van Kampen PN, Boschma H. 1925. Die Rhizocephalen der Siboga Expedition. Siboga-Exped., monogr. 31 bis. Vinogradov LG. 1950. Identification keys for shrimps, prawns and crabs of the Far East. Izvestiya TINRO 33:179–358. (in Russian) Waiho K, Fazhan H, Glenner H, Ikhwanuddin M. 2017. Infestation of parasitic rhizocephalan barnacles Sacculina beauforti (Cirripedia, Rhizocephala) in edible mud crab, Scylla olivacea. PeerJ 5:e3419. doi:10.7717/peerj.3419. Supplementary materials Fig. S1. Bayesian inference analysis of 28S sequences for the Sacculinidae and Polyascidae. Numerals above or below the branches are Bayesian posterior probabilities. (download) Fig. S2. Bayesian inference analysis of COI sequences for the Sacculinidae and Polyascidae. Numerals above or below the branches are Bayesian posterior probabilities. (download) Table S1. 28S uncorrected genetic distances between species of the families Sacculinidae and Polyascidae. Above the diagonal is the SD. (download) Table S2. COI uncorrected genetic distances between species of the families Sacculinidae and Polyascidae. Above the diagonal is the SD. (download) page 16 of 16Zoological Studies 60:54 (2021)