Introduction
The alpheid shrimp genus Betaeus Dana, 1852 (Decapoda: Alpheidae) currently includes 16 valid species, which are distributed across different regions worldwide, with notably higher species diversity observed in temperate zones (Anker and Baeza, Reference Anker and Baeza2012; Hart, Reference Hart1964; Nomura and Komai, Reference Nomura and Komai2000). These 16 species are found in six biogeographical regions, one of which is the North-West Pacific (NWP), extending from the Russian Far East and Japan to Taiwan (Anker and Baeza, Reference Anker and Baeza2012). Within the NWP, three valid species, namely B. gelasinifer Nomura and Komai, Reference Nomura and Komai2000; B. granulimanus Yokoya, 1927; and B. levifrons Vinogradov, Reference Vinogradov1950, are known, while B. pingi Yu, 1930 is considered as a junior synonym of B. granulimanus (Anker and Baeza, Reference Anker and Baeza2012; Miya, Reference Miya1972).
Betaeus gelasinifer and B. granulimanus are free-living species commonly found on intertidal rocky shores, typically found under boulders and in tide pools (Kubo, Reference Kubo1936; Miya, Reference Miya1972; Nomura and Komai, Reference Nomura and Komai2000; Sato and Kuriyama, Reference Sato and Kuriyama2019; Yokoya, Reference Yokoya1936). Both species are mainly distributed in Japan and Korea, although the latter species was also reported from China, Hong Kong, and Taiwan (Miya, Reference Miya1972; Nomura and Komai, Reference Nomura and Komai2000; Yang et al., Reference Yang, Koo and Kim2007). In contrast, B. levifrons was described based on material collected from Zolotoi Rog Bay, Vladivostok, on the Sea of Japan side of the Russian Far East (Vinogradov, Reference Vinogradov1950). Marin (Reference Marin2010) redescribed B. levifrons based on specimens collected from Vostok Bay, located approximately 90 km east of the type locality. Some of Marin’s (Reference Marin2010) specimens were collected from the burrows of the mud-shrimps Upogebia major (De Haan, 1841) and U. issaeffi (Balss, 1913) (Decapoda: Upogebiidae).
Our surveys investigating Upogebia burrows and their associates at two localities in Hokkaido, Japan, led to the discovery of five specimens of B. levifrons in this area. The present study presents the first record of B. levifrons in the Northern Japan and provides supplementary data on this species, including colour variation and DNA sequence data from the mitochondrial 16S rRNA and cytochrome c oxidase subunit I (COI) barcoding regions.
Material and methods
Specimens of B. levifrons were collected using a suction pump (yabby pump) from the crustacean burrows in tidal flats of Akkeshi and Usu Bays, Hokkaido, Japan (Figure 1). A total of 90 and 50 burrows of U. major were surveyed at Akkeshi and Usu Bays, respectively. To check the host specificity of B. levifrons, 35 burrows of Alpheus brevicristatus De Haan, 1844 (Decapoda: Alpheidae) were also surveyed at Usu Bay. At each site, the yabby pumping was conducted three trials per burrow. It is important to note that burrow extraction may have pulled water and possibly organisms from other burrows, especially in the second and third trials. The occurrence rate was calculated as the number of burrows in which a given species was detected (n), divided by the total number of burrows surveyed (N), and expressed as a percentage (n/N, %).
Map showing sites where Betaeus species were recorded in the North-West Pacific region. Closed circles, solid squares, and broken lines represent B. levifrons, B. gelasinifer, and B. granulimanus, respectively (based on Kubo, Reference Kubo1936; Yokoya, Reference Yokoya1936; Vinogradov, Reference Vinogradov1950; Kim & Park, Reference Kim and Park1972; Miya, Reference Miya1972; Kim, Reference Kim1976; Suzuki, Reference Suzuki1979; Hayashi, Reference Hayashi1994; Nomura and Komai, Reference Nomura and Komai2000; Tatsu, Reference Tatsu2002; Motoh and Toyota, Reference Motoh and Toyota2005; Yang et al., Reference Yang, Koo and Kim2007; Motoh, Reference Motoh2008; Sato and Kuriyama, Reference Sato and Kuriyama2019). (A) and (B) show the sites of new records of B. levifrons in the present study: (A) Many burrow openings constructed by Upogebia major in Akkeshi Bay, Hokkaido, Japan; (B) tidal flat in Usu Bay, Hokkaido, Japan. (C) Upogebia major collected from Akkeshi Bay, Hokkaido, Japan.
Carapace length (CL) was measured in millimetres using a Mitutoyo CD-S15C caliper, from the tip of the frontal margin to the posterior margin of the carapace. Photographs of living shrimps were taken using a Canon 8000D digital camera, whereas images of the fixed specimens were taken with a Canon EOS 60D attached to a LEICA S9D. All collected specimens are deposited in the Hokkaido University, Hokkaido, Japan (HUMZ).
Tissue fragments from the individuals collected in Usu Bay were used for DNA extraction and analysis. The COI and 16S rRNA regions of the mitochondrial DNA were amplified by polymerase chain reaction (PCR), and the PCR product was purified using ExoSap-IT. Primer pairs used in this study were LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) for COI (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994), and 16SL2 (5′-TGC CTG TTT ATC AAA AAC AT-3′) and 1472 (5′-AGA TAG AAA CCA ACC TGG-3′) for 16S rDNA (Schubart et al., Reference Schubart, Neigel and Felder2000). The PCR included initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 45°C for 60 s for COI (54°C for 60 s for 16S), 72°C for 60 s, and a final extension at 72°C for 5 min. All PCR products were purified using Exo-SAP-IT (Thermo Fisher Scientific, Tokyo, Japan) and sequenced by Eurofins Genomics using specific PCR primers. The obtained sequences were deposited in the DDBJ, EMBJ, and GenBank databases; the corresponding accession numbers are provided in Table 1. These sequences were compared with previously published B. levifrons sequences from Russia (GenBank accession number for 16S: JX010748) available in public databases (Anker and Baeza, Reference Anker and Baeza2012).
Specimens of Betaeus levifrons Vinogradov, Reference Vinogradov1950 used for the molecular analysis in this study
Deposition institution abbreviations: NTOU, National Taiwan Ocean University, Keelung, Taiwan; HUMZ, Hokkaido University, Hokkaido, Japan.
Results
Family Alpheidae Rafinesque, 1815
Genus Betaeus Dana, 1852
Betaeus levifrons Vinogradov, Reference Vinogradov1950 (Figures 2, 3)
Betaeus levifrons (Vinogradov, Reference Vinogradov1950) collected in Hokkaido, Japan. (A and B) Ovigerous female (HUMZ-C 2419), CL 8.7 mm, Akkeshi Bay; (C) ovigerous female (HUMZ-C 2421), CL 8.1 mm, Usu Bay; (D) female (HUMZ-C 2422), CL 6.6 mm, Usu Bay; (E–G) male (HUMZ-C 2420), CL 8.8 mm, Usu Bay.
Uropod of Betaeus levifrons (Vinogradov, Reference Vinogradov1950) (HUMZ-C 2420) collected from Usu Bay, Hokkaido, Japan. Dorsal view.
(New Japanese name: kakure-teppouebi-modoki)
Material examined. 1 ovigerous female (CL 8.7 mm) [Figure 2A, B], HUMZ-C 2419, Akkeshi Bay (43°03′03.0″N 144°51′26.0″E), tidal flat, yabby pump from burrow of U. major, coll. Y. Henmi & G. Itani, 9 August 2017; 1 male (CL 8.8 mm) [Figure 2E–G, Figure 3], 1 ovigerous female (CL 8.1 mm) [Figure 2C], 1 female (CL 6.6 mm) [Figure 2D], 1 juvenile (CL 5.0 mm), HUMZ-C 2420–2423, Usu Bay (42°31′02″N 140°47′07″E), tidal flat, yabby pump, from burrows of U. major, coll. Y. Henmi & G. Itani, 12 July 2018.
Description
Frontal margin without orbital teeth and rostrum, exceeding the anterolateral margin, forming a well-developed, rounded orbital hood (Figure 2F); pterygostomian angle rounded (Figure 2E). Eyes completely concealed by orbital hood (Figure 2F). First pereopods carried extended anteriorly with the dactylus in ventral position; subsymmetrical and elongate; surface covered with minute conical granules (Figure 2C, E); gap between dactylus and pollex (fixed finger) small, including in the single adult male (Figure 2E); tips crossing, sharp. Uropods distinctly exceeding telson (Figure 2G); protopod bilobed distally (with central concavity), furnished with 12-18 long setae; exopod with long stout distolateral spine; diaresis with 12-18 blunt subtriangular teeth; distolateral margin with sharp, triangular tooth (Figure 3).
Colour in life
In the ovigerous females (Figure 2A–C) and male, all with CL > 8.1 mm, the body and chelipeds are uniformly light brown, whereas the ambulatory legs are pale pinkish in life. The eggs of the ovigerous females are pale green (Figure 2A) or pale orange brownish (Figure 2C), in the latter case possibly being at a more advanced developmental stage. In the smaller female (CL 6.6 mm), the body is greenish to pale brown (Figure 2D).
Ecology
The occurrence rates of each associated species are summarized in Table 2, which presents both the results from the initial sampling and the cumulative data from all three samplings. In Akkeshi Bay, 90 burrows of U. major were sampled using a yabby pump. In the first trial, 8 individuals of Gymnogobius mororanus (Jordan & Snyder, 1901) (Gobiiformes: Gobiidae) (8.9% symbiosis rate); 1 individual of G. macrognathos (Bleeker, 1860) (1.1%); 34 individuals of Cryptomya busoensis Yokoyama, 1922 (Bivalvia) (18.9%); and 8 individuals of Hesperonoe japonensis Hong, Lee, & Sato, 2017 (Polychaeta) (7.8%) were collected. In all three trials, 11 individuals of G. mororanus (12.2% symbiosis rate), 1 individual of G. macrognathos (1.1%), 1 individual of unidentified species of Gymnogobius, 184 individuals of C. busoensis (53.3%), 13 individuals of H. japonensis (13.3%), and 1 individual of B. levifrons (1.1%) were collected. Detailed information on these goby species is available in Henmi et al. (Reference Henmi, Inui, Goto and Itani2018).
Burrow-associated shrimp and goby collected by using a yabby pump
Ninety collections were made in Akkeshi Bay, 50 in Upogebia burrows in Usu Bay, and 35 in Alpheus burrows in Usu Bay.
a Associate fish data for Akkeshi Bay are from Henmi et al. (Reference Henmi, Inui, Goto and Itani2018). Occurrence rate (n/N, %) represents the number of burrows in which the associate species was found (n) out of the total examined burrows (N), with the percentage of occurrence. The first trial (1st) and the cumulative data from all three trials (1st–3rd) are shown.
In Usu Bay, a total of 50 burrows of U. major and 35 burrows of A. brevicristatus were sampled with a yabby pump. During the first sampling, three individuals of B. levifrons (6.0%), three individuals of Acanthogobius lactipes (Hilgendorf, 1879) (Gobiiformes: Gobiidae) (6.0%), one individual of G. breunigii (Steindachner, 1880) (2.0%), and one individual of Athanas japonicus Kubo, 1936 (Decapoda: Alpheidae) (2.0%) were collected from the burrows of U. major. In all three trials, four individuals of B. levifrons (8.0%) were collected from the burrows of U. major. All specimens of B. levifrons were collected separately, i.e., from different burrows. In contrast, the sampled burrows of Alpheus brevicristatus only harboured Acentrogobius virgatulus (Jordan & Snyder, 1901) (Gobiiformes: Gobiidae). In the first trial, two individuals of A. virgatulus (5.7%) were recorded, while a total of three individuals (8.6%) were collected in all three trials.
Molecular analyses
A total of 503 bp in the 16S rRNA region (accession number: LC763506) and 613 bp in the COI region (accession number: LC763505) were obtained from the ovigerous female of B. levifrons (HUMZ-C 2421) collected from Usu Bay. A BLAST search (Edgar, Reference Edgar2010) showed that the sequence obtained in this 16S rRNA region exhibited 99.6% homology with the sequence of B. levifrons collected in the Peter the Great Bay, Russia [accession number: JX010728 (Anker and Baeza, Reference Anker and Baeza2012)] (two base variants), thereby confirming the identity of the Japanese specimens. The COI region of Betaeus species has not yet been recorded (Chow et al., Reference Chow, De Grave, Anker, Poon, Ma, Chu, Chan and Tsang2021).
Distribution
(Figure 1)North-West Pacific Ocean (Hokkaido, Japan) and the Sea of Japan (Gulf of Peter the Great, Russia).
Discussion
The present specimens display all diagnostic characters of the genus Betaeus, such as the frontal margin without orbital teeth and rostrum, and the first pereopods (chelipeds) carried extended anteriorly with the dactylus in ventral position (Anker and Baeza, Reference Anker and Baeza2012; Hart, Reference Hart1964). Marin (Reference Marin2010) redescribed B. levifrons, noting that the uropodal protopod is furnished with 8 long setae and the exopod diaresis has 12 blunt teeth, whereas in our material, the uropodal protopod is furnished with 12–18 long setae and the exopod diaresis has 12–18 blunt teeth. Nevertheless, the overall morphological features align closely with the detailed redescription of B. levifrons provided by Marin (Reference Marin2010), and the DNA sequences were nearly identical. Therefore, these variations in the number of long setae and blunt teeth are likely attributable to intraspecific variability. Betaeus levifrons can be easily distinguished from the other two species recorded from Japan, namely B. granulimanus and B. gelasinifer, by its orbital hood being evenly rounded, i.e., convex (vs. centrally indented in B. granulimanus and B. gelasinifer) (Marin, Reference Marin2010; Miya, Reference Miya1972; Nomura and Komai, Reference Nomura and Komai2000).
Genetic analyses revealed that the Russian and Japanese specimens are not divergent at the species level. As only a single individual of B. levifrons from both Russia and Japan was analysed (Table 1), it remains unknown whether there is any genetic exchange between these populations.
All Japanese specimens of B. levifrons were collected from the burrows of U. major (Table 2), implying that it can be considered as an obligate symbiont of mud-shrimp burrows, as suggested by Marin (Reference Marin2010) and Marin and Antokhina (Reference Marin and Antokhina2020). Ovigerous females were collected in summer – in July from Usu Bay and in August from Akkeshi Bay. Additionally, according to information provided by Mr. Tomonori Sekioka (Hokkaido University) another ovigerous female was collected on June 21, 2024, from Akkeshi Bay. Owing to the limited number of individuals collected, further research is needed to better understand the life history of the Japanese populations of B. levifrons.
Mud-shrimp burrows are known to be inhabited by diverse symbionts (e.g., Henmi and Itani, Reference Henmi and Itani2014; Itani et al., Reference Itani, Kato and Shirayama2002; MacGinitie, Reference MacGinitie1935; Marin and Antokhina, Reference Marin and Antokhina2020). Macrobenthic symbionts inhabiting the burrows or on the body surface of U. major in the West Pacific are listed in Table 3. In Japan, the symbiotic fauna of U. major has been studied in Japanese Main Islands (Honshu, Shikoku, and Kyushu). Previous investigations in Hokkaido have shown that the scale worm Hesperonoe japonensis Hong, Lee, & Sato, Reference Hong, Lee and Sato2017 (Phyllodocida: Polynoidae) (Hong et al., Reference Hong, Lee and Sato2017; Sato et al., Reference Sato, Kato, Seike and Itani2016) and the bivalve Cryptomya busoensis Yokoyama, 1922 (Myida: Myidae) (Seike and Goto, Reference Seike and Goto2020) are symbionts of U. major burrows in Akkeshi Bay. In this study, Athanas japonicus, a facultative symbiont of U. yokoyai and A. brevicistatus (Henmi et al., Reference Henmi, Fujiwara, Kirihara, Okada and Itani2017; Henmi and Itani, Reference Henmi and Itani2021), was found to be symbiotic with U. major, in addition to B. levifrons. However, Stenalpheops anacanthus Miya, 1997, an obligate symbiont of mud-shrimp (Henmi and Itani, Reference Henmi and Itani2021) was not found in Hokkaido. In the burrows of Alpheus brevicristatus, the symbiotic shrimp Athanas alpheusophilus Marin, Reference Marin2017 has been documented in Posjeta Bay, Russia (Marin, Reference Marin2017), but its presence was not observed in this study in Usu Bay.
Macrobenthic symbionts inhabiting burrows or body surface of Upogebia major in the West Pacific
a Judging from the figure in Marin et al. (Reference Marin, Korn and Kornienko2011) and Marin and Antokhina (Reference Marin and Antokhina2020), “Sestrostoma balssi” from Upogebia burrows was identified as S. toriumii (Sato et al., Reference Sato, Jimi, Itani, Henmi and Kobayashi2023).
b Cryptomya busoensis is found in tidal flats where U. major is abundant, although direct observation of its symbiosis with burrows has not been confirmed (Itani and Kato, Reference Itani and Kato2002).
Anker and Baeza (Reference Anker and Baeza2012) showed that five species of the genus Betaeus (B. harrimani Rathbun, 1904, B. longidactylus Lockington, 1877, and B. ensenadensis Glassell, 1938 in the North-East Pacific, B. levifrons in the NWP, and B. jucundus Barnard, 1947 in the South-East Atlantic) are known to inhabit mud- or ghost-shrimp, as well as echiuran burrows. Like B. levifrons, B. ensenadensis, and B. jucundus appear to be obligate symbionts of upogebiid and callianassid shrimps (Anker and Baeza, Reference Anker and Baeza2012; Branch et al., Reference Branch, Griffiths, Branch and Beckley1994; Hart, Reference Hart1964; Marin, Reference Marin2010; Moyo et al., Reference Moyo, Pillay and Baeza2017, Reference Moyo, Pillay and Wright2018). Conversely, B. harrimani and B. longidactylus are facultative symbionts in the burrows of upogebiid and callianassid shrimps and echiuran worms, although they are occasionally observed living freely under rocks (Butler, Reference Butler1980; Chace and Abbott, Reference Chace, Abbott, Morris, Morris, Abbott and Haderlie1980; Hart, Reference Hart1964; Jensen, Reference Jensen2014). Within the genus Betaeus, there has been no documentation of facultative burrow symbionts in the Pacific Ocean. The diversification and evolution of symbioses within this genus represent intriguing research topics.
Acknowledgements
We would like to thank Dr. Fumihito Tashiro (Hokkaido University) for assistance during specimen registration, Dr. Kristian Q. Aldea (Catanduanes State University) for supporting, Mr. Tomonori Sekioka (Hokkaido University), Mr. Seiya Kanai (Kagoshima University), Mr. Hazuki Ono (Ebetsu city), Mr. Keito Tsunoda (Kanazawa University), Dr. Ryutaro Goto (Kyoto University) for sharing valuable information. We also thank the staff of the Akkeshi Marine Station, Hokkaido University, for their support during our stay. We sincerely thank the reviewers for their constructive feedback and valuable suggestions, which have significantly improved the quality of this paper. We also extend our gratitude to the editor, Dr. Antonio Baeza, for his efficient management of the manuscript and insightful guidance throughout the process.
Author contributions
Y. Henmi acquired funding, performed sampling, identified the species, performed DNA experiments, performed measurements and wrote the first draft of this manuscript. G. Itani acquired funding, performed sampling, identified the species and reviewed the manuscript.
Funding
This work was partly supported by a Grant-in-Aid for Fellows from the Japan Society for the Promotion of Science (JSPS Fellows) 15J12531, a Sasakawa Scientific Research Grant from The Japan Science Society to YH, and JSPS KAKENHI Grant Number 16K07233 to GI.
Competing interests
None.
