Journal of the Marine Larval development of the brush-clawed shore Biological Association of the United Kingdom crab Hemigrapsus takanoi Asakura & Watanabe, 2005 (Decapoda, Brachyura, Varunidae) cambridge.org/mbi Jose M. Landeira1,2, Jose A. Cuesta3 and Yuji Tanaka1 1Department of Ocean Sciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo Original Article 108-8477, Japan; 2Graduate School of Biosphere Science Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima, Hiroshima 739-8528, Japan and 3Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), Cite this article: Landeira JM, Cuesta JA, Avenida República Saharaui, 2, 11519 Puerto Real, Cádiz, Spain Tanaka Y (2019). Larval development of the brush-clawed shore crab Hemigrapsus takanoi Abstract Asakura & Watanabe, 2005 (Decapoda, Brachyura, Varunidae). Journal of the Marine The brush-clawed shore crab Hemigrapsus takanoi is a native species from the Western Pacific Biological Association of the United Kingdom and an invader of the European Atlantic coast from northern Spain to southern Denmark. – 1 12. https://doi.org/10.1017/ Despite the increasing concern about its rapid expansion, little is known about the early stages S002531541900002X in its life history. In the present study, the larval morphology of H. takanoi is described and Received: 12 June 2018 illustrated from specimens obtained in the laboratory from its type locality, Tokyo Bay, Japan. Revised: 12 November 2018 Its larval development follows the pattern of Varunidae, that involves five zoeae and one Accepted: 3 January 2019 megalopa. The morphological characters of the larvae of H. takanoi are compared with Key words: those of the other known Hemigrapsus species of the North Pacific. In addition, to facilitate Brachyura; DNA barcoding; Hemigrapsus an early detection of the invasive species of Varunidae inhabiting European Atlantic waters, a takanoi; invasive species; megalopa; summary of the key characters to identify their larval stages is included. morphology; taxonomy; zoea Author for correspondence: Jose M. Landeira, E-mail: [email protected] Introduction The brush-clawed shore crab Hemigrapsus takanoi Asakura & Watanabe, 2005 is a common species of inner bays and estuaries of East Asia, occurring in oyster beds and under boulders and rocks in the intertidal/subtidal zones (Miyajima & Wada, 2017). The native geographic range in East Asia includes the coast of Far East Russia (Marin, 2013), the Korean Peninsula (Lee et al., 2013) and Japan (Asakura & Watanabe, 2005). Moreover, H. takanoi is an invader of the European Atlantic coast. Since this crab was reported for the first time in 1994 in France (Noël et al., 1997), it has expanded its range rapidly in the Bay of Biscay from northern Spain to southern Brittany, and northward in the English Channel, including England (Ashelby et al., 2017) and the southern North Sea coast from the Cotentin peninsula to the Dutch Delta, the whole Wadden Sea, as well as the western Baltic Sea (Geburzi et al., 2015; Wood et al., 2015). Recently, genetic studies have revealed that European H. takanoi populations are at least partly formed by multiple introductions, likely via shipping lines, from genetically differen- tiated Asian populations (Makino et al., 2018). Interestingly, these results also revealed that the H. takanoi populations of Bay of Seine (France) consisted of a genetic admixture between populations in Japan and those in the Yellow Sea region, suggesting a high dispersal capability and connectivity of the European populations. In addition to H. takanoi, there are another five species of the family Varunidae H. Milne Edwards, 1853 inhabiting marine and brackish areas of the North-east Atlantic: Asthenognathus atlanticus Monod, 1933; Brachynotus atlanticus Forest, 1957; Brachynotus sexdentatus (Risso, 1827); Eriocheir sinensis H. Milne Edwards, 1853; and Hemigrapsus sanguineus (De Haan, 1835). The last two varunids, E. sinensis and H. sanguineus are also exotic species that were introduced from Asia (Streftaris et al., 2005). The tropical-temperate species A. atlanticus and B. sexdentatus are native from the African Atlantic coasts and Mediterranean Sea, respectively, but both have been introduced into north European coastal waters through shipping, where they likely have been favoured by the warmer conditions (Streftaris et al., 2005; Jourde et al., 2012). In the case of B. atlanticus, this species is distributed along the north-western coast of Africa and south of the Iberian © Marine Biological Association of the United Kingdom 2019 Peninsula, not being recorded farther to the north along the European Atlantic coast (D’Udekem d’Acoz, 1999; Marco-Herrero et al., 2015). For most crustacean decapods, dispersal is mainly undertaken during a larval phase (Anger, 2006). In this early life history stage, the ocean currents, their own behaviour, and the pelagic larval duration are key factors determining the magnitude of the dispersal capability (Weersing & Toonen, 2009). Therefore, larval studies are important to understand the population dynamics, and the complex patterns of connectivity (Selkoe et al., 2016). In fact, it has been particularly useful for the detection of non-native species in planktonic monitoring pro- grammes conducted in the Mediterranean Sea (Torres et al., 2012; Marco-Herrero et al., 2018). However, to date, these studies are not possible for H. takanoi, because its larval devel- opment is still completely unknown. The present study aims to provide a full description and illustration of the larval morphology of H. takanoi. Moreover, this article includes a summary Downloaded from https://www.cambridge.org/core. Tokyo University of Marine Science and Technology Shinagawa Library, on 16 Feb 2019 at 16:36:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531541900002X 2 Jose M. Landeira et al. of the key characters for the identification of the larval stages for described from proximal to distal segments and in order of endo- Varunidae in European Atlantic waters to facilitate their detection pod to exopod (Clark et al., 1998; Clark & Cuesta, 2015). The first and record their expansion. zoeal stage is described in detail, and only the main differences in subsequent stages are noted. Since only one megalopa was obtained in the culture, the description of this stage was based Materials and methods on both laboratory and field-collected larvae. For the same reason, Location and sampling the minimum duration of the megalopa stage was not possible to estimate. The specimens examined in this study are deposited in The specimens used for this study were collected in Daiba Park ′ ′′ ′ ′′ the National Museum of Nature and Science of Tokyo (catalogue (35°38 04 N 139°46 26 E), Tokyo, Japan. In this locality, placed numbers: NSMT-Cr 25833-25842). in the innermost part of Tokyo Bay, H. takanoi is the dominant intertidal crab, where the likelihood of finding the sibling species H. penicillatus is less than 1% of Hemigrapus spp. in our sam- DNA extraction, amplification and sequencing plings, and potential hybrids are low (Mingkid et al., 2006). In DNA barcode sequences have been obtained for parental females May 2017, ovigerous crabs were collected from the intertidal and megalopae collected in this study. The molecular confirm- zone by hand on substrates comprising a mix of mud, oyster ation and identification of the crab and megalopae were based shells and gravels. Only ovigerous females with embryos in an on partial sequences of the mitochondrial gene 16S. Total gen- advanced stage of development (eyes visible) were collected. In omic DNA was extracted from muscle tissue from the pereiopods the field, the specimens were identified following the key charac- following a modified Chelex 10% protocol by Estoup et al.(1996). ters of pigmentation pattern on the ventral face, the abdominal Target mitochondrial DNA from the 16S rRNA gene was amp- somites, and on the ventral surface of the cephalothorax described lified with polymerase chain reaction (PCR) using the following by Asakura & Watanabe (2005). In the laboratory, the identifica- cycling conditions: 2 min at 95°C, 40 cycles of 20 s at 95°C, 20 s tion was confirmed by DNA barcoding (see below). On 10 June ′ at 45–48°C, 45 s at 72°C and 5 min 72°C. Primers 1472 (5 -AGA 2017, 10 megalopa larvae were collected directly from mussels/ ′ TAG AAA CCA ACC TGG−3 )(Crandall&Fitzpatrick,1996) oysters patches and preserved in ethanol 80% for DNA identifica- ′ ′ and 16L2 (5 -TGC CTG TTT ATC AAA AAC AT-3 )(Schubart tion. After collection, the crabs were transported to the aquarium et al., 2002) were used to amplify 550 bp of 16S. PCR products facilities of the Tokyo University of Marine Science and were sent to Stab-Vida to be purified and then bidirectionally Technology at Shinagawa Campus. sequenced. Sequences were edited using the software Chromas version 2.0. Culturing and larval rearing With the obtained DNA sequences, a BLAST search was executed at NCBI webpage to get the sequence that best matches. All The ovigerous females were placed individually in 2 l plastic buckets sequences are deposited in GenBank under the accession numbers containing 1.5 l of 20°C and 25 salinity seawater (field conditions at MK418855–MK418857. the time of collection), with aeration and under natural daylight conditions. Every day, the water was changed, and the crabs were fed pieces of the seaweed wakame Undaria