Journal of the Marine Biological Association of the United Kingdom cambridge.org/mbi Morphological observations and molecular confirmation of larvae of Levisquilla inermis (Crustacea: Stomatopoda) from the Seto Inland Sea Alyaa Elsaid Abdelaziz Fadl1,2 Original Article Cite this article: Fadl AEA, Yamaguchi S, Wakabayashi K (2021). Morphological observations and molecular confirmation of larvae of Levisquilla inermis (Crustacea: Stomatopoda) from the Seto Inland Sea. Journal of the Marine Biological Association of the United Kingdom 101, 801–810. https:// doi.org/10.1017/S002531542100076X Received: 15 June 2021 Revised: 19 October 2021 Accepted: 21 October 2021 First published online: 24 November 2021 Key words: 16S rRNA; cincinnuli; crustaceans; DNA barcoding; mantis shrimp; stomatopod larvae; taxonomy Author for correspondence: Kaori Wakabayashi, E-mail: kaoriw@hiroshima-u.ac.jp , Shuhei Yamaguchi3 and Kaori Wakabayashi1 1 Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739–8528, Japan; 2Department of Zoology, Faculty of Science, Kafrelsheikh University, Kafr Elsheikh, Egypt and 3School of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739–8528, Japan Abstract Mantis shrimps are commercially important crustaceans in many areas of the world. In contrast to the relatively common studies of adults, limited studies have been attempted of larvae because of a lack of identification keys. The objectives of this study were two-fold: (1) to link wild-caught larval specimens from the Seto Inland Sea to a certain species and (2) to present a detailed morphological description of the last larval stage. The resulting molecular phylogenetic tree based on 16S rRNA gene sequences strongly implies that our larval specimens were linked to Levisquilla inermis, which was barcoded from a newly collected adult specimen with a morphological identification. K2P genetic divergence was found to be 0% among the larval and adult specimens. The congeneric species L. jurichi was the sister group of the L. inermis cluster, but the K2P distance between them was 3.4%, and it was considered a distinct species. Morphological observations provided five major distinguishing characteristics of the last-stage larva of L. inermis: (1) propodus of second maxilliped with three basal spines, (2) all five pleopods possessing an appendix interna with cincinnuli, (3) exopod of uropod longer than the endopod, (4) distal margin of the exopod of the uropod reaching the lateral tooth of telson, and (5) telson with nine intermediate denticles. Overall, our results reveal the importance of combining molecular and morphological analyses for solving stomatopod larval identification. This finding can be used to support and facilitate future research on the taxonomy and biodiversity of stomatopod larvae. Introduction © The Author(s), 2021. Published by Cambridge University Press on behalf of Marine Biological Association of the United Kingdom Mantis shrimps or stomatopods (Crustacea: Malacostraca) are predatory marine crustaceans, which are found mainly in tropical and subtropical coastal waters and are characterized by the remarkably developed second maxilliped that has been modified as a powerful raptorial appendage (Ahyong et al., 2008). Almost 500 species of stomatopods are known and divided into 17 families within seven superfamilies (Ahyong & Harling, 2000; Ahyong et al., 2008; Van Der Wal & Ahyong, 2017; Hwang et al., 2019; WoRMS, 2021). The life history of stomatopods involves a series of larval stages consisting of short propelagic and long pelagic phases. The last-stage larval metamorphosizes into a post-larva and settles into the adult benthic habitat after a single moult (Pyrne, 1972; Feller et al., 2013). Larval forms of stomatopods are highly specialized among crustacean larvae as they possess a remarkable set of morphological characteristics such as large, fully functional raptorial second maxillipeds and often large body size that can be up to 50 mm in total length (Ahyong et al., 2014; Wiethase et al., 2020). Stomatopods can exhibit a greater variety of ecological adaptations in larvae than in adults due to their distinct larval forms (Haug et al., 2016). While all stages of the stomatopod larvae can be clearly recognized from other crustacean larvae, identifying them to the species level based on their morphological characters is still difficult. Only around 10% of known species could be identified in their larval stages (Diaz, 1998; Haug et al., 2016). Traditionally, the identification of stomatopod larvae has been performed either by hatching larvae from a known adult or by cultivating wild-caught larvae to adulthood; however, these laboratory methods are time-consuming and extremely challenging (Provenzano & Manning, 1978; Diaz, 1998; Feller et al., 2013). Descriptions of the entire series of larval stages using those two methods exist for five of the 500 extant species: (1) Neogonodactylus oerstedii (Hansen, 1895) (as Gonodactylus oerstedii) (Manning & Provenzano, 1963; Provenzano & Manning, 1978), (2) Neogonodactylus wennerae Manning & Heard, 1997 (as Gonodactylus bredini Manning, 1969) (Morgan & Goy, 1987), (3) Pterygosquilla schizodontia (Richardson, 1953) (as Squilla armata H. Milne Edwards, 1837) (Pyne, 1972), (4) Heterosquilla tricarinata (Claus, 1871) (Greenwood & Williams, 1984) and (5) Oratosquilla oratoria (De Haan, 1833–1850) (Hamano & Matsuura, 1987). Even partial larval series are known only for a restricted number of species (Gurney, 1946; Alikunhi, 1967; Michel, 1968, 1970; Michel & Manning, 1972; Shanbhogue, 1975; Rodrigues & Downloaded from https://www.cambridge.org/core. IP address: 3.231.220.57, on 13 Dec 2021 at 18:17:26, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531542100076X 802 Manning, 1992; Ahyong, 2002; Veena & Kaladharan, 2010; Feller et al., 2013; Cházaro-Olvera et al., 2018). Therefore, a scarcity of information linking larvae to adult forms clearly exists. To overcome the obstacles and complexities of traditional taxonomic methods, particularly for groups of species with multiple developmental stages, DNA barcoding as a taxonomic method has been applied for the early life stages (Abinawanto et al., 2019). DNA barcoding, proposed by Hebert et al. (2003), is one of the molecular identification methods that is intensely applied in taxonomic study of groups and an effective tool for linking larvae with their adults. DNA barcoding was originally performed by analysing the short nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene from taxonomically unidentified specimens and comparing them with the known sequences stored in public databases, such as the GenBank database and Barcode of Life database. Nowadays, diverse gene regions are successfully used for DNA barcoding in a wide range of both aquatic and terrestrial animal taxa (Hebert et al., 2004a, 2004b; Hajibabaei et al., 2006; Wakabayashi et al., 2006, 2017; Hubert et al., 2008; De Grave et al., 2015; Carreton et al., 2019). A DNA barcode is also useful to identify larvae collected from the field into species by matching with the available sequences from GenBank. Such methods have already been applied to crabs, barnacle larvae, fish larvae and stomatopods (Tang et al., 2010; Chen et al., 2013; Chu et al., 2019; Li et al., 2019; Wei et al., 2021; Wong et al., 2021). In addition, DNA barcoding as a method for identifying stomatopod larvae has been used not only for taxonomy (Barber & Erdmann, 2000; Feller et al., 2013) but also for experimental (Feller & Cronin, 2014; Feller et al., 2019) and diversity or distribution studies (Barber et al., 2002; Barber & Boyce, 2006; Tang et al., 2010; Abinawanto et al., 2019). Although this method has been utilized effectively for stomatopod identification, it is still in the early stages of development since the DNA sequences available for barcoding stomatopods (such as COI and 16S rRNA genes) are limited (Tang et al., 2010). Therefore, as demonstrated in the current study, the assignment of larval specimens to the species level may need the addition of more sequences from the adult specimens with verified morphological identification. Although the diagnostic morphologies of stomatopod larvae are scarce, the late-stage larvae are easily classified into at least the superfamily level using the morphological characters of carapace, abdomen, uropods and telson (Ahyong et al., 2014). The larvae of the superfamily Squilloidea can be distinguished from those of the other superfamilies based on a flatter carapace and a slender abdomen (Ahyong et al., 2014). In Japan, 25 out of 70 recorded species are classified in the Squilloidea (Ahyong, 2012; Osawa & Fujita, 2016; Nakajima et al., 2020) and among them, late-stage larvae have been described in 12 squilloid species (Alikunhi, 1944, 1952, 1975; Manning, 1962; Hamano & Matsuura, 1987; Zheng et al., 2006; Veena & Kaladharan, 2010; Wong et al., 2021). During our recent cruise in the Seto Inland Sea of Japan, late-stage larvae with a pair of large orange spots on the basal part of the fifth pleopods were found. Such larvae have not been described until now. In this paper, we document a detailed morphological description of the squilloid larvae, which were confirmed as Levisquilla inermis (Manning, 1965) via a DNA barcoding technique using an adult specimen found at the same location on the same day. Materials and methods Sampling The larval specimens were sorted from plankton samples collected by an Ocean Research Institute (ORI) net (Omori, 1965; Omori et al., 1965) with a diameter of 160 cm and mesh size of Alyaa Elsaid Abdelaziz Fadl et al. Fig. 1. Levisquilla inermis (Manning, 1965), live: (A) larva (ventral); (B) adult (dorsal). Scale bars: A, 1 mm; B, 5 mm. 0.33 mm that was operated by the crew of the training and research vessel ‘TOYOSHIO MARU’, Hiroshima University. The net was towed in the Seto Inland Sea (33°49′ 59′′ N 132°23′ 41′′ E) at a depth of 30–40 m on 29 October 2020. The adult specimen was found in sediment that was dredged from a depth of 70 m at the same location on the same day. Both live larval and adult specimens were photographed (Figure 1) and then preserved in 99% ethanol for subsequent morphological and molecular analyses. Morphological observation The larval specimens were observed and dissected under a stereomicroscope (SZX-9, Olympus). The stereomicroscope was also used for morphometric characteristics, including the total length (TL) from the tip of the rostrum to the apex of the submedian tooth on the telson, the rostrum length (RL) from the tip to the base of the rostrum, the carapace length (CL) as the distance from the base of the anterolateral spine to the base of the posterolateral spine on the carapace, and the carapace width (CW) as the maximum distance across the carapace. The dissected appendages were preserved in 70% ethanol or mounted on glass slides using a mounting agent (CMCP-10 high-viscosity mountant; Polysciences, Warrington, PA, USA), and then observed under a compound microscope (BX-51, Olympus). The developmental stage of the larval specimens was determined according to Hamano & Matsuura (1987). They hatched larvae from females of the Japanese mantis shrimp and illustrated the entire sequence of larval stages in detail. Illustrations for larval and adult specimens were created with the aid of the camera lucida attachment (U-DA, Olympus). The terminology of the body structure followed the structures of Hamano & Mantsuura (1987), Ahyong (2001) and Feller et al. (2013). An adult male mantis shrimp was also included for the gene analysis to confirm larval identity. This specimen was morphologically identified as Levisquilla inermis based on the dactylus claw with six teeth (Figure 2A), the fifth thoracic somite with short, anteriorly recurved spine (Figure 2B), the absence of supplementary carinae on the dorsolateral surface of the telson (Figure 2C), and the absence of a post-anal carina. The total length measured from the tip of the rostral plate to the apices of the submedian teeth of the telson was 25.4 mm, and carapace length measured along the dorsal midline excluding the rostral plate was 6.1 mm. Downloaded from https://www.cambridge.org/core. IP address: 3.231.220.57, on 13 Dec 2021 at 18:17:26, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531542100076X 803 Journal of the Marine Biological Association of the United Kingdom Fig. 2. Levisquilla inermis (Manning, 1965), adult male: (A) right raptorial claw (lateral); (B) lateral processes of fifth, sixth and seventh thoracic somites; (C) last abdominal somite and telson (dorsal). Scale bars: A–C, 1 mm. Both larval (NSMT-Cr 29221, 29222) and adult (NSMT-Cr 29223) specimens were deposited in the crustacean collections of the National Museum of Nature and Science, Tsukuba, Japan (NSMT). Molecular analysis Genomic DNA was extracted from one pleopod with some pleon muscles of both larval and adult specimens using NucleoSpin® Tissue XS (Cat. no. 740901.50, Machery-Nagel, Germany) according to the manufacturer’s instructions. A polymerase chain reaction (PCR) was used to amplify the partial sequence of the mitochondrial large subunit ribosomal RNA (16S rRNA) gene in 50 μl of reaction volume using the following primers: (1) 1471 and (2) 1472 (Crandall & Fitzpatrick, 1996). The PCR reaction mixture contained 50–100 ng DNA template, 0.2 μM of each primer, 5 μl of 10 × polymerase buffer, 0.2 mM of dNTPs and 1.25 unit of Taq polymerase. PCR was performed using a thermal cycler (Thermal Cycler Dice ® Touch, TaKaRa, Japan) under the following temperature conditions: (1) initial denaturation at 94 °C for 5 min, (2) followed by 35 cycles of denaturation at 94 °C for 30 s, (3) annealing at 50 °C for 30 s, (4) extension at 72 °C for 1 min and (5) a final extension at 72 °C for 10 min. The PCR products were visualized on a 1.5% agarose gel to confirm the size and quality and then purified using the Qiaquick Gel Extraction Kit (Qiagen, Germany). Cycle sequencing for the PCR products was performed using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) under the following temperature regimes: (1) denaturation at 96 °C for 30 s, (2) annealing at 50 °C for 15 s, and (3) extension at 60 °C for 4 min. These steps were repeated for 25 cycles. Nucleotide sequences of the amplified fragments were post-purified and then evaluated with capillary electrophoresis using an ABI PRISM 3130 xl Genetic Analyzer (Applied Biosystems) at the Natural Science Center for Basic Research and Development, Hiroshima University. All sequences were checked on the Sequence Scanner Software v2.0 (Applied Biosystems). Bases expressing ‘N’ were visually checked with the original waveforms. The nucleotide sequences of the studied species were deposited under OK575769–OK575771 through the International Nucleotide Sequence Database Collaboration at the National Center for Biotechnology Information, Bethesda, MD, USA. Multiple sequence alignment was performed using MUSCLE (Edgar, 2004) on MEGA 7 (Kumar et al., 2016). Gblocks v.0.91b (Castresana, 2000) was used to eliminate divergent regions and inadequately aligned positions in the dataset; 306 positions in total were used in the final dataset. Neighbour-joining (NJ) tree with bootstrap analysis (10,000 replications) was constructed based on the Kimura 2-parameter (K2P) model (Kimura, 1980) using MEGA 7. The 16S rRNA gene sequences resulting from the 44 squilloid species are currently available from the GenBank, and 42 species among them were acquired for the molecular analysis (Belosquilla laevis [KR153534] formed a long branch, Cloridina moluccensis [MH168209] completely matched with Busquilla quadraticauda [MH168223]) as shown in Table 1. Up to three sequences were selected to represent a species when several different sequences were available within a species (applied to 12 species in total) in which those sequences derived from the different studies were clustered for all 12 species; thus, the terminal branch of each species was compressed in the final tree (Figure 3, Supplementary Figure S1). Three species from the superfamily Parasquilloidea were selected as the outgroups because of their close relationship to Squilloidea (Van Der Wal et al., 2017, 2019). The analysis involved 67 nucleotide sequences from 46 species of mantis shrimp. Results Molecular identification of stomatopod larvae The K2P genetic distances of the 16S rRNA gene were 1% or below within species (Supplementary Table S1) except for the distances in Alima orientalis and Oratosquilla oratoria (3.7% and 0.3–1.3%, respectively) in which the greater sequence divergences were indicated (Du et al., 2016; Cheng & Sha, 2017; Wong et al., 2021). The constructed phylogenetic tree (Figure 3) placed the two larval individuals with the adult sequence of Levisquilla inermis. The sequences from the two larval individuals were identical, and only one out of 306 base positions was different from the adult L. inermis specimen. The K2P genetic distance was below 0.1% among the three sequences. The larval and adult L. inermis shared the most common ancestor with Levisquilla jurichi (Makarov, 1979) with the K2P genetic distance of 3.4%. Taxonomic account SYSTEMATICS Order STOMATOPODA Latreille, 1817 Suborder UNIPELTATA Latreille, 1825 Superfamily SQUILLOIDEA Latreille, 1802 Family SQUILLIDAE Latreille, 1802 Genus Levisquilla Manning, 1977 Levisquilla inermis (Manning, 1965) (Japanese name: Subesube-shako) (Figures 1, 4, 5) Material examined. Last larval stage, NSMT Cr-29221, 15.95 mm in TL, Seto Inland Sea, Japan, 30–40 m, 29 October 2020. Description Carapace (Figure 4A–C). Elongated; 3.83 mm in CL, 1.90 mm in CW, 3.30 mm in RL, ventral armed with 6 spinules; armed with 2 anterolateral spines extending to the base of the eyestalk, 2 posterolateral spines extending to the second pleomere, and 1 median spine on posterior margin. Posterior margin of carapace reaching thoracic somite 7. Antennule (Figure 4D). Antennular stalk 3-segmented. First stalk segment with 4 setae, the second segment with 6 setae, and the third segment with 1 seta. Antennule with 3 setaceous Downloaded from https://www.cambridge.org/core. IP address: 3.231.220.57, on 13 Dec 2021 at 18:17:26, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531542100076X 804 Alyaa Elsaid Abdelaziz Fadl et al. Table 1. Stomatopod species and the sequence references of 16S rRNA gene used in this study Species Accession Nos. References Squilloidea Squillidae Alima maxima MH168208 Van Der Wal et al. (2019) Alima orientalis HM138813 Porter et al. (2010) MH168229 Van Der Wal et al. (2019) Alima pacifica HM138814 Porter et al. (2010) Anchisquilla fasciata FJ224251 Tang et al. (2010) FJ224252 Tang et al. (2010) MH168211 Van Der Wal et al. (2019) Busquilla plantei HM138815 Porter et al. (2010) Busquilla quadraticauda MH168223 Van Der Wal et al. (2019) Carinosquilla multicarinata MH168221 Van Der Wal et al. (2019) Clorida decorata MH168200 Van Der Wal et al. (2019) FJ224253 Tang et al. (2010) Cloridopsis scorpio MH168234 Van Der Wal et al. (2019) Dictyosquilla foveolata FJ224256 Tang et al. (2010) MW864094 Zhou (2021)a MH168236 Van Der Wal et al. (2019) Erugosquilla graham MH168226 Van Der Wal et al. (2019) Erugosquilla woodmasoni FJ224262 Tang et al. (2010) KU532327 Du et al. (2016) MH168225 Van Der Wal et al. (2019) Fallosquilla fallax HM138821 Porter et al. (2010) Harpiosquilla annandalei MH168202 Van Der Wal et al. (2019) Harpiosquilla harpax AY699271 Miller & Austin (2006) MH168203 Van Der Wal et al. (2019) FJ224264 Tang et al. (2010) Harpiosquilla melanoura MH168201 Van Der Wal et al. (2019) Kempella mikado MH168199 Van Der Wal et al. (2019) FJ871138 Tang et al. (2010) HM138833 Porter et al. (2010) Kempella stridulans MH168213 Van Der Wal et al. (2019) Lenisquilla lata MH168235 Van Der Wal et al. (2019) Levisquilla intermis OK575769 This study (larva, NSMT-Cr 29221) OK575771 This study (larva, NSMT-Cr 29222) OK575770 This study Levisquilla jurichi MH168204 Van Der Wal et al. (2019) Leptosquilla schmeltzii MH168222 Van Der Wal et al. (2019) Lophosquilla costata MH168237 Van Der Wal et al. (2019) MT276143 Zhang et al. (2020) Meiosquilla dawsoni MH168206 Van Der Wal et al. (2019) Meiosquilla swetti MH168210 Van Der Wal et al. (2019) Miyakea nepa FJ224268 Tang et al. (2010) MH168205 Van Der Wal et al. (2019) Miyakea holoschista MH168230 Van Der Wal et al. (2019) Oratosquilla fabricii MH168227 Van Der Wal et al. (2019) (Continued ) Downloaded from https://www.cambridge.org/core. IP address: 3.231.220.57, on 13 Dec 2021 at 18:17:26, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531542100076X 805 Journal of the Marine Biological Association of the United Kingdom Table 1. (Continued.) Species Accession Nos. References KU532325 Du et al. (2016) MH168214 Van Der Wal et al. (2019) FJ224273 Tang et al. (2010) GQ292769 Liu & Cui (2010) Oratosquillina anomala MH168217 Van Der Wal et al. (2019) Oratosquillina asiatica MH168238 Van Der Wal et al. (2019) Oratosquillina inornata MH168220 Van Der Wal et al. (2019) Oratosquillina interrupta MH168219 Van Der Wal et al. (2019) Oratosquillina perpensa MH168218 Van Der Wal et al. (2019) Pterygosquilla schizodontia MH168216 Van Der Wal et al. (2019) Quollastria imperialis MH168207 Van Der Wal et al. (2019) Rissoides barnardi MH168228 Van Der Wal et al. (2019) Squilla empusa DQ191684 Swinstrom et al. (2005) Squilla mantis GQ328956 Koenemann et al. (2010) AY639936 Cook et al. (2005) Oratosquilla oratoria Squilla rugosa HM138854 Porter et al. (2010) Squilloides leptosquilla KR095170 Kang et al. (2015)a MH168224 Van Der Wal et al. (2019) Vossquilla kempi MH168212 Van Der Wal et al. (2019) Triasquilla profunda MH168215 Van Der Wal et al. (2019) Faughnia haani MW632159 Hwang (2021)a Faughnia serenei MH168232 Van Der Wal et al. (2019) Pseudosquillopsis marmorata HM138845 Porter et al. (2010) Parasquilloidea Parasquillidae a Direct submission to GenBank. flagella. Inner flagellum 9-segmented with 1 long seta on the first segment, many short setae on all segments ending with 3 terminal setae. The median flagellum 5-segmented, and end with 1 long seta. Outer flagellum with 17 aesthetascs arranged in 3-3-3-3-3-2 on inner margin. Antenna (Figure 4E). Antennal scale with 40 plumose setae. Flagellum of antenna partially or completely divided into 14 segments. Mandible (Figure 4F). Incisor process with 6 teeth; molar process finely serrated. Maxillule (Figure 4G). Basal endite holding 2 sharp spines and one seta. Coxal endite with 14 marginal spines and 4 mesial spines. Apex with 2 setae. Maxilla (Figure 4H). Partially or completely divided into 4 segments bearing 47 marginal setae and 7 mesial setae, 3-lobed endite. First maxilliped (Figure 5A). 5-segmented with dactylus bearing 6 plumose setae and 4–5 spines; propodus holding 24 hooked setae arranged in 3 + 2 + 2 + 2 + 2 + 1 + 2 + 2 + 2 + 2 + 2 + 1 + 1, 2 mesial setae and 8 terminal setae; carpus with 21 marginal plumose setae; merus bearing 1 distal plumose seta. Second maxilliped (Figure 5B). 5-segmented, with dactylus bearing 4 teeth on occlusal margin; propodus with 48 denticles and 3 proximal large spines on the inner margin. Third maxilliped (Figure 4C). 5-segmented, with dactylus bearing 3 short setae; propodus with 8–9 short setae and 5 spines; carpus with 2 spines and 9 short setae. Fourth maxilliped. 5-segmented, with dactylus bearing 2 short setae; propodus with 14 short setae and 5–6 spines; carpus with 2 spines and 7 short setae. Fifth maxilliped. 5-segmented, dactyl with 2 short setae; propodus with 2 spines and 7–9 short setae; carpus with 2 spines and 4 short setae. Pereiopod I (Figure 5D). Pereopods 1–3, biramous unequally; endopod unsegmented, shorter than exopod; exopod partially two segmented. Pleopod I (Figure 5E). Biramous; endopod with 31 plumose setae, appendix interna with approximately 10 cincinnuli; exopod with 30 plumose setae; 6 gill buds. Pleopod II. Biramous; endopod with 39 plumose setae, appendix interna with ∼10 cincinnuli; exopod with 39 plumose setae; 7 gill buds. Pleopod III. Biramous; endopod with 40 plumose setae, appendix interna with ∼10 cincinnuli; exopod with 39 plumose setae; 7 gill buds. Pleopod IV. Biramous; endopod with 45 plumose setae, appendix interna with ∼10 cincinnuli; exopod with 41 plumose setae; 9 gill buds. Downloaded from https://www.cambridge.org/core. IP address: 3.231.220.57, on 13 Dec 2021 at 18:17:26, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531542100076X 806 Alyaa Elsaid Abdelaziz Fadl et al. Fig. 3. Neighbour-joining tree of 16S rRNA sequences of squilloid stomatopods. Branch length represents the Kimura 2-parameter distance. The terminal branches of the same species are compressed from the original tree (see Supplementary Figure S1). Bootstrap values are shown on the nodes when >50. Arrow indicates the position of the larvae examined. Pleopod V. Biramous; endopod with 38 plumose setae, appendix interna with ∼14 cincinnuli; exopod with 32 plumose setae; 7 gill buds. Uropod (Figure 5F). Protopodite terminating in 2 sharp spines, inner longer; endopodite with 10 plumose setae on distal margin; exopodite slightly longer than endopodite, basal segment of exopodite with 5–6 marginal spines and apical segment with 20 plumose setae on distal margin. Distal margin of exopod reaches over lateral tooth on telson. Telson (Figure 4I). Well-developed with 1 submedian, 1 intermedian and 1 lateral tooth located on each side; 40 submedian denticles, 9 intermedian denticles and 1 lateral denticles. Colouration. The whole body, except for the retinal tissue, was observed as transparent in life. Dark yellow to orange chromatophores were noticed on the mouthparts and a spot (each of which has a combination of dark yellow to orange and dark red chromatophores) was observed on each side of the basal part of the fifth pleopods. The entire specimen turned white when preserved in ethanol. Remarks. The propodus of the second maxilliped with three basal spines; all five pleopods possessing an appendix interna with cincinnuli; the exopod of uropod longer than endopod; the telson with nine intermediate denticles. Discussion The K2P distance of the nucleotide sequences of 16S rRNA and/ or COI genes have been proposed as one of the indicators for species identification of stomatopods (Tang et al., 2010; Wong et al., 2021). The distances to be found within the species are considered below 2–2.4% for the COI gene and below 0.91–2.1% for the 16S rRNA gene (Tang et al., 2010; Wong et al., 2021). The present study also demonstrates that the K2P distance of 16S rRNA gene can be considered 1% or lower within the species of Squilloidea. Based on this indicator, our molecular analysis successfully assigned the two larval individuals to Levisquilla inermis with <0.1% K2P distance. The closest relative of the L. inermis clade was a congeneric species, L. jurichi, which was 3.4% K2P distance from L. inermis. The K2P distance was sufficient to conclude that the two larval individuals are different species from L. jurichi. Downloaded from https://www.cambridge.org/core. IP address: 3.231.220.57, on 13 Dec 2021 at 18:17:26, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531542100076X Journal of the Marine Biological Association of the United Kingdom Fig. 4. Levisquilla inermis (Manning, 1965), last-stage larva: (A) body (ventral); (B) carapace (dorsal); (C) ventral view of carapace (left); (D) left antennule (dorsal); (E) left antenna (dorsal); (F) left mandible (ventral); (G) left maxillule (ventral); (H) left maxilla (ventral); (I) telson (dorsal). Scale bars: A, 5 mm; B, C, I, 1 mm; D–H, 200 μm. Fig. 5. Levisquilla inermis (Manning, 1965), last-stage larva: (A) left first maxilliped with enlarged portion of its distal end; (B) left second maxilliped; (C) left third maxilliped; (D) left first pereiopod; (E) left first pleopod with enlarged cincinnuli; (F) left uropod (ventral). Scale bars: A, E, F, 1 mm; B, C, 0.5 mm; enlarged distal end, 300 μm; enlarged cincinnuli, 40 μm. There are three known species in the genus Levisquilla, namely L. inermis, L. jurichi and Levisquilla minor (Jurich, 1904). Of these, L. inermis is the only species reported to be from Japan, including the Seto Inland Sea (Hamano, 1989, 2005; Ahyong, 2012). This species is also distributed in the other East Asian countries, such as Korea and Taiwan (Ahyong et al., 2008; Hwang, 2019; Hwang et al., 2019) while L. jurichi has also been shown to occur in Korea (Hwang, 2019; Hwang et al., 2019), and a L. jurichi post-larva was recently described as the first record from Taiwan (Wong et al., 2021). These recent records remind us of a possible appearance of L. jurichi larvae in Japanese waters even though adults of the species are not yet recorded there. However, sequences useful for DNA barcoding were available in GenBank only for L. jurichi within the genus. 807 The addition of the sequence obtained from the adult specimen, which morphologically strongly agreed with the previous studies of L. inermis (Manning, 1965; Ahyong, 2001; Ahyong et al., 2008; Ariyama et al., 2014) was the key factor to clearly solving the assignment of the larvae into this species. At the same time, our results support the assignment of the Taiwanese post-larva to a group other than L. inermis even though L. inermis is the only species from which the adult phase is known in Taiwan (Ahyong et al., 2008; Wong et al., 2021). The K2P distance between L. inermis and L. jurichi was confirmed as 3.4% in the current study, which is much greater than the distance of 1.6% (Wong et al., 2021) between their post-larva and the same sequence of L. jurichi used for the analysis in this study. Squilloid species have a propelagic stage with four pairs of pleopods prior to entering the planktonic form of stomatopod larva known as ‘alima’ with five pairs of pleopods (Manning, 1968; Ahyong et al., 2014). Some of the larvae pass through the two propelagic stages before reaching the first pelagic stage (Morgan & Provenzano, 1979). The alima is unique to Squilloidea and is characterized by a telson with four or more intermediate denticles, a propodus from the second maxilliped with three basal spines, a long eyestalk, and the exopod of the uropod longer than the endopod (Gurney, 1942, 1946; Morgan & Provenzano, 1979; Ahyong et al., 2014; Cházaro-Olvera et al., 2018). Studies of pelagic larvae require not only the identification of the species but also the identification of the developmental stages, which makes the process so complicated that the progress of larval biology is hampered. Indeed, the larval duration of stomatopods in the wild is not exactly known, and the number of stages for wild-caught larvae of any provided species is approximate (Pyne, 1972). Our descriptions reveal that several diagnostic features characterize the larvae: (1) well-developed pereiopod characterized by the exopod partially or completely 2-segmented, (2) maxilla partially or completely divided into 3 to 4 segments with a 3-lobed endite, (3) apex of maxillule elongated and holding two setae, and (4) well-developed uropod and distal margin of exopod extend over lateral tooth on the telson. Those features matched closely with the last larval stage of Oratosquilla oratoria described by Hamano & Matsuura (1987). Thus, our larvae were assigned to the last stage. The Seto Inland Sea, which is a semi-enclosed coastal sea in Japan, has various marine ecosystems that support a high level of meroplankton diversity. The stomatopods, which constitute one of the meroplankton communities in the Seto Inland Sea, may play a role in maintaining the pelagic food chain. It is also known that most stomatopod larvae inhabit coral reefs, and they serve as predators of small reef fishes and invertebrates in addition to serving as prey for predatory fishes, which reveal the contribution of stomatopod larvae in maintaining coral reef communities (Reaka et al., 2008; Abinawanto et al., 2019). Besides that, larvae of the stomatopods provide an alternative life stage to study biodiversity. Descriptions of 12 adult stomatopods from the Seto Inland Sea have been well documented by numerous researchers (Ariyama, 1997, 2001, 2004; Hamano, 2005; Ariyama et al., 2014): (1) Lysiosquillina maculata (Fabricius, 1793); (2) Cloridopsis scorpio (Latreille, 1828); (3) Anchisquilla fasciata De Haan, (1833–1850); (4) Harpiosquilla harpax (De Haan (1833–1850)); (5) Lophosquilla costata De Haan, (1833–1850); (6) Oratosquilla oratoria De Haan, (1833– 1850); (7) Acanthosquilla multifasciata (Wood-Mason, 1895); (8) Erugosquilla woodmasoni (Kemp, 1911); (9) Oratosquillina perpensa (Kemp, 1911); (10) Levisquilla inermis (Manning, 1965); (11) Harpiosquilla melanoura Manning, 1968; and (12) Clorida japonica (Manning, 1978). The study of stomatopod larvae from the Seto Inland Sea has been restricted due to the lack of information for accurate taxonomic identification. The larvae of Downloaded from https://www.cambridge.org/core. IP address: 3.231.220.57, on 13 Dec 2021 at 18:17:26, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S002531542100076X 808 many species are yet to be identified, and the majority of work in this area has been done on the commercial Japanese mantis shrimp, Oratosquilla oratoria. Stomatopod larval life cycles usually involve several developmental stages, which are complicated to identify without extensive morphological descriptions. This study has demonstrated the capability of matching one developmental stage of our unknown larvae, obtained from the Seto Inland Sea to species with a high degree of confidence by matching their DNA sequences with the DNA barcodes of the identified adult. The results using this technique enable rapid species identification of stomatopod larvae and may encourage further studies on stomatopod larval communities from the Seto Inland Sea for which information is relatively inadequate. Furthermore, we provide some insights into the ecology of L. inermis since we found both adult and larval specimens at the same location at the same time, a finding that indicates that the Seto Inland Sea is one of the main habitats of species settlement. In conclusion, our data strongly link the larvae to their species and confirm the occurrence of L. inermis in the Seto Inland Sea. In addition, this study demonstrates the validity of the DNA barcoding method using the 16S rRNA gene for stomatopod larvae identification from the Seto Inland Sea. Our study also reveals that morphological identification should be supported with molecular confirmation to accomplish a more accurate and legitimate result. Such a combination may encourage further taxonomic and ecological studies on stomatopod larvae. Supplementary material. The supplementary material for this article can be found at https://doi.org/10.1017/S002531542100076X Acknowledgements. The authors express their gratitude to the captain and crew of the ‘TOYOSHIO MARU’ (School of Applied Biological Science, Hiroshima University) for specimen collection, to Mr Kenta Suzuki (Graduate School of Integrated Sciences for Life, Hiroshima University) for his courtesy of photographing the live materials during the cruise, and Ms Chiho Hidaka (School of Applied Biological Science, Hiroshima University) for her assistance in drawing and molecular work. Sequencing analysis was performed at the Natural Science Center for Basic Research and Development, Hiroshima University. Author contributions. AEAF was responsible for the conceptualization of the study, conceived and designed the experiments; performed the molecular work and drawing; molecular and morphological analysis, interpreted the data, and wrote the manuscript. SY and KW designed the collection of the specimens using the training and research vessel. SY made records of collection. 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