{'fraction_non_alphanumeric': 0.04887860806597606, 'fraction_numerical': 0.03771497535954943, 'mean_word_length': 4.915229030339084, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Species diversity and molecular taxonomy of symbiotic crustaceans on Portunus pelagicus (Linnaeus, 1758) in Vietnam, with remarks on host records and morphological variation

The blue swimming crab ( Portunus pelagicus (Linnaeus, 1758)) is an economically and ecologically important species in Vietnam, and a potential subject for aquaculture as well. More than 400 individual crabs were collected along the Vietnamese coastline and examined for ectosymbiotic crustaceans. Two molecular markers (28S rRNA and COI mtDNA) were applied for species delineation. Seven species were reported and described, of which six are cirripede barnacles (Thecostraca, Thoracia); and one parasitic copepod Choniosphaera indica Gnanamuthu, 1954 (Copepoda, Podoplea). Four species ( Chelonibia testudinaria (Linnaeus, 1758), Semibalanus sp., Octolasmis neptuni (MacDonald, 1869), and Dianajonesia tridens ( Aurivillius, 1894)) were the first records for both host and location. The symbiotic crustaceans occupy specific niches on the crab body, and vary in their infestation status. Molecular taxonomy of symbiotic crustaceans was classified and confirmed based on sequence similarity and phylogenetic analyses.


Species diversity and phylogeny of symbiotic crustaceans
Nauplius, 30: e2022027 intrOductiOn The swimming crab, Portunus pelagicus (Linnaeus, 1758) is a major economic species throughout the Indo-Pacific and to the coast of Africa (Galil and Innocenti, 1999). In Vietnam, it is widely distributed throughout the coastal waters and nearby islands (Vu et al., 2014). Throughout its native range, it is a valued market organism with numerous reports of its commercial value as a multi-million dollar export commodity between Vietnam and Japan and the United States of America (VASEP, 2021). Crabs play ecological important roles, through complex food webs, in coastal and marine ecosystems, especially in mangrove forests, seagrass beds and coral reefs (Kunsook et al., 2014a;2014b). Additionally, their carbonate carapaces are widely known as a living substrate for many epibiont/symbiont organisms (Abelló and Corbera, 1996;Gaddes and Sumpton, 2004;Dvoretsky, 2012;Machado et al., 2013).
Reciprocal selection pressure between the host and its symbiotic species can potentially alter species diversity, ecological function, and community dynamics (Galil and Innocenti, 1999). The diversity of the symbiotic community on P. pelagicus is known to depend on its geographic distribution (Shields, 1992;Shields and Wood, 1993). Among the symbiotic crustacean species, the barnacles frequently found attached to decapod crustaceans have received the most attention. Their symbiotic association depends on the host's biological characteristics, such as distribution, sex, maturity stage, molt cycle, and size (Weng, 1987;Gaddes and Sumpton, 2004;Klinbunga et al., 2007;Babu et al., 2012;Machado et al., 2013). Growing numbers of symbiotic species have been reported on portunid decapod species (Hudson and Lester, 1994;Isaeva et al., 2005;Babu et al., 2012). In Vietnam, several symbiotic species have so far been detected on P. pelagicus and Portunus trituberculatus (Miers, 1876). Among these, two species (Carcinonemertes mitsukurii Takakura, 1910 and Choniosphaera indica Gnanamuthu, 1954), recognized egg eating parasites, are known to have a negative impact on host populations Le et al., 2018a;2018b).
In the past decade, molecular markers have been increasingly applied to investigate species diversity and phylogenetic relationships, including research examining the phylogenetic position and evolution of Cirripedia (Mizrahi et al., 1998;Wu, 2011;Kwiatkowski et al., 2012;Yusa et al., 2012;Filipiak et al., 2016). So far, no research has focused on elucidating the phylogenetic relationships of the symbiotic crustacean community on the blue swimming crab.
The current research conducts the first comprehensive study of species diversity, infestation status, and molecular taxonomy of symbiotic crustacean associations occurring on P. pelagicus distributed along the Vietnamese coastline.

Symbiotic crustacean sampling , identification, and infestation status
Blue swimming crabs (P. pelagicus) were collected from the Vietnamese coastline from Cat Ba Island -Hai Phong, Ha Long Bay -Quang Ninh in the North; Nha Trang and Van Phong Bay -Khanh Hoa, Song Cau and Tuy Hoa -Phu Yen in the Center, and Phu Quoc Island and Rach Gia -Kien Giang in the South. The crabs were transported alive in aerated sea water to the laboratory where they were kept in aquaria until dissected. Sampling information of crab individuals collected is presented in Tab. 1.
Freshly collected symbiotic crustacean species were used for the descriptions and identification. The individuals intended as whole-mounts were transferred to a vial of 70 % alcohol, and those for DNA analysis were preserved in 95 % EtOH, and stored at -20 °C. Symbiotic crustaceans were identified using various keys and species descriptions follow Jeffries and Voris (1996); Jeffries et al. (2005); Cheang et al. (2013). Infestation status of symbiotic species were examined by prevalence and mean intensity, as defined in Margolis et al. (1982) and Rózsa et al. (2000).

Molecular taxonomy
DNA was extracted from individuals of each collected symbiont species using DNeasy Tissue Extraction Kit (Qiagen) in accordance with the manufacturer's instructions. 28S rRNA and COI (Cytochrome c oxidase subunit I) mitochondrial DNA were amplified using primer LSU 5 and 1500R (Olson et al., 2003), and LCO1490 and HCO2198 (Folmer et al., 1994), respectively. PCR reactions were performed using a total volume of 50 µl with components at the following concentrations: 10 µL of DreamTaq buffer 10X (Thermo Fisher Scientific), 2 µL dNTP (10 mM), 2 µL each primer (10 mM), 1.25 unit of DreamTaq polymerase (5U/µl), 5 µl DNA template and distilled water to the final volume. Amplification was implemented using the following PCR profile: a preliminary denaturation at 94 °C for 3 minutes (min), followed by 35 cycles of 94 °C for 45 seconds (s), annealing for 45 s (for 28S rRNA, COI mtDNA genes at 56 °C, 42 °C, respectively), and then 72 °C for 45 s. This was followed by a final extension period at 72 °C for 7 min before the samples were cooled to 4 °C. PCR products were run on a 1.5 % agarose gel for confirmation of equal length against an appropriate size marker. The PCR products were purified using DNA purification kits (Promega) and pre-sequenced using dye-labels dideoxy terminator (Big Dye Terminator 3.1, Applied Biosystems) with the same primer as the PCR reactions at the following temperatures: 96 °C for 30 s, 50 °C for 30 s and 60 °C for 4 min. Sequences of both strands were generated on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) using the amplification primers.
Sequence contigs were assembled using the Geneious Pro 5.5.7 (Kearse et al., 2012). The resulting sequences were confirmed by the Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/ Blast.cgi). The sequences were aligned, analyzed using BioEdit 7.0.5.3 (Hall, 1999), and submitted to GenBank. Sequence identity matrix was used to investigate similarity/identity values. Information on gene application, and Genbank accession numbers are presented in Tab. 2.
To confirm the molecular taxonomy, phylogenetic analyses were conducted using obtained 28S rRNA and COI mtDNA of collected symbiotic crustaceans (7 sequences of two genes and 17 available Genbank sequences (Tab. 2). Phylogenetic trees were constructed using Bayesian inference (BI) approaches. Prior to BI analysis, best-fit models of nucleotide substitution were selected by the Akaike Information Criterion as implemented by MrModeltest 2.2 (Nylander, 2004). The best selected models were GTR+G for 28S rRNA and GTR+I+G for COI data sets, respectively. BI analyses were conducted in MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001) under the selected best-fit models and parameters. Four chains were used, and the analysis was run for 1 million generations, with the sampling frequency of 100. Each analysis was repeated twice to check for similarity of the likelihood plateau. Additionally, parameter values were evaluated for convergence throughout the run by using the ''sump'' command in MrBayes and by examining results in Tracer 1.3 (Drummond et al., 2005). Plots from Tracer were used to determine the appropriate number of trees to be discarded in the ''burn in'' and a final 50 % majority-rule consensus tree was constructed from the remaining trees.   Gray, 1825), and the closely related species Dianajonesia tridens Aurivillius, 1894(WoRMS, 2020Young, 2001) were found to have moderate abundance (> 10 %). The external morphology of these four species (Fig. 1) was consistent with previously published descriptions (Jeffries et al., 2005).
Octolasmis spp. showed the greatest variety of preferred infection sites. Octolasmis warwicki infected most of the body parts of the crabs, except for the gills, while its two congeners (O. angulata and O. neptuni) do not appear on the carapace. Dianajonesia tridens was found on most body parts, but absent from the carapace (Tab. 2, Fig. 1).
Material examined and measured. Ten live specimens, including 4 specimens from a female crab (87 mm CW, 25 August 2016, locality Ha Long, collector T. Q. Sang) and 6 from a male crab (100 mm CW, 14 May 2016, locality Kien Giang, same collector).

Semibalanus sp.
Host and Location. Portunus pelagicus, at Khanh Hoa, Vietnam, new record for the genus to both host and location.
Morphological description (Fig. 3A, B). The shell is ovoid, diameter of 3.35 ± 1.04 (2 -4.5) mm (n = 4), with 6 plates (1 carina, 1 rostrum, 2 carinolateral and 2 lateral) truncated pyramidal, grey-white, thin, fragile, and rather smooth surface. Parietes tubiferous with a single row of major tubes. Carina trapezoidalshaped; lateral borders overlap and do not close up Species diversity and phylogeny of symbiotic crustaceans Nauplius, 30: e2022027 with the carinolateral plates. Rostrum wide, curved trapezoidal-shaped, borders overlap the border of the laterals. Carinolateral narrow with border overlapped by carina and laterals. Orifice wide, diamond-shaped; operculum cover, diamond-shape; scutum wide, triangular with horizontal striations parallel with the basal edge and inconspicuous adductor ridge; tergum narrow and beaked with a narrow and long spur (Fig. 3A). The basis is membranous and very closely cemented to the crab carapace. The tissue inside is white (Fig. 3B).
Infestation status. Semibalanus sp. occurred with low prevalence (0.21 %), and was only found on the carapace of one individual host crab. Pilsbry, 1916 is the genus of acorn barnacle most abundant on tropical intertidal zones. Four species are currently recorded for this genus. The two common ones (Semibalanus balanoides (Linnaeus, 1767), and Semibalanus cariosus (Pallas, 1788)) are found mostly attached to rock (Takeda et al., 1998;Brousseau and Goldberg, 2007;Gyory, 2011), and rarely on crabs (McDermott, 2007); Semibalanus madrasensis (Daniel, 1958) was found on a fishing craft (Daniel, 1958), and Semibalanus sinnurensis (Daniel, 1962) on a mollusc shell (Daniel, 1962). This Semibalanus specimen shared some similar characters with S. balanoides (parallel grooves on the opening surface), and S. sinnurensis (parietes provided with minute calcareous projections), however, the observed external morphological characters (of only two individuals), and the DNA sequences are not enough for species identification. Moreover, the occurrence of Semibalanus sp. on Portunus pelagicus is assumed to be incidental.
Morphological description (Fig. 4A-C). The adult female is seed-like and ellipsoid in outline, size 800 -1200 µm (1014 ± 67.04) × 400 -700 µm (552 ± 119.57) (n = 5), and the posterior part projects with the caudal styles. The abdomen is fused with the cephalothorax, and the esophagus occupies most of the abdominal cavity. Choniosphaera indica body is yellow or light pink with two black eyes visible on the head. The cup-like mouth tube is surrounded by the maxillae and the maxillipeds. The mouth is at the bottom of the cup and has a membranous lip (Fig. 4A1). The maxilliped is a long appendage of four unequal segments with the second one longer than the others; it has two short recurved spines and a long, comb-shaped spine on its fourth segment. Two antennas; the first antennae has five articles. The first article bears a spine, the fourth has three spines, and the terminal article carries three spines (Fig. 4A2). The second antennae are five-segmented. The third article has a spine, and the terminal article carries two long slender spines (Fig. 4A3). The tail forms two branches, assisting attachment to the crab egg (Fig. 4A, B).

Species diversity and phylogeny of symbiotic crustaceans
Nauplius, 30: e2022027 Infestation status: Choniosphaera indica occurred with low prevalence (0.29 %), and moderate intensity (20.90 ± 4.97), and was found strictly on the egg mass of the female crab host (Tab. 2, Fig. 4C), no larvae of this species were found.
Remarks. Choniosphaera spp. are specific egg eating parasites on decapod crabs. Three species have so far been recorded (Costello et al., 2001;Connolly, 2010), and among them C. indica is the most common. Choniosphaera indica was previously reported on Portunus pelagicus (Shields, 1992;Vo et al., 2013), and considered as a significant contributor to mortality of the crab host, and affecting the host population (Shields and Wood, 1993). Despite being one of the parasites capable of affecting the portunid host species population, no gene sequence of this species is available on GenBank for reference. The present study provides descriptions and images of this species, and also registers two gene sequences (28S rRNA and COI mtDNA) into GenBank.

Molecular taxonomy
In total, seven sequences were generated from each gene region of seven species of symbiotic crustacean on P. pelagicus distributing along the Vietnamese coastline. The aligned data contained unambiguous 598 bp and 921 bp of COI mtDNA and 28S rRNA genes, respectively. Compared to GenBank sequences (28S rRNA and COI mtDNA), two species (O. warwicki and C. testudinaria) showed a 100 % (EU082328 and KC138501) and 99.7 -99.8 % (AB723914 and AY174338) identity, respectively. As for O. angulata, there is no 28S rRNA reference sequence on Genbank, however, the current COI mtDNA sequence shows 100 % matching. The remaining four species have no comparable sequences available in GenBank, therefore, comparisons were conducted with species of the same genus, and/or different genera with high proportion of similarity in a blast search (as is the case with C. indica) (see Appendix, Tab. A1). Conflicting results were obtained for the two genes for Semibalanus sp., with the COI sequence showing a 100 % identity match to S. cariosus (MH753555 and KM611728), but the 28S rRNA gene showing a match of only 93.4 % between the two species (MH727740 and AY520593).
Phylograms from obtained sequence data sets (28S rRNA and COI mtDNA) provided broader view of molecular taxonomy of studied symbiotic crustaceans. The BI approach applied to both data sets produced almost similar tree topologies, except the unidentified position of C. indica (Copepoda, Siphonostomatoida) from the COI tree, while it is clustered with Siphonostomatoid species (Asterocheres Boeck, 1859 andParabrachiella C.B. Wilson, 1915) on the 28S rRNA phylogram (Fig. 5). In the 28S rRNA topology, three Octolasmis Gray, 1825 species (O. angulata, O. warwicki, and Octolasmis cor (Aurivillius, 1892)) were clustered together, as a sister clade to O. neptuni + Octolasmis sp., and Octolasmis unguisiformis Kobayashi and Kato, 2003 + D. tridens (Fig. 5A). Minor differences were observed in the COI tree, such as O. angulata, O. warwicki, and O. neptuni grouped as sister species, and O. cor and O. unguisiformis forming a clade (Fig. 5B). In both cases, D. tridens was grouped within the Octolasmis species, either as a sister clade to O. unguisiformis (28S rRNA) or maintained as separate clade (COI mtDNA).
Moderate differences were also seen between the COI and 28S rRNA trees in the relative position of Semibalanus sp., which was either identical to S. cariosus (COI phylogram) or sister clade to other Semibalanus species (28S rRNA phylogram). The sequence differences were 7.6 % and 7.9 % to S. cariosus, and S. balanoides, respectively (Appendix, Tab. A2). The current C. testudinaria specimen clustered in the same clade with previous C. testudinaria and conspecifics C. patula, and C. manati. They all formed a clade with Chelonibia caretta (Spengler, 1790).

Species diversity and phylogeny of symbiotic crustaceans
Nauplius, 30: e2022027 discussiOn Seven symbiotic crustaceans (belonging to 2 suborders, 3 families, and 5 genera) were detected on P. pelagicus in Vietnam. Of these, four species (Semibalanus sp., C. testudinaria, D. tridens, and O. neptuni) are recorded in Vietnam for the first time on P. pelagicus. Despite the intensive sampling (over 400 individual crabs), the rhizocephalan, Sacculina Thompson, 1836, was not found. This is similar to the previous reports by Vo et al. (2013) on the same crab species, and Le et al. (2018b) on Portunus sanguinolentus (Herbst, 1783).
Octolasmis spp. and the genus Dianajonesia have been documented in previous studies on various host crustaceans (Shields and Overstreet, 2003;Dvoetsky and Dvoretsky, 2010;Dvoretsky, 2012;Ekanem et al., 2013) including swimmer crabs (Weng, 1987;Shield, 1992;Jeffries et al., 2005). These species were found to mainly colonize the outer surface of the host, such as the carapace, gills and gill chamber. The high density of symbionts is thought to affect the respiration and movement of hosts, exposing them to predators (Gaddes and Sumpton, 2004;Yusa et al., 2012). Although in the current study, Octolasmis species were found on almost every part of P. pelagicus body surface (Tab. 2), definite niche(s) were observed among the different species. The specific distribution may be related to the external body structure of each species. For example, O. angulata and O. neptuni have capitular plate coverage of 10 % and 16.4 %, with 3 and 5 reduced capitular plates, respectively, are found primarily on the gills (Fig. 1A, C), while O. warwicki and D. tridens, which are covered by 5 robust capitular plates (coverage 43 % and 71 %, respectively) are mainly distributed on the carapace. These findings are concordant with previous studies in terms of the number of Octolasmis species detected on swimming crabs (4 species), in the specific infestation sites, and in the structure and percent coverage of the capitular plates/capitulum (Jeffries et al., 2005).
The Semibalanus species was morphologically similar to S. balanoides; however, the molecular analysis shows it as a different taxon. We could not identify it, or describe it as a new species, due to the conflicting results from 28S rRNA and COI mtDNA sequences and limited taxonomic characters. We also found C. testudinaria, which has a well-known global distribution, and a wide variety of substrate-hosts species. Intraspecific morphological variation has previously been considered to be due to host substrate habitat differences (Rawson et al., 2003;Torres-Pratts et al., 2009;Cheang et al., 2013), and in fact, the three species of this genus reported here (C. testudinaria, C. patula, and C. manati), usually from different host taxa (marine turtles, decapod crustaceans, snakes, and Sirenia), have been combined by some (Zardus et al., 2014).
The current study provides new insights on the host and symbiont associations of swimming crabs in Vietnam and further studies could focus on the ecology of these ectosymbiont species.