Morphological and molecular characterization of the thunder crab Myomenippe hardwickii Gray, 1831 (Brachyura: Menippidae) from the Malabar coast, India

In the present study, a total of five specimens (two males and three females) were collected from two intertidal to shallow subtidal sites (<6 m) of the Cochin estuary, Kerala, Malabar Coast, India: (1) Thevara (9°55′27.6″N 76°18′01.8″E); (2) Marine Science Boat Jetty, School of Marine Sciences, CUSAT (9°57′50.0″N 76°16′54.6″E) (Fig. 1). Specimens were collected during October and November 2022, either by handpicking at low tide or incidentally captured using a van Veen grab sampler during benthic sample collection.

Figure 1

Map indicating the site of collection (red circle), in Cochin Estuary, Kerala, west coast of India.

All of the specimens were transported to the lab for morphological analyses, and preserved in a 4% buffered formaldehyde solution. Tissue samples of leg muscle were preserved in 95% ethanol solution for molecular analysis. Photographs were taken using a Nikon D610 DSLR camera attached to a Nikkor 40 mm macro lens (Nikon Corporation, Japan) or to an Optika SLX-3 stereomicroscope (Optika S.R.L., Italy). Figures were edited using Adobe Photoshop 2021 (version 22.0). The morphometric measurements were taken with a digital Vernier caliper. CW was measured as the maximum distance across the carapace, including lateral teeth, while carapace length (CL) was taken from the frontal margin to the posterior margin along the midline. Abdominal width (AW) and abdominal length (AL) were measured on the ventral side of the crab. Propodus length (PL) and propodus height (PH) were measured from the major cheliped (crusher claw), from the carpus–propodus joint to the base of the fixed finger and at the maximum palm height, respectively. The identification process relied on key taxonomic characteristics outlined in the existing literature (De Man, 1887; Gray, 1831; Ng, 1998). The morphological terminology is in accordance with Davie et al. (2015). The following abbreviations are used: CW, carapace maximum width; CL, carapace length at midline; mxp3, third maxilliped; P5, fifth pereopod; G1, first male gonopod; and G2, second male gonopod.

Three females (CW × CL: 42 mm × 31 mm, 56 mm × 42 mm, 64 mm × 47 mm), two males (CW × CL: 35 mm × 26 mm, 83 mm × 62 mm), Cochin estuary, coll. P. Hari Praved & K.V. Neethu, October & November 2022. The specimens examined were deposited in the collections of the Marine Biology Museum, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology (CUSAT), Kochi, Kerala, India (MH/HPP1—HPP5/12-2022).

For the molecular study, total genomic DNA was extracted from the pereiopod muscle using the QIAamp Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Partial fragments of mitochondrial cytochrome c oxidase subunit I (COI) gene were amplified using universal primers LCO1490 5′ GTCAACAAATCATA AAGATATTGG-3′ and HCO2198 5′- TAAACTTCAGGGTG ACCAAAAAATCA -3′ (Folmer et al., 1994). The PCR mix included 12.5 μL of EmeraldAmp GT PCR Master Mix (Takara Bio), 0.4 μM primers, and 5 ng of template DNA in a 25 μL reaction volume. The amplification conditions for COI involved an initial denaturation step at 95°C for 180 s, followed by 35 cycles of 95°C for 30 s, 55°C for 60 s, and 72°C for 60 s, with a final extension of 72°C for 180 s. A mitochondrial DNA (mtDNA) region of 550 base pairs from the large subunit rRNA (16S rRNA) was amplified using the universal primers 16Sar (59- CGCCTGTTTATCAAAAACAT-39) and 16Sbr (59-CCGGTCTGAA CTCAGATCACGT-39) (Palumbi, 1996). The amplification conditions for 16S involved an initial denaturation step at 94°C for 180 s, followed by 35 cycles of 94°C for 30 s, 50°C for 60 s, and 72°C for 60 s, followed by a final extension of 72°C for 180 s. The PCR products were purified with the GFX PCR DNA and Gel Band Purification Kit (Cytiva Life Technologies, Wilmington DE, United States). Bidirectional sequencing was performed with an ABI PRISM Big Dye Terminator v3.1 cycle sequencing kit in an AB 3730 DNA analyzer (Life Technologies).

Raw Sanger chromatograms were inspected and trimmed for base-calling errors in SeqScanner, followed by the sequences being compiled, analyzed, and edited using BioEdit v7.2.5, Ibis Therapeutics (Hall, 1999). DNA sequences of 631 bp and 527 bp were derived from the COI fragments, and approximately 449 bp from the 16S fragments. The sequences were compared with the available entries in GenBank (National Center for Biotechnology Information, NCBI) using the standard nucleotide basic local alignment search tool (BLAST). To confirm species identification and assess the genetic divergence among closely related taxa using the COI gene, we retrieved sequences for other representatives of Menippidae from GenBank (accession numbers provided on the phylogenetic tree). Based on the phylogeny of Lai et al. (2014), Carpilius convexus was selected as an appropriate outgroup, representing a lineage closely related to but outside Menippidae, enabling reliable alignment and stable rooting of the phylogenetic tree. Multiple sequence alignments for COI were generated using MAFFT v7.505 (Katoh et al., 2019) under default parameters, following a conservative alignment strategy to preserve positional homology. Phylogenetic tree for the COI dataset were constructed using the maximum likelihood (ML) method in MEGA 11 using the Tamura Nei model (T93), the best-fit nucleotide substitution model identified by the Bayesian Information Criterion, and implemented with 1000 bootstrap replicates (Tamura & Nei, 1993; Tamura et al., 2021). The 16S rRNA sequences were used solely to support species identification and were not included in phylogenetic reconstruction due to the limited availability of comparable reference sequences. Pairwise genetic distances between species for COI were also calculated using the Tamura-Nei (T93) model. The obtained mtDNA COI and 16S rRNA sequences were deposited in GenBank (COI: OQ948136, OQ954126, and 16S: OQ955252).

Family Menippidae Ortmann, 1893

Genus Myomenippe Hilgendorf, 1879

Myomenippe hardwickii (Gray, 1831) (Figs 2–4; Table 1)

Synonymized names

Cancer Hardwickii Gray, 1831

Menippe (Myomenippe) duplicidens Hilgendorf, 1879

Menippe granulosa A. Milne-Edwards, 1867

Carapace is dorsally granulated and is broader with a short length (CW/CL 1.33–1.36 times) (Table 1), appearing transversely ovate; the coloration is dirty brown to brownish yellow. The carapace regions are well defined, characterized by shallow depressions and adorned with numerous small granules. The cardiac region is distinctly separated from the gastric region; the gastric region is distinct and is subdivided into three portions—two anterolateral and one posterior (Fig. 3A). The surfaces of the two anterolateral gastric regions show a division into two slight, granule-covered convex lobes. The frontal margin is bilobed, separated from the orbit by a marked concavity, and the lobes are separated from each other by a median notch; each lobe possesses three teeth (Fig. 3D). The anterolateral margin is granular, thin, and sharp, and is rugose; it bears four broad lobiform crested teeth on each side. The first three teeth are broad and anteriorly acuminate, whereas the most posterior tooth is short and narrow (Fig. 3E). The orbits (Figs 3B and 3C) are wide, ovate, and closed internally; the eyes are large, filling the entire orbital cavity, and the corneas are pigmented green and ringed with red in life (Fig. 3C). The infraorbital margin is granular, and an exorbital tooth is present; the large internal lobe of the infraorbital margin extends slightly more forward than the third frontal tooth. The antennular fossae are subrectangular; the antennules are robust and fold transversely and obliquely; the antennary flagellum lies outside the orbital gap. The third maxilliped is large and fits tightly into the buccal cavity (Figs 3H and 4C);it is finely granular externally, and the inner and outer margins of the ischium and merus are sparsely setose. The merus of the third maxilliped is subquadrate, with the distolateral angle projecting and rounded; the ischium is subrectangular and about twice as long as the merus. The palps are thick and subcylindrical, and hairy tufts occur on the inner apical region of the carpus, propodus, and dactylus. The exopod is elongate and not tapering; its distal tip reaches three-quarters of the lateral margin of the merus, and the flagellum is long and well developed. The male thoracic sternum (Fig. 3F) is broad, with a smooth, lightly setose surface; a distinct suture separates the sternites. The abdomen is seven-segmented (Fig. 3G); the pleon is sparsely granulose and setose, with all somites and the telson freely articulating. The lateral margin is convex, and the telson is subtriangular, bluntly rounded distally, with the base as broad as it is wide. The female pleon (Fig. 4A) is much wider than that of the male; somite 3 is the widest in males, whereas somite 5 is the widest in females. The male first gonopod (G1) is stout, with the apical process gently curved; a fine line of setae is present distally along the margins (Figs 4F and 4G). The male second gonopod (G2) is elongate, subequal in length to the G1, and its distal segment is coiled inward (Fig. 4H). The vulvae in females are located 2–3 mm laterally on the sixth thoracic sternite and are ovate in outline (Fig. 4B). The chelipeds are massive and robust, slightly unequal, with the right chela larger in all specimens; males have proportionally larger and more robust chelipeds than females; the carpus is curved, and a small inner spine extends anteriorly from it. The digits are stout and black; the base of the dactylus of the larger chela bears a prominent molariform tooth (Fig. 4D). The dorsal surfaces of the carpus and palm of the chelipeds are granulose. The walking legs are slender; the margins of all legs are fringed with setae, with the thicker setae occurring on the propodus and dactylus.

Figure 3

M. hardwickii, male, (35 mm × 26 mm), MH/HPP4/12-2022: (A) Dorsal view displaying three sections of the gastric region; (B) cephalothorax, frontal view; (C) green pigmented cornea ringed with red and extended infra orbital margin; (D) bilobed frontal margin with three teeth each; (E) anterolateral margin with four teeth; (F) anterior thoracic sternum and telson, ventral view; (G) detached pleon, ventral view; (H) mxp3, external view; (I) thoracic sternum and sternopleonal cavity. mxp3, third maxillipeds; N, notch.

Figure 4

M. hardwickii, female (42 mm × 31 mm), MH/HPP1/12-2022: (A) detached female pleon, external view; (B) female thoracic sternum and vulvae, ventral view; (C) left third maxilliped, ventral view; (D) right cheliped with molariform tooth; (E) right P5, ventral view; (F) entire first male left G1, ventral view; (G) distal tip of left G1, ventral view; (H) entire second male right G2, ventral view; (I) distal tip of G2, ventral view.

We successfully amplified and sequenced two mtDNA COI fragments of 631 bp and 527 bp from Myomenippe specimens collected from the Cochin Estuary. The nucleotide BLAST analysis showed that both fragments exhibited 100% sequence identity with M. hardwickii reference sequences in GenBank (accession numbers: PX457434 and PQ536003), confirming a perfect match across the full sequence length. In addition, a 449 bp fragment of the 16S rRNA gene was generated, which displayed >98.89% sequence identity to the only available M. hardwickii 16S reference sequence in GenBank (accession number: HM637977) from Singapore. The sequences obtained in this study are deposited in GenBank under accession numbers OQ948136, OQ954126 (COI), and OQ955252 (16S).

The evolutionary relationships of M. hardwickii to other menippid crabs were examined using an ML phylogenetic tree constructed from mtDNA COI sequences (Fig. 5). The ML phylogeny divides the taxa into two major clades within Menippidae. The two sequences generated in this study (OQ954126 and OQ948136) cluster tightly with previously published M. hardwickii sequences from Thailand (PX457434), Malaysia (PQ536003), and Singapore (HM638052), forming a strongly supported monophyletic group with a bootstrap value of 100%. M. fornasinii forms the immediate sister clade, represented by sequences PV053328, LK391943, and NC024437, confirming its close evolutionary proximity to M. hardwickii. Other menippid species, including M. rumphii (OL960453), Menippe nodifrons (MW264437), Menippe adina (MW094166), and M. mercenaria (MW094171), form distinct and well-separated lineages outside the Myomenippe clade. The tree is appropriately rooted with C. convexus (HM638025). Pairwise TN93 distances illustrate the same phylogenetic patterns (Table 2). Genetic divergence between M. hardwickii from India and its GenBank representatives was found to be low (0.01). The mean TN93 distance between M. hardwickii and M. fornasinii within Myomenippe was 0.118, whereas distances between Myomenippe and Menippe species were notably higher (mean = 0.176). Net TN93 distances also revealed clear species boundaries: the net divergence between M. hardwickii and M. fornasinii was 0.114, compared with 0.116 between Myomenippe and the other Menippe species, indicating deeper intergeneric separation. Together, the phylogenetic topology and genetic distance metrics robustly confirm the identity of the present specimens as M. hardwickii and demonstrate their strong genetic affinity with the Southeast Asian populations.

Figure 5

Molecular phylogenetic tree inferred using the ML method based on the Tamura–Nei model (T93) showing the relationships among M. hardwickii, related Menippe species, and the outgroup Dromia personata. The analysis is based on a COI gene alignment generated after sequence trimming and multiple sequence alignment using MAFFT v7.505. Bootstrap support values (>50%) are indicated at the nodes and branch lengths drawn to scale as substitutions per site. Sequences generated in this study are represented by a red circle. ML, maximum likelihood.

M. hardwickii was found on submerged structures, decaying aquatic weeds, and mangroves in the Cochin Estuary. The species is usually abundant during the pre-monsoon period (January–May) at sites with a salinity of 10–33 ppt and depth of 2–6 m. During the sampling period (October & November 2022), the organic content in the sediment was 1.7%–2.8%. The habitat is muddy to clayey–silty, the pH of the sediment ranged from 7.5 to 8.3, and the average redox potential was from –135 to –275 mV. The total carbon of the sediment ranged from 29 to 37 g/kg. In the present observation, they were found to occur together with encrusting communities such as clumps of mussels (Mytella strigata and Perna virdis), oyster bed of Magallana bilineata, gastropods (Nassodonta insignis), amphipods (Grandidierella gilesi), and other brachyuran crabs such as Thranita crenata, Neorhynchoplax alcocki, Neorhynchoplax demeloi, and Aniptumnus bijoyi.

M. hardwickii is morphologically most similar to its sister species M. fornasinii (Fig. 6A), in the general form of the carapace, male pleon, and pereopods. Nevertheless, M. hardwickii is readily distinguished from M. fornasinii by a combination of carapace sculpture, anterolateral tooth morphology, cheliped ornamentation, and gonopod structure. The dorsal surface of the carapace in M. hardwickii is distinctly granulose, whereas it is smooth in M. fornasinii (Figs 6A–6C). In M. hardwickii, the first two anterolateral teeth are strongly produced and closely spaced, the second tooth is relatively robust, and the supraorbital margin is continuous, lacking visible fissures (Fig. 6B). By contrast, M. fornasinii possesses weakly produced first and second anterolateral teeth, with the second tooth more elongate and more widely separated from the third (Fig. 6C) (De Man, 1887, 1899), and a supraorbital margin divided by two distinct fissures (Fig. 6C) (Haswell, 1881; Milne-Edwards, 1867). Chelipeds of M. hardwickii are dorsally granulose, with coarse granules on the propodus and carpus and faint punctation on the mesial surfaces (Fig. 2) (De Man, 1887), whereas those of M. fornasinii are smooth and distinctly punctate, with the punctae being larger and more densely arranged along the dorsal margin of the palm (Fig. 6A) (De Man, 1899; Miguel et al., 2025). Earlier accounts of M. hardwickii are limited. Gray (1831) provided only a brief description of the type material, lacking detailed information on diagnostic characters, such as the male gonopods (G1, G2) or the position and shape of the female vulvae. Subsequent treatments by Milne-Edwards (1867) and Hilgendorf (1879) added little beyond Gray’s original account. Serène (1984) presented a key to the genus Myomenippe and illustrated the gonopods of M. fornasinii, noting the apparent similarity of gonopods in both species, while Ng (1998) largely reiterated earlier observations without additional morphological detail. The present study documents and illustrates, for the first time, the male gonopods (G1, G2) and the position and shape of the female vulvae in M. hardwickii. The first gonopod (G1) of M. hardwickii is stout, with the apical process more gently curved than in M. fornasinii (Fig. 6D); in M. fornasinii, the G1 bears denser marginal setation (Fig. 6E). The distal segment of the second gonopod (G2) in M. hardwickii is singly coiled (Fig. 6F), whereas it is doubly coiled in M. fornasinii (Fig. 6G). In females of M. hardwickii, the vulvae are ovate in outline and located laterally on thoracic sternite 6 (Fig. 4B).

Figure 6

M. hardwickii, male (35 mm × 26 mm), MH/HPP4/12-2022: (B) carapace, dorsal view; (D) left first pleopod (G1), ventral view; (F) right second pleopod (G2), ventral view. M. fornasinii, male: (A) dorsal habitus; (C) carapace, dorsal view; (E) left first pleopod (G1), ventral view; (G) left second pleopod (G2), ventral view. Panels (A) and (C) are adapted and redrawn from Miguel et al. (2025); panels (E) and (G) are redrawn from Serène (1984).