Morphological Description and Molecular Characterisation of Glyptothoa gen. nov., a Fish Parasitic Deep-sea Cymothoid (Crustacea: Isopoda) from the Indian Ocean, with Four Species, Including One New Species

© 2023 Academia Sinica, Taiwan Open Access Morphological Description and Molecular Characterisation of Glyptothoa gen. nov., a Fish Parasitic Deep-sea Cymothoid (Crustacea: Isopoda) from the Indian Ocean, with Four Species, Including One New Species Ameri Kottarathil Helna2,3,*,§ , Panakkool Thamban Aneesh1,*,§ , Appukuttannair Biju Kumar3, and Susumu Ohtsuka1,4 1Fisheries Laboratory, Blue Innovation Division, Seto Inland Sea Carbon-neutral Research Center, Hiroshima University, 5-8-1 Minato-machi, Takehara, Hiroshima 725–0024, Japan. *Correspondence: E-mail: [email protected] or [email protected] (Aneesh) E-mail: [email protected] (Ohtsuka) 2Regional Forensic Science Laboratory, Kannur, Kerala, India, 670002. *Correspondence: E-mail: [email protected] (Helna) 3Department of Aquatic Biology & Fisheries, University of Kerala, Karyavattom, Thiruvananthapuram-695 581, Kerala, India. E-mail: [email protected] (Kumar) 4Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Terengganu, Terengganu, Malaysia §AKH and PTA contributed equally to this work. Received 22 November 2022 / Accepted 4 September 2023 / Published 26 October 2023 Communicated by Benny K.K. Chan Glyptothoa sagara gen. and sp. nov. is described from the host fish Glyptophidium macropus Alcock, 1894 (Ophidiidae), at depths 300 to 650 metres from the southwest coast of India. The mitochondrial cytochrome c oxidase subunit I (COI) gene of the species was sequenced and compared with other closely related branchial cymothoid genera. Both morphological and molecular data corroborate the inclusion of this parasitic isopod as a new genus, and we describe Glyptothoa sagara gen. and sp. nov. The following combinations of characters characterise the genus: cephalon immersed in pereonite 1; dorsum vaulted; all coxae visible in dorsal view; coxae shorter than or as long as pereonites; pereonites 4–7 slightly decrease in width towards one side, slightly asymmetrical, lateral margins slightly constricted, in hunched side; relatively wide pleon, with large lateral gaps between pleonites; antennula narrowly separated by rostrum, slender, shorter than antenna; antenna with 13 articles, buccal cone obscuring antennal bases; brood pouch arising from coxae 1–4, 6; oostegite 1 bilobed; pleopods rami all simple, without proximomedial lamellar lobe, without folds or thickened ridges. The adult life stages, such as females (ovigerous and non-ovigerous), males and transitional stage of the new species are described. The species is currently known only from the type locality and the type host. The ecological remarks of the newly described taxon are also provided. The following species are transferred from Elthusa Schioedte and Meinert, 1884: Glyptothoa myripristae (Bruce, 1990) comb. nov., Glyptothoa propinqua (Richardson, 1904) comb. nov. and Glyptothoa caudata (Schioedte and Meinert, 1884) comb. nov. Key words: Marine fish parasite, Branchial cavity, Cytochrome c oxidase subunit I, Cymothoidae, New genus, Indian Ocean, Phylogeny Citation: Helna AK, Aneesh PT, Kumar AB, Ohtsuka S. 2023. Morphological description and molecular characterisation of Glyptothoa gen. nov., a fish parasitic deep-sea Cymothoid (Crustacea: Isopoda) from the Indian Ocean, with four species, including one new species. Zool Stud 62:51. doi:10.6620/ZS.2023.62-51. Zoological Studies 62:51 (2023) doi:10.6620/ZS.2023.62-51 1 © 2023 Academia Sinica, Taiwan BACKGROUND The deep sea is seen by many as one of the most fascinating ecosystems on earth; it remains one of the least explored eco-regions of the world. The deep-sea ecosystem is also severely hampered by human activities, so there is a great need to document biodiversity, the ecosystem structure, and their functional interactions. Globally, information on parasitic cymothoids infesting commercial fishes is well documented, with over 100 publications since 2000 (Smit et al. 2014; Ravichandran et al. 2019; Aneesh and Kappalli 2020; Aneesh et al. 2022 2023; Fujita et al. 2023). On the other hand, studies specifically on parasitic crustaceans of deep-sea fishes are still meagre, especially at depths greater than 500 metres (Yamauchi 2009; Smit et al. 2014; Aneesh et al. 2020c). Knowledge of the cymothoid fauna of the Indian coast began with the work of Brunnich (1783), Miers (1880) and Barnard (1936). Later, Pillai (1954 1963 1964) and Tiwari (1952) added further records and described two new genera and seven new species from India. There was then an extended period of nearly five decades when there was no research on Indian cymothoids until Rameshkumar et al. (2011) described two new species from Indian waters, followed by subsequent revision of the Indian Cymothoidae by Ravichandran et al. (2019). Apart from taxonomy, few studies have been done on the seasonal occurrence of cymothoids (Aneesh et al. 2013; Helna et al. 2019). Since that review (Ravichandran et al. 2019), a further five genera, including one new genus, and ten new species from the southwest coast of India, have been reported by Aneesh et al. (2019 2020a b c 2021a b c 2022 2023). A few attempts have been made to study the reproductive biology and life history of some cymothoids (see Aneesh et al. 2022). The family Cymothoidae currently includes 385 accepted species in 43 genera; of these, only 57 species from 18 genera are known from India (Aneesh et al. 2022; Nashad et al. 2022), with only 12 species known from the deep sea, including the recently described Brucethoa bharata Aneesh, Hadfield, Smit, and Kumar, 2020 (Aneesh et al. 2022). The present study describes a new genus and species of deep-sea fish parasitic cymothoid based on the specimens collected during the recent studies on deep-sea fish parasitic cymothoids of the Indian coast initiated by the authors. The host fish Glyptophidium macropus Alcock, 1894 (Ophidiidae), was captured at a depth between 300 to 650 meters from the southwest coast of India, and an undescribed parasitic isopod was recovered from the branchial cavity. During the identification process of this isopod, it was clear that it belonged to the group of genera including Brucethoa Aneesh, Hadfield, Smit and Kumar, 2020; Elthusa Schioedte and Meinert, 1884, Mothocya Costa, in Hope, 1851 and Ichthyoxenos Herklots, 1870. Morphological differences excluded the inclusion of the new isopod in any of these genera (see Table 1). Further, of the new Indian specimens and three species of Elthusa, two are incertae sedis and one is apparently not so, but all share the following characteristics: cephalon immersed in pereonite 1, pereonites 4–7 slightly decrease in width towards one side, slightly asymmetrical, lateral margins slightly constricted on the hunched side, relatively wide pleon; 1.00 to 1.20 times as wide as greatest pereon width, with lateral gaps between pleonites, buccal cone obscuring antennal bases, pleopods rami all simple, without folds or thickened ridges. The three Elthusa species transferred to Glyptothoa are: E. myripristae Bruce, 1990, E. propinqua (Richardson, 1904), and E. caudata (Schioedte and Meinert, 1884). MATERIALS AND METHODS Sampling site Fresh specimens were collected from the branchial cavity of the deep-sea fish Glyptophidium macropus Alcock, 1894 (Ophidiidae), obtained from the commercial trawlers operating from Neendakara (08°30.0'N, 76°53.30'E), Kollam district, Kerala state, southwest coast of India at a depth between 300 to 650 meters. Parasite identification The collected cymothoids were preserved in 95% ethanol for DNA studies, and the remaining specimens were processed following the techniques described in Aneesh et al. (2019 2021c). One ovigerous female was designated as the holotype, and one paratype was minimally dissected to conserve the specimens (the dissected appendages were kept in separate vials along with the said specimen). Methods for dissection, mounting, and drawings of appendages followed the techniques described in Aneesh et al. (2019). The specimens were microphotographed using a multifocusing dissection microscope Leica-M205A and image capturing software (Leica Application Suit). Drawings were digital-inked using Adobe Illustrator and a WACOM CTL-472/K0-c drawing pad. Sources for the fish taxonomy and host nomenclature were Fish Base (Froese and Pauly 2023) and Catalogue of Fishes (Fricke et al. 2023). Classification of the cymothoid followed Brandt and Poore (2003). The type specimens page 2 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Table 1. Character differences between the closely related branchial cymothoid genera, Glyptothoa gen. nov., Brucethoa Aneesh, Hadfield, Smit & Kumar, 2020, Elthusa Schioedte & Meinert, 1884, Catoessa Schioedte & Meinert, 1884, Ichthyoxenos Herklots, 1870 (marine) and Mothocya Costa, in Hope, 1851 Characters Glyptothoa gen. nov., Brucethoa Aneesh, Hadfield, Smit & Kumar, 2020 Elthusa Schioedte & Meinert, 1884 Catoessa Schioedte & Meinert, 1884 Ichthyoxenos Herklots, 1870 (marine) Mothocya Costa, in Hope, 1851 Cephalon, anterior margin with acute ventrally directed rostrum with acute ventrally directed rostrum truncate (s. str). truncate sub-acute or rounded; with acute ventrally directed rostrum rounded, ventrally folded Body pereonites 4–7 slightly decrease in width towards one side, slightly asymmetrical, lateral margins slightly constrict, in hunched side slightly asymmetrical asymmetrical (most) bilaterally symmetrical bilaterally symmetrical weakly to moderately asymmetrical Body, dorsum moderately vaulted medially vaulted not or weakly vaulted vaulted not or weakly vaulted not or weakly vaulted Buccal “cone” anteriorly positioned, overriding antennal bases anteriorly positioned, overriding antennal bases not anteriorly positioned, not overriding antennal bases not anteriorly positioned, not overriding antennal bases not anteriorly positioned, not overriding antennal bases not anteriorly positioned, not overriding antennal bases Pereonites 6 and 7 posterolateral margin not much expanded posterolateral margin laterally expanded posterolateral margin not expanded posterolateral margin not expanded posterolateral margin not expanded posterolateral margin not expanded Pereonites 6 and 7, coxae narrow, visible in dorsal view not visible in dorsal view wide, visible in dorsal view visible in dorsal view visible in dorsal view visible in dorsal view; often large Pleonite 1 the lateral margins of pleonite 1 strongly extend laterally; moderately narrower than pleonite 2 as wide as pleonite 2 as wide or slightly narrower (s. str) narrower than pleonite 2 narrower than pleonite 2 slightly narrower Pleonites all visible all visible pleonites partly concealed, or all visible all visible all visible pleonites 2–5 or 3–5 visible Pleonites, free lateral margins pleonites 1–5 pleonites 2–5 or 3–5 pleonites 2–5 or 3–5 pleonites 2–5 pleonites 1–5 or 2–5 visible pleonites 2–5 or 3–5 visible Pleon width wider than widest pereon: 1.00 to 1.20 times as wide as pereon max. width wide: 0.87 pereon max. width greater than 0.7 pereon max. width (s. str.) variable: 0.64–0.84 narrow: 0.59–0.69 variable: 0.52–1.01 Pleonite gaps long gaps (60%; as width of widest pleon) present between all pleonites long gaps present (50%; as width of widest pleon) between most or all pleonites without gaps with short gaps without gaps without gaps Antennula length shorter than antenna shorter than antenna shorter than antenna longer than antenna longer than antenna longer than antenna Antennula size slender (= antenna) slender (= antenna) slender (= antenna) slender (= antenna) slender (= antenna) robust (thicker than antenna) Oostegites proximally thick, oostegite 1(of pereonite 2) bilobed proximally thick not proximally thick not proximally thick not proximally thick not proximally thick Pleopods not visible in dorsal view large, conspicuously visible in dorsal view not large, not visible in dorsal view not large, not visible in dorsal view not large, not visible in dorsal view not large, not or slightly visible in dorsal view Uropods short, not reaching posterior of pleotelson short, not reaching posterior of pleotelson short, not reaching posterior of pleotelson variable, reaching to between half-length of pleotelson to posterior margin short, not reaching posterior of pleotelson variable, long or short Maxilliped oostegite lobe present present absent absent present absent Note: Elthusa characters based on Elthusa sensu stricto (s. str.) as defined by Aneesh et al. (2020b) and other genera updated based on Aneesh et al. (2020c). page 3 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan were deposited in the Western Ghat Field Research Centre of the Zoological Survey of India, Kozhikode (ZSI/WGRC) and remaining few non-types were placed in PTA’s & AKH’s personal collection in India (CAH). Molecular analysis Genomic DNA was extracted from the cymothoid pereopods and pleopods following the protocol for animal tissue extraction of the NucleoSpin® Tissue Genomic DNA Tissue Kit (MachereyNagel, Düren, Germany). A targeted part of the mitochondrial cytochrome c oxidase subunit I (COI) gene (approximately 680 bp) of these specimens was subjected to PCR amplification with the aid of a ProFlex™ PCR thermal cycler (Applied Biosystems by Life Technologies) and universal invertebrate primers LCO1490 (5'-GGTCAACAAATCATAAAGATATT GG-3') and HC02198 (5'-TAAACTTCAGGGTGACC AAAAAATCA-3') (Folmer et al. 1994). PCR reactions were performed with volumes of 25 µl, using 12.5 µl Thermo Scientific DreamTaq PCR master mix, 1.25 µl of each primer, 7 µl of PCR-grade nuclease-free water and 3 µl of DNA. Conditions for the PCR were as follows: initial denaturation at 94°C for 5 min; followed by 35 cycles of 94°C denaturation for 30 s, annealing at 47°C for 50 s with an end extension at 72°C for 2 min; and ending with a final extension of 72°C for 10 min. The PCR amplification was performed in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems). Polymerase chain reaction products were purified for sequencing with USB ExoSAP-IT (GE Healthcare) and sequenced in forward and reverse directions with the PCR primers by Dideoxy Sanger standard method with BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems Inc., Foster City, USA) on an ABI sequencer (Applied Biosystems Inc., Foster City, USA). The obtained sequences were edited and aligned with BioEdit v.7.0.9.0. (lbisBiosciences, Carlsbad, USA., Hall, 1999). Phylogenetic analysis and sequence divergence were estimated using the Kimura 2-Parameter distance model of the MEGA (Version 11.0) Package (www.megasoftware.net/, Tamura et al. 2021). The maximum likelihood tree was constructed and was bootstrapped 1,000 times to provide percentage bootstrap values for branch points. The genetic distance of each species was done based on pair-wise distance analysis using the Maximum Composite Likelihood method (MEGA 11, Tamura et al. 2021). A comparison of the candidate sequence to the most similar sequences was carried out with the available data from GenBank (http://www.ncbi.nlm. nih.gov/genbank/). Comparative sequences of other cymothoids (sequences from each of eight different branchial cymothoid genera) from GenBank were downloaded and aligned to one sequence from the current study. These sequences included: LC159567 (Elthusa sp. female); MK652487 (Elthusa raynaudii); LC160320 (Cterissa sakaii); LC159570 (Ichthyoxenus tanganyikae); LC159578 (Ryukyua globosa); Livoneca redmanii MZ208985; MF628260 (Norileca indica); KC896399 (Joryma hilsae); MK652485 (Mothocya renardi); MW002498 (Catoessa boscii). Nucleotide genetic divergence in percentage (p-distance) and base-pair differences among the different species were determined using MEGA11 (Tamura et al. 2021). RESULTS TAXONOMY Suborder Cymothoida Wägele, 1989 Superfamily Cymothooidea Leach, 1814 Family Cymothoidae Leach, 1814 Genus Glyptothoa gen. nov. urn:lsid:zoobank.org:act:05AE1C92-B513-41BC-850E5864488B6EC6 Type species: Glyptothoa sagara sp. nov.; original designation. Etymology: The new generic name is the abbreviation of the host genus name (Glyptophidium) – “Glypto” combined with the ending – thoa indicating the family affinity. Gender is feminine. Diagnosis: Ovigerous female (bold = key features): Body dorsally vaulted, two times as long as wide, widest at pereonite 3. Cephalon partially immersed in pereonite 1, anterior margin with acute ventrally directed rostral point. Pereonites 2–7 coxae visible in dorsal view, all coxae shorter than pereonite; pereonites 4–7 slightly asymmetrical, lateral margins slightly constricted, in hunched side. Pereonites 4–7 slightly decrease in width towards one side. Pleon short, c. 15% BL, pleonites all visible, the lateral margins of pleonite 1 strongly extend laterally; moderately narrower than pleonite 2, becoming progressively wider posteriorly, with large lateral gaps (0.6 times the pleon width) between pleonites; pleonites 1–4 medially subequal in length, pleonite 5 longest. Pleon at pleonite 5 wider than widest pereon: 1.00 to 1.20 times as wide as greatest pereon width at pereonite 3. Pleotelson 0.9 times as wide as pleonite 5. Antennula narrowly separated by rostrum, slender, with 8 articles, shorter than antenna. Antenna with 13 articles. Buccal cone obscuring antennal bases. Brood pouch arising from page 4 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan coxae 1–4, 6 proximally thick; oostegite 1 bilobed. Pleopods not visible in dorsal view. Pleopod rami all simple, without proximomedial lamellar lobe, without folds or thickened ridges; peduncle lateral lobes absent. Uropods short, extending about halfway along pleotelson lateral margin. Additional features: Mandible palp articles all slender, article 2 longer than article 3, both with small spines, Maxilla mesial lobe distinct (not fused), both lobes with two acute apical RS. Maxillula with 1 large and 3 small acuminate terminal RS. Maxilliped with oostegital lobes; mouthparts partially covered by oostegites of pereopod 2. Pereopods basis without prominent carina, without setae; articles not dilated or expanded. Variation: pleonite 1, narrowest, 2–4 subequal, pleonite 5 widest in most specimens, whereas slight varied in one specimen with pleonite 1 is narrowest and pleonites 2–5 are progressively wider. Adult male: Similar to females in general morphology. Body lateral margins sub-parallel. Coxae dorsally visible. Pleotelson proportional longer and narrower than female. Penial process acute, separated by 20% width of sternite 7. Pleopods not extending beyond pleotelson margins. Pereopods with acute RS. Species included: Glyptothoa sagara sp. nov.; Glyptothoa propinqua (Richardson, 1904) comb. nov., Glyptothoa myripristae (Bruce, 1990) comb. nov., and Glyptothoa caudata (Schioedte and Meinert, 1884) comb. nov. Remarks: Glyptothoa gen. nov. can be distinguished from all other cymothoid genera and identified by the following combination of ovigerous female characters: cephalon partially immersed in pereonite 1, dorsum vaulted, all coxae shorter than or as long as pereonite, visible in dorsal view; pereonites 4–7 slightly decrease in width towards one side, slightly asymmetrical, lateral margins slightly constricted in hunched side; relatively wide pleon, with large lateral gaps between pleonites; antennula narrowly separated by rostrum, slender, shorter than antenna; antenna with 13 articles; buccal cone obscuring antennal bases; brood pouch arising from coxae 2–6, proximally thick; oostegite 1 (of pereonite 2) bilobed; pleopods large, not visible in dorsal view; pleopod rami all simple, without proximomedial lamellar lobe, without folds or thickened ridges; peduncle lateral lobes absent. Glyptothoa sagara sp. nov. (Figs. 1–19) urn:lsid:zoobank.org:act:505AF3AB-A7D5-4147-B7ED70A2653FEC10 Type material: Holotype: 1 ovigerous female [36.0 mm L, 18.0 mm W (maximum width)], from Glyptophidium macropus Alcock, 1894, recorded from ~300 to 650 m depth, off Neendakara coast (08°30.0'N, 76°53.30'E), Kerala, India, 29 December 2019, coll. PT Aneesh & AK Helna (Reg. No. ZSI/WGRC/ IR.INV./24781). Paratypes: Same data as holotype with the following measurements and registration details: 1 mature male (15.0 mm TL; 7.0 mm W), partially dissected (Reg. No. ZSI/WGRC/IR.INV./ 24782); 1 female (non-ovigerous) (31.0 mm TL; 15.5 mm W), partially dissected (Reg. No. ZSI/WGRC/IR.INV./ 24783); 1 female (ovigerous) (28.0 mm TL; 14.0 mm W), partially dissected (Reg. No. ZSI/WGRC/IR.INV./ 24784); 1 late transitional (16.0 mm TL; 8.5 mm W), (Reg. No. ZSI/WGRC/IR. INV./ 24785); 1 female (ovigerous) (27.0 mm TL; 13.5 mm W), (Reg. No. ZSI/ WGRC/IR.INV./ 24786). Non-types: 1 young male (12.0 mm TL; 5.5 mm W) (Reg. No. CAH/INV/ISO 0300); 1 early transitional (17.0 mm TL; 8.0 mm W) (Reg. No. CAH/INV/ISO 0301). Etymology: The specific name is derived from the Sanskrit word “sagara”, literally meaning the “gathering together of waters,” i.e., the ocean. Further, it is a reminder of the importance of the ocean for the sustainable development of life underwater and its conservation. Description of ovigerous female (Figs. 1–10): Body 2 times as long as greatest width, dorsal surfaces weakly vaulted, widest at pereonite 3, most narrow at pereonite 1. Cephalon 1.8 times wider than long, partially immersed in pereonite 1. Frontal margin with acute ventrally directed rostral point. Eyes oval with distinct margins, one eye 0.15 times the width of cephalon. Pereonite 1 smooth; anterior border medially straight, slightly curved laterally; anterolateral angles with a small distinct produced point. Coxae 2–6 narrow, all shorter than respective pereonite, coxae 7 slightly shorter or equal to pereonite 7. Pereonites 2–7, posterolateral angles little produced, pereonite 4–7 posterolateral margins slightly asymmetrical, lateral margins slightly constricted in hunched side, pereonite 7 extending posteriorly to pleonite 1 or 2. Pereonites slightly increase the width from 1–3; pereonites 4–7 slightly decrease in width. Pereonite 1 longest, 7 shortest, pereonites decreasing in length from 1–3, pereonites 3–6 sub equal in length. Pleon short, ~15% BL, pleon 1.25 times as wide as maximum pereon width; pleonites progressively increasing in width towards posterior; pleonites 1–4 medially subequal in length, pleonite 5 longest. Pleonite 1 slightly narrower, subequal in length to pleonite 2. Pleotelson 1.10 times wider than pereonite 7; 1.6 times as wide as long; posterior margin, broadly rounded, lateral margins page 5 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Fig. 1. Glyptothoa sagara gen. et sp. nov. A–C, ovigerous female on the branchial cavity of the host fish Glyptophidium macropus Alcock, 1894. The arrow indicates ovigerous female. page 6 of 31 Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Fig. 2. Glyptothoa sagara gen. et sp. nov. ovigerous female holotype (Reg. No. ZSI/WGRC/IR. INV/24781). A, dorsal view. B, ventral view. C, dorso-lateral view. page 7 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan convex, dorsal proximal surface with medial furrow. Antennula narrowly separated by rostrum; article 1 widest, 1.2 times as wide as long; article 3 longest; terminal article shortest; 4–7 with 2–3 simple setae, article 8 with few terminal aesthetes. Antenna, terminal article shortest, with few short simple setae, extending to anterior margin of pereonite 1; article 1 widest, 1.5 times as wide as long; article 2 as wide as long; article 4 longest; articles 4–5 lateral margin with one plumose seta each; articles 5–13, decreasing the width; articles 10–12 sub-equal in width; terminal article 1.5 times as long as wide. Mandible palp article 1 longest, 3.3 times as long as wide; article 2 longer than article 3; palp article 2 with 5–7, article 3 with many 18–25 simple marginal setae. Maxillula with 4 unequal acuminate terminal RS. Maxilla with distinct mesial lobe and lateral lobe, each with 2 RS. Maxilliped with oostegital lobes, article 3 with three recurved RS; mouthparts not covered by oostegites of pereopod 2. Pereopod 1, basis large, 1.7 times as long as greatest width; ischium, 1.5 times as long as wide, 0.7 times as long as basis; merus 0.4 times as long as wide; carpus 0.7 times as wide as merus; propodus 1.5 times as long as wide, 0.5 times as long as ischium; dactylus 1.3 times as long as propodus, 3.3 times as long as proximal width. Pereopod 2 basis 1.7 times as long as greatest width; ischium, twice as long as wide, 0.7 times as long as basis; propodus 1.6 times as long as wide; dactylus 1.1 times as long as propodus. Pereopods 3 similar to pereopod 2. Pereopod 4 basis 1.2 times as long as greatest width; ischium as long as basis, 1.6 times as long as greatest width; propodus 1.3 times as long as wide; dactylus 1.2 times as long as propodus, 3.0 times as long as greatest width. Pereopods 5 similar to pereopod 4. Pereopod 6 basis 1.6 times as long as greatest width, ischium as long as basis, 1.8 times as long as greatest width; merus 1.2 times wider than ischium; propodus 1.4 times as long as wide; dactylus 1.1 times as long as propodus, 2.9 times as long as greatest width. Pereopod 7 basis 1.9 times as long as greatest width; ischium 0.9 times as long as basis; merus 1.2 times as wide as ischium, 0.4 times as long as wide; carpus 0.5 times as long as wide, 0.4 times as long as ischium; propodus 1.5 times as long as wide, 0.5 times as long as ischium; dactylus 1.4 times as long as propodus, three times as long as basal width. Brood pouch moderately bulged ventrally. Pleopod peduncle lateral lobes absent. Pleopod 1, Fig. 3. Glyptothoa sagara gen. et sp. nov. ovigerous female paratype (Reg. No. ZSI/WGRC/IR. INV/24786) A, dorsal view. B–C, lateral views. page 8 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan one side is slightly larger than other side; exopod 1.1 times as long as wide, lateral margin convex, distally broadly rounded, mesial margin convex; endopod 0.9 as long as exopod, 1.3 times as long as wide, lateral margin weakly convex, distally broadly rounded; peduncle twice as wide as long. Pleopod 2 without appendix masculina. Pleopod 2–5, similar. Pleopod 5 exopod as long as wide, lateral margin convex, distally Fig. 4. Glyptothoa sagara gen. et sp. nov. ovigerous female paratype (partially dissected) (Reg. No. ZSI/WGRC/IR. INV/24784). A, dorsal view. B, ventral view. C, lateral view. page 9 of 31 Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan one eye 0.3 times width of cephalon. Coxae all dorsally visible, shorter than pereonites. Pereonites 1–6 posterolateral angles not produced. Pereonite 7 posterolateral margin extending beyond the lateral margin of pleonite 1. Pereonites 3–5 more or less equal in width, 1 narrower than others. Pereonite 1 longest, 7 shortest. Pleon wide, 1.1 times as wide as pereon; pleonites progressively increase in width from 1–5. Pleotelson posterior margin broadly rounded, 1.7 times as wide as long. Antennula length shorter than antenna, with eight articles, narrowly separated by rostrum; article 1 widest, 2.0 times as wide as long; article 3 longest; terminal article shortest, articles 3–8 with few setae. Antenna with 13 articles; terminal article shortest, with few short, simple setae; article 1 widest, 1.2 times as wide as long; article 5 longest; articles 5–13, progressively decreasing in length and width. Mandible, maxillula, maxilla similar to that of non-ovigerous female. Maxilliped article 1, basally narrow without lobes; article 2 0.4 times as long as article 1, article 3, 0.8 times as long as article 2. All pereopods with acute RS. Pereopod 1 basis 1.5 times as long as greatest width; ischium 0.7 times as long as basis; merus 0.4 times as long as wide; propodus 1.6 times as long as wide, inner lateral margin with 3 acute RS; dactylus, 1.2 times as long as propodus, 3.3 times as long as proximal width. Pereopod 2 basis 2.0 times as long as greatest width; ischium 0.4 times as long as basis; merus 0.7 times as long as wide; propodus 1.2 times as long as wide, inner lateral margin with 8 acute RS; dactylus, 1.4 times as long as propodus, 3.2 times as long as proximal width. Pereopod 3 basis 2.0 times as long as greatest width; ischium 0.6 times as long as basis; propodus 1.4 times as long as wide, inner lateral margin with 6 acute RS; dactylus, 1.2 times Fig. 11. Glyptothoa sagara gen. et sp. nov. early transitional (Reg. No. CAH/INV/ISO 0301) A, dorsal view. B, ventral view. page 16 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Fig. 12. Glyptothoa sagara gen. et sp. nov. late transitional (Reg. No. ZSI/WGRC/IR. INV/24785). A, dorsal view. B, ventral view. C, lateral view. D, dorso-frontal view. page 17 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan as long as propodus. Pereopod 4 carpus inner lateral margin with 2, propodus with 3 acute RS. Pereopod 5 basis 2.0 times as long as greatest width; ischium 0.8 times as long as basis; carpus inner lateral margin with 2 acute RS; propodus as long as wide, inner lateral margin with 4 acute RS; dactylus 1.4 times as long as propodus. Pereopod 6 2.0 times as long as greatest width; ischium 0.8 times as long as basis; carpus inner lateral margin with 2 acute RS; propodus 1.4 times as long as wide, inner lateral margin with 6 acute RS; dactylus 1.2 times as long as propodus. Pereopod 7 basis 1.8 times as long as greatest width; ischium 0.9 times as long as basis; carpus inner lateral margin with 3 acute RS; propodus 1.7 times as long as wide, inner lateral margin with 5 acute RS; dactylus 1.2 times as long as propodus. Penial process acute, 1.8 times as long as basal width, separated by 30% width of sternite 7, visible on sternite 7, basally mutually adjacent. Fig. 13. Glyptothoa sagara gen. et sp. nov. transitional (A–D), early transitional (Reg. No. CAH/INV/ISO 0301). A, cephalon dorsal view. B, pleotelson and uropods. C, abdominal sternites with penes. D, penes. (E–J), late transitional (Reg. no ZSI/WGRC/IR. INV/24785). E, cephalon dorsal view. F, pleotelson and uropods. G, uropod. H, abdominal sternites with penes. I, rudimentary penes. J, pleon ventral view. page 18 of 31 Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Pleopods not extending beyond pleotelson margins, not visible in dorsal view. Pleopods 1–5 rami simple, without proximomedial lamellar lobe, folds or thickened ridges; endopod of all pleopods slightly shorter than exopod. Pleopod 1 exopod 1.5 times as long as wide, distally broadly rounded; endopod 0.9 times as long as exopod; peduncle 2.3 times as wide as long. Pleopod 2 exopod 1.1 times as long as endopod; appendix masculina of pleopod 2 straight and narrow, 0.9 as long as endopod. Pleopod 4 exopod as long as endopod. Uropod 0.9 times as long as pleotelson; peduncle 0.5 times as long as exopod, lateral margin without setae; rami not reaching the distal margin of pleotelson, marginal setae absent, apices narrowly rounded, exopod 1.2 times as long as endopod, 2.7 times as long as Fig. 14. Glyptothoa sagara gen. et sp. nov. young male (Reg. No. CAH/INV/ISO 0300). A, dorsal view. B, ventral view. C, lateral view. page 19 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Fig. 15. Glyptothoa sagara gen. et sp. nov. male (Reg. No. ZSI/WGRC/IR. INV/24782). A, dorsal view. B, ventral view. C, lateral view. page 20 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Fig. 16. Glyptothoa sagara gen. et sp. nov. male (Reg. No. ZSI/WGRC/IR. INV/24782). A, dorsal view. B, cephalon frontal view. C, cephalon ventral view. D, antennula. E, antenna. F, mandible. G, maxilla. H, maxillule. I, maxilliped. page 21 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Fig. 17. Glyptothoa sagara gen. et sp. nov. male (Reg. No. ZSI/WGRC/IR. INV/24782). A–G, pereopods 1–7. H, penes. page 22 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan Fig. 18. Glyptothoa sagara gen. et sp. nov. male (Reg. No. ZSI/WGRC/IR. INV/24782). A–E, pleopods 1–5. F, uropods and pleotelson of young male. G, uropods and pleotelson. H, uropod. page 23 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan greatest width. Endopod 2.2 times as long as greatest width, lateral margin convex. Colour: pale tan. Distribution: Known only from the type locality. Host: Known only from the type host Glyptophidium macropus Alcock, 1894. Remarks: Glyptothoa sagara sp. nov. can be identified by the following features: cephalon partially immersed in pereonite 1; antenna with 13 articles; pleotelson 1.9 times longer than pleon; pleotelson 0.6 times as long as wide; uropod exopod longer than endopod; and uropods extending half the length of pleotelson. The inter-specific character between Glyptothoa sagara sp. nov., and three new combinations are listed in table 2. Molecular analysis (Table 3; Fig. 19): A 680 bp long COI sequence for Glyptothoa sagara gen. et sp. nov., was newly generated to maintain a gene library. The sequence was compared to the representatives from other nine branchial cymothoid genera, such as Elthusa, Cterissa, Ichthyoxenus, Ryukyua, Livoneca, Norileca, Joryma, Mothocya and Catoessa sequences available on GenBank (Table 3; Fig. 19). The alignment was 610 bp, no stop codons, translation on frame 2, using invertebrate mitochondrial code. Nucleotide genetic divergence (p-distance) among Glyptothoa sagara gen. et. sp. nov. and other nine available branchial cymothoid genera is 25.5% (Elthusa) to 40.9% (Norileca). Ecological remarks: We have closely examined 45 species of deep-sea fishes (from November 2017 to November 2021) and the parasite Glyptothoa sagara gen. et. sp. nov. was recovered only from Glyptophidium macropus Alcock, 1894 (family: Ophidiidae) signifying its oligoxenous host specificity. In the present study, we have examined the host fish collected from different localities along the southwest coast of India. A total of 96 individuals of G. macropus were examined from four available localities along the southwest coast of India. Of these, 27 individuals were infested with Glyptothoa sagara sp. nov. with a prevalence of 28.1%, and all were obtained from the type locality, the Neendakara coast. Twenty females (16 ovigerous and 4 non-ovigerous), 7 transitional stages (4 early and 3 late), and 12 males were recovered from these 27 infested host fish. Parasites were usually found in pairs in the host fish, one in each branchial cavity; mostly male-female pairs were found; the relatively large ovigerous female was found settled in the floor of the branchial cavity, facing the cephalon anteriorly (see Fig. 1). Males were found Fig. 19. Maximum likelihood tree of the branchial cymothoids including Glyptothoa sagara gen. et sp. nov., based on mitochondrial cytochrome oxidase I (COI) gene. Numbers at nodes indicate bootstrap support values. The accession numbers in GenBank are shown before each scientific name. page 24 of 31Zoological Studies 62:51 (2023) © 2023 Academia Sinica, Taiwan to occupy the opposite gill chamber in more or less the same position. Similar to our previous observations on other branchial cymothoids, the hunching of the body is also seen in the female and late transitional stage of Glyptothoa sagara either towards the left or right according to their occupation of the right and left branchial cavity, respectively. Glyptothoa propinqua (Richardson, 1904) comb. nov. (Fig. 20) urn:lsid:zoobank.org:act:47F8F44F-233C-461B-AEE47D3FA0282081 Lironeca propinqua Richardson 1904: 37, figs. 6, 7; Richardson 1909: 87; 1910: 23; Thielemann 1910: 42; Nierstrasz 1931: 43; Barnard 1936: 170. Elthusa propinqua Bruce 1990: 262–263, figs. 8, 9; Saito and Yamauchi 2016: 64; Kazmi, Schotte and Yousuf, 2002: 102, fig. 85; Ravichandran, Vigneshwaran, and Rameshkumar 2019: 25. Remarks: Glyptothoa propinqua (Richardson, 1904) comb. nov. was described initially from moderately deepwater fishes off Japan as Lironeca propinqua. Later Bruce (1990) transferred this species into the genus Elthusa. The recent revision of the genus Elthusa by Aneesh et al. (2020b) regarded Elthusa as incerta sedis, since it did not wholly conform to the generic characters of Elthusa (see the generic remarks in Aneesh et al. 2020b). Based on the following characters: cephalon immersed in pereonite 1, coxae shorter than or as long as pereonites, pereonites 4–7 slightly decreasing in width towards one side, slightly asymmetrical, Table 2. Interspecific morphological character comparison between Glyptothoa sagara sp. nov., Glyptothoa propinqua (Richardson, 1904) comb. nov., Glyptothoa myripristae (Bruce, 1990) comb. nov., and Glyptothoa caudata (Schioedte & Meinert, 1884) comb. nov. collated from original descriptions and, where applicable, redescriptions (see Schioedte and Meinert 1884; Richardson 1904; Bruce 1990) Characters G. sagara sp. nov. G. propinqua (Richardson, 1904) comb. nov. G. myripristae (Bruce, 1990) comb. nov. G. caudata (Schioedte & Meinert, 1884) comb. nov. Cephalon Partially immersed in pereonite 1 Deeply immersed in pereonite 1 Deeply immersed in pereonite 1 Partially immersed in pereonite 1 Antenna With 13 articles With 11 articles With 11 articles With 12 articles Pleonite 1 Visible in dorsal view Visible in dorsal view Not visible in dorsal view Visible in dorsal view Pleotelson length to pleon length 1.9 times longer than pleon 1.2 times longer than pleon 2.9 times longer than pleon 1.3 times longer than pleon pleotelson 0.6 times as long as wide 0.46 times as long as wide 0.7 times as long as wide 0.6 times as long as wide Uropod Exopod longer than endopod Exopod longer than endopod Endopod slightly longer than exopod Endopod slightly longer than exopod Uropod length to pleotelson Uropods extending half the length of pleotelson Uropods nearly reaching the margin of pleotelson Uropods less than half the length of pleotelson Uropods nearly half the length of pleotelson *Glyptothoa caudata (Schioedte & Meinert, 1884) comb. nov., characters are based on non-ovigerous female (holotype). Table 3. Nucleotide genetic divergence among COI sequences of Glyptothoa sagara gen. et sp. nov., and other branchial cymothoid genera available in GenBank. Values are expressed in percentage (p-distance) 1234567891011 1Glyptothoa sagara gen. et sp. nov. 2 LC159567 Elthusa sp. female 26 3 MK652487Elthusa raynaudii 24 8 4 LC160320 Cterissa sakaii 25 27 26 5 LC159570 Ichthyoxenus tanganyikae 35 39 34 34 6 LC159578 Ryukyua globosa 39 37 37 37 32 7 MZ208985 Livoneca redmanii 39 39 35 39 41 33 8 MF628260 Norileca indica 41 41 40 35 36 16 36 9 KC896399 Joryma hilsae 38 33 32 34 32 29 37 29 10 MK652485 Mothocya renardi 35 35 32 32 29 26 34 26 27 11 MW002498 Catoessa boscii 37 32 31 33 31 28 35 28 2 25 page 25 of 31Zoological Studies 62:51 (2023)