New records of cheilostome Bryozoa from the eastern coast of India encrusting on the exoskeleton of live horseshoe crabs of Indian Sundarbans

Bryozoans are common commensal on hard surfaces and cover slow-moving animals like molluscans, sea turtles, brachyuran crabs, and horseshoe crabs. A total of six species of bryozoans belonging to four genus under three families of order Cheilostomatida were recorded encrusting on the carapaces of horseshoe crabs collected from Indian Sundarbans along the east coast of India and two among them, viz., Biflustra savartii (Audouin, 1826) and Sinoflustra arabianensis (Menon & Nair, 1975) are reported for the first time. Additionally, Jellyella tuberculata (Bosc, 1802) is reported for the first time from West Bengal coastal waters, previously known only from the Odisha coast of India. Both male and female horseshoe crabs were found to have been encrusted with bryozoan mats, although adequately not known about the life stages of their encrustation.


Study area
Field surveys have been conducted in the Sagar Island and Patiboni areas of the Indian Sundarbans. The Sagar Island (21.791°N, 88.131°E) is situated at the western part of Indian Sundarbans and is the largest island of the Sundarban deltaic complex ( Figure 1). Hoogly river borders north and west with Muriganga River in the east and Bay of Bengal in the south. It is a tidal dominated island and characterized by tidal creeks, mud flats/salt marshes, mangroves and sandy beaches/ dunes. The Patiboni in Frezerganj (21.578°N, 88.246°E) is well known for its fishing activities, located eastward to the Sagar Island and having a more sandy substrate at the intertidal zones ( Figure 1). The estuarine area of the Sagar Island (Tripathy et al. 2018) and Patiboni area of Frezerganj are considered as potential habitats for both species of horseshoe crabs.

Methods
The present study was conducted from March to December 2019 as part of the first authors doctoral research. Sampling was done during the end of high tide and the beginning of low tide, keeping a gap of two hours during the full moon/new moon period to avail the maximum exposed intertidal zone. C. rotundicauda and T. gigas were observed carefully on the mudflats and wherever encountered on horseshoe crabs, the bryozoan colonies were scraped off from the exoskeleton (Cephalothorax, telson, appendages, gills, and eyes) using a scalpel blade (Tan et al. 2011). The bryozoan specimens were preserved using 70% ethanol in a glass/plastic container and labelled properly in the field itself. The specimens were brought to the base camp and washed thoroughly with freshwater for automatic removal of any debris. In the base camp laboratory, collected bryozoan specimens were soaked with sodium hypochlorite (0.5%) for eight hours to remove the organic tissue and later soaked in distilled water for four hours (Shrinivaasu et al. 2015) and then dried for identification and thereafter photographed with Nikon Journal of Threatened Taxa | www.threatenedtaxa.org | 26 October 2021 | 13 (12): 19773-19780 19775 J TT D7000 with 105 mm VR lens, post-processing with Adobe Photoshop CS6. The specimen was brought to ZSI HQ, Kolkata, for comparing with other museum specimens of the same families and genus, present in the Zoological Survey of India, which is part of the National Zoological Collections. In the field, fouled horseshoe crabs were counted, sexed and measured. After data collection and sampling of bryozoan specimens, horseshoe crabs were released back to the sea. The bryozoan colonies were observed under a stereomicroscope (Leica EZ4), for which the identified colonies were given a goldpalladium coating under vacuum condition and scanning electron micrographs were prepared with a Zeiss Evo 18 special edition SEM, using the "Smart SEM version 5.09" image processing software.

RESULTS
A total of 58 Carcinoscorpius rotundicauda (Image 2) and six Tachypleus gigas were observed for bryozoan encrustation examination during the study period. Out of 58, 11 C. rotundicauda (six male and five female) and five T. gigas (four male and one female) were found encrusted with bryozoan mat. A total of six bryozoan species belonging to five genera under three families of order Cheilostomatida were documented encrusting on the exoskeleton of horseshoe crabs from the Indian Sundarbans. The study further confirmed the presence of two bryozoan species, viz., Biflustra savartii (Audouin, 1826) and Sinoflustra arabianensis (Menon & Nair, 1975), on the carapaces of horseshoe crabs, reported to be recorded for the first time from the Bay of Bengal, previously known from the Arabian sea (Menon & Menon 2006). Jellyella tuberculata (Bosc, 1802), previously known only from the Odisha coast of India (Menon & Menon 2006), was reported for the first time from the West Bengal coast during this study. Description: Colony encrusting, forming a unilaminar sheet on the substratum arranged in longitudinal rows. Zooids sub-rectangular or sub-hexogonal, curved and raised distally and angular at the two proximal corners, separated by a raised ridge with a distinct mural rim.

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Opesia occupying most of the frontal area, deep and oval, slightly smaller than the frontal membrane, nearly occupying two-thirds of the frontal area. Distribution: It is a very common species worldwide in the tropical and sub-tropical seas reported from Indonesia and all along the Pacific coast. Earlier, it was reported from Cape Comorin (Menon 1967) and the Mangalore coast (Thornely 1907) in India.
Description: Colony encrusting, zooecia elongated, aperture occupying three fourths of the front, separated by a delicate calcareous mural rim. Distal portion of the zooid overarching the pre-seeding zooid. Operculum semi-circular, straight at its proximal border, much wider than long. Cryptocyst marginally developed, granular on its surface, serrated coarsely on its inner margin. Ovicells and avicularia are wanting.
Remarks: Earlier, a colony of encrusting Biflustra hugliensis was identified from the posterior of the carapace of Lepidochelys olivacea (Olive Ridley Sea Turtle) from the Gulf of Kachchh, Gujarat (Frazier et al. 1992).
Distribution: Although a species of tropical and subtropical seas, this species was first identified from the mouth of the Hugli River, Bay of Bengal (Robertson 1921) and subsequently reported from the Gulf of Kachchh, Gujarat (Frazier et al. 1992). Except for these two records, there is no report of this species from anywhere else in India.

Image 1C Location: Bankimnagar (Sagar Island) and Patiboni (Frezerganj)
Substratum: Encrusted on ventral side of prosoma of a male Tachypleus gigas as well as encrusted on the shell of a mollusc found on the right prosoma of a female Carcinoscorpius rotundicauda.
Diagnosis: Colony encrusting, multi-serial. Zooids rectangular to sub-rectangular, quincuncially arrangement, opesia elongate-oval, bordered by a very narrow cryptocystal rim laterally and a cryptocystal shelf proximally; cryptocyst sparsely tubercular. Gymnocyst proximally, starting at the corners of the zooid, then as a thin continuous proximal rim, the gymnocyst arches forward, forming small pockets beneath, especially at the corners; in fully calcified zooids the gymnocystal tubercles can be stoutly developed, completely concealing the proximal cryptocyst.
Distribution: A widely distributed species of the major oceans, this species is reported from North Carolina to Brazil along the Atlantic coast, California to up to Peru along the Pacific coast. Among the Indian Ocean countries, it is reported from Japan and Bangladesh and in India, it has been earlier reported from the coast of Odisha (Menon & Menon 2006 Description: Encrusting, colonies appear as whitish patches with uneven growing margin. Zooecia quincuncially arranged, chitinous outline distinct. Shape of zooecia variable, but generally longer than wide, very much elongated in certain cases. Cryptocyst tuberculated, developed all-round the opesia with tubercles projecting into the opesia. The tubercles are more or less of the same length, small tubercles are present in the proximal region of the cryptocyst. In certain Zooids the proximal region of the opesia is broader than the distal region. Remarks: This species is known to be found on fouling organisms which have been previously identified from the carapace and appendages of the Neptunus pelagicus (Swimming Crab) caught in a trawl net in Cochin (Menon 1967).
Distribution: Conopeum reticulum is a warm water Indo-Pacific species. This is recorded from Tortuges Island, Florida (Osburn 1950); Indonesia (Harmer 1926); Java, Sumatra, and Myanmar (Marcus 1937). In India, it has been reported from the Arabian Sea along with the Lakshadweep Islands and the Cochin coast (Menon 1967) as well as the Bay of Bengal from Chilka Lake (Annandale 1915). (Sagar Island) Substratum: Encrusted on prosoma of a male Carcinoscorpius rotundicauda and and also found encrusted on hardened sediments found on the right side of the prosoma of a female Carcinoscorpius rotundicauda.
Description: Colony encrusting, white. The zooecia are moderate in size and very delicate and chalk like, zooids elongated rectangular, arranged in quincuncial series, and separated by a distinct fine groove. The mural rim is thin, raised and smooth on its edge. No gymnocyst. Frontal membrane large, occupying the whole of the frontal area. Cryptocyst marginal, narrowest distal to the opesia, developed laterally and proximally, smooth and granular in younger colonies, and granular on its surface in older colonies, with strong cryptocystal spinules. It contains six strong cryptocystal spinules on each side equidistant from each other, on its inner border proximal to the orifice. Opesia elongate and reduced by the cryptocystal spinules. A strong conical spine is present on each distal corner of every zooid.
Distribution: This species has been reported to have its presence since the Pliocene era and distribution range in the Indo-Pacific region. It has been originally collected from Amoy of China; in India, this species has the report of its presence in the Holocene rocks of the west coast of Maharashtra, Ernakulam channel from Cochin, and also from the coast of West Bengal (Menon & Menon 2006).
Description: Colony encrusting. Grows flat, disk-like structures in the absence of any hindrance. Zooecia elongated, quadrangular the distal portion of the preceding zooecium slightly over arch the proximal portion of the succeeding zooid. Opecia occupying threefourths of the front, being narrowed distally. Gymnocyst present, slightly extensive proximally. Cryptocyst with spinules, the size of the spinules decrease at the distal portion of the cryptocyst. Ancestrula possesses a pair of branched spines.
Distribution: It has been reported only from Cochin along the coast of the Arabian Sea (Menon 1967). This is the first report from the Bay of Bengal and also from the Indian Sundarbans region.

DISCUSSION AND CONCLUSION
Bryozoans are important macro fouling community in the coastal waters of India. So far, very little is known on the bryozoan species diversity and their association with horseshoe crabs and other organisms with hard surfaces and substratum. In India, the upper eastern coast is a preferred breeding and spawning ground for two species of horseshoe crabs: Tachypleus gigas and Carcinoscorpius rotundicauda. Both the species are in the data deficient category of the IUCN Red List; however, placed in the Schedule IV category of the Indian Wildlife Protection Act, 1972. The Mangrove Horseshoe Crab Carcinoscorpius rotundicauda (Latreille, 1802) is more common on the mudflats of the Indian Sundarbans than the Indian Horseshoe Crab Tachypleus gigas (Müller, 1785) although occurring in a sympatric habitat. In the present study, it was observed that adult male and female horseshoe crabs are host for bryozoan mats and the reason could be multiple. As most marine organisms compete for substrate space (Paine 1974;Jackson 1977;Connell & Keough 1985) to attach with suitable host species for their dispersal and gene flow (Wahl 1989), unoccupied and clean, bare exoskeletons of horseshoe crabs may act as an ideal surface for colonization of bryozoan species and probably help them to expand their range of distribution. Currents generated by the movement of hosts, respiration and feeding of the host (Bowers 1968;Wahl 1989;Gili et al. 1993) may help in capture of suspended food particles to the bryozoans. Additionally, host species may also protect bryozoan species from predators like amphipods, annelids, echinoids, isopods, nudibranchs, pycnogonids, and gastropods and in return, bryozoans help the host species via camouflage (Key et al. 1996(Key et al. , 2000. As studies elsewhere (Renouf 1932;Cadee 1991) suggest bryozoan encrustation can reduce the effectiveness of the host's organs, hence, it can be inferred that epizoic bryozoans may impair the sight of horseshoe crabs as bryozoan mats were found encrusting on the compound eyes of horseshoe crabs during the present study, although bryozoan growth was also found on the mouth, gills, legs and telson of horseshoe crabs. Therefore, these aspects need further investigation to study the occurrence of any parasitic organisms of Bryozoa, which may impair the movement/ function of organs of horseshoe crabs. Overall, the interaction between a horseshoe crab and epizoic bryozoan is found non-symbiotic and facultative (Key et al. 1996) and as epizoic bryozoans have a less negative impact on horseshoe crabs, both co-exist.