In vitro and in silico analysis unravelled clinically desirable attributes of Bacillus altitudinis L-asparaginase.

AIMS: To identify a marine L-asparaginase with clinically desirable attributes and characterize the shortlisted candidate through in silico tools. METHODS AND RESULTS: Marine bacterial strains (number = 105) isolated from marine crabs were evaluated through a stepwise strategy incorporating the crucial attributes for therapeutic safety. The results demonstrated the potential of eight bacterial species for extracellular L-asparaginase production. However, only one isolate (Bacillus altitudinis CMFRI/Bal-2) showed clinically desirable attributes, viz. extracellular production, type-II nature, lack of concurrent L-glutaminase and urease activities, and presence of ansZ (functional gene for clinical type). The enzyme production was 22.55 ± 0.5 µM/mg protein/min within 24 h without optimization. The enzyme also showed good activity and stability in pH 7-8 and temperature 37°C, predicting the functioning inside the human body. The Michealis-Menten constant (Km) was 14.75 µM. Detailed in silico analysis based on functional gene authenticating the results of in vitro characterization and predicted the nonallergenic characteristic of the candidate. Docking results proved the higher affinity of the shortlisted candidate to L-asparagine than L-glutamine and urea. CONCLUSION: Comprehensively, the study highlighted B. altitudinis type II asparaginase as a competent candidate for further research on clinically safe asparaginases.

A novel ferritin subunit gene from Asian green mussel, Perna viridis (Linnaeus, 1758).

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5' untranslated region (UTR) of 112 bp and 3' UTR of 165 bp. The 5' UTR and 3' UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3' loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.

Morphological and molecular characterization of Neoechinorhynchus (N.) cephali n. sp. (Acanthocephala: Neoechinorhynchidae) Stiles and Hassall 1905 infecting the flathead grey mullet Mugil cephalus (Linnaeus, 1758) from the southwest coast of India.

The present paper describes Neoechinorhynchus (Neoechinorhynchus) cephali n. sp., an acanthocephalan parasite infecting the intestine of the flathead grey mullet Mugil cephalus from the southwest coast of India. The parasite exhibited a prevalence of 7.40%, mean intensity of 18.5 and abundance of 18-19 worms/infected host. Morphologically, N. (N.) cephali n. sp. is sexually dimorphic, small, cylindrical, slightly curved and creamy white in colour. Females are larger than males, measured 8.87 × 0.88 mm and 5.65 × 0.66 mm, respectively. Proboscis is armed with three circles of six hooks each, which progressively decreases in size posteriorly. Hooks are backwardly curved and robust and tapering with a sharp, pointed tip, striations on the surface and a manubrium at its base. The body is aspinose, trunk surface with micropores and pits and proboscis surface with papilliform structures. The body wall is with five dorsal and two ventral hypodermal nuclei, along with lacunar canals connected by circular anastomoses. Lemnisci are subequal, small lemnisci are uninucleated, and large ones are binucleated. The cement gland is oval, with four giant nuclei; bursa is with many sensory cells. Eggs are elliptical, with concentric shells, and polar prolongation is absent. In the molecular and phylogenetic analyses based on the 18S ribosomal DNA region, the present species stands out with a high bootstrap value and is positioned as a sister branch of N. (N.) dimorphospinus. Based on the differences in morphology, morphometry and molecular and phylogenetic analyses, the present species of acanthocephalan infecting M. cephalus is considered as new, and the name Neoechinorhynchus (Neoechinorhynchus) cephali n. sp. is proposed.

Morphological and molecular characterization of Ceratomyxa xanthopteri n. sp. (Myxosporea: Ceratomyxidae) from the marine ornamental fish Acanthurus xanthopterus Valenciennes 1835 (Acanthuridae) off Vizhinjam coast, Kerala.

A new species of Ceratomyxa infecting the gallbladder of the marine ornamental fish Acanthurus xanthopterus collected from the Vizhinjam coast of Kerala is described. The parasite exhibited a prevalence of 100%. Mature spores recovered from the gallbladder were slightly crescentic with rounded lateral extremities and possessed convex anterior and slightly concave to straight posterior margins. Spore valves two, equal, joined by a straight and prominent suture. Myxospores measured 5.5 ± 0.6 µm in length and 15.9 ± 2.3 µm in thickness. Polar capsules two, equal, spherical, positioned anteriorly on either sides of the suture, 2.3 ± 0.2 µm long and 2.2 ± 0.2 µm wide. Polar filament with four to five coils, 21.2 ± 0.6 µm when extruded. Posterior angle 173.6 ± 5.2°. Early sporogonic stages and monosporic, disporic, and multisporic plasmodial stages were spherical to irregular in shape, with or without filopodia. Histopathologic analysis revealed that spores and developing stages were attached to the gallbladder wall as well as found free in the lumen. Morphologic and morphometric comparison of the present parasite with known species of Ceratomyxa indicated significant differences. In molecular and phylogenetic analyses, the present myxosporean revealed high divergence with related forms and occupied an independent position within the Ceratomyxa clade with high nodal support. Considering the morphological, morphometric, molecular, and phylogenetic dissimilarities with the previously described species of Ceratomyxa and the differences in host and geographic locations, the present species of myxosporean is treated as new and is named Ceratomyxa xanthopteri n. sp.

Characterization of Filisoma argusum n. sp. (Acanthocephala: Cavisomatidae Meyer, 1932) infecting the spotted scat, Scatophagus argus (Linnaeus, 1766) from the Indian coast.

The present paper describes Filisoma argusum n. sp. (Cavisomatidae), an acanthocephalan parasite infecting the intestine of the spotted scat, Scatophagus argus (Linnaeus, 1766), in the south-west coast of India. The prevalence is 18% (mean intensity: 1.61 and abundance: 1-4 worms/host). Filisoma argusum n. sp. is morphologically characterized by a creamy-white, cylindrical, elongate, aspinose, and robust trunk; a collar-like neck; and a cylindrical proboscis with 18-20 longitudinal rows of hooks, with 19-22 hooks/row. Proboscis receptacle long, double-walled. Lemnisci digitiform, equal, longer than proboscis receptacle. Females 79.14 ± 33.69 × 0.593 ± 0.19 mm; males 32.62 ± 2.98 × 0.46 ± 0.071 mm. Males with four cement glands; bulbous muscular copulatory bursa with six digitiform rays. SEM studies revealed smooth hooks, sensory pits, and epidermal micropores. Histopathological changes at the site of parasite attachment included inflammation, hemorrhage, sloughing of epithelium, and detachment of mucosal layer of the intestine. In molecular and phylogenetic analyses, the parasite occupied an independent position within the Cavisomatidae clade with high bootstrap values for both ITS1-5.8S and ITS2, and mt-CO1 regions. Considering the morphologic and morphometric differences with previously described species of Filisoma along with its phylogenetic positioning, the present acanthocephalan is treated as a new species and the name Filisoma argusum n. sp. is proposed.

A novel myxozoan parasite, Ellipsomyxa boleophthalmi sp. nov. (Myxozoa: Ceratomyxidae) in the brackishwater fish, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae) from India.

A novel myxozoan parasite is identified and described from mudskipper, Boleophthalmus dussumieri, collected from a brackishwater ecosystem in Maharashtra, India. Ellipsomyxa boleophthalmi sp. nov. was found in the gallbladder of 58 of 60 fish examined (96.7%). The parasite formed disporous plasmodia that varied in size and shape, and the thin-walled, ellipsoidal and elongated myxospores measured 9.0-10.7 × 6.0-7.8 µm. The two, spherical polar capsules measured 2.7 µm in diameter and enclosed 3-4 coils of polar tubules. Histological observations of infected gallbladder revealed the attachment of disporous plasmodial stages of the parasite to the gallbladder wall with fine pseudopodia. Under the scanning electron microscope (SEM), the myxospores showed a distinct central sutural line and two distinct depressions on the opposite sides at the openings of polar capsules. SEM also revealed the engulfment of microvilli of gallbladder wall by pseudopodia of the plasmodial stages. Analysis of the partial fragment of the SSU rDNA region (1386 bp) showed less than 98% sequence similarity with the other reported Ellipsomyxa spp. In the phylogenetic tree, the present species formed as a distinct subclade within the major clade of Ellipsomyxa spp. The unique morphological and morphometric features of the myxospore, together with the molecular analysis, allowed us to conclude that the present myxozoan is a new species and is named Ellipsomyxa boleophthalmi sp. nov., after the generic name of the host. This is the first report on the occurrence of the genus Ellipsomyxa in B. dussumieri.

Ellipsomyxa ariusi sp. nov. (Myxosporea: Ceratomyxidae), a new myxosporean infecting the gallbladder of threadfin sea catfish Arius arius in India.

The present study describes a new species of myxosporean, Ellipsomyxa ariusi sp. nov., infecting the gallbladder of the threadfin sea catfish Arius arius (Hamilton, 1822). E. ariusi sp. nov. is characterized by bivalvular, ellipsoid or elongate-oval myxospores with smooth spore valves and a straight suture, arranged at an angle to the longitudinal spore axis. Mature myxospores measured 10.1 ± 0.8 µm in length, 6.8 ± 0.5 µm in width and 7.7 ± 0.7 µm in thickness. Polar capsules are equal in size and oval to pyriform in shape. They are positioned at an angle to the longitudinal myxospore axis and open in opposite directions. Polar capsules measured 2.8 ± 0.3 µm in length and 2.5 ± 0.4 µm in width; polar filaments formed 4-5 coils, and extended to 32.2 ± 2.1 µm in length. Monosporic and disporic plasmodial stages attached to the wall of gallbladder. Molecular analysis of the type specimen generated a 1703 bp partial SSU rDNA sequence (MN892546), which was identical to the isolates from 3 other locations. In phylogenetic analyses, genus Ellipsomyxa appeared monophyletic and E. ariusi sp. nov. occupied an independent position in maximum likelihood and Bayesian inference trees with high bootstrap values. The overall prevalence of infection was 54.8% and multiway ANOVA revealed that it varied significantly with location, year, season, sex and size of the fish host. Histopathological changes associated with E. ariusi sp. nov. infection included swelling, vacuolation and detachment of epithelial layer, reduced mucus production and altered consistency and colour of bile. Based on the morphologic, morphometric and molecular differences with known species of Ellipsomyxa, and considering differences in host and geographic locations, the present species is treated as new and the name Ellipsomyxa ariusi sp. nov. is proposed.

Epizootics of epizootic ulcerative syndrome among estuarine fishes of Kerala, India, under post-flood conditions.

Epizootic ulcerative syndrome (EUS), primarily caused by the water mold Aphanomyces invadans, is an OIE-notifiable disease, having potential impacts on fisheries. We report EUS epizootics among estuarine fishes of Kerala, India, during 2018, under post-flood conditions 3 decades after its primary outbreak. Six fish species (Mugil cephalus, Platycephalus sp., Scatophagus argus, Arius sp., Planiliza macrolepis and Epinephelus malabaricus) were infected, including the first confirmed natural case in E. malabaricus and P. macrolepis. Salinity, surface temperature, dissolved oxygen and pH of resident water during the epizootic were <2 ppt, 25°C, 4.1 ppm and 7.0. The presence of zoonotic bacterial pathogens (Aeromonas veronii, Shewanella putrefaciens, Vibrio vulnificus and V. parahaemolyticus) in tissues of affected fish indicates that EUS-infected fish may pose a public health hazard if not handled properly. Lack of clinical evidence in the region during the last 3 decades, a high number of affected fishes, including 2 new fish species, the severity of skin lesions and very low water salinity (<2 ppt) during the outbreak in contrast to historical water salinity records suggest relatively recent invasion by A. invadans. Phylogenetic analysis based on the internal transcribed spacer region of the rRNA gene showed that the same clone of pathogen has spread across different continents regardless of fish species and ecotypes (fresh/estuarine environments). Altogether, the present study provides baseline data which can be applied in EUS management strategies within brackish-water ecosystems. We recommend strict surveillance and development of sound biosecurity measures against the disease.

Perkinsus olseni in the short neck yellow clam, Paphia malabarica (Chemnitz, 1782) from the southwest coast of India.

Parasites of the genus Perkinsus predominantly infect bivalves, and two species among them, P. olseni and P. marinus, are notifiable to OIE. P. olseni infections are known to cause extensive damage to wild as well as farmed bivalves globally with enormous implications to its fishery. Consequent to the initiation of a surveillance programme for aquatic animal diseases in India, Perkinsus infections were observed in many species of bivalves. The present paper describes P. olseni infections in the short neck yellow clam, Paphia malabarica from the southwest coast of India. Diagnosis of the parasite was carried out using Ray's Fluid Thioglycollate Medium culture, histology, in-situ hybridisation and molecular taxonomy. Pathology of infection and development of zoospores is also described. This forms the first report of a P. olseni infection in P. malabarica. High prevalence and intensity of infection of Perkinsus in clams raises concerns, as clam reserves in this geographical area sustain fisheries and the livelihoods of local fishing communities.

Morphological and molecular characterization of Chloromyxum argusi n. sp. (Myxosporea) infecting the urinary bladder of Scatophagus argus Linnaeus 1766 (Scatophagidae) from the southwest coast of India.

The present paper describes a new species of Chloromyxum infecting the urinary bladder of the estuarine fish, Scatophagus argus, from the southwest coast of India. The parasite exhibited an overall prevalence of 41.93%; the prevalence is influenced by host size and seasons. Mature spores are subspherical, measure 9.40 ± 0.66 by 9.32 ± 0.87 µm, and are characterized by the presence of sutural and extra-sutural ridges, binucleated sporoplasm, and a pair of caudal extensions. Four pyriform, unequal polar capsules with raised polar filament discharge pores and ribbon-like polar filaments are present. Polar filament coils numbered four to five in large polar capsules and three in small polar capsules. Pansporoblast is irregular with granulated cytoplasm and has fine villosites on its surface. Plasmodia are spherical/irregular with monosporic and polysporic forms. In molecular and phylogenetic analysis, the myxosporean stands out with a high bootstrap value and was positioned as a sister branch of Chloromyxum kurisi. In view of the morphologic, morphometric, and molecular differences with the existing species of Chloromyxum, and considering the differences in hosts and geographic locations, the present species is treated as new and the name Chloromyxum argusi n. sp. is proposed.

Molecular and morphological descriptions of Ceratomyxa collarae n. sp. and Ceratomyxa leucosternoni n. sp. from marine ornamental fishes of Indian waters.

Two novel species of Ceratomyxa infecting marine ornamental fishes from Indian waters are described. Marine ornamentals, Chaetodon collare and Chaetodon decussatus, collected from Vizhinjam, along the southwest coast of India and Acanthurus leucosternon collected from Lakshadweep islands of Arabian Sea revealed Ceratomyxa infections in their gall bladders. Mature spores of Ceratomyxa from Chaetodon collare and Chaetodon decussatus were elongate and slightly crescentic, with rounded ends, and measured 5.20 ± 0.32 µm in length and 16.32 ± 1.29 µm in thickness. Polar capsules spherical, equal in size and measured 2.23 ± 0.16 µm long and 2.24 ± 0.20 µm wide. Posterior angle measured 157.75 ± 8.650. Principle Component Analysis and molecular analysis using partial SSUrDNA sequences showed the isolates from these two hosts to be identical. Morphological, morphometric and molecular analysis using partial SSUrDNA sequences revealed the taxonomic novelty of isolates and are hence treated as Ceratomyxa collarae n. sp. Mature spores of Ceratomyxa from Acanthurus leucosternon were elongate, slightly tapering with rounded ends, and measured 7.34 ± 0.92 µm in length and 24.37 ± 2.34 µm in thickness. Shell valves were equal, joined by a narrow suture line. Polar capsules spherical in shape, equal in size, 2.59 ± 0.32 µm long and 2.46 ± 0.32 µm wide, and polar filament measured 18.68 ± 2.54 µm. Based on morphological, morphometric and molecular analyses, the present species of Ceratomyxa is distinct, considered as a new species and named Ceratomyxa leucosternoni n. sp. The paper also discusses the prevalence of the recovered parasites and host specificity of Ceratomyxa collarae n. sp.

Parasites and pathological conditions in the edible oyster, Crassostrea madrasensis (Preston), from the east and west coasts of India.

A detailed pathological survey was carried out on the commercially important edible oyster, Crassostrea madrasensis (Preston), from two distinct coastal/brackish water ecosystems of south India. Samples were collected twice a year during wet and dry seasons from 2009 to 2012. Bacterial colonies in the form of prokaryotic inclusions, protozoans (Perkinsus beihaiensis, Nematopsis sp. and ciliates Sphenophrya sp. and Stegotricha sp.), metazoans (trematodes, turbellaria, cestodes and crustaceans) and shell parasites (Polydora spp. and Cliona spp.) along with various pathological conditions (digestive tubule atrophy, ceroid bodies, haemocytic infiltration, tissue necrosis and neoplastic disorders) were observed in C. madrasensis collected from two sites. Intensity, spatial and seasonal variations in infection prevalence and pathological effects on the host were studied. The protozoan parasite, P. beihaiensis; shell parasite, Polydora spp. and pathological condition, digestive gland atrophy were most prevalent in occurrence. High-intensity infections with P. beihaiensis, larval trematodes and Polydora spp. were found to cause significant impact on host physiology. All other parasites were observed with low mean prevalence and intensity. Karapad in Tuticorin bay, the site reported with marked pollution levels, exhibited higher number of parasitic taxa and high mean prevalence and intensity for pathological conditions.

Morphometric and molecular characterisation of Tenuiproboscis keralensis n. sp. infecting marine and brackish water fishes from the south-west coast of India with a note on morphological plasticity.

A new species of acanthocephalan infecting marine and brackish water fishes from the south-west coast of India is described. The parasite belongs to the genus Tenuiproboscis, and the fish hosts include Lutjanus argentimaculatus, L. ehrenbergii, Siganus javus, Epinephelus malabaricus, E. coioides, Scatophagus argus, Parascolopsis aspinosa, Caranx ignobilis, Gerres filamentosus and Lates calcarifer. The parasite inhabits mid- and hindgut regions and is characterised by an elongated, cylindrical, bulbous and posteriorly tapering metasoma and a claviform proboscis having 14-15 rows of 14-15 hooks each. Females larger than males, measured 3898.16-10,318.00 µm (6430.00 ± 1417.30) in length and 458.93-1435.68 µm (929.81 ± 250.39) in width. Males measured 3234.89-8644.20 µm (5729.50 ± 1176.60) in length and 388.30-1584.61 µm (795.88 ± 184.12) in width. Parasites recovered from different host species showed morphological/morphometric variations. However, principal component analysis (PCA) revealed significant overlapping of characters indicating their similarities. Proboscis profiling based on variations in size and position of hooks also yielded similar results. Further, in molecular phylogenetic analysis, parasites from different fish hosts formed a monophyletic clade with strong bootstrap support, again indicating their conspecific nature. These morphological/morphometric variations can be ascribed to differences in host species. Morphology and morphometrics in combination with PCA, proboscis profiling and molecular analysis suggest the present acanthocephalan parasite is different from other described species of Tenuiproboscis. Hence, it is considered as a new species and named T. keralensis n. sp. Prevalence, intensity and abundance of the parasite in different hosts are also discussed.

Emergence of carp edema virus in cultured ornamental koi carp, Cyprinus carpio koi, in India.

A disease outbreak was reported in adult koi, Cyprinus carpio koi, from a fish farm in Kerala, India, during June 2015. The clinical signs were observed only in recently introduced adult koi, and an existing population of fish did not show any clinical signs or mortality. Microscopic examination of wet mounts from the gills of affected koi revealed minor infestation of Dactylogyrus sp. in a few koi. In bacteriological studies, only opportunistic bacteria were isolated from the gills of affected fish. The histopathological examination of the affected fish revealed necrotic changes in gills and, importantly, virus particles were demonstrated in cytoplasm of gill epithelial cells in transmission electron microscopy. The tissue samples from affected koi were negative for common viruses reported from koi viz. cyprinid herpesvirus 3, spring viraemia of carp virus, koi ranavirus and red sea bream iridovirus in PCR screening. However, gill tissue from affected koi carp was positive for carp edema virus (CEV) in the first step of nested PCR, and sequencing of PCR amplicons confirmed infection with CEV. No cytopathic effect was observed in six fish cell lines following inoculation of filtered tissue homogenate prepared from gills of affected fish. In bioassay, the symptoms could be reproduced by inoculation of naive koi with filtrate from gill tissue homogenate of CEV-positive fish. Subsequently, screening of koi showing clinical signs similar to koi sleepy disease from different locations revealed that CEV infection was widespread. To our knowledge, this is the first report of infection with CEV in koi from India.

Histology and ultrastructure of male reproductive system of the Indian Spiny lobster Panulirus homarus (Decapoda: Palinuridae).

The spiny lobster Panulirus homarus, distributed along the Southeast and Southwest coasts of India, is an important commercial species having mariculture potential. Despite its importance, the structural and ultrastructure features of male gonads from this species have received scarce attention. Hence this study was aimed to describe the male reproductive tract of the species, using standard histological and electron microscopy techniques. Gonads from 94 specimens of P. homarus ranging in carapace length 37mm-92mm from Vizhinjam (Southwest coast of India.) were obtained and processed for the study (Histology-70 numbers & ultrastructure-24 numbers). The male reproductive system consists of paired testis and vas deferens located in the cephalo-thoracic region. Macroscopically, the reproductive tract was observed in lobsters > 35mm carapace length. In immature testis, spermatogonia were seen which measured 6.9-13.8 microm in diameter and in the mature testis primary (5.4-5.9 microm) and secondary spermatocytes (2.8-3 microm) and spermatids (2.2-2.4 microm) were present. Each vas deferens consists of proximal and distal portions. The spermatophoric mass begins formation in the proximal vas deferens. In the distal vas deferens the spermatophoric mass containing the spermatozoa are arranged in packets towards the periphery by the gelatinous matrix produced by the typhlosole. Ultrastructurally, the spermatogonia have lamina, nucleus and mitochondria like bodies, the primary spermatocytes have nucleus, dense chromatin and vacuolated cytoplasm and the spermatids have mitochondria, endoplasmic reticulum and centrioles. The endoplasmic reticulum and the nuclear envelope in the spermatids form the acrosome. The radial arms with microtubules are formed in association with the dense endoplasmic reticulum, near the nucleus. The sperm has a spherical structure with the nucleus, lamellar region, spikes and acrosome. This is the first comprehensive report of the structure of the male gametes and spermatogenesis in P. homarus from Indian waters.

Morphological and Molecular Characterization of Two New Species of Ceratomyxa Thélohan, 1892 (Cnidaria: Myxosporea) from the Marine Ornamental Fish Zanclus cornutus (Linnaeus, 1758) off Lakshadweep Islands, Arabian Sea.

PURPOSE: The present study provides the complete morphological and molecular description of two new species of myxosporeans, Ceratomyxa zancli n. sp. and Ceratomyxa cornuti n. sp. infecting the gallbladder of Zanclus cornutus from the Lakshadweep Islands, Arabian Sea. METHODS: Zanclus cornutus were screened for the presence of myxosporeans, and the recovered myxospores were morphologically characterized using Nomarski Differential Interference Contrast (DIC) optics. The sequences of SSU rDNA were employed for molecular and phylogenetic studies. RESULTS: Both the parasites exhibited a prevalence of 21% each. C. zancli n. sp. is characterized by broadly cresentic myxospores with convex anterior and slightly concave to straight posterior margins and rounded ends. Spore valves two, unequal, measured 9.6 ± 0.7 µm × 25.2 ± 1.3 µm. Polar capsules two, unequal, spherical, measured 4 ± 0.6 µm × 3.5 ± 0.6 µm. Polar filament exceptionally long and arranged irregularly. Myxospores of C. cornuti n. sp. are elongated with convex anterior and slightly concave to straight posterior margins. Spore valves two, unequal, measured 7.00 ± 0.4 µm × 26.56 ± 1.8 µm. Polar capsules spherical, unequal, measured 3.52 ± 0.2 × 3.36 ± 0.35. Molecular analysis of C. zancli n. sp. (ON818297) and C. cornuti n. sp. (ON818298) resulted in 1469 and 1491 bp long SSU rDNA sequences, respectively. Molecularly C. zancli n. sp. is close to C. diplodae and C. barnesi with 91.39% similarity, while C. cornuti n. sp. appears closer to C. robertsthomsoni with 97.46% similarity. In phylogenetic analyses, C. zancli n. sp. branched separately within the Ceratomyxa clade while C. cornuti n. sp. clustered with C. robertsthomsoni and C. thalassomae. CONCLUSION: Based on the differences in morphological, morphometric, molecular, and phylogenetic characteristics, as well as differences in the host and geographic location, the above two species of myxosporeans are considered novel. The study forms the first report of a species of Ceratomyxa from Z. cornutus.

First report of Perkinsus beihaiensis in Crassostrea madrasensis from the Indian subcontinent.

Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in many wild and farmed bivalve populations. The present study was initiated to screen populations of the Indian edible oyster Crassostrea madrasensis, a promising candidate for aquaculture along the Indian coasts, for the presence of Perkinsus spp. The study reports the presence of P. beihaiensis for the first time in C. madrasensis populations from the Indian subcontinent and south Asia. Samples collected from the east and west coasts of India were subjected to Ray's fluid thioglycollate medium (RFTM) culture and histology which indicated the presence of Perkinsus spp. PCR screening of the tissues using specific primers amplified the product specific to the genus Perkinsus. The taxonomic affinities of the parasites were determined by sequencing both internal transcribed spacer (ITS) and actin genes followed by basic local alignment search tool (BLAST) analysis. Analysis based on the ITS sequences showed 98 to 100% identity to Perkinsus spp. (P. beihaiensis and Brazilian Perkinsus sp.). The pairwise genetic distance values and phylogenetic analysis confirmed that 2 of the present samples belonged to the P. beihaiensis clade while the other 4 showed close affinities with the Brazilian Perkinsus sp. clade. The genetic divergence data, close affinity with the Brazilian Perkinsus sp., and co-existence with P. beihaiensis in the same host species in the same habitat show that the remaining 4 samples exhibit some degree of variation from P. beihaiensis. As expected, the sequencing of actin genes did not show any divergence among the samples studied. They probably could be intraspecific variants of P. beihaiensis having a separate lineage in the process of evolution.

Hepatic microsporidiosis of mudskipper, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae), due to Microgemma sp.

The present study reports a case of hepatic microsporidiosis caused by Microgemma sp. in brackishwater fish, Boleophthalmus dussumieri (Valenciennes, 1837) (n = 60), from the west coast of India. An eight-month study from September 2017 to April 2018 revealed a prevalence of 11.7% for this parasite. The microsporidian showed tissue-specific infection and did not reveal any gross pathology in infected fish. Small whitish cysts containing microspores of size 0.3-0.5 mm were observed in the liver of fish. The range of pyriform microsporidian spore size varied from 2.9-3.77 × 1.85-2.67 µm. Scanning electron microscopy of the spores showed a distinct groove on the anterior end of the spore for polar tube extrusion. Polymerase chain reaction (PCR) amplification of the DNA extracted from the microsporidian-infected liver tissue using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340 bp amplicon and the genetic distance analysis showed a 0.2% variation with the reported M. tilanpasiri. Accordingly, in the phylogenetic tree, the present species of Microgemma clustered with M. tilanpasiri. Even though, the morphomeristic characters of the present Microgemma sp. was marginally different from the reported M. tilanpsasiri; the SSU rDNA showed considerably higher similarity with M. tilanpasiri. Thus, we report the species of Microgemma as Microgemma aff. tilanpasiri from a new host. This is the first report of a microsporidian from B. dussumieri and the first record of the genus Microgemma from India.

Characterization of novel L-asparaginases having clinically safe profiles from bacteria inhabiting the hemolymph of the crab, Scylla serrata (Forskål, 1775).

L-asparaginase (ASNase) is the principal chemotherapeutic agent against different blood cancers. The risks associated with current clinical preparations demand screening for novel ASNases. Accordingly, the study was conducted to shortlist ASNases having clinically safer profiles from a novel niche, namely, microbes in the gut and hemolymph of apparently healthy Scylla serrata. A four-step strategic approach incorporating the essential requirements for clinically safer profiles was followed. The initial step through plate assay showed five (9.61%) potential ASNase producers. The relative prevalence of ASNase producers was higher in hemolymph (13.33%) than gut (4.5%). The positive isolates were identified as Priestia aryabhattai, Priestia megaterium, Bacillus altitudinis, Shewanella decolorationis, and Chryseomicrobium amylolyticum. Quantitative profiles revealed high ASNase production (114.29 to 287.36 U/mL) without any optimization, with an added advantage of the extracellular production. The second step for substrate specificity studies revealed the absence of L-glutaminase and urease activities in ASNases from C. amylolyticum and P. megaterium, the most desirable properties for safe clinical applications. This is the first report of glutaminase and urease-free ASNase from these two bacteria. The third step ensured type II nature of selected ASNases, the targeted form in clinical applications. The fourth step confirmed the activity and stability in human physiological conditions. Altogether, the results revealed two potential ASNases with clinically compatible profiles.

Morphological and molecular description of a new species of Myxobolus (Myxosporea: Myxobolidae) infecting Planiliza macrolepis (Smith, 1846) from India.

The present paper describes a novel species of Myxobolus parasitizing the gill filaments of the largescale mullet, Planiliza macrolepis from Cochin backwaters, Kerala, India. The parasite develops in the gill filaments; plasmodia elongated, milky white, measured 1.37-2.18 (1.78 ± 0.35) mm × 0.07-0.12 (0.10 ± 0.02) mm in size. Mature myxospores ovoid in valvular view, biconvex in sutural view with smooth shell valves and measured 6.24-7.02 (6.63 ± 0.23) × 5.01-6.18 (5.68 ± 0.25) µm in size. Polar capsules equal, oval with pointed anterior ends, 3.07-3.58 (3.33 ± 0.12) × 1.68-2.42 (2.09 ± 0.18) µm in size. Polar filaments with 4 coils, measured 29.61 ± 4.75 µm in length when extruded. Sporoplasm binucleate with a rudimentary nucleus and a vacuole. A comparison with related Myxobolus species revealed significant morphological and morphometric differences. In BLASTN and genetic distance analysis, the present parasite showed high divergence with other myxosporean sequences, indicating its molecular uniqueness. In Maximum Likelihood and Bayesian Inference analysis, the present species stands out with M. ramadus as sister branch within the Myxobolus clade. In infected gill filaments, the plasmodia caused swelling/deformation, compression of lamellae and reduction in respiratory surface area. Three of 222 P. macrolepis screened were infected, indicating a prevalence of 1.3%. Considering the morphological, morphometric, molecular and phylogenetic differences with the previously described species of myxosporeans, along with the dissimilarities in host and geographical locations, the present parasite is treated as a new species and the name Myxobolus cochinensis n. sp. is proposed.