Identification and analysis of pathogenic bacteria causing outbreaks in Indian major carp aquaculture of Tripura.

OBJECTIVE: The objective of this study was to investigate bacterial disease outbreaks in Indian major carp from aquaculture systems in Tripura, India, and identify the bacterial species associated with those outbreaks. METHODS: A 3-year surveillance was conducted in eight districts of Tripura, during which nine bacterial disease outbreaks were recorded. Fourteen bacterial strains isolated from diseased Indian major carp were selected and identified using phenotypic, molecular (16S ribosomal RNA gene), and phylogenetic analyses. In vitro pathogenicity studies were performed to assess the potential pathogenicity of the isolated bacteria. RESULT: The selected isolated strains were preliminarily identified under the genera Aeromonas (9 isolates), Acinetobacter (1 isolate), Citrobacter (3 isolates), and Pseudomonas (1 isolate). Molecular and phylogenetic analyses confirmed the species of the isolated bacteria, including Aeromonas jandaei (strains COF_AHE09 and COF_AHE61), Aeromonas veronii (strains COF_AHE13, COF_AHE52, COF_AHE55, COF_AHE56, and COF_AHE62), Aeromonas hydrophila (strains COF_AHE51 and COF_AHE58), Acinetobacter pittii (strain COF_AHE14), Citrobacter freundii (strains COF_AHE20, COF_AHE57, and COF_AHE59), and Pseudomonas aeruginosa (strain COF_AHE54). Behavioral and clinical signs observed in the diseased fish, such as lethargy, skin hemorrhaging, ulcers, fin and tail rot, exophthalmia, distended abdomen, scale loss, and skin discoloration, indicated the presence of bacterial septicemia. The in vitro pathogenicity studies highlighted the potential role of these bacteria in disease development, especially under environmental stress. CONCLUSION: This study provides valuable insights into the diversity of bacterial species associated with bacterial disease outbreaks in Indian major carp from aquaculture systems in Tripura. It serves as the first comprehensive investigation of its kind, contributing to our understanding of bacterial infections in Indian major carp.

Alteration of gut microbiota composition and function of Indian major carp, rohu (Labeo rohita) infected with Argulus siamensis.

Gut microbiome homeostasis is critical in preventing diseases. However, the effect of disease on gut microbiota assembly remains unclear. At present, there are no reports on the composition and functional analysis of intestinal microbiota of Indian major carp, rohu (L. rohita) infected with ectoparasite, Argulus. In this study, we analysed and compared the intestinal microbiota of healthy and Argulus-infected rohu by 16S rRNA amplicon sequencing. Argulus infection could significantly influence the diversity and richness of the gut microbiota. However, abundance of Actinobacteria and Patescibacteria were enriched significantly in Argulus-infected fish. Venn diagram revealed that there were many more unique genera in the infected group as compared to control fish. The genera, Stenotrophomonas and Pirellula were significantly increased in infected fish while the abundance of Reyranella was decreased. LEfSe analysis showed a significant enrichment in abundances of 11 taxa in healthy group and 17 taxa in infected group. Furthermore, genera Rubellimicrobium, Dielma, Hyphomicrobium, Reyranella, Streptomyces and Cloacibacterium performed the best in differentiating between both the groups. Predicted microbiota function by PICRUSt revealed that the gut microbiota of infected fish was mainly associated with enriched synthesis of chitinases, chitin binding proteins, osmoprotectant proteins and sulfatases enzymes. There was a positive association between the structural and functional composition of the gut microbiota. The results indicated that the Argulus infection could affect the intestinal microbiota composition and function of rohu.

De novo assembly, transcriptome characterization and marker discovery in Indian major carp, Labeo rohita through pyrosequencing.

Labeo rohita, one of the Indian major carps, is the most popular culture species in Indian subcontinent due to its consumer preference and delicacy. A selective breeding program for harvest body weight has resulted in an average genetic gain of 17% per generation. Transcriptome resource for this species is scanty. Here, we have characterized the liver and muscle transcriptomes of rohu using Roche 454 GS-FLX next generation sequencing platform. In total, 1.2 million reads were generated, de novo assembly and clustering resulted in 4171 transcripts. Out of these, 4171 had significant blast hit against NCBI nr database, and 2130 transcripts were successfully annotated. In total, 289 SSRs were identified with an identification rate of 5.8%, and dinucleotide repeat motifs were observed to be the most abundant SSRs. Further, 2231 putative SNPs were identified with high confidence. Validation of eight putative SNPs using Sanger sequencing resulted in 100% true SNPs. Significant allelic imbalance of M1, M4 and M5 loci between growth selected and control individual were observed. Furthermore, 13 transcription factors were identified in the present study belonging to six different transcription factor families. The present study demonstrated the utility of RNAseq to develop genomics resources in non-model fish species, and the marker resources developed would support the genetic improvement program of this species.

Molecular characterization and expressional modulation of IRAK1 as downstream signaling adaptor molecule of TLR-signaling pathways in Labeo rohita following PAMPs stimulation and bacterial infections.

Interleukin-1 receptor associated kinase (IRAK1) is one of the crucial signal transduction mediators in TLR/IL-1R signaling pathways in host immune system. To investigate about it in rohu (Labeo rohita), one of the economically important freshwater fish species in the Indian subcontinent, we cloned, characterized and analyzed its expression following bacterial infection and pathogens associated molecular patterns (PAMPs) stimulation. The full-length cDNA of rohu IRAK1 (LrIRAK1) consisted of 2765 nucleotide (nt) having an ORF of 2115 nt encoding a polypeptide of 704 amino acids (aa) with a molecular mass of 70.4 kDa. Structurally, LrIRAK1 consisted of twenty-nine helix, twelve strands and forty one coils making one N-terminal death domain (19-94 aa) and a central serine threonine kinase catalytic domain (or kinase domain) (188-489aa). In addition to these two prominent domains, LrIRAK1 also contained highly conserved amino acids viz., lysine 215 and aspartic acid 314 and threonine 185, 361 which were reported to be important for kinase and phosphorylation activity respectively in other animals. Similar to higher vertebrates, LrIRAK1 also consisted of CDK1 (cyclin-dependent kinase1) at 338-352 aa; NEK2 (NIMA-related kinase 2) at 47-61 aa; NEK6 (NIMA-related kinase 6) at 581-595 aa and AMPK (AMP- activated protein kinase) motif at 518-538 aa. Phylogenetically, LrIRAK1 is closely related to cave fish, common carp exhibiting high similarity (~95%) and identity (~90%). In the uninfected fish, the LrIRAK1 expression was highest in liver (~11.5 fold) and lowest in blood. In response to Aeromonas hydrophila, Edwardsiella tarda and Bacillus subtilis infection and various TLR and NLR-ligands stimulation, the expression of LrIRAK1 was markedly enhanced at various time points in almost all the tested tissues. These results together suggest the key role of LrIRAK1 in pattern recognition receptors (PRRs)-mediated host defense against pathogenic insults.

Extreme warm acclimation temperature alters oxygen consumption, micronucleus formation in erythrocytes, and gill morphology of rohu (Labeo rohita) fingerlings.

Experiencing the seasonal variation and rapid global warming in the tropical climate is a common phenomenon which challenged the aquatic organisms to adapt the physiology and behavior. To investigate the effect of high-temperature acclimation, we selected Indian major carp, rohu (Labeo rohita), a commercially important freshwater aquaculture species. Oxygen consumptions, micronucleus formation in erythrocytes, and gill histopathology were observed in L. rohita fingerlings acclimated at three temperatures (30, 33, and 36 °C) for 30 days. Results showed that the highest acclimated temperature (36 °C) induced higher oxygen consumption and increased frequency of micronucleus formation in erythrocytes. Severity of different histological alterations (hyperplasia, epithelial necrosis, telangiectasis, epithelial lifting, and hypertrophy of chloride cells) in the gills was found to be increased in the highest acclimated temperature (36 °C). These findings indicate the temperature induced adaptive responses and climate vulnerability in a changing environment.

Biometric Indices, Physio-Metabolic Responses and Carcass Quality in Rohu (Labeo rohita) during Feed Deprivation.

Understanding changes in biometric indices and metabolism in fish exposed to feed deprivation may be useful in aquaculture. The present study elucidates the effect of feed deprivation on physio-biochemical responses, such as changes in biometric indices, nutrient mobilization patterns, and enzyme activities in rohu (Labeo rohita). Experimental fish (av. wt. 3.41 ± 0.07 g) were deprived of feed and sampled at intervals of 0, 15, 30, 45, and 60 days to measure weight, length, body composition, and the activities of enzymes involved in digestion, metabolism, and antioxidation. A decrease in body weight, condition factor (CF), hepatosomatic index (HSI), and gastrosomatic index (GSI) was observed during the initial stage of feed deprivation (15 to 30 days) but remained unchanged thereafter. The total carbohydrate and lipid content also decreased rapidly up until 30 days, then stabilized. However, the reduction in tissue protein content (% wet weight) continued gradually with the duration of feed deprivation from 12.85 ± 0.36 at 0 days to 10.04 ± 0.67 at 15 days, 8.79 ± 0.59 at 30 days, 6.95 ± 0.69 at 45 days, and 6.16 ± 0.8 at 60 days, which was lower, compared to the other two body constituents. Amylase, protease. and lipase activities significantly reduced up until 30 days, but then stabilized. Although G6PDH enzyme activity decreased, gluconeogenic (LDH, AST, and ALT) and antioxidative (SOD and catalase) enzyme activities increased during initial feed deprivation (up to 30 days). A tissue-specific difference in amino acid metabolism with a major role of ALT in liver and AST in muscle was observed. This study revealed that rohu fingerlings adapted well to feed deprivation up until 30 days, beyond which there was an overall deterioration in the metabolic functions.

The draft genome of Labeo catla.

OBJECTIVE: Labeo catla (catla), one of the three Indian major carps, is native to the Indo-Gangetic riverine system of India as well as the rivers of Pakistan, Bangladesh, Nepal and Myanmar. Its higher growth rate and compatibility with other major carps, specific surface feeding habit, and consumer preference have increased its popularity in carp polyculture systems among the fish farmers in Indian subcontinent. Recent advancement in sequencing technology coupled with massive parallel sequencing platforms has facilitated accelerated genetic improvement in aquaculture species through integration of genomics tools. A draft genome and allied resources are lacking in catla. Therefore, in the present study, we have performed de-novo assembly of Labeo catla for the first time. DATA DESCRIPTION: A male farm reared catla was used for extracting high molecular weight genomic DNA followed by sequencing in Oxford Nanopore and Illumina platforms. Approximately, 80× coverage of sequence data was assembled adopting the hybrid assembly strategy. The assembled genome size of catla was 1.01 Gb containing 5345 scaffolds with N50 value 0.7 Mb and more than 92% BUSCO completeness. Gene annotation resulted in 25,812 predicted genes.

Effect of heavy metals on tissue-specific antioxidant response in Indian major carps.

Concentrations of heavy metals (Cu, Ni, Zn, Cd and Pb) were measured in sediments, water and liver and kidney tissues of three Indian major carps (Labeo rohita, Catla catla and Cirrhinus cirrhosus), belonging to two different weight groups (250 and 500 g), collected from ponds at two different sites (Nalban bheri and Diamond Harbour). The tissues were analysed for the levels of different antioxidant defence systems such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRd), glutathione S-transferase (GST), glutathione (GSH) and malondialdehyde (MDA). Concentrations of all the metals were significantly higher (P < 0.05) in sediment, water and the tissues from Nalban bheri compared to those in Diamond Harbour. Metal concentrations were the lowest in C. cirrhosus, which increased with an increase in fish weight, and the liver accumulated higher amount of metals than the kidney. Activities of all enzymatic and non-enzymatic antioxidant parameters except GPx and GRd were significantly higher (P < 0.05) in the tissues from Nalban bheri than those in Diamond Harbour. Significant multicollinearity was found in the values of SOD, CAT, GST, GRd, GPx and MDA with Pb, Cu and Ni in all three fish species at Nalban and with Cd in L. rohita and C. catla. Principal component analysis results revealed that stress response in a polluted site was directly regulated by an amalgamation of GSH profile and the levels of MDA in a synchronized manner. The study indicated a tissue-specific and species-specific difference for heavy metal-induced oxidative stress response in fish and a correlation between different heavy metals and individual oxidative stress markers.

Potential probiotics from Indian major carp, Cirrhinus mrigala. Characterization, pathogen inhibitory activity, partial characterization of bacteriocin and production of exoenzymes.

The study explored antagonistic activity of the cellular components of potential probiotic bacteria from mrigal (Cirrhinus mrigala) against fish pathogens with a basic insight of the chemical nature of the antagonistic compound. Totally 208 autochthonous gut bacteria were isolated, of which 22 strains revealed antagonism towards ≥2 of the six common fish pathogens. Zones of inhibition (halo diameter) were presented as score and the four most promising strains were selected as putative probiotics based on the cumulative score assigned. Further, evaluation of different cellular components exhibited bactericidal activity against the fish pathogens. Verification of other probiotic properties revealed that each of the selected strains produced diverse extra-cellular enzymes. The selected strains grew better in intestinal mucus than skin mucus, were resistant to diluted bile juice (2-20%) and safe for the target fish. The extracellular product used as crude bacteriocin revealed thermostability (up to 90°C) and activity over wide pH range (4-9). Partial loss of activity through treatment with proteinase-K and trypsin indicated proteinaceous nature of the antibacterial compound produced by the probiotic strains. 16S rRNA partial gene sequencing revealed that the four strains CM1FG7, CM1HG5, CM3FG19 and CM3HG10 were similar to Bacillus stratosphericus (KM277362), Bacillus aerophilus (KM277363), Bacillus licheniformis (KM277364) and Solibacillus silvestris (KM277365), respectively.

Myxobolus nanokiensis sp. nov. (Myxozoa: Bivalvulidae), a new pathogenic myxosporean parasite causing haemorrhagic gill disease in cultured Indian major carp fish, Labeo rohita (Hamilton 1822) in Punjab, India.

The plasmodia of Myxobolus nanokiensis sp. nov. were found infecting gills of Labeo rohita (Hamilton 1822) The infection rate was found to be 36.67 % (30 fishes were examined and 11 fishes were infected) in the Nanoki pond in Patiala district Punjab. Numerous minute plasmodia each filled with 150-200 spores were detected. Smear of scrapped blood-tinged mucous from gills exhibited millions of spores. Histological sections also indicated numerous plasmodia measuring 38.33-40.33 µm in diameter in the blood vessels of gill filaments. Spores of M. nanokiensis sp. nov. were elongate pyriform in shape and morphologically unique in having sharply pointed beak-like anterior end. Spores measured 9.28 µm × 5.71 µm in size. Polar capsules were equal, pyriform, 5.71 × 2.73 µm in size, each having polar filament with 7-9 coils. The present species has been proposed as new on the basis of its peculiar shape and morphometrics. This is the first report of any myxobolid infection in the farmland fishes in Punjab (India). The plasmodia in the gill filaments were of intralamellar vascular type (LV) and were present within the entire length of the filament. These plasmodia caused hemorrhage, necrosis of the blood vessels and cellular infiltration.

Tyrosine hydroxylase in the olfactory system, forebrain and pituitary of the Indian major carp, Cirrhinus cirrhosus: organisation and interaction with neuropeptide Y in the preoptic area.

Dopamine (DA) inhibits, whereas gonadotrophin-releasing hormone (GnRH) stimulates, luteinisiing (LH) cells in the pituitary of some but not all teleosts. A reduction in the hypophysiotropic dopaminergic tone is necessary for the stimulatory effect of GnRH on LH cells. Neuropeptide Y (NPY) has emerged as one of the potent, endogenous agent that modulates LH secretion directly or indirectly via GnRH. Involvement of NPY in the regulation of hypophysiotropic DA neurones, however, is not known, but there is good evidence suggesting an interaction in the mammalian hypothalamus. DA neurones, identified by tyrosine hydroxylase (TH)-immunoreactivity, were observed widely throughout the brain of the Indian major carp, Cirrhinus cirrhosus. The granule cells and ganglion cells of terminal nerve in the olfactory bulb, and cells in ventral telencephalon and preoptic area (POA) showed conspicuous TH immunoreactivity. In the POA, the nucleus preopticus periventricularis (NPP), divisible into anterior (NPPa) and posterior (NPPp) components, showed prominent TH-immunoreactivity. The majority of TH neurones in NPPa showed axonal extensions to the pituitary and were closely associated with LH cells. The NPPa also appeared to be the site for intense interaction between NPY and DA because it contains a rich network of NPY fibres and few immunoreactive cells. Approximately 89.7 ± 1.5% TH neurones in NPPa were contacted by NPY fibres. Superfused POA slices treated with a NPY Y2 -receptor agonist, NPY 13-36 resulted in a significant (P < 0.001) reduction in TH-immunoreactivity in NPPa. TH neurones in NPPa did not respond to NPY Y1 -receptor agonist, [Leu(31) , Pro(34) ] Neuropeptide Y treatment. We suggest that, by inhibiting DAergic neurones in NPPa via Y2 -receptors, NPY may contribute to the up-regulation of the GnRH-LH cells axis. The microcircuitry of DA and NPY and their interaction in NPPa might be a crucial component in the central regulation of LH secretion in the teleosts.