Jatindra Nath Saha

Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita.

Seven innate immune parameters were investigated in 64 full-sib families (the offspring of 64 sires and 45 dams) from two year-classes of farmed rohu carp (Labeo rohita). Survival rates were also available from Aeromonas hydrophila infection (aeromoniasis) recorded in controlled challenge tests on a different sample of individuals from the same families. Due to strong confounding between the animal additive genetic effect and the family effects (common environmental+non-additive genetic), reliable additive (co)variance components and hence heritabilities and genetic correlations could not be obtained for the investigated parameters. Therefore, estimates of the association of challenge test survival with the studied immune parameters were obtained as product moment correlations between family least square means. These correlations revealed statistically significant (p<0.05) negative correlations of survival with bacterial agglutination titre (-0.48), serum haemolysin titre (-0.29) and haemagglutination titre (-0.34); and significant positive correlation with ceruloplasmin level (0.51). The correlations of survival to aeromoniasis with myeloperoxidase activity, superoxide production and lysozyme activity were found to be not significantly different from zero (p>0.05). Assuming that the negatively correlated candidate traits are not favourable as indirect selection criteria, the results suggest that ceruloplasmin level could potentially be a marker for resistance to aeromoniasis in rohu. The use of this immune parameter as an indirect selection criterion for increased resistance to aeromoniasis in rohu will, however, require that the parameter shows significant additive genetic variation and a significant genetic correlation with survival. Further studies are therefore needed to obtain a reliable heritability estimate for ceruloplasmin and its genetic correlation with survival from aeromoniasis.

Expressed Sequences and Polymorphisms in Rohu Carp (Labeo rohita, Hamilton) Revealed by mRNA-seq

Expressed genes and polymorphisms were identified in lines of rohu Labeo rohita selected for resistance or susceptibility to Aeromonas hydrophila, an important bacterial pathogen causing aeromoniasis. All animals were grown in a common environment and RNA from ten individuals from each line pooled for Illumina mRNA-seq. De novo transcriptome assembly produced 137,629 contigs with 40× average coverage. Forty-four percent of the assembled sequences were annotated with gene names and ontology terms. Of these, 3,419 were assigned biological process terms related to “stress response” and 1,939 “immune system”. Twenty-six contigs containing 38 single nucleotide polymorphisms (SNPs) were found to map to the Cyprinus carpio mitochondrial genome and over 26,000 putative SNPs and 1,700 microsatellite loci were detected. Seventeen percent of the 100 transcripts with coverage data most indicative of higher-fold expression (>5.6 fold) in the resistant line pool showed homology to major histocompatibility (MH), heat shock proteins (HSP) 30, 70 and 90, glycoproteins or serum lectin genes with putative functions affecting immune response. Forty-one percent of these 100 transcripts showed no or low homology to known genes. Of the SNPs identified, 96 showing the highest allele frequency differences between susceptible and resistant line fish included transcripts with homology to MH class I and galactoside-binding soluble lectin, also with putative functions affecting innate and acquired immune response. A comprehensive sequence resource for L. rohita, including annotated microsatellites and SNPs from a mixture of A. hydrophila-susceptible and -resistant individuals, was created for subsequent experiments aiming to identify genes associated with A. hydrophila resistance.

Selection for improved resistance to Aeromonas hydrophila in Indian major carp Labeo rohita: survival and innate immune responses in first generation of resistant and susceptible lines.

Selection for disease resistance in fish may be performed directly on basis of survival data obtained in controlled challenge trials, or indirectly using information from immunological or molecular markers linked to differential survival. In the present study, several key innate immune parameters were measured in aeromoniasis resistant and susceptible lines of rohu Labeo rohita to assess their suitability as immune markers for use in indirect selection for increased resistance. Experimental infection with Aeromonas hydrophila (9.55 × 10(6) cfu g(-1) fish) through the intraperitoneal route produced higher survival in the resistant line (73.33%) as compared to the susceptible line (16.67%). Blood and liver tissue samples from both lines were collected to study some of the innate immune parameters and immune-related gene expression. The respiratory burst activity of blood phagocytes, serum myeloperoxidase activity and ceruloplasmin level were significantly (p < 0.05) higher in the resistant line compared to the susceptible line. Lower level of blood glucose and serum natural haemolysin titre were marked in the resistant line as compared to the susceptible line. No significant difference was measured in total serum protein concentration, antiprotease activity and bacterial agglutinin level between two lines, while the expression of transferrin, complement factor C3 and TLR 22-like transcripts were significantly (P < 0.05) higher in liver samples of the susceptible line. However, no such difference was found in β(2)-microglobulin and lysozyme gene expression between lines. The study demonstrated the possibility of using some of the investigated innate immune parameters as indirect marker traits for selection for improved resistance to aeromoniasis in rohu.

Growth and survival of six stocks of rohu (Labeo rohita, Hamilton) in mono and polyculture production systems

One farmed stock (Local) and five riverine stocks of Rohu (Labeo rohita, Hamilton) from 2-year classes (1993 and 1994) were evaluated for growth and survival under monoculture and polyculture systems at the Central Institute of Freshwater Aquaculture (CIFA), India. In the 1993 year-class, 29 full-sib groups of Local and 28 full-sib groups of river Ganga were tested while in the 1994 year-class the stocks tested were of Local and of rivers Brahmaputra, Ganga, Gomati, Sutlej and Yamuna with 6, 8, 7, 7, 18 and 11 full-sib groups, respectively. For each year-class, individually tagged fingerlings were stocked into three 0.1 ha monoculture ponds (9 fish/full-sib group/pond) and two 0.4 ha polyculture ponds (13 fish/full-sib group/pond). In polyculture, Rohu were stocked together with Catla (Catla catla, Hamilton) and Mrigal (Cirrhinus mrigala, Hamilton) in the ratio of 1.2:1:1, respectively. The stock effect on body weight at harvest was not significant in the 1993 year-class but highly significant in the 1994 year-class. The stock effect on survival from tagging to harvest was not significant in any of the year-classes. The interaction between production system and stock on growth and survival was significant but low, particularly compared to the significant and important full-sib effect. Thus, the growth performance of the stocks was relatively consistent in monoculture and polyculture. The significant full-sib effects for growth and survival suggest that these traits can be improved through selective breeding. The implications of the results for the development of a selective breeding program for Rohu in India are discussed.

First evidence of comparative responses of Toll-like receptor 22 (TLR22) to relatively resistant and susceptible Indian farmed carps to Argulus siamensis infection.

Toll-like receptor 22 (TLR22) is present in teleost but not in mammals. Among Indian farmed carps, Catla catla is relatively more resistant than Labeo rohita to Argulus siamensis lice infection. TLR22 is believed to be associated with innate immunity against ectoparasite infection. To investigate the TLR22 mediated immunity against argulosis, we have cloned and characterized TLR22 genes of L. rohita (rTLR22) and C. catla (cTLR22). The full-length cDNAs of rTLR22 and cTLR22 contained an open reading frame of 2838 and 2841 nucleotides, respectively; bearing the typical structural features. Phylogenetically rTLR22/cTLR22 was most closely related to Cyprinus carpio (common carp) counterpart, having highest sequence identity of 86.0%. The TIR domain remained highly conserved with 90% identity within freshwater fishes. The sequence information of cDNA and genomic DNA together revealed that the rTLR22/cTLR22 genes are encoded by uninterrupted exons. The co-habitation challenge study with A. siamensis infection confirmed that C. catla is comparatively more resistant than L. rohita. Further, comparative mRNA expression profile in immune relevant tissues also suggested about the participatory role of TLR22 during lice infection. However, TLR22 might not solely be involved in conferring relative resistance among carp species against argulosis.

Analysis of immune-related ESTs and differential expression analysis of few important genes in lines of rohu (Labeo rohita) selected for resistance and susceptibility to Aeromonas hydrophila infection

A total of 137,629 contigs generated via de novo transcriptome assembly from resistant and susceptible lines of rohu (first generation) raised against aeromoniasis were further analyzed in terms of defence-related genes. Out of 1,939 contigs showing homology to genes involved in immune processes, 1,866 were further categorised into different functional subgroups. Comparative analysis revealed five genes for the first time in any carp species out of which apolipoprotein h, septin 4 isoform 3 and septin isoform cra_c were identified for the first time in fish. Differential expression analysis of ten genes viz., heat shock proteins (Hsps) (Hsp30, Hsp70 and Hsp90), serum lectin isoform 1 (SLI1), linker histone H1M (LHH1M), NAD(P)H quinone 1 (NQO1), zona pellucida 2 (ZP2) and three unknown genes that were highly up-expressed in first generation resistant line fish from mRNA-seq coverage data, was carried out using susceptible and resistant individuals of the second generation selected populations in eight different tissues viz. liver, kidney, intestine, gill, brain, spleen, skin and muscle using qPCR. Significant up-regulation in Hsp90, NQO1, C_116914 and C_22454 in specific tissues of resistant line and variable expression in Hsp30 and LHH1M genes in different tissues of both lines were noticed. The expression of Hsp70 was lower in many tissues of the resistant line than in susceptible line rohu. The expression of ZP2, SLI1 and C_94589 genes was not significantly different in terms of fold difference between the two lines. Differentially expressed genes need further characterisation to explore their role in resistance to Aeromonas hydrophila infection in rohu.

Cavernous haemangioma in cultured olive barb, Puntius sarana (Hamilton)

Neoplasms of fish are classified similarly to those of higher animals (Hochwartner et al. 2010). The primary neoplasms of the skin of cultured fish are uncommon (Schoniger et al. 2008). Haemangiomas are benign tumours of vascular endothelial cells or their progenitors. In humans, they are most frequently located in the skin, but have also been also described in other tissues and organs (Weiss & Goldblum 2001). These tumours are common in dogs and rare in cats, horses and other domestic animals (Goldschmidt & Hendrick 2002). They may be classified histologically as capillary, cavernous, arteriovenous, lobular, spindle cell or epithelioid subtypes (Vos et al. 1986; Calonje & Fletcher 2007; Gross et al. 2007; Warren & Summers 2007). Tumours of vascular origin have also been reported from a number of fish species (Fournie, Overstreet & Bullock 1985). Rare occurrences of capillary, cavernous haemangiomas, haemangioendotheliomas and haemangio-endotheliosarcomas affecting integument and visceral organs have been recorded in fish from the USA by Harshbarger (1965). Microscopically, haemangiomas are characterized by proliferating, vasoformative mesenchymal tissue forming capillary and cavernous vessels and, less often, arterial and venous structures. Endothelial cells in these tumours may be surrounded by pericytes and fibroblasts (Calonje & Fletcher 2007; Gross et al. 2007). Cavernous haemangioma is a frequent benign vascular neoplasm that can occur in multiple tissues in humans and animals including fish (Harshbarger 1965–1981; Goldschmidt & Hendrick 2002; Zhang et al. 2009). Cavernous haemangioma consists of vascular malformation, that is, the vascular walls with no basement membranes and rare pericytes, singlelayer lining endothelial cells (ECs) with fenestrated luminal surfaces and large gaps at intercellular junctions between ECs (Tu et al. 2005). This communication reports cases of multiple cavernous haemangiomas from the olive barb, Puntius sarana (Hamilton). The first case observed was in a 2-year-old, pondcultured Puntius sarana, which developed mediumsized, exophytic dermal masses at the base of the pelvic fin and caudal fin. The masses (1–4 cm size) were dark red and fluctuant on palpation. Subsequently, similar lesions were observed in three specimens out of a total of 250 one-year-old fish, maintained in a wet laboratory for experimental purpose. The lesions were observed as small nodular lesions at the base of the fin and operculum. Following physical examination, the fish were killed by overdose of anaesthesia (MS222). A complete post-mortem examination was carried out in all cases. All tumours were well-demarcated, multilobular reddish brown masses mostly occurring at the base of the fins and lower jaw (Fig. 1). The tumours were soft, friable and contained a small amount of sero-sanguinous fluid. The excised tumour masses were removed, fixed in 10% neutral buffered formalin and processed for Journal of Fish Diseases 2012 doi:10.1111/j.1365-2761.2012.01370.x

Differential resistance to edwardsiellosis in rohu (Labeo rohita) families selected previously for higher growth and/or aeromoniasis-resistance

Selection in fish for disease resistance is one of the most useful approaches to solve disease problems. Genetic variation in resistance to edwardsiellosis in fullsib families of rohu, Labeo rohita was investigated in the present study. A large variation in the susceptibility pattern (0 to 94.74 percent survival) against Edwardsiella tarda challenge was observed among 57 families. Additive genetic variation showed a heritability of 0.38 ± 0.08 across the year-class survival. The apparent resistant families showed more delayed mortality than the apparent susceptible ones. The cross-protection provided by aeromoniasis-resistant lines of rohu to edwardsiellosis was also studied to evaluate the possibility of selection for both diseases simultaneously. Challenge of F1-generation aeromoniasis-resistant and -susceptible lines with E. tarda showed significant difference in survival between the lines with higher percent survival in resistant line. This study suggests that direct selection method may be used reliably in selection programs and selection for multiple diseases simultaneously can be considered for rohu.