Microsatellite markers from the Indian major carp species,Catla catla

The Indian major carp Catla catla is indigenous to the major river systems of Northern India, Pakistan and Bangladesh, and is an important contributor to aquaculture production in India and Bangladesh. Five polymorphic microsatellite loci, developed from Catla catla are described and their utility in other major carp and cyprinid species is tested.     

Loss of genetic variation in hatchery-reared Indian major carp, Catla catla

The hypothesis that effective population sizes are low in hatchery-reared catla ( Catla catla ) from Bangladesh, possibly leading to inbreeding and loss of variation, was tested. The study was based on analysis of seven microsatellite loci in three samples of hatchery-reared catla and four samples representing wild populations. Pair-wise estimates of genetic differentiation between samples were low between wild samples (θ ranging from 0·012 to 0·034), but high between hatchery samples (θ ranging from 0·153 to 0·185), suggesting strong genetic drift in hatcheries. Genetic variation, both in terms of expected heterozygosity and allelic richness, was significantly lower in hatchery samples than in samples of wild catla. Application of a method for reconstructing families among offspring without parental genetic data showed that the hatchery samples consisted of very few half- and full-sib families, whereas the wild samples consisted of a high number of families, suggesting that most individuals were unrelated. Finally, estimation of the effective number of parents ( N _b) in the largest sample of hatchery fish confirmed that effective population size was low ( N _b= 14·9 for multiallelic loci and N _b= 10·6 if alleles were pooled into two composite alleles). The results show that low effective population sizes leading to loss of variation and possibly inbreeding depression should be a matter of serious concern in aquaculture production of catla.     

Epithelium of lips and associated structures of the Indian major carp,Catla catla

Structural organization of the epithelium of the lips and associated structures of the Indian major carp,Catla catla, is described. The upper lip is thin and is associated on its dorsal side with a membranous fold of skin and the rostral cap. In contrast, the lower lip is thick and very conspicuous. It is associated on its ventral side with a fold of skin between it and the ventral head skin. The lower lip is divided into a non-projectile portion, a projectile portion and an intermediate groove region. The projectile portion remains folded covering a part of the ventral head skin when the mouth is closed. Their role in relation to the formation of the characteristic feeding tube is discussed. The epithelium of the lips and associated structures is stratified in nature and is composed of the epithelial cells, mucous cells, club cells, lymphocytes and the taste buds. The mucous cells are small, few or even absent and do not appear to secrete profusely at the surfaces of the upper and the lower lips. This suggests that the lips inCatla catla, which feeds on micro-organisms, do not need extra lubrication for protection against abrasion during feeding. In the epithelium at the folds of skin, the voluminous mucous cells secrete profusely and provide extra lubrication to their surface. This reduces the resistance to surface drag during stretching and enables the jaws to protrude with increasing efficiency and swiftness. The club cells are developed additionally to complement the mucous cells in the rostral cap and the upper lip epithelium. Their primary function appears protective in some way, which needs further confirmation. The taste buds, though few in the lower lip, are located in a good number in the upper lip on the characteristic epithelial papillae-like projections, and are projected at the surface. These have been associated with the acute gustatory sense of the fish. The taste buds are absent on the folds of skin where they may not be of much significance.     

Homologous radioimmunoassay and radioreceptorassay of gonadotropic hormone for an Indian carp Catla catla

A homologous radioimmunoassay (RIA) for Indian carp (C. catla) gonadotropin (GtH) was developed by using purified Catla GtH and its specific antiserum. In Ouchterlony agar diffusion, antisera raised against purified Catla GtH (cGtH), showed clear crossreaction. Catla-anti GtH (anti-cGtH) did not cross-reacted with Catla TSH enriched fraction. Immunocrossreaction was further confirmed with competitive binding inhibition studies where displacement of radiolabelled cGtH was precisely linear against increasing concentrations of cGtH, hence this was later used as standard curve for RIA. Competitive binding inhibition was also observed with purified murrel GtH, silver carp GtH and Cyprinus GtH, using varied doses. Both murrel and silver carp GtH showed clear parallelism with cGtH, while Cyprinus GtH inhibition slope was less steeper. Mammalian GtHs (hCG, oLH, oFSH), bTSH and bPRL had no crossreaction with anti-cGtH. Radioreceptorassay (RRA) for cGtH was developed by preparing plasma membranes from Catla oocytes. Binding of 125I-cGtH to oocyte plasma membranes showed saturability with high affinity (Ka = 0.11 X 10(13)M-1) and low capacity (17 fmol/mg protein). Displacement of 125I-cGtH in receptorassay by cold cGtH was linear and therefore served as standard curve. The interassay and intrassay variability in RIA was 7.9% and 3% while that of RRA was 5% and 3% respectively. Sensitivity of RIA was in the picogram level whereas it was in nanogram level by RRA. Determination of carp pituitary and serum GtH content by RIA and RRA showed the consistency, precision and validity of these assays. Although RRA was comparatively less sensitive than RIA, it was convenient, quick and less expensive.     

Photoperiodic regulation of annual testicular events in the Indian major carp Catla catla

The importance of photoperiods in the regulation of annual testicular events in the carp Catla catla was evaluated by subjecting them to either long (16 h light : 8 h dark) or short (8 h light : 16 h dark) photoperiods for 30 days during the preparatory, prespawning, spawning and postspawning phases of an annual gonadal cycle. In each reproductive phase, testicular responsiveness to subjected photoperiods was determined by comparing the gonadal status in corresponding groups of control or natural photoperiodic fish. The values of testicular weight, gametogenic index, as well as testicular activity of two steroidogenic enzymes (Δ⁵3β‐, and 17β‐hydroxysteroid dehydrogenase), and the serum titre of testosterone were considered as the indices of functional status of the testis in the fish concerned. During the prespawning phase, exposure of fish to a daily long photoperiod schedule resulted in precocious maturation of testis, while retardation of testicular growth was noted under the influences of short photoperiod. However, none of the employed photo‐schedules could influence the gametogenic and steroidogenic functions of the testis in the remaining part of the gonadal cycle. Collectively, the present study provides evidence for the first time that in the case of a commercially important carp, Catla catla, artificial, long photoperiods may be used for advanced testicular maturation, while reductions in maturation‐associated growth and deterioration in flesh quality may be avoided by submitting the fish to shorter day lengths during the prespawning phase of the reproductive cycle.     

Adenohypophysial cell types in an Indian freshwater major carp, Catla catla (Hamilton)

The different adenocytes in an Indian freshwater major carp, Catla catla (Hamilton) have been studied by employing various modern staining techniques. Altogether, 8 different cell types have been reported in the adenohypophysis of the aforesaid fish. Variations in the staining intensities of the cell types with the different techniques adopted have been detailed.     

Stimulation of immunity in Indian major carp Catla catla with herbal feed ingredients

Catla catla, catla (150 +/- 20 g) were fed a diet containing seed of Achyranthes aspera (0.5%) and control diet without A. aspera for four weeks prior to and after ip injection with chicken erythrocytes. Fish were sampled for four consecutive weeks after immunization. Hemagglutination antibody titers were significantly higher in the test group of fishes compared with the control group. Serum globulin levels were significantly (P(t-test) < 0.05) higher in the test group than control group on days 14 and 21. Anti-trypsin activity due to total serum protease inhibitors and alpha1-antiprotease was also significantly (P(t-test) < 0.05) higher in the test group of fishes than the control. RNA/DNA ratio of spleen and kidney was also significantly (P(t-test) < 0.05) higher in test group than the control group. All these results confirm that A. aspera enhances the immunity of catla.     

Isolation and characterization of polymorphic microsatellite loci in Indian major carp,Catla catla using next-generation sequencing platform

Catla catla, the second most important Indian major carp, is gaining its popularity among Indian fish farmers due to its high growth rate and consumer preferences. Simple sequence repeats (SSRs) are rapidly evolving, versatile, co-dominant and highly informative molecular markers used in genetic research. However, the time and cost involved in developing such resources has limited their extensive use. Advent of massive parallel sequencing technology has considerably eased these limitations. In the present investigation, we used Ion Torrent sequencing platform to identify potentially amplifiable microsatellite loci for catla. A modest sequencing volume generated approximately 5.7 MB of sequence data. Out of 29,794 sequences generated, 21,477 contained simple sequence repeats. Only 81 sequences had enough flanking sequences for primer designing. Out of 81 loci, 51 were successfully PCR amplified in a panel of five unrelated individuals. Out of 15 loci randomly checked for polymorphism, 13 loci were polymorphic with allele number ranged from 3 to 6 and two loci were found to be monomorphic. The observed and expected heterozygosities ranged from 0.565 to 0.870 and 0.483–0.804, respectively. These markers will be useful for studying genetics of wild populations, breeding programs of C. catla and closely related species.     

Temporal relationship between the daily profiles of gut melatonin,oxidative status and major digestive enzymes in carp Catla catla

The present study demonstrated a temporal relationship between the concentrations of melatonin, oxidative status and digestive physiology in the gut of a tropical carp Catla catla. We measured the levels of gut melatonin, malondialdehyde (MDA) – a faithful marker of intracellular stress, different antioxidants and major digestive enzymes in the carp gut at four different clock hours in a daily cycle under natural photo-thermal conditions. A correlation between the gut variables was sought to point their possible functional relationship. Gut melatonin titers displayed significant diurnal variations with a peak at midday. An identical temporal pattern with the highest value at midday and nadir at midnight was noted in the activities of superoxide dismutase, catalase and glutathione peroxidase. In contrast, levels of MDA and reduced glutathione (GSH) were highest at midnight and lowest at midday. The activity of all the studied digestive enzymes (α-amylase, cellulase, protease and lipase) showed significant daily variations with a peak at midday. Gut melatonin concentrations by showing a positive correlation with the activity of both enzymatic antioxidants and digestive enzymes, and a negative correlation with the levels of GSH and MDA indicated their possible physiological interplay in a daily cycle. Collectively, our study presented the first information on the daily profiles of oxidative stress, different antioxidants and digestive enzymes in the gut tissues of any fish species, and suggested their functional relationship with the concentrations of gut melatonin in carp Catla catla.     

Environmental correlate of the testicular events in a major carp Catla catla in an annual reproductive cycle

Current communication describes annual testicular events in free-living Indian major carp Catla catla and their probable environmental synchronizer(s). The study was initiated with month-wise evaluation of gametogenic and steroidogenic status of the testis, and thus dividing the annual testicular cycle into the preparatory spawning (November to March), the pre-spawning (April to June), the spawning (July to August) and the post-spawning (September to October) phases. An exhaustive statistical analysis of the data on the studied variables of testicular functions and various components of the environment indicated seasonal fluctuations of photoperiod as the major environmental factor associated with the seasonal reproductive activity of this carp. Ambient temperature appeared as a dependent variable of photoperiod, and thereby, may have substantial influences on the development of testis in Catla catla. Rainfall, on the other hand, showed significant correlation only with the peak reproductive activity, i.e. the act of spawning. Collectively, it appears logical to surmise that photo-thermal conditions may act as proximate and rainfall may play a role of ultimate environmental factor in the regulation of annual testicular events in Indian major carp Catla catla.     

Glycosylated molecular variants of C-reactive proteins from the major carp Catla catla in fresh and polluted aquatic environments

Elevated level of pollutant specific glycosylated molecular variants of C-reactive protein have been purified to electrophoretic homogeneity from the sera of major carp, Catla catla confined in freshwater (CRP_N) and water polluted with nonlethal doses of cadmium (CRP_Cd), mercury (CRP_Hg), phenol (CRP_Ph) and hexachlorocyclohexane (CRP_Hex). These CRPs differ amongst themselves in electrophoretic mobility, and in their carbohydrate content ranging from 20–50%. CRPs interact with pneumococcal C-polysaccharide (CPS) showing different binding constants. Both phosphorylcholine (PC) and calcium are indispensable for binding. Studies on amino acid compositions, electrophoretic analysis, isoelectric focusing, binding to PC & CPS and secondary structures of the purified CRPs indicate, that, they differ from each other. However, they share the common properties of a CRP, including pentraxin structure revealed by electron microscopy. Taken together, our results provide a new structural insight regarding the connection between the presence of unique molecular variants and probably the toxicity therein combated.     

Bioactive forms of gonadotropin releasing hormone in the brain of an Indian major carp,Catla catla (Ham.)

Two forms of biologically active gonadotropin releasing hormones were isolated from the hypothalami ofCatla catla. Gonadotropin releasing hormone activity was studiedin vitro using enzymatically dispersed carp pituitary cell incubation system. Gonadotropin released into the medium was measured by carp gonadotropin-radio immuno assay. Acetic acid extracted hypothalamic material was subjected to acetone fractionation. Among the three protein pellets obtained at different time periods (ACI, ACII and ACIII), AC II exhibited the gonadotropin releasing hormone activity. Gel filtration of AC II through Sephadex G-25 column showed three protein peaks (SG I, SG II SGIII) and only S G II demonstrated strong gonadotropin releasing hormone activity. Elution of SG II through FPLC Mono Q column (an anion exchanger) in NaCl gradient programme showed one unadsorbed (MQ I) and three adsorbed (MQ II, MQ III and MQ IV) protein peaks. MQ III, which was eluted with 51% NaCl, exhibited gonadotropin releasing hormone activity. Surprisingly, unadsorbed fractions, MQ I, also showed gonadotropin releasing hormone activity. MQ 1 was therefore subjected to FPLC Mono S (a cation exchanger) column chromatography where a highly active gonadotropin releasing hormone enriched peak, i.e., MS III, could be eluted with 45% NaCl. These findings show thatCatla catla hypothalamus has two forms of gonadotropin releasing hormones one anionic (carp gonadotropin releasing hormone I) and another cationic (carp gonadotropin releasing hormone II). These two forms of gonadotropin releasing hormones were also active in heterologous carp species, rohu(Labeo rohita), mrigal(Cirrhinus mrigala) and an exotic common carp(Cyprinus carpio). Combined activity of two forms of gonadotropin releasing hormones was significantly greater as compared to any of the single form.     

Carcass characteristics of marketable size farmed catla,Catla catla (Hamilton, 1822)

The present study investigates carcass traits of farmed freshwater Catla catla for important information in calculating yield and/or providing data for programming machine/manual handling. For this purpose specimens of C. catla ranging from 1880 to 2150 g were collected from grow‐out culture ponds of the Central Institute of Freshwater Aquaculture, Odisha State, India. Carcass yield, offal yield and carcass cutability were assessed. The percentage of head yield was highest (31.2%) in comparison to other carp species. Gutted yield and final dressed yield of 2 kg market class catla amounted to 85.4 and 54.1%, respectively. The average meat : bone ratio in filleting was reported to be 3 : 9. The middle cut of catla had both the highest total yield percentage and highest meat yield. Dry matter, ether extract and protein percentage was highest in the fore cut followed by the middle and hind cuts.     

Influence of serotonin on the action of melatonin in MIH-induced meiotic resumption in the oocytes of carp Catla catla

The influences of serotonin (5-hydroxytryptamine) on the action of melatonin (N-acetyl-5-methoxytryptamine) in MIH (maturation inducing hormone)-induced meiotic resumption were evaluated in the oocytes of carp Catla catla using an in vitro model. Oocytes from gravid female carp were isolated and incubated separately in Medium 199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 microg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 microM) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 microM) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. Control oocytes were incubated in the medium alone. Oocytes were incubated for 4, or 8, or 12, or 16 h and effects were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). At the end of 16 h incubation, 93.24+/-1.57% oocytes underwent GVBD following incubation with only MIH, while incubation with only MEL or only SER resulted in 77.15+/-1.91% or 14.42+/-0.43% GVBD respectively. Interestingly, incubation with MEL 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD (92.58+/-1.10% at 12 h). In contrast, SER, irrespective of its time of application in relation to MIH, resulted in a maximum of 64.57+/-0.86% GVBD. While L was found to reduce the stimulatory actions of melatonin, M suppressed the inhibitory actions of serotonin. In each case, both electrophoretic and immunoblot studies revealed that the rate of GVBD was associated with the rate of formation of maturation promoting factor (a complex of two proteins: a regulatory component--cyclin B and the catalytic component--Cdk1 or cdc2). Collectively, the present study reports for the first time that SER not only inhibits the independent actions of MIH, but also the actions of MEL on the MIH-induced oocytes maturation in carp.     

Genetic variation of wild and hatchery populations of the catla Indian major carp (Catla catla Hamilton 1822: Cypriniformes, Cyprinidae) revealed by RAPD markers

Genetic variation is a key component for improving a stock through selective breeding programs. Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic variation in three wild population of the catla carp (Catla catla Hamilton 1822) in the Halda, Jamuna and Padma rivers and one hatchery population in Bangladesh. Five decamer random primers were used to amplify RAPD markers from 30 fish from each population. Thirty of the 55 scorable bands were polymorphic, indicating some degree of genetic variation in all the populations. The proportion of polymorphic loci and gene diversity values reflected a relatively higher level of genetic variation in the Halda population. Sixteen of the 30 polymorphic loci showed a significant (p < 0.05, p < 0.01, p < 0.001) departure from homogeneity and the F(ST) values in the different populations indicated some degree of genetic differentiation in the population pairs. Estimated genetic distances between populations were directly correlated with geographical distances. The unweighted pair group method with averages (UPGMA) dendrogram showed two clusters, the Halda population forming one cluster and the other populations the second cluster. Genetic variation of C. catla is a useful trait for developing a good management strategy for maintaining genetic quality of the species.     

Reproductive performance of carp, Catla catla (Ham.), reared on a formulated diet with PUFA supplementation

Effect of dietary supplementation of n‐3 and n‐6 polyunsaturated fatty acids (PUFA) on reproductive performances in both sexes of an Indian major carp, Catla catla (Ham.), were investigated in terms of sperm quality, spawning response, fecundity and spawn recovery over a consecutive 2‐year period. Sperm count and spermatocrit value were in the range of 3.2–3.8 × 10⁷ ml⁻¹ and 80–82%, respectively, in fish fed the PUFA‐enriched test diet, and significantly higher (P < 0.01) than in fish fed control diet (2.3–2.5 × 10⁷ ml⁻¹ and 64–70%). Spawning response in the test diet group was quite high (96%) compared to 76% in the control. Egg and larval quality were superior in the test diet group as evidenced by the significantly higher fertilization rate and larval survival in the range of 91–92% and 93.3%, respectively. Fatty acid profile of fertilized eggs and larvae showed that the quantities of 18:3 (n‐3), 20:5 (n‐3) and 22:6 (n‐3) were significantly (P < 0.01) increased in the test group while 18:1, 18:2 (n‐6) and 20:4 (n‐6) remained at higher levels in control. The results indicated that enrichment of n‐3 PUFA in the broodfish diet not only improved the quality of eggs and larvae in terms of recovery, but also enhanced the physiological response of broodfish to induced spawning. Oil enrichment in the broodfish diet should therefore be mandatory to ensure the optimum n‐3 PUFA content for warmwater carp for quality seed production.