Possible Causes Of Selective Lernaea Attack On Different Fish Species
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Publisher: 2013 Dissertation note: The present study was managed to investigate the possible causes of selective Lernaea attack in different fish species. Planned studies were conducted in five trials. During the first trial lernaea susceptibility and infestation were observed in indigenous major and exotic Chinese carps. Studies were conducted in 4 earthen ponds with two ponds per experimental group. Fishes in the both groups were fed isonitrogenous diet containing 40% crude protein formulated with different feed ingredients. Fishes were identified and examined for the presence of Lernaea species. The parasites were removed and preserved in vials containing 5% formalin. Other growth parameters i.e. average weight gain, average increase in length while physico-chemical analysis including dissolved oxygen, pH, electrical conductivity, water temperature, salinity and total dissolved solids, Chlorides, Phosphates ions (PO4-2), nitrates (NO3) and secchi disk visibility were recorded. The fishes were treated with Tender, an organophasphate (DDVP (Dichlorvos) or 2, 2-dichlorovinyl dimethyl phosphate) for the control of lernaea.
The results showed that C. catla is the most susceptible fish to L. cyprinacea infestation and its appropriate control for conservation of this precious and declining fish is of extreme importance. Thunder is biologically safe as it is biodegradable and degenerated after 36 h without causing any negative effect on the water quality parameters and other water flora and fauna. Treatment of L. cyprinace with 'Thunder' (0.10-0.25ppm) gives promising results without adverse effect on fish life. Though DDVP worked well in its control but its heavy infestation really weaken fish incapacitating its feeding and competing abilities with its counterparts.
In the 2nd trial, comparison of bio-chemical profile was observed on healthy and infected Indian and Chinese major carps. The experiment was managed in four earthen rearing ponds (59m x 30.5m x 1.8m). There were two-groups, one group without any treatment served as control and the second as treatment. At the outset of trial all the fish species were weighed, measured and comprehensively examined for presence of L. cyprinacea and general health condition. Fishes from both groups were dealt uniformly except administration of regular applications of DDVP (0.25ppm in treatment group while no any medication in control group. Water quality parameters DO, pH, electrical conductivity, water temperature, salinity and total dissolved solids, chloride ion (Cl-1), phosphate ions (PO4-2) nitrates (NO-3) and light penetration were recorded. Samples of healthy and infected fishes of each species were collected for proximate composition (moisture, ash, crude protein, crude fat) and minerals (Iron, Zinc, Calcium, Copper and Magnesium) and phosphorous analysis.
The results showed that dry matter, fat and crude protein percentages were significantly decreased in lernaeid fishes. Moisture and ash contents of fishes increased in infected fishes as compared to healthy and treated fish. A slightly lower level of protein (12.65±0.49) and fat (7.30±0.28) in C. catla was observed than rest of the species. The protein was the highest (26.00±4.24) in L. rohita while the fats were the highest (10.55±0.92) in C. mrigala and C. carpio the second highest. Similarly looking at mineral profile there is not much difference, so it is hard to say that level and type of nutrients are solely responsible for L. cyprinacea attack. Mineral composition of infected fish indicated that minerals balance upsets during disease condition. Pathogenicity is a complex of so many factors, which encompass environmental, biological, and physiological so still lot remains to be explored before issuance of any concrete conclusion and recommendation that which factor is more active and critical in inviting and attracting this parasite.
In the third experiment, various blood indices were compared among Chinese and Indian major carps from the perspectives of their resistance against lernaeaosis that included probable role of blood and its components in reception of L. cyprinacea in some commercially important locally culturable herbivorous fish varieties while repulsion in others living under similar environment.
Healthy samples of each fish species were selected and blood was immediately drawn by puncturing gill lamellae, caudal vein and heart of both male and female of each representative experimental species. Blood parameters including Red Blood Cells (RBC, 10 -6/µL), White Blood Cells (WBC, 10-3/µL) and Platelets (10-3/µL) in blood from experimental fish species were counted by placing sample on haemocytometer grids. Differential Leucocyte Count (DLC), red blood indices, blood chemistry and ESR were determined for males and females of experimental fishes.
The results revealed that females of each species have relatively higher values for blood indices as compared to males. C. carpio has maximum number of granulocytes that may support the fish against the parasitic attack. C. catla has the lowest values for the immunoglobulin Ig M as compared to the species which showed less susceptibility.
In the 4th trial, healthy fishes of major and Chinese carps were collected from commercial rearing and grow out ponds. Each fish weighed 830 ± 316 g on the average. For mucus collection, fishes were bathed in Potassium permanganate (KMnO4) solution (8.0 ppm) to remove microbial or fungal infection/infestation. Samples were centrifuged at 12000×g at 4 ?C for 10 minutes and stored at -40?C in biomedical freezer. Bradford Micro Assay technique was applied to determine protein contents. Bradford protein solution (50 ?L) was added to each well and absorbance was recorded at 595nm. Standard curve was drawn from various but consecutive dilutions of BSA solution and protein concentrations in different samples were calculated. Electrophoresis was carried out with slight modifications. 15% separating and 4% staking buffer were used to run the SDS- PAGE under constant voltage of 120. Fermentas PageRuler™ protein ladder was used as the standard marker for non reducing protein. The gel was stained with PageBlue™ (Fermentas) stain for identification of protein bands for molecular weight determination. Lectin activity and Alkaline Phosphatase test were determined. Mucus was incubated with 4 mM p-nitrophenyl phosphate in ammonium bicarbonate buffer (100 mM) with 1 mM MgC12 (pH 7.8) at 30°C. The increase in optical density (OD) was measured continuously for 2 to 3 hours at 405 nm using a micro plate reader.
The results showed that lectin activity was the highest in C. idellus (109) indicative of low resistance while it was the lowest (21) in H.molitrix which was completely parasite free. Alkaline phosphatase level was the highest in C. catla, C.idella was the second highest and was the lowest in C. carpio. Protein concentrations were the highest in C. idella (3.29 ± 0.13 mg/ml) and C. catla (3.02 ± 0.57 mg/ml) while it was the lowest in C. carpio (1.80 ± 0.09). C. catla contained the highest molecular weight proteins (100 kDa) while C. carpio has one unique protein band of 14.13 kDa not present in any other species in current setup.
In the 5th experiment, the lernaea were observed in the month of June to August. Lernaeied infestation was observed in all experimental fishes except in C. carpio. After appearance of infestation fishes were treated with Thunder (DDVP- an organophasphate) to eliminate the parasite. Parasite free C. idellus, H. molitrix, L. rohita, C. mrigala, C. catla and C. carpio were collected with an average weight of 830 ± 316 g each were used for studies on whole-body amino acid composition. Triplicate samples of each species were over dried after evacuating their gut contents. Dried samples were then finely powdered, sieved and vacuum hydrolyzed in 2 ml of 6 N HCl at 1lO°C for 24 hours. Total amino acid composition was determined by o-phthaldialdehyde (OPA) method using an Agilent chromatograph, revers-phase high performance liquid chromatography (HPLC). The results showed that the essential amino acids (arginine, lysine, phenylalanine, tyrosine, histidine and leucine) play a major role in the immune system. It is revealed that amino acids will widely become cost-effective neutraceuticals for improving health and preventing infectious disease in animals. C. carpio have no infestation due to increased numbers and concentrations of essential amino acids when compared to other species of Indian and Chinese major carps. C. catla, C. idella and H. molitrix have the lowest number and concentration of essential amino acids and hence appeared more susceptible to lernaea attack.
Finally it was concluded from the entire study that C. carpio may have high resistance for the L. cyprinaceaea as compared to the other experimental fishes. It possesses higher values for Ig M-immunoglobulin as compared to C. catla that indicated its high immunity against the parasite. Similarly C. carpio has maximum number of granulocytes (WBCs, esinophils, basophils and lymphocytes) that may support the fish against the parasitic attack. SDS-PAGE analysis of mucus revealed that C. carpio has one unique protein band of 14.13 kDa not present in any other species in current setup. This protein band may indicate the presence of lysozyme enzyme that actively participates against the invading pathogen. Essential and non-essential amino acids concentrations were also higher in the C. carpio that play a vital role in immunity especially arginine, lysine, phenylalanine, tyrosine, histidine, leucine, glutamic acid and aspartic acids.
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