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Identification of Causative Agent for Fungal Infection and Effect of Disinfectants on Hatching and Survival Rate of Common Carp (c. Carpio) Larvae

Syeda Maksuda Yeasmin
Department of Fisheries and Marine Bioscience, Faculty of Biological Science and Technology, Jessore University of Science and Technology, Jessore-7408, Bangladesh.

Md. Anisur Rahman
Department of Fisheries and Marine Bioscience, Faculty of Biological Science and Technology, Jessore University of Science and Technology, Jessore-7408, Bangladesh.

Md. Mer Mosharraf Hossain
Department of Fisheries and Marine Bioscience, Faculty of Biological Science and Technology, Jessore University of Science and Technology, Jessore-7408, Bangladesh.

Md. Habibur Rahman
Department of Fisheries and Marine Bioscience, Faculty of Biological Science and Technology, Jessore University of Science and Technology, Jessore-7408, Bangladesh.

Abdulla-Al-Asif
Department of Aquaculture, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

Common carp (Cyprinus carpio) is one of the commercially important and commonly cultured fish. In the hatchery intensive incubation leads to microbial overgrowth in C. carpio eggs that hamper egg development, hatchability and larval survivability. The aim of this study is to find out causes of mass mortality in C. carpio eggs during peak- breeding season between March to May 2015 at Mafatema fish hatchery, Chanchra, Jessore sadar upazilla. In the present study three disinfectants with three different concentrations in each such as methylene blue 1, 3 and 5mg/L., malachite green 1, 3 and 5mg/L., sodium chloride 1, 2 and 3g/L were used to observe the hatching rate of fertilized eggs and survival rate of larvae. Bacterial load of culture water was examined during the induced breeding of C. carpio with mycological examination of egg samples with different disinfectants. The total bacterial count fluctuated from 3.4 x 108 CFU/ml to 32.7 x 108 CFU/ml during the period of fertilization to 4 days of hatching. The fertilized eggs infected by Saprolegnia spp. were appeared as tuft hairy like balls with a white cottony envelop. Among all the treatment 1mg/L methylene blue, 3mg/L malachite green and 1g/L sodium chloride showed significantly better (P<0.05) hatching rate 95·33±2·08, 88.00±2.64 and 92.33±4.04% respectively. The same concentration of methylene blue, malachite green and sodium chloride showed significantly better (P<0.05) better survival rate 95·00±4.35, 75.00±3.00 and 87.00±6.24% respectively. Finally among all the treatment 1mg/L of methylene blue showed significantly better (P<0.05) hatching and survival rate 95·33±2·08% and 95·00±4.35 % respectively. So 1mg/L of methylene blue is the best disinfectant for C. carpio fertilized egg treatment.

Keywords: Common carp larvae, Fungal infection, Saprolegnia spp., Disinfectants, Survival rate

Location: Mafatema fish hatchery, Chanchra, Jessore and laboratory of fisheries and marine bioscience, Jessore University of Science and Technology.

Start Date: 00-03-2015

End Date: 00-05-2015

Program Area: Pest Management

Commodity: Carp fish

Objectives:

To determine the bacterial load before and after the disinfection treatment,
To identify the causative agent of fungal disease of C. carpio fertilized eggs and identify the best disinfection treatment of fertilized eggs for increasing hatching rate and survival rate of C. carpio.

Methodology:

The study was carried out during peak- breeding season between March to May 2015 at Mafatema fish hatchery, Chanchra, Jessore and laboratory of fisheries and marine bioscience, Jessore University of Science and Technology. Water sample was collected in aseptically sterilized glass container (250 ml capacity). Egg samples were collected in a sterilized screw cap tube. Samples were transmitted to laboratory for examination within 2-3 hours of collection. Bacterial load of incubation tank water was observed two times. First observation was conducted before transferring the fertilized eggs in to the bottle incubator. Final observation was conducted after four days of disinfection treatments. At first glass wares (petridishes, test tubes, L-sticks, mortar , conical flasks, vials, measuring cylinder etc.) were washed very nicely after that dry and sterilized at 170°C for 1 hour by a dry sterilizer. Time was maintained very carefully. The plastic materials were autoclaved at 121°C for 15 minutes. An amount of 0.85g NaCl was weighed and kept in a measuring flask. It was then filled with distilled water to make the volume 100 ml. This was called physiological saline (PS = 0.85% NaCl). Then preparation was mixed nicely by vortex mixer. All the PS was autoclaved at 121°C for 15 min and kept at 4°C for future use. TSA media was mainly used as a nutrient medium for bacteria culture. TSA medium was prepared by mixing at the rate of 40 g/L of distilled water in conical flask. Required amount of distilled water was measured in a cylinder. The mixture was heated on a hot plate for few minutes and then autoclave at 121°C. After autoclaving it was placed in clean chamber waited up to cooling to 60°C and then poured to sterile petridishes at an amount of 20 ml. After completion of cooling and solidification, all the TSA Plates were turned upside down. Bacteria commonly grow up to densities around 10⁹ CFU/ml, although the maximum densities vary tremendously depending on the species of bacteria and the media they are growing in. In the present study a 10-fold serial dilutions of the bacteria was prepared to cover the entire probable range of concentrations. Then 0.1 ml of each dilution was transferred to an agar plate, which in effect makes another 10-fold dilution, since the final unit is CFU/ml and only 0.1 ml was streaked. Spreading in this technique is done using a bent glass rod. Bacterial suspension at 0.1ml was placed in the center of the plate using a sterile pipette. The glass rod is sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through the Bunsen burner flame. The burning alcohol sterilizes the rod at a cooler temperature than holding the rod in the burner flame. Then streak the rod back and forth across the plate working up and down several times. The cover of the petridishes was marked into three divisions corresponding to the designated serial dilution. Each petridishes was replicated three times. The petridishes were incubated in an inverted position at 37°C for 24 hours. The average count of colonies from the designated division of each of the replicates was taken as mean ± standard deviation. The infected egg was compressed with a drop of normalsaline between two slide examined at low power magnification. Eggs with hyphae were taken for fungal isolation using Potato dextrose agar (PDA). After three days incubation period at 25°C the fungal growth was observed. Wet smears from the isolated colonies were done and stained with Lactophenol cotton blue for microscopic study. A total of 1g of fertilized common carp eggs (1500 egg g^-1) was used for each treatment in triplicate. The number of eggs was calculated from the weight of eggs and expressed as number/g eggs. Three different egg disinfectants with their three different concentration treatments and a control treatment were assigned for that experiment. All treatments were 30 minute bath 1 time/day for four days after fertilization. Disinfection treatment trials of fertilized eggs of C. carpio are shown. The results obtained in the experiment were subjected to analysis. Qualitative and quantitative analysis of all kinds of data were carried out. MS Excel was also used for presentation of the tables and graphs obtainable from different types of data. Analysis of variance (One way), Tukey-Kramer Test for differences between means were used to analyze the effect of different disinfectants treatment on hatching and survival rate of common carp (C. carpio) eggs by using SPSS 11.5 software. The significance of the data from disinfectants treatment effect were considered significantly different at P<0.05.

Source of Information: Asian J. Med. Biol. Res. December 2015, 1(3): 578-588, ISSN 2411-4472

Article Link (Online Journal): DOI: http://dx.doi.org/10.3329/ajmbr.v1i3.26481

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The fungi Saprolegnia spp. fungus commonly attack the C. carpio fertilized eggs during the incubation period. High egg densities, organic matter in fish hatcheries beside the regular removal of dead eggs, as they are considered the basic environment for microbial overgrowth which hamper egg development and subsequently affect hatching and larval survival rates. Treatment of methylene blue at 1mg/L, malachite green at 3mg/L and sodium chloride at 1 g/L for 30 min. bath daily for 4 days showed significantly (P<0.05) higher hatching rate and survival rate.

Knowledge Management: Journal