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Mass Larval Mortality in a Giant Freshwater Prawn Macrobrachium Rosenbergii Hatchery: an Attempt to Detect Microbes in the Berried and Larvae

MD SAMSUL ALAM
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

MD ABDUR RAHMAN-AL-MAMUN
Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

MUHAMMAD TOFAZZAL HOSSAIN
Department of Microbiology and Hygiene Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

MOHAMMED YEASIN
BRAC Fisheries Enterprise, BRAC, 75 Mohakhali, Dhaka 1212

MD MUSHARRAF HOSSAIN
BRAC Fisheries Enterprise, BRAC, 75 Mohakhali, Dhaka 1212

Abstract/ Executive Summary:

Early mass mortality of Macrobrachium rosenbergii larvae is a big problem of hatchery failure. Vibrio spp. and/or M. rosenbergii nodavirus (MrNV) have been reported to be the major causes of mass mortality of M. rosenbergii larvae. An attempt was made to detect the reported bacterial and viral pathogens in the berried and larvae of M. rosenbergii. The berried were collected from the Kocha river, Pirojpur and brought to the hatchery. For detection of bacterial pathogen, five berried were collected before the treatment with disinfectant and five berried were collected after the treatment and release of the hatchlings. Larvae samples were collected from the larvae rearing tanks after five days and 15 days post-hatching. The samples were processed, inoculated in TCBS agar media and PCR was done using primers designed from groEL and 16S rRNAgenes for Vibrio sp. MrNV detection kit was used to detect MrNV. We have identified two bacterial species namely Morganella morganii and Citrobacter freundi in the berried samples. However, no Vibrio spp. nor MrNV was detected in either of the samples of berried and larvae. However, mass mortality was observed in the hatchery resulting in a poor PL production. Therefore, other factors might be responsible for the mass mortality in the prawn hatchery that demands further in depth investigations.

Keywords: Macrobrachium rosenbergii, Bacteria, Virus, Larvae

Location: the Kocha river, Pirojpur, Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Prawn, Bacteria

Objectives:

The objective of the study is to identify most common pathogenic bacteria and virus from M. rosenbergii berried and larvae.

Methodology:

Collection of berried and larvae samples: A total of 350 berried were collected from the Kocha river, Pirojpur from artisanal fishermen, brought to the BRAC Prawn Hatchery, Barisal by truck in 200-L drums equipped with aerators for one cycle of hatchery operation. For the treated group, the berried were acclimatized to 6 ppt salinity for 2-3 hrs and then treated with formalin at a dose of 15 ml/50L for 30 min. The berried were then treated with povidone-iodine for 45 sec and transferred to the hatching tank. For this study, five treated and five untreated apparently healthy berried were randomly selected and preserved in freezer. The samples were aseptically transported to the Microbiology Laboratory of the Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh maintaining cool condition using flacked ice. The larvae samples were collected from the larvae rearing tank after 5 and 15 days post-hatching and preserved in 95% ethanol. Tissue preparation and culture of bacteria: The brain, hepatopancreas and intestine of each prawn was removed aseptically, placed in brain heart infusion (BHI) broth containing 2% NaCl and incubated at 37°C for 8 hrs with a view to enriching the growth of Vibrio spp. Each broth culture was streaked on thiosulfate-citrate-bile salts-sucrose (TCBS) agar media and overnight incubated at 37oC. Subculture was done on same media to obtain pure culture. Extraction of chromosomal DNA and PCR amplification: One milliliter of overnight broth culture of each isolate was centrifuged at 12000 rpm for 3 min. Supernatant was discarded and total DNA from the cells was isolated following phenol: chloroform: isoamyl alcohol extraction method (Sambrook et al. 1989). One set of primer designed from groEL gene was used for the specific detection of Vibrio genus (Hossain et al. 2014) and for further confirmation the universal primer set (27f and 1492r) (Lane 1991) was used to amplify 16SrRNA gene fragment. Sequencing of DNA fragment generated by PCR: Sequencing of the 16SrRNA gene fragments of the isolates was done from Macrogen, South Korea. For bacterial identification, 16SrRNA gene sequences were submitted to the Gene Bank database of National Center for Biotechnology Information (NCBI), USA and homology with the closest known relatives were estimated using the BLAST program (Altschul et al. 1990). The 16SrRNA gene sequences of the isolates were used to construct a neighbor-joining tree following Saitou and Nei (1987) using the software MEGA7.0 (Kumar et al. 2016). Detection of MrNV infection: Samples were collected from one untreated and one spent berried; and larvae of first and second cycle and analyzed for MrNV infection using the MrNV detection kit IQ 2000 (GeneReach Biotechnology Corp, Taiwan). Water Quality Parameters Monitoring: Early life stages are the most sensitive phase in the complex life cycle of aquatic invertebrates and physicochemical parameters of water play important roles on embryonic and larval development and survival of M. rosenbergii. Among the physicochemical parameters temperature, pH and ammonia are very crucial and were monitored throughout the operation cycle. pH of the LRT waters was estimated using a benchtop pH Meter (Mi 150, Milwaukee). AQUA AM Ammonia Test (Made in Thailand) was used for estimation of Ammonia. A thermometer was used for reading the water temperature of the LRTs.

Source of Information: Bangladesh J. Fish. (2019) 31(1) : 41-48

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Temperature management is a critical and difficult job in a Macrobrachium hatchery. The optimum range of temperature for Macrobrachium larvae is from 29-31^OC. Temperature may go up beyond the upper limit of the optimal range in hot sunny days. On the other hand, on rainy cool days, it may go down the lower limit of the optimal range. Either situation can cause heavy mortality of the larvae. During the cycle, the LRT temperature went below 26^OC due to heavy rains that might be the crucial factor for the mass mortality and non-conversion of larvae into PL in the hatchery. We have observed that larvae survived more than 30 days but PL conversion was very low. So, not only the potential pathogens, we have to look for other reasons for mass mortality of galda larvae in the hatchery. However, mass mortality was observed in the hatchery resulting in a poor PL production. Therefore, other factors might be responsible for the mass mortality in the prawn hatchery that demands further in depth investigations.

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