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December 27, 2013
Biofloc/EMS Conference, December 2013
Workshop on Biofloc Technology and Shrimp Diseases
Ho Chi Minh City, Vietnam, December 9, 2013,
Organizers: Yoram Avnimelech, Hoang Tung, Craig Browdy, and John Hargreaves
On December 9, 2013, a two-day workshop on the link between biofloc technology and and acute hepatopancreatic necrosis disease (AHPND, also called early mortality syndrome, EMS) was held in Ho Chi Minh City, Vietnam. The workshop was motivated by the large economic losses in Vietnam amounting to $1 billion per year over the last three years. The workshop specifically focused on characteristics of biofloc technology systems that reduce the risk of the incidence and severity of this disease. There were over 200 participants and 21 presentations, and at least 15 shrimp farmers attended the workshop.
Biofloc and Shrimp Immunity
Evidence is accumulating that exposure to bioflocs stimulates the non-specific immune system in shrimp. Constituents of bacterial cell walls (lipopolysaccharides, peptidoglycans and β -1, 3-glucans) activate the non-specific immune system in shrimp. Specifically these components activate a cascade of reactions leading to the production of prophenoloxidase, leading ultimately to melanization. Other biochemical pathways that are part of the shrimp immune system are likewise stimulated by contact with or consumption of biofloc.
At the workshop, Julie Ekasari and co-workers reported that phenoloxidase activity increases in response to organic carbon loading from different sources (molasses, tapioca, tapioca by-product and rice bran). Biofloc systems contribute to the enhancement of immune response and survival of Penaeus vannamei after IMNV challenge regardless of carbon source.
Bioflocs can be viewed as a mechanism that provides shrimp with pattern recognition and other molecules that lead to stimulation of the non-specific immune system. These molecules are provided to shrimp constantly. There is an energetic cost associated with constant immunostimulation although it is difficult to conclude whether or not this effect is deleterious. Biofloc “primes” the immune system but it is not fully activated until a pathogen is encountered.
Avnimelech presented results indicating significantly lower infection of tilapia by Streptococcus iniae released to the water from challenged fish in biofloc systems as compared to clear water. This may be related to antagonism between the pathogen and other bacteria that limits the pathogen. It is possible that a similar antagonism occurs between dense heterotrophic bacteria and Vibrio parahaemolyticus, the causative agent of AHPND.
Su-Kyoung Kim and In Kwon Jang measured mRNA expression of six genes that are involved in the innate immune response of shrimp [proPO1 (prophenoloxidase 1), proPO2 (prophenoloxidase 2), PPAE (prophenoloxidase activating enzyme), SP1 (serine protease), mas (masquerade-like serine proteinase), ran (ras-related nuclear)]. Gene expression measured in mysid, postlarvae and adult P. vannamei is enhanced in the presence of biofloc. Gene expression is greater in P. vannamei than in other shrimp species (P. chinensis, P. japonicus), possibly related to differences in morphology of the third maxilliped, which affects the ability of shrimp to capture and use bioflocs as food.
The Effect of the Microbial Ecology of Biofloc Technology
Systems on Shrimp Disease
Water in biofloc technology systems contains a large number of bacterial species. Jang found 351-773 operational taxonomic units (essentially equivalent to “species”) in water from biofloc systems. Others reported as many as 2,000 species. The most dominant group is Bacteroidetes, a common constituent of wastewater in treatment plants.
Vibrio is an opportunistic “early successional” species that is controlled in “mature” or aged water with a more diverse assemblage of bacteria. One supporting piece of evidence was provided by Victoria Alday-Sanz, who reported that AHPND outbreaks have occurred a few days after water exchange in shrimp ponds in Mexico. This would be expected if the bacterial community were simplified to an early successional state by water exchange. Characteristics of “mature” and stable water that confer control of V. parahaemolyticus is not known with certainty. Better characterization of the microbial composition of flocs, especially the bacteria that have protective effects, is needed.
Oliver Decamp, citing the Ph.D. dissertation of R. Crab (2010) provided additional evidence on the capacity of biofloc systems to control Vibrio. Shrimp were fed either an artificial diet or an artificial diet partially replaced with biofloc. Bioflocs were grown on different carbon sources with or without the addition of a Bacillus-based probiotic. Treatments were feed only, feed + sucrose, feed + sucrose + Bacillus, feed + glycerol, feed +glycerol + Bacillus. With both types of carbon source, with or without Bacillus addition, the density of Vibrio was less than the feed-only control. Within either type of organic carbon source, adding Bacillus reduced Vibrio cell density.
Questions about Microbial Community Management
• What is the optimal biofloc concentration?
• How to manage/control biofloc community composition for optimal shrimp health?
• How to measure functionality of system in terms of disease control?
• Biofloc systems are quite unstable at the species level; what is the optimum balance of species?
• How to establish biofloc quickly?
• What is the best way to “feed” the biofloc?
1. Manipulate C:N ratio?
2. Continuous or intermittent inputs?
Information was presented regarding normal practice and methods to accelerate aging or maturity of water with respect to microbial community composition. AHPND occurs early in the culture period. In newly started systems, 30-40 days are normally required before flocs develop. Thus, there is a pressing need to develop flocs quickly before AHPND occurs. Development of bioflocs that are target oriented, considering feed composition, immune effects, shrimp growth rates and other properties is presently taking place in academic institutions as well as by commercial companies. We hope that these efforts will improve bioflocs advantages.
The Effect of Co-culture of Fish and Shrimp
Data on the effect of co-culturing shrimp and tilapia and the effect on decreasing the incidence of shrimp diseases was presented, based on experiences in the Philippines and Thailand. Including tilapia or other fish in a shrimp production system appears to confer some protection from AHPND in shrimp, although the mechanism is not clearly understood. The possibilities for the mechanism include:
1. Some zooplankton may serve as concentrators of Vibrio in ponds. Consumption of these zooplankton by shrimp may lead to infection. Grazing of zooplankton by filter-feeding fish may reduce the density of the zooplankton that concentrates the Vibrio.
2. There appears to be some association between Vibrio and blue-green algae. Grazing of blue-green algae by filter-feeding fish may reduce the importance of this association in shrimp ponds.
3. There may be some link between antibiotic effects of fish mucus and the suppression of Vibrio.
More research is needed to elucidate these mechanisms.
Shrimp farmers in the Philippines, Vietnam, and China co-culture shrimp with fish, although a farmer from China reported that results were not very good. In Thailand, an equal biomass of shrimp and tilapia are produced in co-culture ponds. Fish used in co-culture systems include tilapia, silver carp and grass carp.
Attributes of Biofloc Technology System Management
That Reduce Disease Risk
1. Biofloc technology systems are characteristically operated with very low rates of water exchange. Inherently this improves biosecurity because exclusion of pathogens is enhanced by limiting contact with water from external aquatic ecosystems adjacent to farms. Low rates of water exchange is only one aspect of farm biosecurity, which also includes using postlarvae that have been evaluated and certified as disease-free, filtering incoming water (250 microns), erecting barriers to crustacean carriers (crab fences), and maintaining a clean pond bottom.
2. Biofloc technology systems are typically operated with high levels of aeration and mixing. This characteristic creates a stable water quality environment with respect to dissolved oxygen concentration and pH, conditions that are favorable for good shrimp growth and elevated immunocompetence.
3. Removal of accumulated sludge is seen as essential to reduce the risk of AHPND outbreaks. Pond areas with accumulated sludge deposits are areas of impaired water quality. These areas are the location of active production of sulfide, a potent toxicant of shrimp, and other growth-inhibiting chemicals. Furthermore, bacterial population densities in the fluid sediment layer near the pond bottom are very high. Grazing by shrimp in this area increases exposure to high bacterial density. It appears that the lethal dose of V. parahaemolyticus is quite high (108 CFU/mL), a density that could be encountered in the fluid sediment layer. Thus, regular removal of waste solids is viewed as essential. Tung and co-workers recommend solids removal two hours after every feeding.
Biofloc systems can restrain the development of shrimp or fish diseases. This conclusion is based on the results of controlled research and a significant number of field observations. Due to the severity of the shrimp disease problem, the use of biofloc technology can be recommended.
• Efforts should be taken to grow shrimp in “mature” water with a diverse and active micro-biota in the pond. As a practical matter, this calls for the development of mature water prior to shrimp stocking, through inoculation protocols, and minimizing water exchange during the production cycle.
• Probiotics and feed additives can increase shrimp resistance to diseases. Only additives that have been properly tested with demonstrated efficacy should be used.
• Prevent bottom sludge accumulation to reduce shrimp stress and possibly disease. Plan ponds in a way to enable drainage and washout of sludge when it accumulates.
• Biosecurity, selection of healthy stock and other best management practices and technologies deployed on modern commercial shrimp farms should be implemented.
Priority Research Areas
The economic losses caused by shrimp diseases are huge and the probability that implementation of biofloc technology improves the situation is quite high. Research costs are relatively low compared to economic losses. Research oriented to develop, optimize and ascertain means to minimize disease outbreaks and severity is essential. Thus, investment in research oriented toward further development of the biofloc technology approach is recommended in the following areas:
Short-Term Research Topics
• Evaluation of the effects of dense heterotrophic population on V. parahaemolyticus survival
• Establishing the essential parameters to define biofloc systems.
Longer-Term Research Topics
• Develop methods to establish a diverse and stable microbial community.
• Identification of the best tools for measuring and describing the complex microbial floc community.
• Define the optimal microbial community composition in biofloc systems
1. For competitive exclusion of pathogens
2. For target crop growth
3. For water quality management
• Develop methods for maintaining the microbial community at its optimal composition of:
4. Inoculation, probiotics
To summarize this discussion, the main attributes of biofloc systems that reduce the risk of shrimp disease are:
• Low rates of water exchange improve pathogen exclusion (biosecurity).
• Continuous aeration provides stable water quality (DO and pH).
• Regular removal of accumulated sludge controls biofloc concentration to moderate levels.
Information: Yoram Avnimelech (Professor Emeritus), Civil and Environmental Engineering, Technion, Israel Institute of Technology, Haifa, 32000 Israel (phone 972-0-3-7522406, mobile 972-0523-511702, email email@example.com, webpage http://www.technion.ac.il/en/).
Source: Email to Shrimp News International from Yoram Avnimelech (above). Subject: Report on Workshop. December 26, 2013.
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