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What role does the health of shrimp post larvae (PLs)play in farm outcome?

The terms microbiome and microbiota are frequently confused with each other. The microbiota is a subset of the microbiome. It refers to the microorganisms that are present in each environment. The term microbiome refers to the total genome and gene products of all the bacteria, bacteriophage, fungi, protozoa, and viruses that are present in a specific environment. This can be in and on an animal or a plant. It can also

refer to any element of the environment including objects. As applied to aquaculture most often it is used in reference to what is present internally as well as the external surfaces of a given aquatic animal. It also refers to the specific composition of individual elements of a production environment, such as the sediments, the water column, etc. A given microbiome is really just a subset of a much larger ecosystem, just as the microbiota are components or elements of the microbiome.

Not all that long ago much of the microbiome was hidden from science. It was widely held that we could culture most of the bacterial and fungal components on artificial media. Many workers reported broad changes in the microbiome based on observations of how experimental protocols altered what could be cultured. Using much more sophisticated tools today we can characterize many of the elements of a given microbiome, the vast majority of which, we now know, cannot be cultured. This, for the first time, has allowed us to see how complex the microbiota/microbiome is and how it can be affected by the use of many different materials in the feed and in the environment. We have a much clearer, albeit evolving, idea of what microbiota are elements of the microbiome.

There are some indications that specific microbiota may be used as indicators of animal fitness. Given the extreme variability of production paradigms and environments, any generalization of this nature is questionable, although there is data that suggests that healthy animals may have microbiomes that one can correlate with the overall health of the population. Whether we will be able to link specific elements of the microbiome causally with health, disease resistance and/or tolerance is the challenge. Correlation is not causation.

Shrimp hatchery production

Currently, the exact tonnage of farmed shrimp being produced globally each year can only be approximated. Production is somewhere between 3.8 and 6 million MT with the numbers being source dependent. Using the higher figure, to produce the 6 million MTs of shrimp that will be farmed globally in 2023, an estimated 600 billion post larvae (PLs) would have had to be produced (assumes 20 g average weight at harvest, 50% survival in the hatchery). This production is spread across thousands of hatcheries in dozens of countries.

The state-of-the-art production of post-larval shrimp remains, for the most part, relatively primitive. Larger operations may produce a billion or more PLs a month although this is the exception with most production coming from smaller hatcheries. Biosecurity is weak in general regardless of the size of the facility and those who operate them all too often neglect the tools of science (Newman personal observations). Widespread use of disinfectants can damage the microbiota and create niches for rapidly reproducing bacteria, such as Vibrios, to dominate.

A major source of diseases in farmed shrimp is a result of the carry-over of obligate pathogens from broodstock. PCR testing of populations using the standard statistical approach can result in animals carrying pathogens below the threshold. For example, when there is a 98% chance of finding a given pathogen when screened for using non-pooled samples, appropriate primers, and conditions conducive to maximum sensitivity, 2% of the population can still be carriers, i.e. 20,000 out of a million PLs. These will continue to spread pathogens, both those that are present historically and potentially new ones that are cropping up with a consistent frequency. Broodstock should be held under lifelong quarantine, screened repeatedly by sensitive and specific PCR primers with moribund animals examined clinically including thorough examination by histopathology to determine the cause. Endogenous viral elements (EVEs) are increasingly being reported and this can lead to costly false positives. It is critical that the right primers are used. Eliminating obligate pathogens from the broodstock ensures that nauplii, pre-larval and PLs are not carrying them into their respective production systems.

Vibriosis in shrimp culture

One of the most common reported problems in hatcheries is vibriosis due to infections with Vibrio bacteria. Vibrios are ubiquitous in marine and, to a lesser extent, freshwater environments. They serve an important function in the recycling of chitin, the structural component of crustacean exoskeletons, among other things. Some are human pathogens (Vibrio parahaemolyticus, Vibrio cholerae) and others are aquatic animal pathogens (Vibrio parahaemolyticus, Vibrio anguillarum, etc.). As of this writing, some 149 distinct species have been identified with many thousands of variant strains. Note that many bacteria can cause disease in farmed shrimp, not just in the hatchery, but on the farm as well. These include Aeromonas, Pseudomonas, Clostridium, Propigenium, Streptococcus and many others.

Focusing exclusively on Vibrios as being the source of problems, although widespread, is not consistent with optimum results. Attention needs to be paid to known pathogens and ensuring that their presence is minimized by breaking the cycle between broodstock and the farm and minimizing the presence of known vectors in farms as well as creating a production environment that ensures reduced stress on the animals.

Perhaps the most widely Vibrio-linked problem in shrimp hatcheries is known as the zoea syndrome. Zoea are the first feeding stage of larval shrimp and large mortalities can occur at this highly sensitive stage if Vibrio loads are not adequately controlled. The first effort to control the impact of Vibrios in shrimp hatcheries via the use of microbiota manipulation was developed by Giovanni Chasin and published by Garriques in Ecuador in the early 1990s. They reported that Vibrio strains from the wild that were able to degrade sucrose on the widely used selective media for Vibrios, thiosulfate-citrate-bile salts-sucrose agar (TCBS), i.e. they formed yellow colonies when added in large numbers to hatchery tanks were able to dramatically reduce the severity of the disease. This approach has some challenges when applied to the field. Unfortunately, the assumption was that these Vibrios, typically Vibrio alginolyticus, were not virulent. Animals were routinely tested for susceptibility to verify this. However, failure to understand how diverse this taxon is and how readily they exchange genetic material has resulted in hatcheries losing entire production runs using a similar approach. The ability to degrade sucrose has nothing to do with virulence.

Other important pathogens are toxigenic strains of V. parahaemolyticus. The combination of PirA/PirB toxins has wreaked havoc globally and continues to be problematic. These toxins have been found in several other species and are highly specific for the tubules in the hepatopancreas. Damage can range from being barely evident to death. Often secondary bacterial infections kill. Recently, a relatively new syndrome, transparent shrimp disease, has been associated with other strains of toxigenic V. parahaemolyticus. Given the ubiquitous nature of Vibrios, this strain is quite likely to spread as much as the PirA- and PirBproducing strains have.

Microbiota manipulation

We are still in the early stages of this being a truly science-based protocol. Knowing what to add, where to add it, how much to add and how often are only a few of the challenges. Perhaps the greatest single challenge is that many of the companies that sell bacteria as probiotics are selling products for bioremediation. Probiotics are living bacteria, ingested orally, that alter the microbiome (the microbiota) and have a positive impact on animal health. While there are some claims that this is what is occurring the field results do not seem to verify that there is indeed a probiotic effect as defined above. Shrimp consume large amounts of bacteria as their natural food source.

This practice of adding any number of a wide variety of bacteria continues despite potential hazards. There is a widespread failure to appreciate that bacteria readily exchange genetic material, sometimes at high frequencies, and that this can lead to acute problems as a result of the exchange of genetic material between virulent obligately pathogenic strains and currently benign strains. Typically, this is between bacteria with some genetic similarity, but this is not always the case. The practice of adding as many different sources of bacteria as there are vendors poses a risk to the stability of the pond and hatchery ecology as well. Bacteria compete against each other and the strains of VP that cause AHPNS have a very effective tool for doing this.

With this in mind, the ability of several spore-forming gram-positive bacteria (Bacillus species) were tested for their ability to impact Vibrio loads in a shrimp hatchery in India. A tableted product (PRO4000X) containing equal proportions of specifically selected proprietary strains of Bacillus subtilis and B. licheniformis was added to hatchery tanks and the impacts on total Vibrio loads determined (Fig. 1, 2).

These tests demonstrated that these proprietary Bacillus strains when added daily to shrimp hatchery tanks were able to dramatically lower the total Vibrio loads and keep them low. This was an example of microbiota manipulation in a production environment

that had a dramatic impact on the presence of presumptive pathogens that routinely have a negatively impact on shrimp hatchery production. The hatchery personnel noted that the tanks were much cleaner, that they were able to stop exchanging water and that the animals in the experimental tanks were cleaner, stronger, grew better and were free from vibriosis problems.

These examples of microbiota manipulation show a clear cut, although not permanent, impact. The Bacillus spores had to be added daily to ensure that levels did not decline to the point where they no longer resulted in the desired outcome, i.e. reduction of Vibrio loads (Habeeb Rahman-personal communication). When the Bacillus spores are no longer being added the impact will disappear. It is not likely that the Bacillus have become a stable component of the microbiota. One would expect that daily addition would not be needed if the added Bacillus strains were able to become stable elements of the microbiota/microbiome.

These tests demonstrate that in shrimp hatchery tanks it is possible to alter the microbiota in a manner that has a beneficial impact on the overall production, significantly altering the composition of the bacteria normally present. While the exact mechanisms remain to be elucidated, the evidence suggests that we are looking at a simple case where the added Bacillus spores upon germination compete against resident species for nutrients. This effect has also been noted in shrimp ponds as well (Habeeb Rahman personal communication).

Similar experiments have been carried out in production ponds demonstrating that this approach can impact the presence of potential pathogens in grow-out ponds. The reduction of potential pathogens is a valuable tool and an important step in minimizing the impact of animal health challenges on production. However, this does not occur in a void. Broodstock must be produced in a manner that ensures that no pathogens are present. Biosecure production protocols must be present throughout the PL production process to ensure that Artemia and algae culture are not introducing pathogens. Open to the air systems can easily be contaminated. Stocking PLs that do not carry high loads of obligate and opportunistic pathogens is essential for sustainable shrimp production.

Stocking protocols must be geared toward minimizing stress and the production environment should be free of accumulated organic matter, and well aerated with automatic feeders programmed to minimize waste, feeds should be designed to minimize waste by unnecessary grinding of feed before ingestion with highly digestible ingredients and adequate levels of micro and macronutrients. The goal should be to create an environment that is conducive to high survival and allows the animals to realize their genetic potential, growing as rapidly as they can. Shorter cycles allow for more cycles per year.

Knowing what is being stocked both quantitatively and qualitatively is the only way to know what the true outcome is. Efforts to alter the microbiota have been successful using Bacillus species in the hatchery and the farm with favorable results. These are short-term changes. High-quality, pathogen-free PLs are an integral part of sustainable production.

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