Selective Breeding in Shrimp Farming

Elastomer tagging is one method that is used to identify which characters have passed onto the next generation in breeding programmes. (Photo courtesy: Kona Bay vannamei breeding program, Hawaii) Selective Breeding in Shrimp Farming A. Victor Suresh United Research (Singapore) Pte. Ltd. & Growel Feeds Pvt. Ltd., India E-mail: avictor.suresh@gmail.com 32 Selective breeding has been used for ages by mankind for improving plant and animal production. In shrimp farming, it has a relatively short history of about 20 years and the gains are already impressive. Production of 15 g vannamei in 120-135 days at a stocking density of 20 animals/sq.m was the norm twenty years ago. Today 28 g shrimp can be produced in the same duration at a stocking density of 50-100 animals/sq.m. While factors such as disease control, pond technology, partial harvest, better feeds and improvements in management knowledge have contributed to the phenomenal improvement in shrimp farm productivity, selective breeding has played the most important role. Further improvements in productivity will occur as we continue to select shrimp for growth, disease resistance, and other desirable traits. The purpose of this article is to provide a broad understanding of selective breeding programs so that farmers can make educated decisions concerning the seed stocks they buy. FISHING CHIMES APRIL - MAY 2015

HISTORY Selective breeding requires that the lifecycle of target animals should be closed under captivity, i.e. animals should be able to live under human management from their birth/hatch onward and be able to breed and produce offspring that will survive to produce another generation of animals. Without this ability, broodstock needs to be taken from wild stocks every generation and selection of animals for improvement cannot occur.hence, closing of the lifecycle under captivity is an essential part of selective breeding, which is defined as the process whereby a population of living organisms is changed at the genetic level, through generations of controlled reproduction, to accentuate traits that ultimately benefit the interests of humans. Necessity for Breeding:For a period of time in shrimp farming history, seed stocks were sourced from the wild. Later on, due to increasing demand broodstock were sourced from the wild to produce offspring in captivity. The third stage was selective breeding, which occurred when the shrimp were domesticated. The species of the Litopenaeus genera, namely L. vannamei and L. stylirostris (blue shrimp) were the first ones to be domesticated widely due to the limited geographic range and seasonal reproduction of the wild populations and the ease of breeding them under captivity. L. vannamei are native to the Pacific coast of the Americas between Mexico and Peru, so farms located on the Caribbean and Atlantic coasts were forced to adopt domestication programmes. Even within the geographic range of L. vannamei, wild adults often were not available, due to seasonal factors such as cool offshore currents during La Nina periods off Ecuador. On the other hand, Penaeus monodon, has much wider geographic range throughout the IndoPacific, which enabled farms throughout the region to use wild stocks without the necessity of domestication. In terms of ease of breeding, L. vannamei reaches maturity when they were relatively small in size (30-50 g) and are open thelycum breeders. Nevertheless, it was a challenge to induce reproduction in the wild adults under captive conditions. Now, populations of L. vannamei have been domesticated for about 30 generations and they reproduce easily in captivity, even when the diet and environmental conditions are not optimal. While it has been possible to rear the more popular tiger shrimp, Penaeus monodon, from egg to maturity and breed them, successful domestication to full commercial impact has been limited to only a handful of groups till date. This is partly due to the late start of tiger shrimp breeding programmes and the limited number of generations in captivity. Undoubtedly, selective breeding of tiger shrimp will become easier with successive generations. Domestication and selective breeding are also limited in other penaeid species due to the lack of widespread commercial interest in those species. So this article considers breeding of vannamei shrimp only. U.S Government funded breeding programme: Efforts to develop selectively bred stocks of vannamei shrimp can be broadly divided into two, based on where the efforts occurred. One is the large and highly successful effort in the United States of America initially funded by the US government in what was known as the US Marine Shrimp Farming Programme (USMSFP). The primary objective of the program was to develop Specific Pathogen Free (SPF) stocks of vannamei shrimp for shrimp farming in the US. Concerted efforts by multiple institutions since 1989 led to the collection of adult vannamei from the wild, which were taken through a systematic quarantine and disease surveillance system, after which they were bred and the domesticated. The disease-free stocks were deposited in a Nuclear Breeding Center (NBC) located at the Oceanic Institute (OI) in Hawaii. The stocks were perpetuated at the NBC and high health larvae (larvae free from listed pathogens) were supplied to the farms. The programme screened for nine viruses, Concerted efforts by multiple institutions since 1989 led to the collection of adult vannamei from the wild, which were taken through a systematic quarantine and disease surveillance system, after which they were bred and the domesticated. The disease-free stocks were deposited in a Nuclear Breeding Center (NBC) located at the Oceanic Institute (OI) in Hawaii. Acknowledgement: The author is grateful to DrJoão L. Rocha, a leading shrimp geneticist associated with many successful vannamei breeding programmes around the world (Genearch, Brazil; PrimaGen, Indonesia; Texcumar, Ecuador; CAMACO, Panama)for providing study materials and going through the first draft of the manuscript and correcting many errors. Dr George W. Chamberlain, whose companies Kona Bay Marine Resources and iaqua run breeding programmes on vannamei and monodon, respectively, provided valuable inputs on the second draft of the manuscript. VOL. 35 NO. 1 & 2 FISHING CHIMES 33

Steps in the development of selectively-bred shrimp stocks one bacteria and three parasites and assured that the high health larvae did not have the listed pathogens when they were released from the NBC. Once SPF was established, efforts went into selective breeding of stocks. The breeding programme primarily focused on growth improvement and development of Specific Pathogen Resistant (SPR) stocks. The programme accomplished the development of fast growing stocks, and stocks resisting the Taura Syndrome Virus (TSV). Broodstock developed by USMSFP were sold from the NBC to US companies that wished to start their own SPF programmes. These companies were located in the states of Hawaii and Florida. These companies then sent out their stocks to shrimp farming countries around the world for propagation and use in shrimp farming. Consolidation of these organisations also occurred over time resulting in only a handful of breeding companies. While all vannamei stocks used in Asia can be traced to the founder stocks developed by USMSFP, the commercial breeding programmes have become more advanced and have achieved higher acceptance by farms throughout Asia. About the time USMSFP was initiated, many shrimp farming countries in Latin America, notably Mexico, Honduras, Panama, Ecuador, Colombia, Venezuela and Brazil embarked on their own vannamei domestication programmes. The Pacific white shrimp, vannamei are non-native species in Venezuela and Brazil While all vannamei stocks used in Asia can be traced to the founder stocks developed by USMSFP, the commercial breeding programs have become more advanced and have achieved higher acceptance by farms throughout Asia. Vannamei shrimp breeding programmes that are on the Atlantic Ocean. So, domestication was required for independence from the need to frequently import broodstock from other countries. In other Latin American countries, hatcheries found it logistically easier and more seasonably reliable to source adult animals from farms than from the ocean. However, no systematic efforts were taken to exclude pathogens when the broodstock were sourced. This led to the development of stocks with pathogens. Some breeding programs intentionally took animals that survived disease outbreaks and used them for breeding with the belief that they could produce stocks that were resistant to pathogens. According to some industry insiders, the approach of sourcing broodstock with pathogens was counterproductive and resulted in the rapid evolution of the pathogens ability to cause harm.however, this assertion has been disputed by others and trends 34 FISHING CHIMES APRIL - MAY 2015

emerging in the last few years show that the Latin American approach to building disease resistance by direct exposure to pathogens may have some merits. Breeding programmes in Latin America are more focused on the development of stocks that are tolerant of pathogens because the large pond size (5-20 ha) does not allow for intensive management of ponds. Tight biosecurity, heavy aeration and periodic sludge removal are not realistic in large ponds, therefore farmers preferred pathogen-resistant stocks to pathogen free stocks. Some breeding programmes have achieved notable success in producing pathogenresistant stocks, the most important one being against the white spot syndrome virus (WSSV). The recent industry growth trends in Ecuador and some other Latin American countries have been attributed to the development of resistance to WSSV in some stocks. Whether these stocks have truly attained complete resistance against WSSV is not confirmed, but these countries are able to coexist well with the occurrence of the pathogen but without major catastrophes. In conclusion, the brief history of vannamei breeding has seen two major approaches: one was oriented to the development of fast-growing, pathogen-free stocks while the other targeted the development of pathogenresistant stocks. The strengths and limitations of both approaches are now better understood and the obvious path for the future is to combine the efforts to produce fast-growing, pathogen-free stocks that are also resistant to pathogens....trends emerging in the last few years show that the Latin American approach to building disease resistance by direct exposure to pathogens may have some merits. DOMESTICATION, SPF, SPR & BREEDING PROGRAMMES The terms Domestication, SPF, SPR & Breeding Programmes are often misunderstood in the shrimp farming sector, so distinguishing each term is important. Domestication, as explained in the previous section, involves the completion of the life cycle of an animal under human management. Domestication paves the way for production of stocks generation after generation from one or more parental lines. Some degree of genetic selection intended or inadvertent occurs during the domestication process, but without scrupulous VOL. 35 NO. 1 & 2 FISHING CHIMES Laboratory disease challenge trials indicate which families from a given genetic line have greater resistance to specific diseases. This data can be used as one of criteria for selecting which families to use for producing the next generation. In this trial, survival of 30 L. vannamei families exposed to a 5-day WSSV challenge ranged from 8-64%. The average survival of the top 15 families was 51%. [data courtesy of Kona Bay breeding program in Kauai, USA]. management, genetic gains might be minimal or even negative. Targeted selective breeding programmes are a way of accelerating gains in desirable traits and avoiding the negative effects of inbreeding. Specific Pathogen Free (SPF) refers to the production of animals that are free of a specified list of pathogens. The process involves biosecurity controls and systematic surveillance and exclusion of pathogens as per a set of protocols. Typically, the protocol requires primary and secondary quarantine steps spanning at least two generations before a population can be declared SPF. Technically SPF status is guaranteed only until an animal is within the premises of the Nucleus Breeding Typically, the protocol requires primary and secondary quarantine steps spanning at least two generations before a population can be declared SPF. Center that operates the SPF program. Once it leaves the facility, it should be protected against infections along the way and in every facility that receives, holds or handles the animal until it is harvested. Since facilities like multiplication centers and hatcheries that handle SPF animals are not likely to be as biosecure as the NBC, distinction has in the past been by what is referred to as High Health animals. These are animals that are offspring of SPF broodstock produced in a hatchery with a good pathogen control programme. 37

Studying performance of different families in net cages suspended in ponds is a common practice in selective breeding (photo courtesy: Generach, Brazil) The term is not commonly used at present because of the practical difficulties in defining a High Health program for universal implementation. Breeding programme means that-animals within the programme are mated as per a plan devised by a geneticist who wants to achieve specific traits in the target organisms. The traits could be related to the appearance of the organism or its ability to resist one or more pathogens, growth, feed efficiency, reproductive capacity, etc. Development of a single trait is a long and painstaking process involving more than one generation of breeding, so breeding programs require investment of time and money to be rewarding. At the same time, breeding is a process that builds value in the stocks continuously that the rewards are generally more than worth the efforts invested. As stated above, ability to resist pathogens can be achieved through selective breeding programmes or through natural selection. The term Specific Pathogen Resistant (SPR)means that stocks are resistant to specified pathogens. As stated earlier, SPR stocks are commonly available for TSV resistance from most commercial SPF vannamei breeding companies. Additionally, some SPF vannamei breeding companies have developed stocks with resistance to IHHNV, IMNV, and WSSV. Efforts are also underway to develop stocks resistant to EMS. In a form of convergent evolution, South American breeding programmes are also developing stocks resistant to a wide range of pathogens, but not necessarily SPF. These South American stocks are yet to be available in the open market but used within programmes that have developed the stocks. BASICS OF GENETICS Aquaculturists need to understand what certain genetic terms mean to understand breeding programmes better. Traits are characteristics that are targeted for 38 FISHING CHIMES APRIL - MAY 2015

Graphs illustrating Genetic x Environmental Interactions. The graph on the left shows that performance of families for growth (as measured by harvest weight) at low stocking density is highly correlated to performance at high stocking density. In other words, families performing well at low stocking density would also perform well at high stocking density, therefore there is no interaction between genetics and stocking density for growth. The graph on the right shows that there is poor correlation between survival of families in low stocking environment and high stocking density environment. In other words, high survival of a family in low stocking density does not mean that the same family would survive equally well in high stocking density. This indicates there is interaction between genetics and stocking density for survival (Data courtesy: Dr.João L. Rocha) attainment or improvement through breeding. Traits should be identifiable or quantifiable. Examples of traits commonly used in today s breeding programmes are: growth rate, multiple pathogen resistance, and reproductive performance. Traits targeted in a breeding program must be heritable, i.e. passable from parents to offspring. Heritability of a trait ranges from 0 to 100%. When it is 0%, the trait is not heritable at all. When it is 100%, the trait is fully heritable. The higher the heritability, the fewer the generations required for breeding to achieve its targets. Geneticists consider heritability levels above 20% to be suitable for attainment through breeding programmes. Traits are coded by one or more genes. Each gene has one or more forms called alleles. To understand let us take a simplified hypothetical example: eye colour in a population may be black or brown coded by an allele for whatever factor that causes eye black or brown respectively. An individual will have two alleles of each gene, one derived from the father and other one derived from the mother and will pass one of the two alleles to the offspring. The alleles may be the same (homozygous, for example Black, Black) or different (heterozygous, for example, Black, Brown) and expression of the gene will depend on whether one allele dominates over the other or both alleles contribute equally to the expression. If the allele for black eye colour dominates over allele for brown eye colour, for example, an individual will have black eye colour even if he derives an allele for brown eye colour from one of the parents. Two alleles of brown eye colour will be needed for the expression of brown eye colour. The allele for brown eye colour is called a recessive allele because it requires two alleles for expression. In cases where both alleles contribute equally, the offspring will have brownish black eyes. SELECTIVE BREEDING PROGRAMMES: HOW DO THEY WORK? Selective breeding is the means by which one or more traits is attained or improved through crossing of selected individuals or families. In agriculture,target traits can be divided into two types: one that is qualitative and another that is quantitative.qualitative traits(for example, a fish with red eye)are typically controlled by a single gene and can be achieved with a mating between individuals that possess the trait. It is also easy to identify the fish with the allele that codes for the trait (any fish that has a red eye will have the allele) and use it in breeding. In two rounds of breeding, the way the alleles are expressed, i.e. dominant or recessive, can be established to develop a sound breeding program. Quantitative traits, for example, weight gain, are controlled by many, many genes, so identifying fish or shrimp that has superior alleles for all the genes is not practically possible. Further, redistribution of alleles occur every generation, so there is no guarantee that an The higher the heritability, the fewer generations required for breeding to achieve its targets. Geneticists consider heritability levels above 20% to be suitable for attainment through breeding programs. VOL. 35 NO. 1 & 2 FISHING CHIMES 39

Table 1: Some prominent SPF vannamei breeding companies that derived parental stocks from USMSFP Company/Brand name Location Status of Permission for import into India Shrimp Improvement Systems USA, Singapore On the Approved List Kona Bay Marine Resources USA On the Approved List Oceanic Institute USA On the Approved List SyAqua (acquired by Gold Coin) Thailand On the Approved List CP Thailand On the Approved List Global Gen Indonesia On the Approved List Prima Gen Indonesia Never applied Genearch Brazil Never applied 40 individual with superior alleles will pass all superior alleles to its offspring. So, rather than relying only on individual selection for performance traits, geneticists also rely heavily on what is known as family selection. As the name implies, families instead of individual animals are selected for breeding based on their performance record in a family selection programme. Families are created by mating a single male with a single female and then identifying the offspring by placing in tagged containers or tagging the animals with a mark. Injectable elastomer marking is the most common method of tagging penaeid shrimp, but more sophisticated breeding programs use genetic markers to increase selection intensity. Breeding programmes depend on geneticists who are adept in the use of sophisticated computer programs to manage selective breeding as data need to be compiled on thousands of individuals belonging to multiple families and analysed for decisions to be made on what families should be selected for further development. Breeding programmes must maintain a sufficient number of families and carefully select mating pairs to minimise the rate of inbreeding. One of the key considerations in a selective breeding program is the environment in which the selection occurs. Performance of shrimp is dictated by many environmental factors such as temperature, salinity, stocking density, etc. Shrimp selected for performance in one set of conditions may not perform well in other conditions. There is also need for breeding programmes to develop stocks that perform well in a specific environment, for example, low temperature. Consequently geneticists need to study performance of their animals under different variables of environmental conditions and calculate what is known as Genetic x Environmental Interaction to guide their breeding programmes. Quantitative traits, for example, weight gain, are controlled by many, many genes, so identifying fish or shrimp that has superior alleles for all the genes is not practically possible. SPF VANNAMEI BREEDING COMPANIES & SELECTIVE BREEDING All SPF vannamei breeding companies have derived their founding stocks from the USMSFP and many have implemented selective breeding programmes to improve growth, disease resistance, and reproductive performance. These programmes have been active over dozens of generations and at different locations (including some in Asia and Brazil), so it is reasonable to expect that the stocks sold by different companies have distinct performance characteristics. There has also been some consolidation in the breeding companies likely resulting in the amalgamation of stocks. Companies hold the details of their programmes secret, so the performance of the stocks at the hatchery level or at the farm level are currently known only through subjective feedback from the hatcheries and farms. Objective evaluation of stocks of different companies is quite limited at this time. In today s world, the majority of shrimp production is still in the hands of independent farmers who are dependent on independent hatchery operators for their seed stocks and the hatchery operators, in turn, rely on broodstock suppliers for the spawners. The breeding efforts of many broodstock suppliers are oriented to two objectives. One is to keep running their selection programme. A large number of families are perpetuated in this program. The second objective FISHING CHIMES APRIL - MAY 2015

is to produce stocks that will be reared into adult size and sent out to hatcheries for spawning (or the PL may be sent out to multiplication centers in the recipient country for rearing to adult size). Typically only a few families are used for this purpose due to space as well as proprietary considerations. By limiting the number of families sent out, the breeding company limits the chances that the recipients of the stocks can establish their own breeding programmes. If a recipient begins a breeding programme with members of the same family, the programme will suffer from inbreeding depression in a few generations. In most countries that import SPF vannamei, local programmes exist that mature and spawn the first or second generation offspring of genuine SPF broodstock. These programmes generally rely on shrimp reared in farm ponds to develop their broodstock. These programmes present a great threat to the sustainability of shrimp farming because on one hand they perpetuate One of the key considerations in a selective breeding program is the environment in which the selection occurs. Performance of shrimp is dictated by many environmental factors such as temperature, salinity, stocking density, etc. VOL. 35 NO. 1 & 2 FISHING CHIMES and broadcast pathogens from one area to a wider area, and on the other, create inbred stocks that are likely to be more sensitive to pathogens. WHAT CAN FARMERS DO TO ENSURE THAT THEY GET QUALITY PL? Currently India allows only SPF vannamei broodstock to be imported into the country and stipulates that only such imported broodstock be used for seed production. The broodstock suppliers are authorised after going through a process to ascertain the origin and SPF status of the stocks. The stocks are quarantined upon receipt in the country. These steps reduce the likelihood of defective stocks imported into the country. But, clandestine programmes that illegally use pond-reared broodstock are more difficult to control. So, farmers can only use vigilance in their stock selection. 1. Develop close and long-term relationships with their hatchery suppliers 2. Select hatcheries that are quality oriented and genuinely interested in the long-term survival of shrimp farming 3. Insist on quality and pathogen testing of the seeds they buy 4. Avoid purchase of PL that are priced suspiciously below general market prices. 41