Shrimp Genomics: Making the Most of Limited Resources

Kurt R. Klimpel

Super Shrimp Inc.

1545 Tidelands Avenue, Suite J

National City, California 91950

Klimpel@supershrimp.com

ABSTRACT

Successful farming of marine shrimp in the United States faces a number of biological, environmental and economic challenges. Despite these challenges the number of shrimp farms in the United States is increasing dramatically. The economic models for many of these ventures are considered marginal and rely on achieving very good growth in relatively short growing cycles. Current molecular genetic techniques are available to assist the farmer select strains of shrimp that can grow more quickly, resist disease and utilize nutritional resources more efficiently. A number of research groups have been collecting information about important regions of the shrimp genome relative to their area of interest. It would be of great benefit to bring the work of these separate groups of individual researchers and institutions together to promote a better understanding of the relationship of different phenotypic traits, genetic markers and gene functions.

KEYWORDS: Shrimp, Genetics, Growth, Disease, Nutrition, DNA, PCR, Marker analysis, Selection

INTRODUCTION

The potential for application of molecular genetic techniques to shrimp farming has been recognized by a number of academic and industry groups throughout the world (1). Applying traditional husbandry practices have resulted in a slow development of improved strains showing only very modest improvements with respect to growth and disease resistance. Utilization of current molecular tools for screening, identification and linking genetic markers to desired traits promises to rapidly develop novel strains of shrimp which can out perform currently available stocks with respect to three key areas: utilization of feed, growth and disease resistance. There is a fundamental lack of understanding of the shrimp genome at the current time. Several factors including the large genome size and limited resources make it unreasonable to expect any major advancement in our understanding in the foreseeable future. Fortunately, application of current molecular techniques, when used appropriately, can enable the researcher, and ultimately the farmer, to economically grow a healthy profitable crop without additional specific knowledge of the shrimp genome. Stocks with improvements in these three key areas will lead directly to a more productive and more commercially viable business. The challenge is to use the limited resources available from government, academic and industry sources to efficiently develop useful strains for the shrimp farmer. Much of the ongoing current research, while well intended, does not efficiently target these key areas of improvement needed to make shrimp farming in the United States an attractive agricultural concern.

Starting to understand the shrimp genome

A realistic approach to shrimp stock improvement means knowing that there is much we do not know about the basic organization of the shrimp genome, including the actual genome size, the number of chromosomes, the splicing frequency or the natural mutation frequency. Each of these items is associated with a host of important basic scientific questions, which because of lack of adequate resources, will likely only slowly be addressed. We do know that penaied species have a high degree of genetic diversity (2,3). This implies that developing a genomic map for one farmed species, such as P. monodon, may not likely benefit other important farmed species, such as P. vannamei or P. stylirostris. Knowing our limitations it appears obvious that embarking upon untethered mapping of the shrimp genome with various marker systems is not a prudent option.

How do researchers and farmers get a handle on the important aspects of the shrimp genome without the requisite knowledge of its basic organization? The starting point will be one of several molecular genetic tools. Many of these methods do not require any previous knowledge of the genome to be studied. There are several basic techniques, each with a number of variations that are used to learn more about the genetic structure of the host.

Microsatellite analysis (MSA) looks at repeated short DNA sequences (1-6 base pair tandem repeats) found throughout the non-coding portions of the genome. Microsatellites are found in all organisms, however the structure and frequency varies greatly. The short repetitions are prone to mutations as the enzymes replicating the genome "slip" and lengthen or shorten the motif. The high rate of mutation along with the lack of selective pressure since these sequences are in non-essential or "junk" regions of DNA make them ideal candidates for identifying individuals. MSA is most suited for pedigree analysis, but can also be used in forensic science, to compare suspects to crime scene stains, to determine the paternity or maternity of individuals, to estimate population size for wild populations, to determine if populations have suffered genetic bottlenecks and for phylogenetic studies in applications involving very closely related subspecies. Like other molecular genetic techniques MSA requires only a small tissue sample for DNA extraction and the process is amenable to automation. MSA does however rely on knowledge of the primary DNA sequence of the target genome and requires a significant amount of DNA sequencing to identify potential satellites. This makes MSA fairly time consuming and expensive. MSA can be used for linkage analysis for certain traits, but there are other more suitable methods available.

Randomly amplified polymorphic DNA analysis (RAPD) examines the presence of DNA sequences within an animal directed by a specific DNA primer set with the polymerase chain reaction (PCR). This process requires no prior knowledge of the target genome. Once the appropriate primer set is chosen RAPD can be used to identify specific banding patterns. These patterns can be used like MSA, but have several advantages. First, since these sequences are not based on repeating nucleotides and are more stable. RAPD examines both coding and non-coding regions of the genome and can be used to assess genetic diversity, hybrid speciation, and be associated directly with specific traits. RAPD is inexpensive, fast and not technically demanding.

Expressed sequence tags (EST) are derived from messenger RNA (mRNA) of genes expressed in the target organism. EST’s can be used in a number of ways including determining the expression level of the mRNA under experimental conditions. Sequencing of EST’s can identify novel genes or can often give insight into what the mechanism of action is by suggesting a homology with a know protein. EST’s can be used to determine the level of expression of the gene within a particular tissue or organ in the animal, suggesting a further mechanism of action. As such, EST’s are a valuable research tool for studying the physiology, genetics, evolution and gene mapping of a species. Compared to MSA or RAPD however, EST’s are labor intensive, technically demanding and expensive.

How can we use these tools most effectively? The most fruitful course would be to begin with a selected population. We have developed a number of markers for sex determination using RAPD analysis and hemolymph from male and female shrimp. This population was, of course, pre-selected for the desired trait and marker development was quick. To develop other markers researchers must start with a highly specific goal. Once a specific goal is determined it is a fairly straightforward process to devise a method to apply the appropriate pressure to select the desired trait. Penaied species are amenable to the selection process for several reasons. First, shrimp show a very high fecundity; females often produce 500,000 or more eggs each time they spawn. Second, some of our research work and that of other groups indicate that shrimp mutate a relatively quick pace (2,4). The combination of large numbers of offspring and a high frequency of mutation lead to the development of desired traits or adaptation to the selective conditions relatively quickly. The selection process can rely on the natural mutation rate within the species or it can be "enhanced" using several standard techniques (chemical or biological) and even directed with transgenic methods.

As an example Super Shrimp Inc, has selected a population of P. stylirostris that can grow rapidly, independent of the population density. These animals were developed by grossly overcrowding larva from a single spawn and visually selecting animals that grew more quickly. 50,000 animals were housed in a 400-liter tank for 50 days. At the end of the selection process there were two distinct populations of shrimp in addition to the average size animals, very large animals (shooters), 200-400% larger than the bulk of the population, and small animals (runts) less than one quarter the size of the bulk of the population (figure 1). Shooters and runts were collected, weighed, tissue samples were removed and DNA extracted from the tissue. The animals were then housed together at normal density. The DNA from the two populations was compared to the DNA from the mother and father that produced the initial population. RAPD was used to identify distinct genetic markers that were associated with each trait (shooters and runts, figure 2). It was observed that some of these markers were not always shared with either parent. The fact that the parents did not have some of the makers identified in the growth enhanced animals supports two important concepts. First that growth can be linked to a genetic locus, independent of environmental factors. And second that the unenhanced mutation rate is such that it allows the appearance of multiple individuals with the same mutation in the population. The selected animals are being raised to maturity so that appropriate crosses can be produced. The markers identified that are associated with increased growth can be used to screen potential broodstock animals for this trait and positive animals selected for breeding. Similar selection process can de used to select for animals that are able to utilize specific alternate proteins in feeds or for disease resistance.

How do we approach gathering additional knowledge in the three key areas (nutrition, growth, and disease resistance) important to the improvement of shrimp stocks used in the United States? First we need to make access to current research plans and results readily available to researchers and farmers. This can prevent duplication of efforts, streamline design of work plans, and accelerate the pace of development and discoveries to help the shrimp industry. There are a number of groups in the United States and within the worldwide research community that are working towards similar goals. Bringing these groups together can accelerate the progress made each year. Second, we can emphasize the need for applied research over basic research. While many basic research questions are interesting to us as scientists, the needs of the U.S. shrimp farmer for direct application of our efforts must be paramount. Finally, since the resources available for this work is limited, more care needs to be expended on deciding where the resources are applied.

CONCLUSIONS AND RECOMMENDATIONS

Successful shrimp farming in the United States will rely on the development of more efficient methods to produce shrimp. Current molecular genetic tools are being used to address a number of critical issues including utilization of feed, enhanced growth and disease resistance. A vigorous effort to foster the coordination of ongoing research programs, stimulate new research and promote the exchange of data would advance current shrimp farming practices.

Short-term (1-3 years):