DURING THE PAST YEAR, the USMSFP focused research on three major objectives: Stock Improvement, Disease Control, and Sustainable Culture Technologies. Important milestones were achieved in all objectives and the program continues to adjust to changing needs and challenges of the domestic industry. In addition, the USMSFP assisted industry directly under some of these objectives by providing broodstock, postlarvae, education and training, disease diagnostics, and information dissemination. Following are highlight thrusts and achievements of the USMSFP conducted under each major activity.

The Stock Improvement objective this year continued to evolve the breeding program to improve the efficiency of selective breeding and performance evaluation efforts, include new approaches to genetic line development to address emerging industry issues, and research the genetic basis of disease (TSV) resistance. Pedigree tracking and analysis of genetic diversity supported this work, while genomics research emphasized continued development of the linkage map for L. vannamei. Quality control with inbreeding analysis was delayed due to a water main break at Tufts University which destroyed the majority of the genomics lab and equipment. Fortunately, samples were not affected and the work will continue once the lab and equipment have been restored. Work on cryopreservation of male gametes also continued this year as well as upgrades to the Nucleus Breeding Center at the Oceanic Institute to expand the capabilities of the program.

The selective breeding program reached an important milestone during FY05 in the development of the computerized pedigree database (Task 1.5.1). Sufficient information has now been incorporated that for the first time, the database was used to draw specific inferences about estimated breeding value (EBV) of Consortium lines to a specific trait of interest using the BLUP/REML system. Survival data from TSV-resistant line shrimp subjected to multiple TSV isolates (Task 1.2.4) generated EBVs for TSV-resistance from Oaxaca (0.14) and Chiapas (0.16) that were higher than those of other founder stocks (rng. 0.04 – 0.08). This indicates the Oaxaca and Chiapas founders contribute the better portion of TSV-resistance to USMSFP stocks. The information confirmed the steady progress of the selective breeding efforts of the Consortium for TSV-resistance since EBVs have also increased with time. The computerized pedigree also identified the effects of inbreeding on survival are more harsh, the harsher the environment. For each 10% increase in inbreeding coefficient (F), there was found only a 3.2% reduction in survival in growout, while survival decreases 18 and 36 percent, respectively, in animals exposed to TSV-A and the more virulent, TSV-C. The database is nearing completion and currently has 130,000 individual records from Batches 8 through 22 incorporated.

The breeding program also continued to develop and evaluate its TSV-Resistant (Task 1.2.2.1), Growth (Task 1.2.2.2), Inbred (Task 1.2.2.3), and Sentinel (Task 2.2.6) lines. Evidence is building that selection for TSV resistance does not adversely affect growth, as was previously thought. Very little difference was found between growth of shrimp from Batch 19 (TSV-resistant line) and Batch 20 (Growth line) shrimp during FY05. This may indicate that lines can be bred for both TSV resistance and growth. Plans for development of eight hybrid families using four different TSV-resistant families and two different growth line families are moving forward (Task 1.2.2.5). This initial screening experiment should provide evidence of the value for hybridization of the two traits. This possibility will be further evaluated using the computerized database.

Plans also moved forward on utilization of USMSFP microsatellite markers to conduct walk-back selection of Consortium lines to improve selection intensity (Task 1.2.2.1). Blind samples of 50 shrimp of known pedigree were sent to Tufts for testing and identification of parentage. If identification is 100% accurate, the program will begin transition from the use of physical tags to genetic tags for family identification in its selection efforts. Although pedigree tracking and allele association analyses were slowed due to damage of Tufts facilities, work was completed for the 50 families of Batch 19 shrimp received in July 2004 and challenged with the three TSV serotypes (Task 1.2.2.1).

Research on the genetic basis of TSV resistance also reached significant milestones during FY05. The program established a bioinformatics database and search capability to identify genes associated with traits of economic importance (Task 1.2.3.1). Approximately 5,000 ESTs were entered into the Tufts’ database (ShrimpESTbase). A first-generation, microsatellite-based, low-density linkage map (ShrimpMap) was also produced that can be used for mapping QTLs of economic importance, and construction on a second-generation map based on ESTs and SNPs was begun (Task 1.2.3.2).

DNA from the shrimp mapping families is available to any researcher interested in contributing markers to the development of a high density linkage map. A total 100 microsatellite primer sets are being tested for polymorphism with the mapping panels. Primers that did not show polymorphism with the USMSFP linkage mapping family may be applicable to other domestic breeding programs with different gene pools, and are currently available for industry use.

A genome-wide scanning was also initiated to identify candidate QTLs associated with resistance to TSV with 14 markers geneotyped thus far. The program is currently at a critical crossroad in linkage mapping development to determine if additional families are needed to supplement the existing LMRF and if RMFs for QTL analysis are appropriate candidates. Addition of new families with higher levels of polymorphism may help speed up linkage map development.
Work on cyropreservation of gametes re-initiated last year continued during FY05 by profiling sperm morphology and egg water-sperm reaction studies by stabilizing testing conditions (Task 1.1.2). Apparent differences were found in morphology of chromation fibers. Research is currently ongoing to determine if those changes were artifacts of the cyropreservation process. Changes in the thelycum after mating are also being documented, which may prove important in obtaining high fertilization after cyropreservation.

Disease Control

Disease research continued to take a proactive approach to protect the industry by improving the precision of diagnostic methods and tools, and gaining insight into the mechanisms and transmission of disease.

Particular attention this year was again focused on TSV and NHP due to continued industry concern, and infectious myonecrosis virus (IMNV), an emerging and significant disease of L. vannamei that has not been identified in the US, but has been added to the USMSFP working list of specific (C-1,2) pathogens. The USMSFP has recommended IMNV be included on the OIE list of notifiable pathogens.

For the past four years, the USMSFP has continued to develop its TSV- resistant line of animals for the domestic broodstock industry and evaluated the effectiveness of technology transfer. During FY05, TSV challenge studies of commercially available families (Task 1.2.4.1) indicated USMSFP stocks have had a significant impact on development of US broodstock companies, which continue to rely on Consortium stocks for their base. It is now common to find commercial companies producing families that exhibit up to 100% resistance to TSV (A serotype). US broodstock companies have made tremendous inroads in selling product both in the US and overseas. It can be estimated that L. vannmaei now accounts for 40% of all shrimp produced in Thailand, Vietnam, Indonesia, and India, up from less than 10% in 2002.
While work continues on TSV line development for industry, research has evolved to utilize Consortium lines as a tool to understand the genetic and physiological basis of disease resistance in shrimp.

Results on the mechanisms of TSV resistance (Task 2.5.1.4) confirmed suppositions made last year that TSV pathology and resistance is related to viral loads. TSV-resistant animals apparently do not prevent a build-up of levels of the virus in their bodies; rather they build up levels much more slowly than non-resistant animals. The same level of virus was found to kill both resistant and non-resistant animals, and all animals have some viral load when exposed. Work last year allowed the Consortium to transition to TSV load instead of survival as the key metric for evaluating response and examining the genetic basis of disease resistance. Other research indicated an additive effect of the pesticide, endosulfan, on mortality of TSV-infected shrimp (Task 2.5.1.5). Mortality increased in both TSV-infected and non-infected shrimp, but was independent of molting stage.

Work also continued this year on the multi-institutional collaborative effort of the USMSFP in Texas to discern the causes and origins of a TSV outbreak that occurred in early June 2004. The viral strain (04 USTX) is closely linked phylogenetically to the Southeast Asian group of TSV isolates, but there have previously been no connections to its origins. A particularly significant finding this year was identification of a linkage between the Asian group with isolate 98-HO (Task 2.3.1.1.1). This indicated that TSV was brought to Asia from Honduras. As of this writing, samples taken from south Texas during 2005 did not contain TSV (Task 2.2.4), indicating no genetic persistence of the disease. Sampling will continue on specific sites, with additional funding secured through APHIS.

Significant progress was also made on the epidemiology and transfer mechanisms of NHP. Research confirmed that multiple strains among geographic isolates of NHP exist based on sequences from the gryB region of the genome (Task 2.3.1.2.1), and that NHP can be transferred through culture water using the USMSFP chronic infection assay method (Task 2.4.3.2.2). There appears a flagellated form of the bacterium, indicating a mode of transmission independent of a specific host or vector. However, it was also found that the infection is not transferred by co-habitation with infected live shrimp (Task 2.4.3.2.3). Cannibalism of dead shrimp caused infection and transmission was lowest at the lowest (10 ppt) and highest (40 ppt) salinities tested. Survival was independent of salinity.

The USMSFP continued efforts on two diseases that are not present in the US, but which have potential to spread rapidly if diagnostic and prevention methods are not in place. Rapid progress has been made on IMNV, which was first characterized last year by the USMSFP. Additional detection methods for IMNV were developed during FY05 (Task 2.3.1.1.6), one of which — a highly specific nested PCR — was transferred to IntelliGene Corp. for commercial application. Challenge studies against IMNV were also initiated with 42 families from Sygen International (Task 1.2.4.1). Survival of these families ranged from 20 to 100 percent, indicating the potential for development of stocks resistant to this rapidly emerging and devastating disease. Challenge models developed last year for sprioplama penaeii also were used to determine that L. vannamei were much more susceptible than L. stylirostris to this bacterium (Task 2.4.3.1).

The need to remain vigilant has also been highlighted by the number of new diseases and new strains of existing ones that appear to be emerging. During FY05, outbreaks of WSSV were identified in Guam, Brazil, Mexico, and Saudi Arabia. Some TSV variants arose in Venezuela and Indonesia, and TSV-C was detected in Nicaragua (Task 2.2.2). NHP was also identified in Jamaica. New diseases found included haplosporidian and intranuclear mirosporidian in L. vannamei in Central America, a new enteric parvovirus in Fennenopenaeus indicus and Penaeus monodon in East Africa, and a new HPV strain in P. monodon from Southeast Asia and East Africa.

Research on sustainable culture technologies continued to focus on the development of superintensive production systems and methods to improve system operations and cost-effectiveness.

Researchers continued to use the USMSFP economic model to better refine their estimates and to devise innovative ways to use the model to guide research. This effort was emphasized this year with a special workshop on the bioeconomic model and system operations in conjunction with Aquaculture America 2005 in New Orleans, January 2005. Work also continued on diet development and the physiological effects of low-salinity culture and methods to improve production in earthen ponds.

For the past two years, the Consortium has diligently used the bioeconomic model to guide research and development efforts of its superintensive production system. During FY05, one of the most promising advancements was achieved in identifying the potential for use of partial harvesting (Task 3.1.1.1.3). Modeling indicated the net return on existing systems could be increased significantly with partial harvest, but the optimum cropping rates and schedules are sensitive to growth rate of shrimp between 20 to 30 g and biomass loading. If too few shrimp are harvested, the remaining animals do not reach the final size desired since the targeted carrying capacity is reached first; harvest too many and there is not enough shrimp left to reach the carrying capacity.

Importantly, cash flow, a critical factor to commercial firms, is enhanced by partial harvesting. The model indicated research is needed to confirm growth rates from 20 to 30 g, and to larger sizes (circa. 40 g), and to examine the effect of different densities, particularly on growth. Use of the partial harvest concept to improve competitiveness also emphasizes the importance for selective breeding efforts to target faster growth rates in shrimp.

Research also continued to improve operations of the superintensive raceway systems. Managing wastes and controlling solids and particles is essential, particularly to manage microbial communities. All Consortium systems are converging toward simplicity to become more efficient. Changes to the prototype (282 m2) system at WMC enhanced efficiency of oxygen delivery, minimized flow impairment, measure¬ment of electricity usage, use of the propeller-washed bead filter for solids management with incorporation of an external filtration system that allow for maximum water recovery, and concentration of sludge (Task 3.1.1.1.1).

Filters are now being used for particle removal to manage microbial communities; turbidity is kept near 100 NTU. Survival and FCR of shrimp stocked this year at 4 g (83.5%, 1.36, respectively) was better than of shrimp stocked at 1 g (54.3%, 2.59) last year, but final biomass was not improved (6.35 kg/m3 in 2004 vs. 6.66 kg/m3 in 2004). In a series of small tank trials using water from the prototype raceway (Task 3.1.1.2.1), CO2 was found to reduce growth and FCR, but not survival as pH falls from 7.0 to 5.1. The response was due to an increase in CO2 and not the fall in pH per se.

Continued collaboration between WMC and NSU (Task 3.1.1.1.4) found no need to add heat to reduce sludge volumes, and that microbes adapt to salinity changes from 5 to 40 ppt. Aeration was more efficient than mixing to desalinate the sludge and a majority of the salt can be reclaimed for water reuse. Overall sludge volume reduction is 19.5 percent, and the waste produced by shrimp does not contain human pathogens.
Commissioning of the large-scale, 12-unit prototype growout facility at GCRL was completed with tests of three levels of light transmittance and two levels of solids waste removal (Task 3.1.1.1.2). While much variability still remained from tank to tank, there was indication that lower levels of light transmission and higher levels of solids removal were beneficial. Importantly, some raceways reached harvest densities up to 3.5 kg/m3, near the 4.0 kg/m3 targeted last year for commissioning.

High variability among survival and growth results were due to fluctuating and high temperature levels (>31°C) in raceways, but the causative factors have largely been resolved and operations of the facility are appropriate for more extensive, replicated experimental trials. As of this writing, the effects of Hurricane Katrina are still being assessed at the Cedar Point facility. Raceway covers were destroyed as well as shrimp in the majority of units in an ongoing trial. It is anticipated, however, the facility will be fully functional within the next year.

The semi-purified research diet is now ready for use by the USMSFP. Trials this year confirmed the effectiveness of the diet at a stocking density of 50 shrimp/m2 (Task 3.1.3.1). Research also quantified the digestible protein and energy requirements for growth and maintenance of shrimp based on daily intake which can be used to develop specific feed management strategies based on diet composition.

Pond trials (Task 3.1.5.2) demonstrated shrimp fed commercially-available low protein feeds (30 and 31% crude protein) grow as well as shrimp fed high protein feeds (35 and 41% crude protein) if fed on an isonitrogenous basis, although FCR is higher on the low protein feeds. Funds to conduct research on diet attractability (Task 3.1.3.3) were reprogrammed to assist analysis of the TSV outbreak on south Texas farms.

The USMSFP commitment to information dissemination to the domestic shrimp industry has been reflected in its workshops, training and coursework as well as the program’s website, brochures and newsletter, Industry Briefs.

During FY05, quarterly issues of Industry Briefs were published, making this the second year in a row of consistent quarterly output. The format and scope continued to target both the shrimp farming industry and research partners, with more timely and in-depth analysis of industry issues and challenges of individual farmers. The general theme this year was identifying opportunities for industry in the evolving world market, with topics ranging from bait shrimp production (Vol. 10, No.4), organic shrimp farming (Vol. 11, No. 1), domestic broodstock production (Vol. 11, No. 2), a special issue on industry perspectives of the future of shrimp farming in the US (Vol. 11, No. 3), and an issue dedicated to a farmers' survey about the potential use of OTC (Vol. 11, No. 4). Readership is currently more than 300 and restricted to only US researchers, farms, and industry professionals.

Work also continued to expand the USMSFP website, www.usmsfp.org, to enhance its value to the industry and researchers. Additional linkages were provided to shrimp farming classes and other sites with relevant shrimp farming information. Also included this year was a listing of all broodstock and post-larvae sold by the Consortium to domestic breeders and farmers. This was an attempt to assist the domestic broodstock industry in marketing their products by providing a key reference to origins of their stocks.

The USMSFP has earned a top ranking based on website traffic on major search engines. The website continues to provide a high level of statistical and qualitative feedback on industry trends and concerns with its “headline news” feature. The site consistently receives inquiries from established and prospective shrimp farmers in addition to responses from online surveys. Both the newsletter and website continue to actively reach out to farmers to seek their input and involvement to enhance communication between the industry and the Consortium.

In addition to its publications and website, information is distributed by the USMSFP through conference meetings and farm site visits. In January 2005, the USMSFP conducted a Special Session at the Aquaculture America 2005 conference in New Orleans, Louisiana, to update the domestic industry on progress. It also included a question-and-answer period. For the past three years, the director has been an invited speaker at the TAA conference at which updates on USMSFP activities are presented. The director also presented, “Opportunities for SPF L. vannamei in an evolving world market” at the Hawaii Aquaculture Association meeting in Hawaii, the state where the majority of domestic broodstock suppliers are located. The presentation included projections on the growing demand for L. vannamei stocks worldwide, estimate of broodstock needs, and potential market for domestic suppliers. Farm site and Consortium member institutional visits were conducted in Texas, Arizona, and Massachusetts.

Of special note was the USMSFP’s participation at the JSA National Aquatic Animal Health Task Force meeting in Arizona. Topics included a review of crustacean disease list, value and challenges of aquaculture zones for crustaceans, necessary sampling protocols for surveillance, and disease prevention measures. Biosecurity protocols of Consortium institutions were forwarded to the task force to help develop national guidelines on disease management. The task force was also provided USMSFP protocols for testing SPF shrimp.