The highlights and accomplishments of this report are a continuation of activities developed for the FY06 funding period (June 1, 2006-May 31, 2007) which were designed to extend into the next funding cycle of June 1, 2007-May 31, 2008 (FY07). Unfortunately, the congressional earmark for continued funding was not obtained and the project needed to carry on using remaining FY06 funds. To ensure continuation of the work, a first-time, no-cost extension under the “General Terms and Conditions – A (Administrative Waivers)” was also enacted, extending the FY06 grant through May 31, 2008. This report covers overlapping activities from September 16, 2006, through September 15, 2007. Research focused on three major objectives: Stock Improvement, Disease Control, and Sustainable Culture Technologies.

Despite the loss of FY06 funding, considerable progress was made during the reporting period in moving forward the key objectives of the Consortium. Essential was the continuation of the selective breeding program to maintain the years of investment in genetic resources for research and industry. OI was able to consolidate its Kona line stocks to its Makapuu facility to reduce costs (Task 1.1.1) and used sales from excess broodstock and postlarvae to maintain the program at a minimum level. The last batch of animals sent to Kona was in September 2006. The location of back-up stocks at the OI Makapuu site represents a risk to the breeding program if a catastrophe strikes, since the Nucleus Breeding Center is also at the Makapuu site. OI is currently seeking alternative sites if (FY08) funding is restored.
During the reporting period, OI was able to complete a commercial-scale growout of Growth Line animals in a 337-m2 round pond recirculation unit (Task 1.2.4.1) that yielded the highest density (10.3 kg/m2) ever achieved from the highest stocking density (828 shrimp/m2). Growth was 1.5 g, and only 325 L of water was required to produce 1 kg of shrimp. Significant was that production costs were $1.66/lb, which is a dramatic reduction in cost compared to previous Consortium runs. This one run indicates the potential for a competitive U.S. shrimp farming industry. Importantly, too, was the fact that biofiltration was accomplished by in situ microbes rather than within an external biofilter. This approach resulted in a significant reduction in capital and operating costs. A foam fractionator was used to remove fine particles and dissolved organic matter from raceways stocked in parallel with Growth Line animals (401shrimp/m2) in the OI NBC. Stable ammonia and nitrite concentrations achieved throughout the trial, including the initial acclimation period, were from improved management strategies. In addition, there was no phenotypic correlation between stocking weight and final weight or stocking weight and weight gain (r = 0.18 and r = –0.04, respectively), and no correlation between mean family weight gain and survival (r = -.04; b = –0.12). This suggests that selection for growth will not negatively affect survival.

• Work on development of the TSV Line of shrimp continued to demonstrate the power of the computerized breeding program to maximize response while minimizing inbreeding depression. In April 2007, 49 families from the TSV Line were produced (Batch 28) that included animals from the ATP-A4 line (Task 1.2.1.2). The ATP line was from a separate project designed to develop lines bred specifically to accommodate high density culture. Despite assortative mating strategies designed to increase selection intensity for TSV survival, but which could have increased inbreeding, the mean inbreeding level for Batch 28 was <7.4%. This is below the target level of the program (10%) and approximately the same as for Batch 25 (7.3%) produced a year earlier and using rotational or minimal kinship mating strategy, which is designed to minimize inbreeding, but not necessarily to maximize the response. The ability of the Consortium to conduct assortative mating strategies with minimal effect on inbreeding should allow for more rapid progress with all lines.

To add to the genetic diversity of Consortium stocks, OI also recruited a population of candidate SPF shrimp from the Instituto Technologicl y de Estudios Superiores de Monterrey (ITESM) Campus in Guaymas, Sonora, Mexico (Task 1.3.2). These shrimp originated from wild stocks collected from Venezuela and El Salvador and were outbred with domesticated stocks from Columbia (as well as other broodstock of unknown origin).
TUFTS researchers continued to refine the market kit, Shrimp24TM to help differentiate between Consortium stocks and wild shrimp stocks (Task 1.3.1). Based on a blind sample of 50 animals, previous data indicated that a minimum of 12 markers were needed for 100% identification. All 20 markers tested over this reporting period in the new kit amplified in all parental broodstock of TSV-R (Batch 22) and Growth (Batch 23) lines as expected. However, preliminary data indicated that some markers did not amplify in some samples of wild shrimp from Ecuador, Peru, and Mexico. The lack of PCR amplification could be due to null alleles (mutations in the primer sequences), but technician error, equipment failure, quality of reagents, etc., cannot be ruled out. Efforts will move toward using these marker panels to conduct walk-back selection of our lines as confidence is gained with the method and costs and timeliness improved.
Progress was also made toward the construction of the linkage map for L. vannamei (Task 1.2.3). The sex locus was located on linkage group #4 of ShrimpMap and researchers evaluated some ESTs to develop polymorphic EST-SSR markers for linkage mapping. Five ESTs have already been located onto ShrimpMap, the first ESTs mapped for L. vannmei. A less expensive protocol for preliminary test and development of polymorphic markers was also developed. This protocol is useful for initial development of markers, but is not reliable when the allele size difference is very small (<20 base pairs).

Disease research at UAZ was also hampered by the lack of funds and the need to release technical staff, and at GCRL because of loss of space and facilities due to Hurricane Katrina. During the report period, however, challenge studies were conducted at UAZ in 176 family lines of L. vannamei from OI, Kona Bay Marine Resources, Agromarina de la Costa, and Maricultura del Pacifico. These lines were challenged with the Hawaii (HI), Belize (BZ), Thailand (TH), and Venezuela (VE) isolates of TSV (Task 1.2.4.2). Survival in the TSV-challenged L. vannamei families, regardless of TSV isolate, ranged from 0% to 100%. The percentage of family lines with resistance to the Belize isolate of TSV was low, but some family lines were identified that showed 80-100% resistance to this isolate. In addition, WSSV challenge studies were performed on one family of Fenneropenaeus indicus for a Saudi Arabian company. No survivors were collected from either the study or Kona Line controls. The search for lines of animals resistant to WSSV remains elusive.
Much effort over this reporting period concentrated on procedure development for viral identification. A new monoclonal antibody was produced at UAZ that recognizes the Belize isolate of TSV (TSV-BZ-01) by immunohistochemisty and immuno-dot blot (Task 2.3.1.1.2). In addition to TSV-BZ-01, the new TSV monoclonal antibody, designated 2C4, recognizes the USA-Hawaii (1994) and Sinaloa Mexico (1998) isolates of TSV, but it does not react with the Venezuela (2005) isolate of the virus. New primers were also developed that distinguish between the infectious form of IHHNV, which produces viable particles and disease upon transfer to susceptible penaeid shrimp hosts, and an IHHNV-related sequence that occurs in certain strains of Penaeus mondon from Africa and Australia, but does not produce viable virus particles or disease when transferred to IHHNV-susceptible hosts (Task 2.3.1.1.3). The multiplex PCR assay using the two primer sets (309F/R and MG 831F/R) to distinguish between the two forms of IHHNV was validated and shown to be as sensitive as the standard diagnostic PCR primer set.
The genome sequence of the Korean isolate of HPV was extended by almost 2,400 bases at UAZ APL, which allowed for the identification of the major open reading frames (ORFs) of the Korean isolate and to compare them to other geographic isolates of HPV that are published in GenBank (Task 2.3.1.1.4). The highest degree of identity among various isolates was seen with the middle ORF that may encode the non-structural protein 1 with 91% identity to both the Australia and Thailand isolates of HPV. The 3¢ ORF encoding the capsid protein showed the highest degree of diversity, with 75% identity to the Thailand isolate, 78% identity to the Australia isolate, and 87% identity to the HPV isolate from Madagascar. This high degree of diversity in the genome of HPV is unusual for parvoviruses that generally show 4-5% diversity among different isolates. Two other isolates of HPV, from Madagascar and Tanzania, are being sequenced by UAZ. These two isolates also show a high degree of genetic diversity in comparison with other sequenced isolates. This work is crucial in order to design molecular tests that will identify all isolates of the virus simply, unambiguously, and inexpensively.
The disease that caused mynecrosis of L. vannamei from Brazil (IMNV) and the etiologic agent was also identified (Task 2.3.1.1.5). Several methods were developed to identify the agent in clinical specimens, including in situ hybridization, nested RT-PCR, and real-time RT-PCR. The commercial kit for RT-PCR was developed by a Taiwan company and is commercially available.
Research on diseases of bacterial origin also continued in earnest. Persistent, low-grade mortality that occurred at a shrimp farm in Texas in late 2006 was determined to be caused by Vibrio parahemolyticus, which demonstrates resistance to oxytetracycline (OTC) (Task 2.3.3.1). The farm in question had permission to treat for NHP with OTC under an INAD and has applied the drug at the site for several years. The implication of these findings suggests that use of a single antibacterial compound for NHP control can lead to selection for OTC resistance, and potentially pathogenic bacteria in ponds receiving OTC medicated feed. An additional note is that UAZ suspended indefinitely serving as active INAD Sponsor for OTC use in shrimp, effective June 1, 2007. Efforts by the Consortium director and Dr. Lightner to convince Philbro Animal Health to transfer sponsorship were unsuccessful. It was felt the leverage of a commercial company to push forward approval would be the best route after the Target Animal Safety study was rejected by the FDA and resolution on design could not be made. Philbro indicated that despite the exclusive use of their product in Texas for 10-15 years, OTC sale in the U.S. did not justify costs associated with approval. With reduced funds, the Consortium discontinued its contract with Mr. Ron Williams, the INAD monitor from UAZ and transferred the Sponsorship to Mr. Kieth Gregg of Harlingen Shrimp Farms in May 2007.
NHP was also demonstrated to cause disease in SPF P. monodon (Task 2.5.2.1). Significant was the finding that NHP is transmissible through the water without the need for indicator shrimp to ingest infected carcasses (Task 2.4.3.2.1). The ability to transmit NHP through a water exchange is being further investigated to develop a challenge model that can be used to assess family line susceptibility/resistance to the disease. As production systems become more intensive, the potential for bacterial related diseases becomes more apparent and there may be need to develop lines resistant to key bacterium such as NHP or pathogenic vibrios.
Research on immune enhancers (Task 2.6.2.1) and pre- and probiotics (Task 2.6.2.2) continues to indicate there is no scientific evidence these products work, despite the marketing and continued use in industry.

Updated March 2, 2008