IMPROVING LOUISIANA'S AQUACULTURE PRODUCTION AND FOOD INDUSTRIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SPECIAL GRANT
• Reporting Frequency: Annual
• Accession No.: 0218948
• Grant No.: 2009-34310-19921
• Proposal No.: 2009-04187
• Project Start Date: Sep 1, 2009
• Project End Date: Feb 14, 2012
• Grant Year: 2009
• Program Code: [CF]- Aquaculture, LA

Project Director
Morrison, D. G.

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
Administration

Non Technical Summary
Louisiana supports one of the most diverse aquaculture industries in the nation. Numerous challenges require development of new technologies to ensure that the state's aquaculture industries remain viable and profitable. Crawfish production, the leading aquacultural enterprise, relies on baited, wire-mesh traps to harvest the crop and harvesting comprises 50-60% of the annual production costs. Numerous issues related to bait-crawfish-trap interactions need to be further researched for the development of more cost-effective trapping strategies. In addition, seasonality of crawfish harvest, which occurs primarily in winter-spring, is a major constraint to expanding the market. Availability of crawfish for peeling and specialty markets is needed in summer and fall. Therefore, determination of the potential of alternative species, such as the shrimp crawfish (O. lancifer), to commercial culture conditions is needed. Louisiana is the largest producer of farm-raised alligators in the nation and little research on the nutritional needs of alligators has been conducted. Baseline research on how alligators process common feedstuffs will begin an initiative to develop nutritionally sound diets at low cost. The development of an aquaculture industry centered on the culture of marine baitfish, such as the Gulf Killifish (F. grandis), represents a potential new economic market within Louisiana. Therefore, the application of air incubation as a hatchery and larval rearing technique for the Gulf Killifish will be investigated with an ultimate goal of providing a consistent source of this species to Louisiana anglers. Vibrio vulnificus is a naturally occurring estuarine pathogen frequently transmitted via raw oysters and it is important to reduce the prevalence rate of its infection to humans. Chitosan is a non-toxic biopolymer with antimicrobial and gel-forming properties produced from crawfish waste, which may have the ability to maintain the sensory quality of raw oysters while enhancing their safety and shelf-life. In addition, the need to determine the most cost effective freezing method for aquacultural food products, a major expense in the operation of a processing plant and a growing segment of the market, is greatly needed. So, cryogenic freezing methods will be compared to blast-freezing in relation to yield, quality, and shelf-life of various aquacultural food products. Finally, Photobacterium damsela subspecies piscicida is a serious and important disease of marine fish worldwide and has greatly affected several hybrid striped bass farms on the Gulf Coast. Using molecular biology procedures, important virulence factors of this disease will be determined to provide insight for planning future research concerning pathogensis, control, and prevention.

Animal Health Component

90%

Research Effort Categories

Basic

10%

Applied

90%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3799	1010	10%
304	3713	1040	10%
307	3729	1060	10%
307	3715	1060	20%
307	3720	1060	30%
503	3799	1000	10%
712	3723	1100	10%

Knowledge Area
302 - Nutrient Utilization in Animals; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 304 - Animal Genome; 307 - Animal Management Systems; 503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
3723 - Oysters; 3713 - Striped bass; 3729 - Other cultured shellfish; 3720 - Crawfish; 3799 - Cultured aquatic animals, general/other; 3715 - Baitfish;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1010 - Nutrition and metabolism; 1100 - Bacteriology; 1000 - Biochemistry and biophysics;

Keywords

crawfish, catch efficiency, traps, bait, shrimp crawfish, stocking

density, alligator, diet, feedstuffs, digestibility, baitfish, air

incubation, temperature, salinity, hybrid striped bass, p. damsela,

gap closure, sequencing, oysters, ozonation, chitosan, safety,

shelf-life, food products, cryogenic freezing, blast freezing

Goals / Objectives
1) To quantify catch efficiency and retentive ability of commercial crawfish traps and improve bait effectiveness on catchability of crawfish in commercial ponds, 2) To determine the adaptability of the shrimp crawfish (O. lancifer) to commercial culture conditions, 3) To determine the digestibility of some common aquatic animal feedstuffs in young of year American alligators, 4) To investigate the application of air incubation as a hatchery and larval rearing technique for the marine baitfish, Gulf Killifish (F. grandis), 5) To close any gaps in the sequence of Photobacterium damselae subsp. piscicida remaining after automated sequencing and to annotate the genome. 6) To explore the feasibility of using chitosan solution from crawfish waste and ozonation to maintain the sensory quality of raw oysters while enhancing their safety and shelf-life. 7) To determine whether cryogenic freezing methods are superior to blast-freezing in relation to yield, quality, and shelf-life of various aquacultural food products.

Project Methods
This proposal contains seven component projects, each with the goal of enhancing Louisiana's aquaculture industry. 1) Experiments in monoculture and rice/crawfish rotation ponds with crawfish marked with a permanent, water insoluble marker will determine the capture efficiency of different commercial crawfish traps, quantify the catch efficiency of traps in various crawfish populations, increase the efficacy of baited traps with decreased bait use, identify alternative baits that may be effective attractants in cold/cool waters and evaluate potential bait-free trapping alternatives. 2) Juvenile shrimp crawfish will be stocked into pools with soil and aeration at densities that will allow comparison of previous research with the red swamp crawfish. Experiments will be conducted on stocking density, vegetation and planting date, and benefits, if any, from supplemental feeding. 3) Twenty young-of-year American alligator will be obtained from a single clutch of eggs and stocked in tanks (one per tank) in a wet laboratory. Each feed ingredient, mixed with chromic oxide (indicator) and force-fed in slurry form, will be assigned to four tanks using a completely randomized design. Diets and fecal samples will be subjected to proximate analysis to determine levels of crude protein, lipid, fiber, ash and carbohydrate. 4) To identify the optimum temperatures and salinity for incubation protocols, batches of newly fertilized Gulf Killifish eggs (about 500) will be incubated in water and air at temperatures of 19, 22, 25, 28, and 31 degrees C with optimum substrate. Trials at varying salinities (0, 5, 10, 15 ppt) will determine the role of salinity on developmental rate and percent hatch. These experiments will demonstrate the relationship between survival and the degree of development and egg-yolk utilized during the delayed hatch phase of air incubated eggs. 5) An available automated sequence of the P. damselae genome will be used and a large insert (10-20 kb) DNA library constructed in the linear cloning vector pJAZZ- KA for manual paired end sequencing. This will allow for gap closure and ordering of contigs. Final closure will rely on PCR and primer walking. Annotation will utilize services at the J. Craig Ventor Institute, Rockville, MD. 6) Freshly harvested live oysters will be shucked and either (1) soaked in ozonated water and then chitosan solution or (2) soaked in chitosan solution only. They will then be placed in containers and stored at 4 degrees C for up to 21 days. Samples will be drawn every 3 days for V. parahaemolyticus and V. vulnificus counts (CFU/g) and subjected to evaluation of sensory quality. 7) A cabinet type cryogenic freezer capable of utilizing both liquid carbon dioxide and liquid nitrogen will be used for cryogenic-freezing, while a blast freezer will be used for mechanical freezing. Aquaculture food products will be separately placed in the cryogenic freezer or blast freezer and frozen to an internal temperature of -20 degrees C. Initial weight, product temperature, moisture, protein, lipids, and ash will be analyzed. Freezing-rate, moisture-loss, and weight-loss of the seafood for each freezing method will be determined.

Progress 09/01/09 to 02/14/12

Outputs
OUTPUTS: 1. Many materials on marine baitfish spawning and culture have been created and posted on the LSU AgCenter's aquaculture website. 2. Recent findings and recommendations for crawfish production were presented at 6 Louisiana producer gatherings. 3. The superior effectiveness of chitosan against Vibrio spp. demonstrated in this study may prompt future applications of chitosan to control Vibrio spp. in foods, particularly raw oysters. This application will help reduce the number of illnesses and deaths because of Vibrio spp. risks associated with raw oysters. 4. Optimum production practices for this shrimp crawfish and integration into traditional crawfish production was presented to aquaculture scientists. 5. This project generated 3 referred journal articles, four abstracts and LA Agricultural submission. PARTICIPANTS: D. Morrison (PI), C. Lutz, C. Green, R. Reigh, R. Romaire, R. Thune, S. Sathivel, R. McClain, and W. Prinyawiwatkul, LSU AgCenter TARGET AUDIENCES: The aquaculture scientific community and producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Reproduction studies with Gulf killifish have resulted in valuable data on spawning cycles with respect to egg production over the spring, summer and fall months. Outdoor research continued with a series of egg production experiments in large pools. Investigate the effect of different sex ratios (2:1 vs. 4:1 female-to-male), as well as a labor component that examined the effectiveness of only collecting spawning mats during predicted peak spawning periods (based on semi-lunar spawning data from previous experiments) showed no significant differences in eggs collected between the sex ratios. 2. Mark/recapture trials indicated that, on average, less than 50% of crawfish released back to the pond for further growth were subsequently recaptured. The resulting conclusion is that this practice is probably not economically feasible, thus, new recommendations were generated regarding intentional release of small crawfish for further growth. A gelatin-based matrix was also developed that allows effective testing of potential crawfish attractants, which lead to a Southern Regional Aquaculture grant for evaluating alternative attractants for crawfish in cool water. 3. Three methods (agar dilution, broth microdilution, and disk diffusion) were used and compared to determine antibacterial effects of five chitosan compounds against 36 foodborne pathogenic strains. One water-soluble chitosan with molecular weight of 43 kDa was found to be the most effective one and Vibrio spp. strains were most susceptible to chitosan compared to other bacterial genera. The two quantitative methods (broth microdilution, and disk diffusion) had a poor agreement (14.6%) in determining the minimal inhibitory concentrations of chitosan. 4. Grow out trials indicated a range of potential yields for off-season production of shrimp crawfish. In replicated trials, depending on stocking date and forage crop combination, average yields ranged from 136 to 552 kg/ha. Corresponding gross profits ranged, from -$291 to +$694 per ha respectively. On-going research is focusing on further evaluation of optimum production practices for this species and integration into traditional crawfish production crop rotations.

Publications

• Brown, C.A., Galvez, F., and Green, C.C. 2010. Larval developmental timing and metabolic costs from incubation of Gulf killifish at varying environmental salinities. Annual Meeting of the World Aquaculture Society, March 2010, San Diego, California.
• Coulon, M., Gothreaux, C., and Green, C. 2010. Substrate preference, survival, and developmental ontogeny of Gulf killifish eggs incubated in air. Annual Meeting of the World Aquaculture Society, March 2010, San Diego, California.
• Ramalho, R.O. and W.R. McClain. 2011. Capture rate and crayfish movements among experimental crayfish production ponds. Freshwater Crayfish 18(1):7-11.
• McClain, W. R. 2011. Assessing the efficacy of releasing crawfish back to the pond for further growth. Louisiana Agriculture, 54(3):14-15.
• Jiang, L, W. Prinyawiwatkul, H. K. No, and B. Ge. 2011. Comparison of agar dilution and broth microdilution for antimicrobial susceptibility testing of chitosan. Abstr. (198-46) Institute of Food Technologists 2011 Annual Meeting and Food Expo, New Orleans, LA, June 11-14, 2011.
• Green, C. C., Gothreaux, C. T., and Lutz, C. G. 2010. Reproductive output of Gulf killifish Fundulus grandis at different stocking densities in static outdoor tanks. North American Journal of Aquaculture, 72:321-331.
• Brown, C.A., Galvez, F., and Green, C.C. 2010. Larval developmental timing and metabolic costs from incubation of Gulf killifish at varying environmental salinities. Annual meeting of the Louisiana American Fisheries Society Chapter, February 2010, Baton Rouge, Louisiana.
• Lutz, C.G. and Bischoff, J. 2011. Preliminary results from growout trials for the shrimp crawfish, in Louisiana. Aquaculture America 2011 Book of Abstracts, p. 292. World Aquaculture Society, Baton Rouge.
• Green, C.C., Gautreaux, K., Perez Perez, R.A. and Lutz, C.G. 2011. Comparative physiological responses to increasing ambient salinity levels in Procambarus clarkii and Orconectes lancifer. Aquaculture America 2011 Book of Abstracts, p. 193. World Aquaculture Society, Baton Rouge.
• Green, C.C., Gautreaux, K., Perez Perez, R.A. and Lutz, C.G. 2011. Comparative physiological responses to increasing ambient salinity levels in Procambarus (Scapulicambarus) clarkii( Girard 1852) and Orconectes (Tragulicambarus) lancifer (Hagen 1870). Freshwater Crayfish 18(1):87-92.

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: 1. Preliminary 454 sequencing and assembly of the Photobacterium damselae genome resulted in the generation of 624 contigs totaling 3,817,112 bp with an average contig size of 13,369 bp. Funding also enabled sequencing and assembly of the emerging tilapia pathogen, Francisella naotunensis, resulting in only 16 contigs totaling 1,847,126 bp with an average size of 236,944 bp. The P. damselae and F. noatunensis genome coverage were 27 and 24 bp deep, respectively. 2. Digestibility research was conducted with American alligator to determine availability of nutrients in common feed ingredients used in manufactured diets. Results were shared with alligator producers at several monthly meetings of the Louisiana Alligator Farmers and Ranchers Association and the Louisiana Alligator Advisory Council. 3. Gulf killifish air incubation protocols were developed and demonstrated to provide an adequate environment for killifish eggs. 4. Several alterations were made to conventional crawfish traps to investigate their effects on crawfish catch. An effective medium was developed to investigate cool water bait attractants, and several potential attractants were tested. 5. Grow-out trials were conducted to evaluate the adaptability of Orconectes lancifer (shrimp crawfish) to commercial culture conditions. Dressout percentages were notably higher than those of red swamp crawfish. Cumulative mortality and physiological responses were also examined in Procambarus clarkii and O. lancifer juveniles subjected to increasing salinity in daily increments and P. clarkii was more tolerant of increasing salinity. 6. The antibacterial effects of five chitosan compounds against 36 foodborne pathogenic strains were studied and one water-soluble chitosan with molecular weight of 43 kDa was found to be the most effective. Vibrio spp. strains were most susceptible to chitosan compared to other bacterial genera. 7. Energy-removal-rates (ERR) of cryogenic-freezing and air blast-freezing for producing frozen catfish fillets and the effects of both freezing techniques on catfish fillet quality were investigated. This project generated 1 M.S. Thesis, 1 refereed journal article, 1 farm magazine article, 8 abstracts, 1 annual research report, and contributions to 4 newsletters. Recent findings and recommendations were also presented at local parish extension offices and to various clientele groups. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. The availability of the P. damselae and F. noatunensis genome sequences provides the opportunity to examine genes that are potentially involved in virulence and begin to establish their role in pathogenesis. 2. Laboratory experiments with alligators determined the digestibility of crude protein and energy, and the availability of amino acids and selected minerals in menhaden fish meal; de-hulled, solvent-extracted soybean meal; soybean protein concentrate; wheat gluten and yellow corn. Preliminary results indicate that mean crude-protein digestibility (CPD) was 87-94 percent among fish meal, soybean meal, and soybean protein concentrate; while energy digestibility (DE) was 83-92 percent. Measurements of CPD and DE in wheat gluten and yellow corn, and determinations of amino-acid and mineral availability in all ingredients, are in progress. 3. Embryo incubation studies with Gulf killifish have shown significant differences in viability at varying salinities and substrate, with the best hatch occurring at salinities between 7 to 10 ppt and on a synthetic poly foam material. Incubation temperature can be modified between 20 and 30 degrees Celsius to increase or decrease the rate of development with no significant impact on viability upon hatch. 4. Partial entrance funnel restrictions reduced crawfish catch while bait retention wells had no effect. In coldwater bait studies, various flesh derivatives provided results similar to the industry standard (cut fish), but lipid, blood, amino acid mixtures, and grain-based products were less effective. Moreover, mark/recapture trials indicated the relative inefficiency of baited traps for removing crawfish from the population. 5. Average yields of shrimp crawfish ranged from a low of 136 kg/ha to a high of 571 kg/ha, with corresponding average lengths of 82 and 78 mm, respectively. Economic analysis suggested a harvest of 270 kg/ha would result in net returns of $50/ha to $240/ha at typical early-season pond-bank prices. Culture trials for the shrimp crawfish suggested several economically viable options to extend the crawfish production season in Louisiana, with potential farm-gate revenues worth tens of millions of dollars. 6. The variability shown when different susceptibility testing methods were used suggests the need to apply multiple methods when conducting antimicrobial susceptibility testing of chitosans. The superior effectiveness of chitosan against Vibrio spp. demonstrated in this study may prompt future applications of chitosan to control Vibrio spp. in foods, particularly raw oysters. This application will help reduce the number of illnesses and deaths because of Vibrio spp. risks associated with raw oysters in the long run. 7. Freezing time for cryogenic freezing and air blast freezing were 19.3, and 55.1 min, respectively. Cryogenic freezing had higher freezing rate and ERR than blast freezing. Overall, cryogenic freezing produced catfish fillets with the best color values, less relative moisture loss, higher moisture content, and less production of secondary oxidation products as compared to the lower energy removal rates of blast freezing.

Publications

• Coulon, M., C. T. Gothreaux, and C. C. Green. 2010. Substrate preference, survival, and developmental ontogeny of Gulf killifish Fundulus grandis eggs incubated in air. Annual Meeting of the Louisiana Chapter of the American Fisheries Society.
• Coulon, M., C. T. Gothreaux, and C. C. Green. 2010. Substrate preference, survival, and developmental ontogeny of Gulf killifish Fundulus grandis eggs incubated in air. Annual Meeting of the Louisiana Chapter of the American Fisheries Society. 1st Place Student Presentation.
• Green, C., Gautreaux, K., Perez-Perez, R., and Lutz, C.G. 2010. Comparative physiological responses to increasing ambient salinity levels in Procambarus clarkii and Orconectes lancifer. Eighteenth Meeting of the International Association of Astacology, July 2010, Columbia, Missouri.
• Hardee, D.C. 2009. The effects of bait type, trap-soak duration, and trap modification on harvest of red swamp crawfish. Masters Thesis, Louisiana State University, Baton Rouge, Louisiana, USA, p. 47.
• Hardee, D.C., R.P. Romaire, and W.R. McClain. 2010. The effects of bait type, trap-soak duration, and a trap modification on harvest of red swamp crawfish. Abstracts:Annual Meeting of the Louisiana Chapter American Fisheries Society: 2010 January 28; Baton Rouge, Louisiana, USA. p. 53.
• McClain, W.R. 2010. On-board crawfish grader: Good idea or not Lousiiana Farm and Ranch. 6(2):42-43.
• Ramalho, R.O., W.R. McClain, and J.J. Sonnier. 2009. Preliminary assessments of capture rate and crawfish movement in a commercial crawfish pond. Ann. Res. Rpt., Rice Research Station, LSU Agricultural Center, 101:209-213.
• Ramalho, R.O., W.R. McClain, and J.J. Sonnier. 2009. Assessments of capture rate and crawfish movement over time in experimental crawfish ponds. Ann. Res. Rpt., Rice Research Station, LSU Agricultural Center, 101:214-219.
• Ramalho, R.O., W.R. McClain, and P.M. Anastacio. 2010. An effective and simple method of marking crayfish. Freshwater Crayfish 17:57-60.
• Coulon, M., C. T. Gothreaux, and C. C. Green. 2010. Substrate preference, survival, and developmental ontogeny of Gulf killifish Fundulus grandis eggs incubated in air. World Aquaculture 2010, San Diego California.
• Lutz, C.G. and J. Bischoff. 2011. Preliminary results from growout trials for the "shrimp crawfish," Orconectes lancifer, in Louisiana. Aquaculture America 2011, February 28-March 3, New Orleans, Louisiana.
• Sundararajan, S., D.J. Bankston, and S. Sathivel. 2010. Effect of green tea glazing on lipid oxidation of shrimp (Litopenaeus setiferus) during 35-days of frozen storage. Ann. Mtg. of the Institute of Food Technologists, Chicago, IL, abstract no. 092-02, p. 112.
• Espinoza, L., S. Sundararajan, J.D. Bankston, and S. Sathivel. 2010. Effects of energy removal rate during freezing on the quality of catfish fillets. Ann. Mtg. of the Institute of Food Technologists, Chicago, IL, abstract no. 092-01, p. 112.