Progress 09/01/24 to 08/31/25

Outputs
Target Audience:The target audiences reached by efforts during this reporting period included Louisiana Farm Bureau, Louisiana Crawfish Promotion Board members, crawfish farmers in the state of Louisiana,researchers from Auburn University, Nichol's State University, Louisiana State University Agricultural Center, and the United States Department of Agriculture National Animal Germplasm Program. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Tweleve undergraduate student workers from an array of backgrounds were trained to work with crawfish including aquaculture system planning and assembly, animal husbandry, biological research, technology development, and grant management. To date, these students include Gabrielle Bush (Natural Resource Ecology and Management), Owen Plaisance (Accounting), Cameron Bonds (Mechanical Engineering), Honore Miguel Simo (Mechanical Engineering), Alana Fregoso-Castillano (Coastal Environmental Sciences), Caleb Hattier (Mechanical Engineering), Jace Collins (Mechanical Engineering), Nathan Cao (Biology), Ariel Gauthier (Animal Science), Cole Brumfield (Electrical Engineering), Jacqueline Schlamp (Biology), and Nicholas Spoonmore (Mechanical Engineering). How have the results been disseminated to communities of interest?Experiment planning and dissemination of results has occurred through various mechanisms including dozens of emails, phone calls, two virtual meetings, and one in-person fisheries conference. Participants included AGGRC personnel, Louisiana Farm Bureau personnel, Louisiana Crawfish Promotion Board members, researchers from Auburn University, Nichol's State University, Louisiana State University Agricultural Center, and the United States Department of Agriculture National Animal Germplasm Program. The in-person meeting (Louisiana Chapter of the American Fisheries Society) included a much wider audience. An interactive poster was presented at this meeting by an AGGRC undergraduate student. What do you plan to do during the next reporting period to accomplish the goals?Task 1: Develop standardized collection of spermatophore (e.g., by electrical stimulation) Electro-expression of spermatophores will be tested more rigorously during the reproductive season and at higher voltages and frequencies based on literature reported values (35-45V). This will be tested with 9-V batteries connected in series, achieving 36V, and with a signal generator coupled to a signal amplifier to reach the recommended parameters. The feasibility of an open-hardware electro-expression device will be evaluated. We are designing a 3-D printed open-hardware version of the crawfish holding apparatus. Task 2: Establish protocols and standards for sperm quality evaluation Other dyes (e.g., fixable amine dyes or the fluorescent dyes propidium iodide and SYBR-14) will be tested as alternative methods to determine sperm quality. We are evaluating the possibility of developing a 3-D printed microfluidics quality evaluation device for use with crawfish sperm. Task 3: Develop a scalable pathway-level cryopreservation process for crawfish spermatophore We will perform a viability assay on sperm samples exposed to extenders with different osmolalities to understand the range of extender solutions that can be used withP. clarkii. In parallel we will perform viability assays on sperm samples exposed to potential cryoprotectant solutions at different concentrations. If spermatophore expression becomes a viable technique for obtaining sperm samples, we will evaluate the stickiness of spermatophores. This test will be performed at multiple stages during the cryopreservation process including after expression, exposure to cryoprotectants, and after thawing. Task 5: Collect genetic samples from specific locations for submission to NAGP We will use the population structure information and input from farmers across the state to select locations to collect samples for cryopreservation and submission to USDA NAGP. Task 6: Develop a roadmap for sustainable crawfish aquaculture with integrated genetic management Process maps will be developed as roadmaps for sustainable crawfish aquaculture. This will aid in the development of future stocks with minimal bottlenecks and constraints.

Impacts
What was accomplished under these goals? Objective 1 Capacity developed at the AGGRC An indoor recirculating aquaculture system was assembled to house crawfish for the project. The building that houses this system is photoperiod and temperature controlled to facilitate optimal growth and reproductive conditions outside of the normal crawfish reproductive season. The system is equipped with a bubble bead filter and aeration to maintain water quality. Crawfish are fed three times per week, twice with carrot slices and once with Zeigler Maturation shrimp pellets. The system can hold more than 150 individually housed crawfish to reduce mortality and aggression. Task 1: Develop standardized collection of spermatophores Biological Research Electrical stimulation was the primary method tested for spermatophore collection. This involved holding the crawfish including controlling the claws and tail, holding the walking legs out of the way, and exposing the gonopores (near the fifth pair of walking legs). Wires (electrodes) were connected to a power source (battery or signal generator) and electrodes placed on either side of the base of one of the fifth walking legs. Most of the literature available on electrical stimulation of spermatophores from crawfish does not describe the electrical settings used in usable detail. Thus, we have tested a variety of settings. We began our procedure development with two electrodes attached to a 9-V battery. This resulted in twitching of the fifth walking leg but no spermatophore expression. This test was performed early in the crawfish season (April) and needs to be repeated during the breeding season (August/September). There may not have been spermatophores in the crawfish vas deferens. We also tested electrical spermatophore expression by use of a signal generator. We evaluated signals (both sine and square waveforms) up to 20V at 40-60Hz resulting in small movements of the crawfish legs with no spermatophore expression. This test was performed in April and will also be evaluated again in August/September. Open Hardware Three-dimensional (3-D) printing was used to support technology development. A 3-D printed device was developed to provide control of distance and application of electrodes to easily accommodate the variety of sizes of males and ensure operability. This prototype has been used in several experiments. If electrical stimulation becomes a viable technique for spermatophore collection, an improved version of the device is planned. The new version would improve the precision and replicability of the distance between electrodes. An improvised crawfish holding apparatus was prototyped for holding crawfish in place during electro-expression testing and for future artificial insemination testing. This apparatus was assembled from a rubber sheet and Velcro. Eight versions of an invertebrate holding tube were prototyped. The most recent version of the invertebrate tube (fully reusable with a material cost of <$2) is crush-resistant and has an integrated labeling system. The tube also functions as a container to segregate crawfish during husbandry and experiments. The tube is modular allowing multiple tubes to be connected to adjust the space available to the crawfish. The bottom of the tube can be printed with different degrees of air space (i.e., infill setting) so tubes can be engineered to float upright (heavier bottom) or on the side (lighter bottom). The top of the tube has an integrated feeding hole. We are currently adjusting (parameterizing) the design to facilitate 3D printing to accommodate all sizes of crawfish. This device will increase efficiency of husbandry and offers a scalable and standardized solution for shipping and experimentation. This device is also generalizable to other species beyond crawfish. Task 2: Establish protocols and standards for sperm quality evaluation Biological Research To support research on electro-expression of spermatophores we are also developing methods to collect sperm via dissection. Sperm samples from the vas deferens were collected to examine morphology and membrane integrity (a measure of viability).Sperm appeared to exist in two morphological states: planar multistellate and aplanar multistellate.The biological significance of these states is unclear at present. These different states have not been described previously and do not appear to be related to the maturity of sperm. This requires further investigation. A method was developed to reliably evaluate sperm membrane integrity and quality by use of the non-fluorescent dyes, eosin Y and nigrosin. Task 3: Develop a scalable pathway-level cryopreservation process for crawfish spermatophore Biological Research We developed a computer-assisted sperm analysis (CASA) profile to rapidly and accurately assess concentration of crawfish sperm samples. We began to evaluate refrigerated storage of sperm. Based on previous literature on crawfish hemolymph we determined that a suitable extender for sperm samples would be 435 mOsm Hank's Buffered Salt Solution (HBSS). Sperm samples kept in this extender solution for as long as 2 weeks in the refrigerator (4 degrees Celsius) remained viable. The samples became nonviable after 2 weeks. Sperm samples that were not placed in the extender solution within 5 minutes of dissection became nonviable. We tested several experimental conditions to understand what viable and nonviable sperm looked like when using membrane integrity assays. For example, sperm samples placed for as long as 20 minutes in a water bath heated to 40 degrees Celciusremained viable. In other preliminary testing, there have been mixed results between trials. For example, two experimental trials that placed sperm samples in a freezer (-20 degrees Celcius) for 30 minutes resulted in 100% membrane compromised sperm and 81% membrane intact sperm. This experiment requires further trials and exploration. Objective 2 Task 4: Establish genetic screening tools such as SNPpanels and screen genetic variability of representative populations A low-density SNP panel was designed by the Center for Aquaculture Technologies (CAT, San Diego, CA).The panel was designed using publicly available resources. The finalized panel includes approximately 500 polymorphic SNPs selected for their utility in population genetic assessment and parentage analysis. In addition, three SNPs within white spot syndrome virus (WSSV) resistance in red swamp crawfish were included based on published findings. The panel design was submitted for multiplexing and synthesis. Ninety-six samples (crawfish leg stored in 95% ethanol) were collected and shipped to CAT to complete SNP panel validation and performance testing. To choose representative populations and populations of interest in Louisiana we worked closely with industry partners and a geographic information system (GIS) team. We attempted to survey crawfish farmers across the state to help in choosing sites that they were interested in including in the genetic sampling. We have identified farmers in several parishes who are interested in providing samples from their farms for the population structure sampling. We are also incorporating environmental factors in the sampling decisions. In 2023 there was a severe, prolonged drought across Louisiana that almost certainly affected the genetic structure of crawfish in Louisiana. We generated three maps to inform our state-wide genetic sampling efforts: a drought index map, a precipitation map, and a temperature map. To date, we have visited two crawfish farms near White Castle, LA (St. Martin Parish) and collected 50 crawfish from each. Task 6: Develop a roadmap for sustainable crawfish aquaculture with integrated genetic management To date, we had eight scheduled conversations and meetings with crawfish experts, farmers, and biologists to identify the needs and wants of the future crawfish industry in LA, including discussion with Louisiana Farm Bureau.

Publications