Source: NORTH CAROLINA STATE UNIV submitted to
DEVELOPMENT OF A RAPID, NON-INVASIVE, AND LOW COST DNA-BASED METHOD FOR SEX DETERMINATION IN RUSSIAN STURGEON
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1030514
Grant No.
2023-70007-40200
Cumulative Award Amt.
$300,000.00
Proposal No.
2022-06012
Multistate No.
(N/A)
Project Start Date
Jul 1, 2023
Project End Date
Jun 30, 2025
Grant Year
2023
Program Code
[AQUA]- Aquaculture Research
Project Director
Chouljenko, A.
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
(N/A)
Non Technical Summary
Currently, methods used to determine sex in aquacultured Russian sturgeons (Acipenser gueldenstaedtii) are laborious and applicable only for older fish (5-6+ years old) due to slow reproductive organ maturity. Value of its caviar is high (up to $2,000 per female fish) and thus there is a great need for the development of a more efficient caviar production process. A solution would allow Russian sturgeon farmers to raise monosex caviar-producing populations from early on, resulting in increased caviar production. We propose a comparison of 2 potential DNA-based diagnostic tests using 2 different primer sets and to apply the most reliable version in actual field conditions at the sturgeon farm. The main goal is to develop a rapid (8-10 h), non-invasive (swab DNA collection), and cost-effective ($7-10 reagent cost/fish) DNA-based test that will allow sex determination of juvenile (≤2 month old) Russian sturgeons. Successful application of this technology would allow U.S. sturgeon farmers to potentially double their caviar production and enhance the capacity of the United States aquaculture industry to contribute to domestic and global food security and economic growth. Additionally, successful validation of our proposed methodology would open up application opportunities to other species of sturgeon for production and conservation purposes.
Animal Health Component
70%
Research Effort Categories
Basic
10%
Applied
70%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3073719104040%
3043719104040%
3073719108010%
3043719108010%
Knowledge Area
307 - Animal Management Systems; 304 - Animal Genome;

Subject Of Investigation
3719 - Other cultured finfish;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;
Goals / Objectives
The main goal of this project is to develop a rapid (8-10 h), non-invasive (swab DNA collection), and cost-effective ($7-10 reagent cost/fish) DNA-based test that will allow sex determination of juvenile (≤2 month old) Russian sturgeon. Successful application of this technology as a long-term goal would allow U.S. Russian sturgeon farmers to potentially double their caviar production capacity (if there is a roughly equal distribution of males/females) by raising monosex populations. Additionally, successful validation of our proposed methodology would open up application opportunities to other species of sturgeon for production and conservation purposes. The overall objectives of the research are:1. To establish dedicated PCR-based DNA detection capability at CMAST, Morehead City, NC2. To validate VAC-1M male-specific Russian sturgeon DNA fragment together with AllWSex2/Ag49 primer set through a direct side-by-side comparison using DNA collected during ultrasound sex determination of mature sturgeons3. To develop a rapid DNA-based method for sex determination that may be applied to juvenile Russian sturgeons using non-invasive skin swabbing for the DNA samples collection
Project Methods
MethodsPureLink Genomic DNA Mini Kit (Invitrogen) will be used to isolate DNA from at least 24 individual caviar grains from geographically distant sources of Russian sturgeon. VAC-1M primers (previously identified unique and strictly male-specific DNA sequence from Russian sturgeons grown at Marshallberg Farm, Smyrna, North Carolina) will be tested for specificity side-by-side with the DNA from the Marshallberg Farm caviar. If found to be specific and similar for all three groups, VAC-1M-based primers set will be compared directly against AllWSex2/Ag49 multiplex primers using 96 sturgeon skin swab-derived DNA samples that will be collected during routine ultrasound as described below. If not, only AllWSex2/Ag49 multiplex primers will be used for comparing DNA-based sex determination results with the ultrasound method and to complete objective 3. Marshallberg Farm will allow us to collect DNA swab samples during their routine process of ultrasound sex determination for the sturgeons. Very similar to the experiments described in the preliminary results, at least four sets of 24 swab samples each (coinciding with the maximum tube capacity for the centrifuge rotor) will be collected from mature fish (5-7 years old). DNA will be isolated at the PD's lab located within a 30 min drive from the farm. For sex determination, our VAC-1M-based primers set (if successfully validated from caviar analyses) will be compared directly against AllWSex2/Ag49 multiplex primers to routine ultrasound results using an E-gel power snap electrophoresis system with the 48 samples E-gel format.After experimental validation of the DNA-based genetic markers, we plan to apply our DNA-based test to juvenile Russian sturgeons of up to 2 months in age (the actual minimum age/size still to be determined during the future experiments). As part of their regular business activities, Marshallberg Farm will obtain fertilized Russian sturgeon eggs. Marshallberg Farm will be responsible for fish hatching and young sturgeon handling and rearing activities/care. Overall, Marshallberg Farm will house more than 1,000 juvenile Russian sturgeons and make them available in order to conduct the proposed research. The test is designed to use a 96 well PCR plate format and can be potentially scaled up easily by utilizing additional resources if needed. We envision having 96 numbered, cube-shaped netting cages (with sides of around 12 inches) hooked inside and around the large tanks used for sturgeon farming. After swabbing, the juvenile sturgeons will be comfortably separated from each other within these cages until the end of the test (projected to be completed within 8-10 h). Samples transportation, DNA isolation, and PCR set up is estimated to last about 6-8 h plus at least 1 h 30 min for running and documenting results of two sets of 48 PCR samples using 48-well gels (two of the 48-wells E-gels should be used instead of one 96 wells gel that is also available, because of the need to achieve proper bands separation and ultimately producing more accurate results). After testing and relaying results to the farm, the respective female and male juvenile sturgeons can be easily relocated to different holding tanks. The process will be repeated every working day resulting in accurate sex determination of about 1,000 juvenile sturgeons over a 10 working day period using only one 96-well plate PCR format.Marshallberg Farm will provide standard care for all sturgeon fish that are subjects of this proposal until they are ready for typical ultrasound sex determination. They will have the unique opportunity to acquire all valuable information about growing monosex fish populations before deciding about the method of sturgeon sex determination that serves their needs best. We expect to have a total cost of reagents necessary to conduct this test to be within $7-10 range per DNA sample which appears very reasonable considering the fact that one female can produce caviar worth up to $2,000. Also, total time to be able to determine sex can be reduced from at least 5 years to around 2 months (at least 30 times reduction).EvaluationAchievement of specific project objectives will be monitored as follows:1. To establish dedicated PCR-based DNA detection capability at CMAST, Morehead City, NC - Successful completion of objective 1 will rely upon purchase and receipt of equipment and supplies for DNA analysis, and fish holding nets and hooks in a timely manner. This, along with hiring and training of a research associate, will allow us to move on to other objectives.2. To validate VAC-1M male-specific Russian sturgeon DNA fragment together with AllWSex2/Ag49 primer set through a direct side-by-side comparison using DNA collected during ultrasound sex determination of mature sturgeons - Objective 2 will consist of two main procedures. The first part will be to validate VAC-1M DNA fragment together with AllWSex2/Ag49 primer set using DNA collected from differently-sourced Russian sturgeon caviar grains. The second part (main idea) will be to validate primers based on the VAC-1M fragment (if found to be specific for all Russian sturgeons regardless of the origin) and AllWSex2/Ag49 primer set by comparing our DNA-based method vs. ultrasound during routine sex determination of mature Russian sturgeons. If VAC-1M-based primers set is deemed unsuitable for diagnostics with caviar, we will apply only the AllWSex2/Ag49 primer set to the larger number of samples (96 swab DNA samples). This primer set was already validated using a small sample size by us when working with mature Russian sturgeons. Similar to our preliminary results, we expect our DNA-based method of sex determination to reveal significant inaccuracies regarding use of the ultrasound method.3. To develop a rapid DNA-based method for sex determination that may be applied to juvenile Russian sturgeons using non-invasive skin swabbing for the DNA samples collection - Keys to achieving objective 3 will be to maintain/care for enough juvenile fish in the appropriate age range (up to 2 months) for DNA collection. We do not anticipate any other potential issues for objective 3 because up to 1,000 juvenile sturgeon fish can be easily tested within two weeks.EffortsWe plan to present our findings at a national aquaculture conference and publish manuscripts in high impact peer-reviewed journals. We also plan to publish articles related to the study in relevant trade magazines and digital repositories available to everyone, including the general public. Presentations and/or workshops conducted by those involved in this study will target stakeholders and interested parties. Once established and validated, this technology will be available to anyone in the sturgeon aquaculture industry. Priority will be placed on dissemination of information to other sturgeon farmers in the United States. It is important to support U.S. caviar producers who compete with imported caviar which makes up most of the caviar consumed in the U.S. A main goal of this proposal is not only to develop a DNA-based sex diagnostic test using Marshallberg Farm as a base for the test's practical validation, but also to serve as a potential hub providing either direct DNA testing service on a fee-for-service basis or proper training to anybody who is interested.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:A dedicated post-doctoral researcher has been hired to perform day-to-day work related to the successful completion of the project. The post-doctoral researcher, along with industry collaborators at a Russian sturgeon farm, Marshallberg Farm, have partaken in practical and experiential learning related to the project. The knowledge gained from this work contributes to key interests for industry, academia, and the broader public, including seafood genetics and production. Our work has made progress in the goal of providing Russian sturgeon caviar producers with a method to rapidly and non-invasively determine sex in Russian sturgeons using DNA. Changes/Problems:The rate of expenditure was significantly delayed because we have had difficulty in hiring, first a qualified research associate which was budgeted for in the project to conduct significant portions of the research. After some months of searching, we re-budgeted to search for a postdoctoral scholar to hire for conducting the research. Eventually we found a good candidate to hire as a postdoc and they arrived with a start date of June 11, 2024. This has caused some delay in the research. We initially proposed for our collaborator, Marshallberg Farms, to spawn young fish for us to test with our DNA-based sex identification method. Having the young fish to work with is a key component of the project. Marshallberg Farms needs some more time to get the spawning method to work properly. A contingency may be to obtain newly spawned fish of the same species from another source, but still to do everything else as originally planned at Marshallberg Farms. What opportunities for training and professional development has the project provided?A dedicated post-doctoral researcher has been hired to perform day-to-day work related to the successful completion of the project. The post-doctoral researcher, along with industry collaborators at a Russian sturgeon farm, Marshallberg Farm, have partaken in practical and experiential learning related to the project. Some early project results were presented at the 2024 North Carolina Aquaculture Development Conference as part of an NC State University Research Update. How have the results been disseminated to communities of interest?Some early project results were presented at the 2024 North Carolina Aquaculture Development Conference as part of an NC State University Research Update. Attendees of this conference include scientists, current and prospective fish farmers, personnel from regulatory agencies, and the general public. What do you plan to do during the next reporting period to accomplish the goals?Moving forward, we will shift our focus to data collection from the side-by-side comparison during adult sturgeon sex determination by ultrasound at Marshallberg Farm vs. our DNA-based sex determination method using AllWsex2/Ag49 primers. We are also ready to apply this test for the sex determination of juvenile Russian sturgeon as soon as they will become available.

Impacts
What was accomplished under these goals? Goal 1. To establish dedicated PCR-based DNA detection capability at CMAST, Morehead City, NC: After funding became available, we have acquired a SimpliAmp PCR Instrument from Life Technology together with Invitrogen E-Gel Power Snap Electrophoresis System that has integrated nucleic acid electrophoresis and gel imaging capabilities. The instrument works with precast E-Gel agarose gels, which are buffer-less gels containing SYBR safe DNA stains, and ladders. This eliminates the need to pour gels, prepare buffers, handle toxic dyes such as ethidium bromide, and provide a high throughput analysis of multiple PCR products within 25-30 min time frame. Also, a dedicated post-doctoral researcher has been hired to perform day-to-day work related to the successful completion of the project. All major steps of the DNA-based diagnostic test starting from the skin swab DNA collection and purification, PCR conditions and DNA products detection using 48 well E-gels format has been validated at the Center for Marine Sciences and Technology (CMAST), Morehead City, NC location using adult Russian sturgeon of different ages grown at Marshallberg Farm. Goal 2. To validate VAC-1M male-specific Russian sturgeon DNA fragment together with AllWSex2/Ag49 primer set through a direct side-by-side comparison using DNA collected during ultrasound sex determination of mature sturgeons: As expected, all potential female samples generated two bands of predicted size while all male samples only one band, respectively (multiplex of AllWSex2 and Ag49 primer sets). It is important to note that the presence of the female-specific band after using DNA samples derived from the individual unfertilized caviar grains strongly points toward female-specific (wz/zz) type of sex determination in sturgeons. However, PCR using VAC-1M -specific primers and DNA from individual caviar grains produced bands of the sizes that would be expected from the corresponding unchanged 735 bp long male VAC-1M sequence. Furthermore, another sample did not produce any PCR product which is like the data generated previously using DNA samples from some of the confirmed adult female fish. The same DNA using AllWsex2/Ag49 primers identifies this sample as a potential male. Importantly, we have tested DNA isolated from commercially available individual caviar grains that were derived from the fish grown at a fish farm in Israel and Evans Farm, FL with very similar outcome. DNA samples isolated from the skin swabs were from the fish that produced inconclusive results by ultrasound testing at Marshallberg Farm. However, genetic testing using AllWsex2/Ag49 primers were able to pick up 4 potential females that would be, most likely, eliminated from consideration under normal conditions. Therefore, these data do not support the use of VAC-1M sequence as a sex-specific genetic marker and all further genetic testing with regard to adult and juvenile fish will be conducted using AllWsex2/Ag49 multiplex primers set. Manuscript in Preparation Although we did not receive the results we hoped for regarding the highly repetitive VAC-1M sequence, this experience opened us to explore a completely new direction toward the better understanding for the role of the repetitive elements within the sturgeon genome in general. DNA sequencing of 16 male-derived cloned DNA fragments revealed that 13 out of 16 sequences named VAC-1M are the same while 3 others named as VAC-2M were completely different but also highly repetitive sequences of similar size that co-migrated on the gel before using for cloning. Interestingly, a large portion of this DNA fragment aligned significantly to the Acipenser baerii partial IGLV gene for immunoglobulin light chain variable region, exons 1-2 that was first described almost 25 years ago (Gene Bank accession number AJ245365). Currently, we are in the final stages of manuscript preparation with the tentative Title and Abstract shown below. Repetitive and specific for different Acipenser species genomic region aligning to the known A. baerii IGLV gene cluster may serve as a transcription termination element Abstract Specific 218 bp long sturgeon genomic region located upstream of the previously identified A. baerii partial IGLV gene for immunoglobulin light chain variable region was found to be identical 25 years later for A. baerii and A. stellatus (caviar samples from Evans Farm, FL) as well as for A. gueldenstaedtii grown recently at 3 different fish farms (caviar samples from Israel, Evans Farm, FL, and both caviar and skin swab samples from Marshallberg Farm, NC). NCBI blast search results against the best annotated whole genome sequencing data set of A. ruthenus revealed the presence of this DNA fragment within the multiple chromosomes with total number of copies estimated to be at least 800 and with approximately 3% of them to have 100% sequence identity. The respective available A. gueldenstaedtii sequencing data set doesn't provide individual chromosome distribution, but multiple copies of the targeted genomic region were also scattered within an assembled DNA contigs. The use of the same DNA as a template combined with the same forward but different reverse primer (both were designed based on the consensus from the cloned A. gueldenstaedtii DNA sequences) allowed specific amplification of either 168 bp DNA fragment of the predicted size that exhibited much higher degree of variability in comparison to the respective "old" A. baerii region or also a similarly highly mutated 139 bp DNA fragment that contained the same 29 bp deletion specific for all subjects tested. The PCR outcome was very consistent regardless of the fish geographical origin, age, sex, source of DNA isolation (individual caviar grains vs skin swabs) or sturgeon species (A. gueldenstaedtii vs. A. baerii vs. A. stellatus). The results effectively confirmed that different versions of this highly repetitive sequence exist simultaneously within the same organism and the process of selection toward specific mutation is not random and ongoing based on the sequence variations within DNA derived from different caviar grains but originated from the same fish. We have constructed multiple recombinant plasmids containing different versions of the A. baerii genomic region covering ~5.6 kb of the IGLV cluster (starting from the location of the discovered repetitive sequence and up to the initiation codon for IGLV exon 1) cloned in front of the luciferase reporter gene (pBV-Luc plasmid). After transfection of Vero cells with all appropriate controls, the cell lysates were analyzed for the level of luciferase expression. All constructs containing the targeted repetitive region significantly inhibited the level of luciferase expression. Importantly, there are 3 sequence motifs (GATTAT) that can serve as a potential DNA binding site for the transcription termination that were clustered within this highly repetitive sequence. Cloning of the CMV promoter sequence validated to work efficiently within eucaryotic organisms resulted in more than 200 times increase of luciferase expression. Both orientations of the repeat DNA sequence cloned after CMV promoter but before luciferase reporter gene resulted in more than 10 times reduction (by 19 and 14 times for the (-) and (+) strands, respectively) of the level of luciferase expression. These results provide some new information about the role of the repetitive sequences within eucaryotic organisms in general and indicate that they may play an important role in the sturgeon immune system regulation. Specific for different Acipenser species repetitive region within the sequence preceding A. baerii IGLV gene can serve as a transcription termination element.

Publications