Triploid Grass Carp FishBusters
The U.S. Fish and Wildlife Service (USFWS) conducted a National Triploid Grass Carp Inspection and Certification Program (NTGCICP) Workshop in August 2008. One goal of the workshop was to examine the scientific credibility for the foundation of the program. Four commonly asked questions were addressed by professionals at the workshop. These questions were given scientific scrutiny and the subsequent answers were brought before a national group of scientists and resource managers. The findings are expressed below in the form of Fish Busters:
Fish Myth: Triploid grass carp can change to diploid.
Busted: The assertion that triploid carp (grass or black) have the ability to revert to diploid and then sexually reproduce has been suggested without citing experimental or observational data to support this statement (Fuller 2003; Tillitt 2003). The USFWS has completed an extensive fact-finding effort to determine the validity of this assertion. As of 2009, there were no experimental or observational studies to demonstrate that grass carp can change from triploid to diploid. For more information on the scientific evidence and citations, click here.
During normal reproduction, a third set of chromosomes is expelled before the first division of the fertilized egg to produce an egg with two sets of chromosomes (diploid). A variety of treatments (chemical, physical or temperature) can be used to induce the retention of the third set of chromosomes creating a triploid grass carp at four to five minutes after fertilization. Triploid grass carp producers use the most consistently effective practice of pressure shocking fertilized eggs with 7000 to 8000 pounds per square inch for up to two minutes. If successfully induced, all subsequent cells within the animal contain three sets of chromosomes (Dunham 2004; Lutz 2001). Chromosome number, size and shape are a fundamental aspect of an entire organism, since the karyotype is determined prior to the first cell division (except in some induced molluscan polyploids). As a result, "reversion" to a diploid state would require all the cells in the organism (or at least in the gonads) to somehow shed an entire chromosome set - and only one set and one copy of each chromosome. This, of course, is beyond the realm of possibility (C. Greg Lutz, personal communication). After an extensive literature review, no reference or opinion by a scientist knowledgeable in the field of fishery genetics, polyploidy induction, or triploid grass carp could be found to support the assertion that triploid fish revert to a diploid or reproductively active fish.
- Dunham, R.A. 2004. Aquaculture and Fisheries Biotechnology: Genetic Approaches. CABI Publishing. Cambridge Massachusetts.
- Fuller, P. L. 2003. Freshwater aquatic vertebrate introductions in the United States: Patterns and pathways in Invasive Species: Vectors and Management Strategies edited by G.M. Ruiz and J.T. Carlton. Island Press. Washington DC.
- Lutz, C.G. 2001. Practical Genetics for Aquaculture. Fishing News Books, Blackwell Science Ltd. Iowa State University Press, Ames Iowa.
- Tillitt, D. 2003. Invasive and exotic species: Reproductive potential of triploid grass and black carp. U.S. Geological Survey, Columbia Environmental Research Center. (http://www.cerc.usgs.gov/pubs/center/pdfDocs/black_carp.pdf accessed June 4, 2009).
Fish Myth: Triploid grass carp are not really sterile.
Busted: Triploid grass carp are sterile. Induced polyploidy (or producing more than two homologous sets of chromosomes) for the purpose of sterilization has become increasing popular and is the method of choice when creating sterile individuals in normally diploid (i.e., two chromosome set) animals. The presence of three homologous chromosomes disrupts meiosis (i.e., production of single chromosome set sperm or eggs) because they cannot correctly pair to separate as haploid gametes (Piferrer et al. 2009). For more information on induced polyploidy and functional sterility, click here.
Induced Polyploidy and Functional Sterility
Initial attempts to prevent unwanted naturalization of grass carp focused on producing all female populations through gynogenesis (Stanley 1976) and hormonal implants followed by mating XX males to normal females (Boney 1984). However, monosex populations are still fertile and the process of sexing juveniles is tedious and time consuming. Clippinger and Osborne (1984) attempted surgical gonadectomies but failed due to the rapid regeneration of gonads in grass carp. Studies then focused on creating triploid hybrids through grass carp x bighead carp (Hypophthalmichthys nobilis) intergeneric crossing (Marian and Krasznai 1978; Buck 1979), but the low spontaneous production of triploid hybrids and the failure of the cross to survive led directly to the current focus on the production of pure, unhybridized triploid grass carp (Allen and Wattendorf 1987).
Induced polyploidy for the purpose of sterilization has become increasingly popular with aquaculturists since the first trials of the technology in the 1970’s (Purdom 1972). The process involves inhibiting the second maturation division of meiosis in the fertilized egg thereby causing retention of the extra chromosome set contained in the second polar body of the ovum (Allen and Wattendorf 1987) by temperature and pressure shock to the eggs (Cassani and Caton 1985; Cassani and Caton 1986). This causes the triploid condition in grass carp giving them a chromosome number of 72 (3N) instead of the natural 48 (2N) and makes them functionally sterile.
Triploid grass carp have been proposed as a sterile substitute to diploid grass carp for the management of nuisance aquatic vegetation, but the working definitions of sterility for aquaculture and management purposes are very different and must be considered. In aquaculture, reduced development of gonads may suffice as sterility. However, in management the definition of sterility must include an estimate of reproductive likelihood (Allen et al. 1986).
Doroshov (1986) found that female triploid grass carp produce only rudimentary gonads and egg production is non-existent. Later work at the University of California-Davis that examined triploid female grass carp from Florida found ovaries but with no advanced oocyte development (Joel Van Eenennaam, personal communication).
Triploid males produce substantial testes, can be induced to spermiate and will attempt to spawn (Allen and Wattendorf 1987). Allen et al. (1986) determined that for every 109 cells undergoing meiotic reduction in triploid males approximately 60 euploid sperm could be formed. They also noticed triploid male grass carp sperm were abnormal in shape; variable size that were 1.5N (54%), 3N (24%), and 6N (22%); and sperm density was about 2% of diploid milt. They estimated the reproductive likelihood of a single male grass carp is approximately 4 x 10-11 for every meiotic reduction of hexaploid spermatogonia, meaning one F1 adult from a triploid X diploid backcross will result from every .25 x 1011 of these reductions, and a mature population of diploid females must be present due to the complete infertility of female triploids. In summary, Allen et al (1986) concluded that “…in a river into which diploids have already escaped, the additional reproductive potential of triploids seems practically nonexistent. Triploids, in fact, may disrupt spawning, fertilization success, or viability of diploids, decreasing the overall reproductive potential of the population (page 847).”
Van Eenennaam et al. (1990) artificially inseminated diploid female grass carp with triploid male milt. The fertilization rate was one-half to one-third than that for diploid x diploid mating. The diploid x triploid embryos had smaller bodies and yolk-sacs and utilized their yolk slower than diploid x diploid products. The majority (75 to 90%) of the diploid x triploid embryos exhibited notochord deformities, underdeveloped tail rudiments, failed to swim up after hatching, and died before first feeding; however, 0.1 to 0.2% of the embryos survived to five months and were diploid. In summary, Van Eenennaam et al. (1990) concluded, “Although triploid males could potentially participate in mating and spawning under natural conditions, they may be regarded as functionally sterile. Stocking of triploid males will not support natural reproduction of grass carp in the wild. However, this conclusion implies that they compete with diploid males and mate with diploid females (page 124).” As reported by Dunham (2004), Dr. John Cassani in Florida videoed triploid male grass carp in normal courtship with diploid female grass carp but produced no visible ejaculate when the female ovulated.
- Allen, S. K., Thiery, R. G., and Hagstrom, N. T. 1986. Cytological evaluation of the likelihood that triploid grass carp will reproduce. Transactions of the American Fisheries Society, 115(6):841-848.
- Allen, S. K., and Wattendorf, R. J. 1987. Triploid grass carp: Status and management implications. Fisheries, 12(4):20-24.
- Boney, S. 1984. Sex reversal and breeding of grass carp. Transactions of the American Fisheries Society. 113:348-354.
- Buck, H. 1979. Optimism swells with the possibility of a sterile hybrid grass carp. Fisheries, 4(5):31.
- Cassani, J.R. and Caton, W.E. 1986. Efficient production of triploid grass carp (Ctenopharyngodon idella) utilizing hydrostatic pressure. Aquaculture 55:43-50.
- Cassani, J. R. and Caton, W. E. 1985. Induced triploidy in grass carp. Aquaculture, 46: 37-44.
- Clippinger, D. and Osborne, J. A. 1984. Surgical sterilization of grass carp, a nice idea. Aquatics, 6:9-10.
- Doroshov, J. 1986. Comparative gametogenesis in diploid and triploid grass carp. Meeting of World Mariculture Soc., Reno, NV. January, 1986.
- Dunham, R.A. 2004. Aquaculture and Fisheries Biotechnology: Genetic Approaches. CABI Publishing, Cambridge, MA.
- Marian, T. and Krasznai, Z. 1978. Kariological investigation of Ctenopharyngodon idella and Hypophthalmichthys nobilis and their cross-breeding. Aquaculture Hungarica, 1: 44-50.
- Piferrer, F., A. Beaumont, J-C. Falguière, M. Flajšhans, P. Haffray, and L. Colombo. 2009. Polyploid fish and shellfish: Production, biology and applications to aquaculture for performance improvement and genetic containment. Aquaculture 293: 125-156.
- Stanley, J. G. 1976. Production of hybrid, androgenetic, and gynogenetic grass carp. Trans. Am. Fish. Soc. 105:10-16.
- Van Eenennaam, J.P., R.K. Stocker, R.G. Thiery, N.T. Hagstrom, and S.I. Doroshov. 1990. Egg fertility, early development and survival from crosses of diploid female x triploid male grass carp (Ctenopharyngodon idella). Aquaculture. 86: 111-125.
Fish Myth: The USFWS National Triploid Grass Carp Inspection and Certification Program only tests a small percentage of fish.
Busted: The National Triploid Grass Carp Inspection and Certification Program tests 120 randomly-selected fish from each identified lot of alleged 100 percent triploids.
Producers will test each fish (from an identified lot) individually to determine if it is diploid or triploid. Before triploid fish are sold and after the producer tests each fish from identified lot, an inspection is scheduled with a U.S. Fish and Wildlife Service inspector (Griffin and Mitchell 1992). A trained inspector will travel to the facility and observe the producer as they retest 120 fish for ploidy from an alleged lot of 100 percent triploids. If all 120 fish are triploid, the inspection is complete. If a diploid is encountered, the identified lot fails the inspection and all fish in the identified lot must be retested by the producer (Griffin and Mitchell 1992; USFWS, NTGCIC Program Standards Updated 2009). For more information on tests, inspections, and certifications performed by the USFWS, click here.
The USFWS only provides the inspection and certification service to producers that want to cooperate, and participation is completely voluntary. All grass carp in an identified lot, offered for sale, will have been individually tested by the producer using coulter counter techniques before a USFWS Triploid Grass Carp Inspection will be performed. The USFWS Inspection consists of a retesting by the producer, in the presence of the inspector, of 120 individuals randomly selected by the inspector from the identified lot of alleged 100 percent triploid grass carp. The inspector will view the group of fish that is to be certified, verifying that the group is isolated in a containment unit at least 100-ft. away from the production ponds (thus reducing the change of inadvertent mixing of triploids and diploids) and that numbers of fish are appropriate for the orders to be certified. The inspector will channelize (at a minimum) every tenth fish during the inspection of the 120-fish sample of alleged triploid grass carp. Any sample with a questionable monitor reading will also be channelized, and any questionable data resulting from channelization will be considered non-triploid. If all 120 fish tested are triploid, the inspection is complete. If a diploid is found in the course of testing the 120 fish sample, the lot fails certification. All fish in that lot must be retested, individually, by the producer, before another inspection of that lot of fish is rescheduled for certification inspection.
- Griffin, B. R. and A. J. Mitchell. 1992. Standards of the U.S. Fish and Wildlife Service's triploid grass carp inspection program. Aquaculture Magazine, 18(6):73-74.
- USFWS. 1 January 2009. Triploid Grass Carp Inspection and Certification Program. 8 April 2009 http://www.fws.gov/warmsprings/FishHealth/frgrscrp.html.