Transformation methods for the glochidia of the spectaclecase mussel Cumberlandia monodonta
The spectaclecase mussel, Cumberlandia monodonta, was effectively listed as federally endangered in April 2012 (https://federalregister.gov/a/2012-5603). It is endemic to the Mississippi, Ohio and Missouri River basins and historically inhabited 44 streams in these basins (USFWS 2014). Currently, the species is known to inhabit 20 of the historical streams, five of which are represented by one or two specimens (Butler 2002). The three remaining strongholds of the species occur in the Meramec, Gasconade and St. Croix River systems. The existence of small, geographically isolated populations has been identified in the Spectaclecase Recovery Outline (USFWS 2014) as a key threat to the species. As such, the Recovery Outline lists captive propagation and augmentation of remaining populations as a top priority for recovery of the species. Despite numerous attempts and testing of over 60 species of fish, amphibians, crayfish and insects, a suitable host of the spectaclecase has not been identified (Knudsen and Hove 1997, Lee and Hove 1997, Hove et al. 1998, Baird 2000, Hove and Berg 2008, Hove et al. 2008, Hove et al. 2009).
In vitro transformation of glochidia is an alternative method for production of juveniles that has been successful for over 40 species unionids (Lima et al. 2012); but has not been reported for the spectaclecase. One of the major obstacles to successful in vitro transformation of glochidia is contamination from microorganisms, particularly fungus (Owen et al. 2010, Lima et al. 2012). Current protocols for in vitro culture recommend the use of antibiotics and antimycotics to reduce contamination, but none effectively control contamination for the duration of the incubation period and some are toxic to the glochidia (Owen et al. 2010). Hydrogen peroxide has been successfully used to reduce the incidence of Saprolegniasis on fish eggs (Gaikowsld et al. 2003) and inculture of fish cell lines (Eric Leis, La Crosse Fish Health Center, La Crosse, WI, personal communication). A pilot study was conducted in November 2015 in which Lampsilis cardium glochidia were treated with 100, 500 or 1000 mg/L hydrogen peroxide for 10 minutes. The treatment effectively reduced contamination and glochidia and cells remained viable for over 1 week (personal observation). Inthe present study, pretreatment with hydrogen peroxide will be compared with controls (no hydrogen peroxide) to determine differences in glochidial viability and transformation and levels of microbial contamination in culture plates.
We will evaluate in vivo and in vitro techniques to propagate juveniles of spectaclecase mussels by inoculation of potential host species and growth in an artificial medium. Results of the study will be relevant for recovery of the spectaclecase.
Objectives
- To generate and rank a list of potential glochidial hosts based on previous host studies and fish and amphibian survey data from the St. Croix River
- To determine the transformation success of spectaclecase glochidia on potential fish and amphibian species (identified in objective 1) by laboratory inoculation
- To evaluate in vitro culture methods, including culture media and decontamination treatments, for transformation of spectaclecase glochidia in an artificial medium.
References
Baird, M. S. 2000. Life history of the spectaclecase, Cumberlandia monodonta Say, 1829 (Bivalvia, Unionidea, Margaritiferidae). Master of Science. Southwest Missouri State University.
Burch, J. B. 1973. Freshwater unionacean clams (Mollusca: Pelecyopoda) of North America. United States Environmental Protection Agency Identification Manual No. 11. 176 pp.
Butler, R. S. 2002. Status assessment report for the spectaclecase, Cumberlandia monodonta, occurring in the Mississippi River system (U.S. Fish and Wildlife Service Regions 3, 4, 5, and 6). Ohio River Valley Ecosystem Team, Mollusk Subgroup, Ashville, NC.
Cummings, K. and C. A. Mayer. 1992. Field guide to freshwater mussels of the Midwest. Illinois Natural History Survey Manual. 194 pp
Eddy, S., J. Underhill, J. Barnrick, E. Cawley, and W. Jaques. 1978. How to know the freshwater fishes. 3'<l edition. Dubuque, IA, W.C. Brown.
Fritts, AK.,M. Barnhart, M. Bradley, N. Liu, W. G.Cope, E. Hammer, and R. Bringolf. 2013.
Assessment of toxicity test endpoints for freshwater mussel larvae (glochidia). Environmental Toxicology and Chemistry 33:199-207.
Gaikowski, M. P., J. J. Rach, M. Drobish, and 5 others. 2003. Efficacy of hydrogen peroxide in controlling mortality associated with saprolegniasis on walleye, white sucker, and paddlefish eggs. North American Journal of Aquaculture 65:349-355.
Hove, M. C. and M. Berg. 2008. Host fish requirements of upper Mississippi River Basin sheepnose (Plethobasus cyphyus) and spectaclecase (Cumberlandia monodonta). Wisconsin Department of Natural Resources, Madison, WI. 10 pp.
Hove, M. C., J. E. Kurth, and A. R. Kapuscinski. 1998. Brown bullhead suitable host for Tritogonia verrucosa; Cumberlandia monodonta host(s) remain elusive. Trianual Unionid Report No. 15. 13 pp.IS
Hove, M. C., N. Rudh, A. Morantes, J. Stevenson, B. Sietman, A. Crownhart, and B. Dickinson. 2008. Host fish requirements of select upper Mississippi River mussel species. Annual Report for the Minnesota Department of Natural Resources. St. Paul, MN. 9 pp.
Hove, M. C., D. J. Hornbach, B. E. Sietman, A. K. Crownhart, and M. S. Berg. 2009. Spectaclecase (Cumberlandia monodonta) host studies produce more negative results. Ellipsaria 11:22.
Keller, A. E. and S. G. Zam. 1990. Simplication of in vitro culture techniques for freshwater mussels. Environmental Toxicity and Chemistry 9:1291-1296.
Knudsen, K. A. and M. C. Hove. 1997. Spectaclecase (Cumberlandia monodonta) conglutinates unique, host(s) elusive. Trianual Unionid Report No. 11. 2 pp.
Lee, C. and M. C. Hove. 1997. Spectaclecase (Cumberlandia monodonta) host(s) still elusive. Trianual Unionid Report No. 12. 9 pp.
Lima, P., U. Kovitvadhi, S. Kovitvadhi and J. Machado. 2006. In vitro culture of glochidia from the freshwater mussel Anodonta cygnea. Invertebrate Biology 125:34-44.
Lima, R. C. and W. E. P. Avelar. 2010. A new additive to the artificial culture medium for freshwater bivalve culture in vitro. Invertebrate Reproduction and Development 54:89-94.
Lima, P., M. L. Lima, U. Kovitvadhi, S. Kovitvadhi, C. Owen and J. Machado. 2012. A review of the "in vitro" culture of freshwater mussels (Unionoida). Hydrobiologia 691:21-33.
Owen, C. T. 2009. Investigations for the conservation and propagation of freshwater mussels.
Dissertation, University of Louisvi111e, Louisville.
Owen, C. T., J. E. Alexander, Jr. and M. McGregor. 2010. Control of microbial contamination during in vitro culture oflarval unionid mussels. Journal ofinvertebrate Reproduction and Development. 54:187-193.
Piper, R.G.1986. Fish hatchery management. 2nd edition. U.S. Department of the Interior, Fish and Wildlife Service, Washington, D.C. 548 pp.
Roberts, A.D. and M. C. Barnhart. 1999. Effects of temperature, pH and C02 on transformation of the glochidia of Anodonta suborbiculata on fish hosts and in vitro. Journal of the North American Benthological Socieity 18:477-487.
U.S. Fish and Wildlife Service (USFWS). 2014. Recovery outline for the spectaclecase mussel
(Cumberlandia monodonta). January 2014. Region 3, FWS. 18 pp.
The spectaclecase mussel, Cumberlandia monodonta, was effectively listed as federally endangered in April 2012 (https://federalregister.gov/a/2012-5603). It is endemic to the Mississippi, Ohio and Missouri River basins and historically inhabited 44 streams in these basins (USFWS 2014). Currently, the species is known to inhabit 20 of the historical streams, five of which are represented by one or two specimens (Butler 2002). The three remaining strongholds of the species occur in the Meramec, Gasconade and St. Croix River systems. The existence of small, geographically isolated populations has been identified in the Spectaclecase Recovery Outline (USFWS 2014) as a key threat to the species. As such, the Recovery Outline lists captive propagation and augmentation of remaining populations as a top priority for recovery of the species. Despite numerous attempts and testing of over 60 species of fish, amphibians, crayfish and insects, a suitable host of the spectaclecase has not been identified (Knudsen and Hove 1997, Lee and Hove 1997, Hove et al. 1998, Baird 2000, Hove and Berg 2008, Hove et al. 2008, Hove et al. 2009).
In vitro transformation of glochidia is an alternative method for production of juveniles that has been successful for over 40 species unionids (Lima et al. 2012); but has not been reported for the spectaclecase. One of the major obstacles to successful in vitro transformation of glochidia is contamination from microorganisms, particularly fungus (Owen et al. 2010, Lima et al. 2012). Current protocols for in vitro culture recommend the use of antibiotics and antimycotics to reduce contamination, but none effectively control contamination for the duration of the incubation period and some are toxic to the glochidia (Owen et al. 2010). Hydrogen peroxide has been successfully used to reduce the incidence of Saprolegniasis on fish eggs (Gaikowsld et al. 2003) and inculture of fish cell lines (Eric Leis, La Crosse Fish Health Center, La Crosse, WI, personal communication). A pilot study was conducted in November 2015 in which Lampsilis cardium glochidia were treated with 100, 500 or 1000 mg/L hydrogen peroxide for 10 minutes. The treatment effectively reduced contamination and glochidia and cells remained viable for over 1 week (personal observation). Inthe present study, pretreatment with hydrogen peroxide will be compared with controls (no hydrogen peroxide) to determine differences in glochidial viability and transformation and levels of microbial contamination in culture plates.
We will evaluate in vivo and in vitro techniques to propagate juveniles of spectaclecase mussels by inoculation of potential host species and growth in an artificial medium. Results of the study will be relevant for recovery of the spectaclecase.
Objectives
- To generate and rank a list of potential glochidial hosts based on previous host studies and fish and amphibian survey data from the St. Croix River
- To determine the transformation success of spectaclecase glochidia on potential fish and amphibian species (identified in objective 1) by laboratory inoculation
- To evaluate in vitro culture methods, including culture media and decontamination treatments, for transformation of spectaclecase glochidia in an artificial medium.
References
Baird, M. S. 2000. Life history of the spectaclecase, Cumberlandia monodonta Say, 1829 (Bivalvia, Unionidea, Margaritiferidae). Master of Science. Southwest Missouri State University.
Burch, J. B. 1973. Freshwater unionacean clams (Mollusca: Pelecyopoda) of North America. United States Environmental Protection Agency Identification Manual No. 11. 176 pp.
Butler, R. S. 2002. Status assessment report for the spectaclecase, Cumberlandia monodonta, occurring in the Mississippi River system (U.S. Fish and Wildlife Service Regions 3, 4, 5, and 6). Ohio River Valley Ecosystem Team, Mollusk Subgroup, Ashville, NC.
Cummings, K. and C. A. Mayer. 1992. Field guide to freshwater mussels of the Midwest. Illinois Natural History Survey Manual. 194 pp
Eddy, S., J. Underhill, J. Barnrick, E. Cawley, and W. Jaques. 1978. How to know the freshwater fishes. 3'<l edition. Dubuque, IA, W.C. Brown.
Fritts, AK.,M. Barnhart, M. Bradley, N. Liu, W. G.Cope, E. Hammer, and R. Bringolf. 2013.
Assessment of toxicity test endpoints for freshwater mussel larvae (glochidia). Environmental Toxicology and Chemistry 33:199-207.
Gaikowski, M. P., J. J. Rach, M. Drobish, and 5 others. 2003. Efficacy of hydrogen peroxide in controlling mortality associated with saprolegniasis on walleye, white sucker, and paddlefish eggs. North American Journal of Aquaculture 65:349-355.
Hove, M. C. and M. Berg. 2008. Host fish requirements of upper Mississippi River Basin sheepnose (Plethobasus cyphyus) and spectaclecase (Cumberlandia monodonta). Wisconsin Department of Natural Resources, Madison, WI. 10 pp.
Hove, M. C., J. E. Kurth, and A. R. Kapuscinski. 1998. Brown bullhead suitable host for Tritogonia verrucosa; Cumberlandia monodonta host(s) remain elusive. Trianual Unionid Report No. 15. 13 pp.IS
Hove, M. C., N. Rudh, A. Morantes, J. Stevenson, B. Sietman, A. Crownhart, and B. Dickinson. 2008. Host fish requirements of select upper Mississippi River mussel species. Annual Report for the Minnesota Department of Natural Resources. St. Paul, MN. 9 pp.
Hove, M. C., D. J. Hornbach, B. E. Sietman, A. K. Crownhart, and M. S. Berg. 2009. Spectaclecase (Cumberlandia monodonta) host studies produce more negative results. Ellipsaria 11:22.
Keller, A. E. and S. G. Zam. 1990. Simplication of in vitro culture techniques for freshwater mussels. Environmental Toxicity and Chemistry 9:1291-1296.
Knudsen, K. A. and M. C. Hove. 1997. Spectaclecase (Cumberlandia monodonta) conglutinates unique, host(s) elusive. Trianual Unionid Report No. 11. 2 pp.
Lee, C. and M. C. Hove. 1997. Spectaclecase (Cumberlandia monodonta) host(s) still elusive. Trianual Unionid Report No. 12. 9 pp.
Lima, P., U. Kovitvadhi, S. Kovitvadhi and J. Machado. 2006. In vitro culture of glochidia from the freshwater mussel Anodonta cygnea. Invertebrate Biology 125:34-44.
Lima, R. C. and W. E. P. Avelar. 2010. A new additive to the artificial culture medium for freshwater bivalve culture in vitro. Invertebrate Reproduction and Development 54:89-94.
Lima, P., M. L. Lima, U. Kovitvadhi, S. Kovitvadhi, C. Owen and J. Machado. 2012. A review of the "in vitro" culture of freshwater mussels (Unionoida). Hydrobiologia 691:21-33.
Owen, C. T. 2009. Investigations for the conservation and propagation of freshwater mussels.
Dissertation, University of Louisvi111e, Louisville.
Owen, C. T., J. E. Alexander, Jr. and M. McGregor. 2010. Control of microbial contamination during in vitro culture oflarval unionid mussels. Journal ofinvertebrate Reproduction and Development. 54:187-193.
Piper, R.G.1986. Fish hatchery management. 2nd edition. U.S. Department of the Interior, Fish and Wildlife Service, Washington, D.C. 548 pp.
Roberts, A.D. and M. C. Barnhart. 1999. Effects of temperature, pH and C02 on transformation of the glochidia of Anodonta suborbiculata on fish hosts and in vitro. Journal of the North American Benthological Socieity 18:477-487.
U.S. Fish and Wildlife Service (USFWS). 2014. Recovery outline for the spectaclecase mussel
(Cumberlandia monodonta). January 2014. Region 3, FWS. 18 pp.