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Ms. Carol Riegelman, UMCES~MDSG Teacher Research Fellowship 2003 Dr. Allen R. Place Center of Marine Biotechnology Baltimore, MD Investigating the toxicity.

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Presentation on theme: "Ms. Carol Riegelman, UMCES~MDSG Teacher Research Fellowship 2003 Dr. Allen R. Place Center of Marine Biotechnology Baltimore, MD Investigating the toxicity."— Presentation transcript:

1 Ms. Carol Riegelman, UMCES~MDSG Teacher Research Fellowship 2003 Dr. Allen R. Place Center of Marine Biotechnology Baltimore, MD Investigating the toxicity of the dinoflagellate Karlodinium micrum Fish kill. JoAnn Burkholder, North Carolina State University

2 Research Project I worked with Dr. Allen R. Place of the Center of Marine Biotechnology as part of the UMCES~Maryland Sea Grant Teacher Research Fellowship during the summer of 2003. The research project concerned Investigating the toxicity of the dinoflagellate Karlodinium micrum. Classroom Project One of my tasks during the Teacher Research Fellowship was to develop an application for the classroom based upon my research project. The activity I developed is entitled, “What is Killing Maryland’s Fish?”.

3 Investigating the toxicity of the dinoflagellate Karlodinium micrum A. Place SEM of K. micrum collected from Pokomoke River, MD, 1997 Image provided by Florida Marine Research Institute.

4 Background Research 1997 fish kill in Chesapeake Bay, series of fish kills in North Carolina heighten awareness of toxic algae Menhaden with skin lesions Maryland Department of Natural Resources, 1997

5 The dinoflagellate Pfiesteria piscicida is thought to be the culprit.

6 Dinoflagellates Karlodinium micrum and Pfiesteria piscicida are dinoflagellates Type of phytoplankton or algae K. micrum can photosynthesize or graze while P. piscicida is a grazer Use 2 flagella to move around Karlodinium micrum and Pfiesteria piscicida are dinoflagellates Type of phytoplankton or algae K. micrum can photosynthesize or graze while P. piscicida is a grazer Use 2 flagella to move around Light microscopy image of K. micrum in culture showing two flagella, J. Deeds

7 PROBLEM #1 A separate fish kill at HyRock Fish Farm in Chesapeake Bay is examined. Large quantities of both Pfiesteria piscicida and a lesser-known dinoflagellate called Karlodinium micrum on the site. Q: Which one killed the fish? A separate fish kill at HyRock Fish Farm in Chesapeake Bay is examined. Large quantities of both Pfiesteria piscicida and a lesser-known dinoflagellate called Karlodinium micrum on the site. Q: Which one killed the fish? Fish kill at Hyrock Fish Farm, D. Terlizzi, MD Sea Grant Extension Program

8 At COMB, Dr. Allen Place and Dr. Jon Deeds found K. micrum to be dominant species at fish kill sites. K. micrum toxin was isolated using liquid chromatography This showed K. micrum toxin kills small fish by entering through the gills and eroding epidermis At COMB, Dr. Allen Place and Dr. Jon Deeds found K. micrum to be dominant species at fish kill sites. K. micrum toxin was isolated using liquid chromatography This showed K. micrum toxin kills small fish by entering through the gills and eroding epidermis Light microscopy of K. micrum from fish kills in 2003 From the Swan and Canning River estuaries in Perth, Australia

9 PROBLEM #2 It is determined that K. micrum kills fish, but how does it interact with other organisms in the ecosystem? Samples and behavior examined under the microscope. Hypothesize their role in ecosystem. It is determined that K. micrum kills fish, but how does it interact with other organisms in the ecosystem? Samples and behavior examined under the microscope. Hypothesize their role in ecosystem.

10 General Food Chain Diatoms, Dinoflagellates Rotifers Crab zoea, megalopes Fish Humans Diatoms, Dinoflagellates Rotifers Crab zoea, megalopes Fish Humans

11 PROBLEM #3 K. micrum is not lethal to other organisms in the ecosystem. May have some sub-lethal effects. Why does K. micrum kill fish but have little effect on other organisms? K. micrum is not lethal to other organisms in the ecosystem. May have some sub-lethal effects. Why does K. micrum kill fish but have little effect on other organisms?

12 Place lab at COMB is trying to answer that question right now. Current Hypotheses K. micrum toxin is lipid-soluble Toxin attacks specific sterols found in fish and other vertebrate cell membranes Leaves invertebrate sterols alone in self-preservation Place lab at COMB is trying to answer that question right now. Current Hypotheses K. micrum toxin is lipid-soluble Toxin attacks specific sterols found in fish and other vertebrate cell membranes Leaves invertebrate sterols alone in self-preservation

13 Fellowship Research Question: What are the acute and sub-lethal effects of the K. micrum toxin on other organisms? Hypothesis: Grazers agitate K. micrum and cause toxin release. Question: What are the acute and sub-lethal effects of the K. micrum toxin on other organisms? Hypothesis: Grazers agitate K. micrum and cause toxin release.

14 Skills needed: Maintain K. micrum Maintain B. plicatilis Skills needed: Maintain K. micrum Maintain B. plicatilis R. Hanewald

15 Filter and isolate the toxin Determine if toxin is still potent using hemolytic assay Assess acute toxicity and sub-lethal effects Filter and isolate the toxin Determine if toxin is still potent using hemolytic assay Assess acute toxicity and sub-lethal effects R. Hanewald

16 Rotifer Experiment Tested the acute toxicity of K. micrum toxin on rotifers Rotifers slightly effected when exposed to 1.5% and 3% crude toxin Negative control: seawater Positive control: 8% copper Tested the acute toxicity of K. micrum toxin on rotifers Rotifers slightly effected when exposed to 1.5% and 3% crude toxin Negative control: seawater Positive control: 8% copper R. Hanewald

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18 Rotifer Study Rotifers only affected when exposed to extremely concentrated toxin

19 Rotifer Experiment Examined sub-lethal effects of K. micrum on rotifers Toxin had little impact Egg production decreased when K. micrum only food source Examined sub-lethal effects of K. micrum on rotifers Toxin had little impact Egg production decreased when K. micrum only food source R. Hanewald

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21 Crab Experiment Examined acute toxicity on young blue crab, Callinectes sapidus Toxin had no effect on zoea or juvenile crabs Zoea A. Findiesen, COMB

22 Megalope Crab Experiment Toxin killed megalopes Methanol control also killed megalopes Further toxin concentration needed Toxin killed megalopes Methanol control also killed megalopes Further toxin concentration needed A. Findiesen, COMB

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24 Diatom Experiment Using UV-microscope, assessed K. micrum toxicity to Chaetoceros convolutus Immune to filtrant; Affected by high doses of concentrated toxin Using UV-microscope, assessed K. micrum toxicity to Chaetoceros convolutus Immune to filtrant; Affected by high doses of concentrated toxin Jan Rines (http://thalassa.gso.uri.edu/HABChaet/)

25 Hemolytics Conducted hemolytic assay to determine if toxin is active Extracted red blood cells from rainbow trout Determined if toxin lyses red blood blood cells Conducted hemolytic assay to determine if toxin is active Extracted red blood cells from rainbow trout Determined if toxin lyses red blood blood cells

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27 Conclusion K. micrum, toxic dinoflagellate, causes fish kills Toxin had some acute and sub-lethal effects Community can be used as ecology model in the classroom K. micrum, toxic dinoflagellate, causes fish kills Toxin had some acute and sub-lethal effects Community can be used as ecology model in the classroom

28 Classroom Connections Title: What is killing Maryland’s fish? Objective: Students will be able to observe relationships within an estuarine ecosystem in order to better understand ecological concepts Audience: 9 th grade Biology Title: What is killing Maryland’s fish? Objective: Students will be able to observe relationships within an estuarine ecosystem in order to better understand ecological concepts Audience: 9 th grade Biology

29 MD Core Learning Goals Expectation 3.5 The student will investigate the interdependence of diverse living organisms and their interactions with the components of the biosphere. Indicator 3.5.2 The student will analyze the interrelationships and interdependencies among different organisms … Expectation 3.5 The student will investigate the interdependence of diverse living organisms and their interactions with the components of the biosphere. Indicator 3.5.2 The student will analyze the interrelationships and interdependencies among different organisms …

30 Engagement Engagement: Students examine a photograph of a fish kill and brainstorm as a class what might have caused it. Activates and assesses prior knowledge Engagement: Students examine a photograph of a fish kill and brainstorm as a class what might have caused it. Activates and assesses prior knowledge

31 Exploration Exploration: Students observe an estuarine water sample under the microscope. Sample contains algae, dinoflagellates, rotifers, crab larvae, and zebra fish. Students identify the organisms, measure physical characteristics, and describe any relationships visible between organisms (i.e. predation) Exploration: Students observe an estuarine water sample under the microscope. Sample contains algae, dinoflagellates, rotifers, crab larvae, and zebra fish. Students identify the organisms, measure physical characteristics, and describe any relationships visible between organisms (i.e. predation) A. Place

32 Video Clip of Dinoflagellate Ingestion Click here ---> Ingestion of cryptophyts by K. micrum D. Wayne Coates, SERC (http://www.serc.si.edu/protist/images_mixo.htm)

33 Explanation Explanation: Students discuss as a class what they observed in the water sample. Teacher facilitates discussion by putting together student observations and ecological concepts Students fill in graphic organizer of ecological concepts based on class discussion Explanation: Students discuss as a class what they observed in the water sample. Teacher facilitates discussion by putting together student observations and ecological concepts Students fill in graphic organizer of ecological concepts based on class discussion

34 Extension Extension: Students read article “The Rise in Toxic Tides” about toxic dinoflagellates (Science News, Vol. 152, Sep. 27, 1997 or on-line at http://www.sciencenews.org/pages/archives/ft_1997.asp) In groups of 4, students create a food web based on their observations of the marine ecosystem Students illustrate on their food web the effect a toxic dinoflagellate could have on the system as a whole Extension: Students read article “The Rise in Toxic Tides” about toxic dinoflagellates (Science News, Vol. 152, Sep. 27, 1997 or on-line at http://www.sciencenews.org/pages/archives/ft_1997.asp) In groups of 4, students create a food web based on their observations of the marine ecosystem Students illustrate on their food web the effect a toxic dinoflagellate could have on the system as a whole

35 Extension “Above and Beyond” Interested students can research why toxic algal blooms have become more common in recent years. This should lead to an understanding of the human effects on the environment “Above and Beyond” Interested students can research why toxic algal blooms have become more common in recent years. This should lead to an understanding of the human effects on the environment

36 Evaluation Evaluation: Individually, students compose a brief constructed response answering the question “What is killing Maryland’s fish?” Students are assessed based on their understanding of the ecosystem they observed and the ecological concepts learned Evaluation: Individually, students compose a brief constructed response answering the question “What is killing Maryland’s fish?” Students are assessed based on their understanding of the ecosystem they observed and the ecological concepts learned

37 Credits Center of Marine Biotechnology Dr. Allen Place Jon Deeds Carla Berard Odi Zmora Center of Marine Biotechnology Dr. Allen Place Jon Deeds Carla Berard Odi Zmora

38 References Blankenship, K. 2002 Scientists isolate toxin from fish-killing algae in watershed. Bay Journal 12 (8) 6-7. Coates, D. Wayne. Ingestion of cryptophyts by K. micrum, http://www.serc.si.edu/protist/images_mixo.htm Deeds, J.R., Terlizzi, D.E., Adolf, J.E., Stoecker, D.K., Place, A.R. 2002 Toxic activity from cultures of Karlodinium micrum (= Gyrodinium galatheanum) (Dinophycaea) – a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility. Harmful Algae 1 169-189. Greer, J., Leffler, M., Belas, R., Kramer, J., Place, A. Molecular Technologies and Pfiesteria Research: A Scientific Synthesis. Center of Marine Biotechnology, Maryland Sea Grant College, USDA Agricultural Research Service, 1997. John, U., Tillman, U., Medlin, L.K., 2002 A comparative approach to study inhibition of grazing and lipid composition of a toxic and non-toxic clone of Chrysochromulina polylepis (Prymnesiophyceae) Harmful Algae 1 45-57. Kempton, J.W., Lewitus, A.J., Deeds, J.R., Law, J.M., Place, A.R. 2002 Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond. Harmful Algae 1 233-241. Mlot, C., 1997 The Rise in Toxic Tides: What’s behind the ocean blooms? Science News 152 (13), 202-204. Pesticides, Resources Recovery, Hazardous Substances and Oil Spill Responses, Waste Management, Biological Effects. 1992 Annual Book of ASTM Standards. 11.04 Rines, Jan. Inimical Chaetoceros, http://thalassa.gso.uri.edu/HABChaet/ Rosetta, C.H., McManus, G.B. 2003 Feeding by ciliates on two harmful algal bloom species, Prymnesium parvum and Prorocentrum minimum Harmful Algae 2 109-126 Blankenship, K. 2002 Scientists isolate toxin from fish-killing algae in watershed. Bay Journal 12 (8) 6-7. Coates, D. Wayne. Ingestion of cryptophyts by K. micrum, http://www.serc.si.edu/protist/images_mixo.htm Deeds, J.R., Terlizzi, D.E., Adolf, J.E., Stoecker, D.K., Place, A.R. 2002 Toxic activity from cultures of Karlodinium micrum (= Gyrodinium galatheanum) (Dinophycaea) – a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility. Harmful Algae 1 169-189. Greer, J., Leffler, M., Belas, R., Kramer, J., Place, A. Molecular Technologies and Pfiesteria Research: A Scientific Synthesis. Center of Marine Biotechnology, Maryland Sea Grant College, USDA Agricultural Research Service, 1997. John, U., Tillman, U., Medlin, L.K., 2002 A comparative approach to study inhibition of grazing and lipid composition of a toxic and non-toxic clone of Chrysochromulina polylepis (Prymnesiophyceae) Harmful Algae 1 45-57. Kempton, J.W., Lewitus, A.J., Deeds, J.R., Law, J.M., Place, A.R. 2002 Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond. Harmful Algae 1 233-241. Mlot, C., 1997 The Rise in Toxic Tides: What’s behind the ocean blooms? Science News 152 (13), 202-204. Pesticides, Resources Recovery, Hazardous Substances and Oil Spill Responses, Waste Management, Biological Effects. 1992 Annual Book of ASTM Standards. 11.04 Rines, Jan. Inimical Chaetoceros, http://thalassa.gso.uri.edu/HABChaet/ Rosetta, C.H., McManus, G.B. 2003 Feeding by ciliates on two harmful algal bloom species, Prymnesium parvum and Prorocentrum minimum Harmful Algae 2 109-126


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