Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 4-16-2014 Mosquito Larvicides from Cyanobacteria Gerald A. Berry Florida International University, [email protected] DOI: 10.25148/etd.FI14071114 Follow this and additional works at: https://digitalcommons.fiu.edu/etd Recommended Citation Berry, Gerald A., "Mosquito Larvicides from Cyanobacteria" (2014). FIU Electronic Theses and Dissertations. 1449. https://digitalcommons.fiu.edu/etd/1449 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida MOSQUITO LARVICIDES FROM CYANOBACTERIA A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in BIOLOGY by Gerald A. Berry 2014 To: Interim Dean Michael Heithaus College of Arts and Sciences This dissertation, written by Gerald A. Berry, and entitled Mosquito Larvicides from Cyanobacteria, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. _____________________________ Evelyn Gaiser _____________________________ Miroslav Gantar _____________________________ Kathleen Rein _____________________________ John P. Berry, Co-Major Professor _____________________________ Fernando G. Noriega, Co-Major Professor Date of defense: April 16, 2014 The dissertation of Gerald A. Berry is approved. _____________________________ Interim Dean Michael Heithaus College of Arts and Sciences _____________________________ Dean Lakshmi N. Reddi University Graduate School Florida International University, 2014 ii DEDICATION This dissertation is dedicated to my family for supporting me in this long endeavor and to the Universe for being such a fascinating venue inspiring me to learn all that I can. iii ACKNOWLEDGMENTS I would like to thank Dr. Pat Gibbs for his efforts in the DNA analysis. I thank Mayra Exposito for instructions in regards to IR operations. I thank Dr. Wentian Wang for advice and all around help. I thank my committee Dr. Evelyn Gaiser, Dr. Miroslav Gantar, Dr. Kathleen Rein, and Dr. Fernando G. Noriega for their instrumental guidance in the design and overall direction of my dissertation. I would also like to thank members of the Noriega laboratory (Dr. Mario Perez and Dr. Marcela Nouzova) for guidance in regards to rearing mosquito larvae and overall assay setup. Lastly, I would like to thank Dr. John P. Berry and laboratory members (Dr. Asha Jaja, Katherine Walton, and Dalton Steel) for efforts far too numerous to mention here. iv ABSTRACT OF THE DISSERTATION MOSQUITO LARVICIDES FROM CYANOBACTERIA by Gerald A. Berry Florida International University, 2014 Miami, Florida Professor John P. Berry, Co-Major Professor Professor Fernando G. Noriega, Co-Major Professor Cyanobacteria (blue-green algae) produce a diverse array of toxic or otherwise bioactive metabolites. These allelochemicals may also play a role in defense against potential predators and grazers, particularly aquatic invertebrates and their larvae, including mosquitoes. Compounds derived from cyanobacteria collected from the Florida Everglades and other Florida waterways were investigated as insecticides against the mosquito Aedes aegypti, a vector of dengue and yellow fever. Screening of cyanobacterial biomass revealed several strains that exhibited mosquito larvicidal activity. Guided via bioassay guided fractionation, a non-polar compound from Leptolyngbya sp. 21-9-3 was found to be the most active component. Characterization revealed the prospective compound to be a monounsaturated fatty acid with the molecular formula C16H30O2. This is the first evidence of mosquito larvicidal activity for this particular fatty acid. With larvicidal becoming more prevalent, fatty acids should be explored for future mosquito control strategies. v TABLE OF CONTENTS CHAPTER PAGE Chapter 1: Background ....................................................................................................... 1 1.1 Introduction ............................................................................................................. 1 1.2 Mosquitoes as disease vectors ................................................................................ 1 1.3 Targeting aquatic mosquito larvae instead of aerial adults ..................................... 3 1.4 The need for novel insecticides............................................................................... 4 1.4.1 Inadequacies of pyrethroids ........................................................................... 4 1.4.2 Human risk to pyrethroids ............................................................................. 6 1.4.3 Insect resistance to pyrethroids ...................................................................... 6 1.5 Cyanobacteria as a potential source for arthrocidal compounds ............................ 8 1.5.1 Introduction .................................................................................................... 8 1.5.2 Chemical defense against planktivores .......................................................... 8 1.5.2.1 Apparency theory .................................................................................. 8 1.5.2.2 Quantitative defenses of Microcystis aerugunosa against Daphnia ..... 9 1.6 Cyanobacteria as a potential source for mosquito larvicidal compounds ............. 10 1.6.1 Cyanobacteria and mosquito sympatry ........................................................ 10 1.6.2 Grazer choice of food within periphyton ..................................................... 11 1.6.3 Mosquito larvae feeding on periphyton ....................................................... 12 1.6.4 Cyanobacteria: Novel insecticides and candidates for biological control agents .................................................................................................................... 13 1.6.5 Effects of anatoxin-a and microcystin-LR on mosquito larvae ................... 15 Chapter 2: Mosquito Larvicidal Assay ............................................................................. 17 2.1 The larval developmental stage of choice ............................................................. 17 2.2 Development of the mosquito larvicidal assay ..................................................... 17 2.2.1 Treatments and resuspensions...................................................................... 17 2.2.2 Mosquito larvae: Egg production and handling of larvae ............................ 19 2.2.3 Evaluation of mosquito larvae ..................................................................... 21 2.2.4 Evaluation of negative controls and resuspensions on mosquito larvae ...... 21 2.2.5 Evaluation positive controls on mosquito larvae ......................................... 23 Chapter 3: Bioassay Guided Fractionation ....................................................................... 26 3.1 Background ........................................................................................................... 26 3.2 Bioassays............................................................................................................... 26 3.3 Fractionation ......................................................................................................... 27 3.4 Characterization .................................................................................................... 28 3.5 Proof of Principle .................................................................................................. 29 3.5.1 Introduction .................................................................................................. 29 3.5.2 Original purification of sulfated glycolipids ................................................ 29 3.5.3 Re-purification and bioassay guided fractionation of sulfated glycolipids . 31 3.5.4 Identification of re-purified of sulfated glycolipids ..................................... 31 3.5.5 Discussion .................................................................................................... 32 vi Chapter 4: Isolation and Characterization of Mosquito Larvicidal Compounds from Cyanobacteria ................................................................................................................... 34 4.1 Introduction ........................................................................................................... 34 4.2 Methods................................................................................................................. 35 4.2.1 Screening of cyanobacterial isolates ............................................................ 35 4.2.2 Screening of cyanobacterial biomass ........................................................... 35 4.2.3 Bioassay guided fractionation of 3 prospective larvicidal cyanobacteria .... 35 4.2.4 Purification of larvicidal compounds from Leptolyngbya sp. 21-9-3 .......... 37 4.2.5 Characterization of larvicidal compounds from Leptolyngbya sp. 21-9-3 .. 42 4.2.5.1 Liquid chromatography – mass spectrometry ..................................... 42 4.2.5.2 Nuclear magnetic resonance ............................................................... 42 4.2.5.3 Infrared spectroscopy