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ABSTRACT ZHA, CHEN. Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae

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ABSTRACT ZHA, CHEN. Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae ABSTRACT ZHA, CHEN. Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae. (Under the direction of Allen Cohen.) Mold control is one of the most vital issues in insect rearing systems. Mold outbreaks can alter the nutritional value of the diet, do harm to the insects, and even threaten the health of people who work in insectaries. Antifungal agents are widely used in insect diets to suppress the growth of mold; however, the potential detrimental effects of antifungal agents on insects are always a concern. When there is a high level of antifungal agents in artificial diets, the growth, development, survivorship and fecundity of insects are often negatively affected. To study the mechanisms underlying those deleterious effects, nutritional indices were determined for two representative lepidopterans, a butterfly and a moth, Vanessa cardui and Heliothis virescens larvae reared on different concentrations of three widely used antifungal agents: methyl paraben, potassium sorbate and sodium propionate. These antifungal agents were administered in concentrations of 1,000, 5,000, and 10,000 parts per million (ppm). The results show that a high level of antifungal agents in the diets of these insects will suppress nutrient absorption and increase metabolic costs. Relative consumption rates and digestibility increased with increasing antifungal agent concentration, possibly to compensate for the declines in absorption and metabolism. Silk production and frass management by Vanessa cardui larvae were reduced in the presence of the highest level of antifungal agents. Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae by Chen Zha A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirement for the Degree of Master of Science Entomology Raleigh, North Carolina 2013 APPROVED BY: ________________________ ________________________ Allen C. Cohen Yasmin J. Cardoza Chair of Advisory Committee ________________________ ________________________ David Orr Clyde Sorenson © Copyright 2013 by Chen Zha All Rights Reserved BIOGRAPHY Chen Zha was born April 11th, 1989 in Anhui province, China. His love of insects revealed itself during his childhood. He studied life sciences in University of Science and Technology of China. After having received his BS degree, he shifted his major into entomology under the direction of Dr. Allen C. Cohen at North Carolina State University. His first research effort in entomology focused on the influences of antifungal agents on insect nutritional ecology, and he plans on working with chemical ecology of bed bugs and getting a PhD in entomology at Rutgers University. ii ACKNOWLEDGEMENTS I would like to thank everyone who helped me along the way, my advisor Dr. Allen Cohen, fellow graduate students, professors and staff, and my parents who provide me economical support studying abroad. Special thanks go to Drs. Yasmin J. Cardoza, David Orr and Clyde Sorenson for being members of my graduate committee and for providing much help and advice to guide my work. I also thank Dr. Fred Gould for providing H. virescens eggs from his lab colony, and Sandra Paa for egg collection and technical support in rearing H. virescens. Finally, I thank Dr. Consuelo Arellano for helping with the statistical analysis of my data. iii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ v LIST OF FIGURES.......................................................................................................... vi Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae .............. 1 INTRODUCTION ................................................................................................. 2 MATERIALS AND METHODS ......................................................................... 16 RESULTS ............................................................................................................. 27 DISCUSSION ....................................................................................................... 30 REFERENCES .................................................................................................... 46 iv LIST OF TABLES Table 1. The ingredients of YC diet ................................................................................... 63 Table 2. Effects of antifungal agents on V. cardui feeding and growth ............................... 64 Table 3. Effects of antifungal agents on H. virescens feeding and growth .......................... 65 Table 4. Percent V. cardui larvae fed on diet columns contain different concentrations of antifungal agents ............................................................................................................... 66 Table 5. Percent H. virescens larvae fed on diet columns contain different concentrations of antifungal agents ............................................................................................................... 67 Table 6. Percent larvae fed on different diet columns with different pH ............................. 68 v LIST OF FIGURES Figure 1. The effects of different concentrations of 3 antifungal agents on frass production ... .......................................................................................................................................... 69 Figure 2. The effects of different concentrations of 3 antifungal agents on insect weight gain .......................................................................................................................................... 70 Figure 3. The effects of different concentrations of 3 antifungal agents on consumption rate (CR) .................................................................................................................................. 71 Figure 4. The effects of different concentrations of 3 antifungal agents on relative consumption rate (RCR) .................................................................................................... 72 Figure 5. The effects of different concentrations of 3 antifungal agents on approximate digestibility (AD) .............................................................................................................. 73 Figure 6. The effects of different concentrations of 3 antifungal agents on the efficiency of conversion of ingested food to biomass (ECI) .................................................................... 74 Figure 7. The effects of different concentrations of 3 antifungal agents on the efficiency of conversion of digested food to biomass (ECD) .................................................................. 75 Figure 8. Comparison among the 3 antifungal agents on frass production .......................... 76 Figure 9. Comparison among the 3 antifungal agents on insect weight gain ....................... 77 Figure 10. Comparison among the 3 antifungal agents on consumption rate (CR) .............. 78 Figure 11. Comparison among the 3 antifungal agents on relative consumption rate (RCR) ... .......................................................................................................................................... 79 Figure 12. Comparison among the 3 antifungal agents on approximate digestibility (AD) . 80 Figure 13. Comparison among the 3 antifungal agents on the efficiency of conversion of ingested food to biomass (ECI) .......................................................................................... 81 Figure 14. Comparison among the 3 antifungal agents on the efficiency of conversion of digested food to biomass (ECD) ........................................................................................ 82 Figure 15. Freeze dried diet sample of 10,000ppm methyl paraben treatment, showing the presence of white methyl paraben crystals which insects avoided to ingest ........................ 83 vi Figure 16. “Frass management” of a V. cardui larva, showing that the frass was attached with silk and kept away from diet .............................................................................................. 84 Figure 17. A comparison of V. cardui frass management behavior between 1,000 and 10,000ppm potassium sorbate (KS1 & KS3) treatments ..................................................... 85 vii Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae C. Zha Department of Entomology, North Carolina State University Campus Box 7613, Raleigh, NC 27695 1 INTRODUCTION Background on antifungal agents in insect diets Laboratory rearing of insects has significant importance for studying their nutrition, biochemistry, behavior and other biological processes (Vanderzant 1974). Mass production of insects is an important process for biological control and sterile insect technologies (Knipling 1955; Klassen & Curtis 2005), feeding other animals (Versoi & French 1992), production of pharmaceuticals and recombinant proteins as bioreactors (Hughes and Wood 1998), and food for people (DeFoliart 1999). Insect artificial diets have been widely used for laboratory rearing and mass production of insects (Vanderzant 1974). Microbial contamination in artificial diet is one of the most serious problems in insect rearing systems. Aspergillus niger, A. flavus, Cladosporium spp., Fusarium spp., Rhizopus nigricans, Penicillium spp. and some yeasts and bacteria are among the most commonly found microorganisms in insect diets (Cohen 2003; Bell et al. 1980; Ouye 1962).
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