STACKED BT PROTEINS EXACERBATE NEGATIVE GROWTH EFFECTS OF JUVENILE (F. RUSTICUS) CRAYFISH FED CORN DIET Molly West A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE May 2019 Committee: Paul Moore, Advisor Eric Hellquist Helen Michaels ii ABSTRACT Paul Moore, Advisor The adoption of genetically modified (GM) crops has occurred rapidly in the United States. The transfer of GM corn byproducts from agricultural fields to nearby streams after harvest is significant and occurs well into the post-harvest year. These corn leaves, stems and cobs then become a detrital food source for organisms such as shredders in the stream ecosystem. Considering non-target effects of Bt corn have been observed in some terrestrial organisms, we aimed to assess whether Bt toxins affect an important aquatic organism, juvenile F. rusticus crayfish. Juvenile crayfish were fed six distinct diet treatments: two varieties of Bt corn, two varieties of herbicide tolerant corn, and two controls: fish gelatin and river detritus. Juveniles were fed these diets while housed in flow-through artificial streams that received natural stream water from a local source. Specific growth rate and survivorship of the crayfish were measured throughout the study. Juveniles fed corn diets grew significantly less and had reduced survival when compared to juveniles fed fish gelatin or river detritus diets. Furthermore, juveniles fed one Bt variety of corn (VT Triple Pro) exhibited significantly less growth than those fed one of the herbicide tolerant varieties (Roundup Ready 2). Our study shows that corn inputs to streams near agricultural fields may be detrimental to the growth and survivorship of juvenile crayfish and that certain Bt varieties may exacerbate these negative effects. These effects on crayfish will have repercussions for the entire ecosystem, as crayfish are conduits of energy between many trophic levels. iii For my existence-mate and our two precious progeny. iv ACKNOWLEDGMENTS We thank the members of the Laboratory for Sensory Ecology, Bowling Green State University, for their assistance in collection and care of specimens, as well as for reviewing the manuscript. We would also like to thank the University of Michigan Biological Station for funding through the Mariam P. and David M. Gates Graduate Student Endowment Fund to M.E.J.W. and also for the use of facilities. Lastly, thanks to the Bowling Green State University Faculty Research Committee for a Building Strength Award and a Fulbright Fellowship to P.A.M. for help in funding this project. v TABLE OF CONTENTS Page INTRODUCTION ................................................................................................................. 1 MATERIALS & METHODS ................................................................................................ 6 Animals ...................................................................................................................... 6 Experimental Design .................................................................................................. 6 Diets ........................................................................................................................... 7 GM Corn ........................................................................................................ 7 Naturally-Occurring Detritus ......................................................................... 8 Fish Gelatin .................................................................................................... 8 Experimental Treatment Areas .................................................................................. 9 Data Analysis ............................................................................................................. 9 RESULTS .............................................................................................................................. 11 Survival ...................................................................................................................... 11 Growth ....................................................................................................................... 11 DISCUSSION ........................................................................................................................ 12 REFERENCES ...................................................................................................................... 17 APPENDIX A – TABLES ..................................................................................................... 27 APPENDIX B – FIGURES ................................................................................................... 28 1 INTRODUCTION The United States rapidly embraced the implementation of genetically modified (GM) crops (Klerck & Sweeney, 2007). The adoption of these products by farmers is estimated to increase their profits by 69% by reducing pesticide costs and increasing crop yields (Klümper & Qaim, 2014). Up to 80% of processed foods in the U.S. contain GM plants, largely due to corn products such as cornstarch and corn syrup (Hemphill & Banerjee, 2015). The percentage of corn crops that are GM in the United States increased from 25% in 2000 to 92% in 2018 (USDA, 2018). Along with this widespread adoption of GM products came an increase in the number of genetic modifications within single GM plant varieties (Taverniers et al., 2008). Currently, there are two main types of gene insertions in GM corn crops—insecticidal (Bt) and herbicide-tolerant (HT). Insecticidal genes produce Bt toxins that cause lesions in the membranes of cells within the midgut of specific orders of insects (Soberón et al., 2007). HT genes confer crop tolerance to glyphosate and glufosinate herbicides (Firbank et al., 2003). When GM corn was first released on the market, each variety contained one of these gene insertions (i.e. transgenes) (Que et al., 2010). As a result of the development of pesticide resistance in target insects, multiple insect resistances and herbicide tolerances have been inserted into the genome of single plant species. This “stacked” or “pyramided” GM corn now makes up 80% of all corn in the United States (USDA, 2018). As an example, one current GM corn product includes transgenes for controlling multiple varieties of both lepidopteran and coleopteran pests as well as transgenes that provide resistance to glyphosate and glufosinate herbicides (EPA, 2011). Given the increase in both the amount of GM corn being planted and the number of transgenes within that corn, assessing the impact of these products on ecosystems becomes increasingly important. 2 Terrestrial non-target organisms are a main focus of research regarding the safety of Bt crops. However, corn detritus is not confined to agricultural fields or the surrounding terrestrial habitats because wind and rain move detritus significant distances. For example, corn components enter streams through multiple pathways, but the greatest quantity comes from decomposed byproducts left on the field after harvest (Griffiths et al., 2009; Zwahlen et al., 2003). Corn byproducts, such as leaves, stalks and cobs, travel into streams via wind transport and surface runoff (Viktorov, 2011), which mostly occur immediately after harvest but can extend into the next year. Jensen et al. (2010) found that the highest input of corn byproduct into streams was delayed until February or March of the post-harvest year. The presence of detritus (allochthonous material) in streams is a vital source of energy which passes through trophic levels by multiple processes—chemical leaching, physical abrasion, microbial decomposition and the shredding of material by macroinvertebrates (Graça & Canhoto, 2006). The macroinvertebrates that serve as detrital shredders in stream ecosystems include taxa such as amphipods, caddisflies and stoneflies, but also larger invertebrates such as crayfish (Graça, 2001). Since crop detrital inputs are found in streams from October through April of the next year, organisms within these aquatic ecosystems are exposed to transgenic corn as a possible food source for potentially half the year (Jensen et al., 2010). Corn byproducts occurring in streams near agricultural fields in Indiana have been found to range from 0.1 to 7.9 g ash-free dry mass/m2 (Rosi-Marshall et al., 2007), with isotope analysis indicating that 17-22% of terrestrial organic carbon in Midwestern streams originates from corn (Dalzell et al., 2005). Moreover, the active protein in Bt maize, Cry1AB, may persist in watersheds because of various pathways of entry from terrestrial to aquatic habitats (Griffiths et al., 2017). Although 3 accumulation of maize detritus within streams shows no clear spatial pattern, there is potential for exposure to a variety of organisms within these ecosystems due to stream flow (Tank et al., 2010). Multiple aquatic organisms have been shown to exhibit negative effects following exposure to Bt corn. Daphnia magna, an aquatic crustacean, fed transgenic corn was found to have higher mortality rates, lower fecundity and less population growth than those fed isogenic strains (Bøhn et al., 2008, Bøhn et al., 2010). Multiple studies have also found detrimental effects on the growth rates and survival of two species of Trichoptera, a non-target order of insects, exposed to Bt corn (Chambers et al., 2010; Rosi-Marshall et al., 2007), as well as of C. dilutus, an aquatic midge (Li