Stacked Bt Proteins Exacerbate Negative Growth Effects of Juvenile (F. Rusticus) Crayfish Fed Corn Diet

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.

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 et al., 2013; Prihoda & Coats, 2008). Since Bt toxins are exhibiting negative effects on non-target organisms, the mechanism behind its proposed specificity to exclusive orders of insects has been reexamined.

Although different models exist for the cytotoxic mechanism of Bt toxin, all of the models coincide in a crucial way. After ingestion, the protoxin form of the Bt protein is activated to its toxic form by proteases in the insect midgut (Aimanova & Zhuang, 2006; Jurat-Fuentes &

Adang, 2006; Vachon et al., 2012). However, a few studies have found that the protoxin can be more detrimental than the activated toxin (Tabashnik et al., 2015; Gómez et al., 2014), and previously unknown receptors of Bt toxins have been discovered as well (Crickmore, 2005).

Since Bt toxin does not actually require activation within the insect midgut to be effective and extra receptors exist for Bt activation other than those in the target insect, the selectivity of Bt toxin is less defined than previously thought. After activation, several synergisms and antagonisms between Bt toxin variants, as well as with enzymes and proteins from other sources, come into play (Sharma et al., 2004; Broderick et al., 2006, 2009; Dubois and Dean, 1995).

Lastly, toxicity can be amplified by stress factors such as heavy metal exposure and parasitic infection (Koppenhofer and Kaya, 1997). The uncertainties surrounding the Bt mechanism and the detrimental effects of Bt on non-target organisms reinforce the need for continued research of

Bt’s effects on critical aquatic organisms.

Crayfish are critical aquatic species in many ecosystems due to their endangered status and role as ecological engineers (Reynolds et al., 2013; Moore, 2006). Crayfish frequently comprise a large proportion of the biomass produced in aquatic ecosystems (Dorn & Mittelbach,

1999), even exceeding 50% in some areas (Momot, 1995). Crayfish are key prey items for more than 240 predators, including many freshwater fish species, mammals, turtles and birds (Jones et al., 2006). As omnivores and both prey and predator species, crayfish are essential transformers of energy between different levels of the food chain in these ecosystems (Holdich, 2002).

Crayfish are polytrophic and as a result feed on an array of items in food webs including detritus, algae, macrophytes, invertebrates and vertebrates (Dorn & Wojdak, 2004; Holdich, 2002). Since terrestrial plant detritus contributes more than 60% to the production of juvenile and adult crayfish in streams, the effects of corn inputs on this critical species should be investigated

(Whitledge & Rabeni, 1997).

Previous research by Linn & Moore (2014) assessed growth and survivorship effects on adult F. rusticus crayfish fed GM corn containing one Bt toxin (Cry1Ab). In this study, crayfish fed a Bt corn diet had 31% lower survivorship than those fed an isogenic treatment. By controlling for nutritional equivalency between Bt and isogenic strains of corn, the researchers were able to conclude that the Bt strain was having a toxic effect on the crayfish within the GM treatments. Follow up studies are necessary to establish these effects. Since stacked and pyramided strains of corn are much more abundant now than those with single modified traits,

5 assessing their impact on non-target species such as crayfish is critical for ensuring their environmental safety. Moreover, since juvenile mortality in particular is one of the most important parameters in assessing the survival of threatened crayfish populations (Meyer et al.,

2007), we utilized crayfish in the juvenile stage when measuring growth and survivorship differences among diet treatments.

MATERIALS & METHODS

Animals

Juvenile crayfish (Faxonius rusticus) were caught in the Portage River in Bowling Green,

OH (41.37ºN, 83.65ºW) in late May 2015 by kick-seining and in Little Carp River in Pellston,

MI (45.55ºN, 84.68ºW) in late June 2015 with aquarium nets. Juveniles from both locations were randomized when assigned to treatments to minimize the possibility of pre-exposure to corn detritus affecting the results of the study. Juveniles used in this study had a carapace length of

0.474 ± 0.08 cm (mean ± SEM). Juveniles were stored in separate cylindrical mesh containers

(radius: 1.9 cm; height: 10.2 cm) in 37.9 L glass community aquaria filled with Maple River water and aerated with bubblers until experimental use. Crayfish were fed Purina© rabbit pellets composed mostly of Alfalfa, wheat and soybean twice a week during this time. One week before use, all crayfish were moved to separate 59 ml BPA-free Diamond mini-cups in the artificial stream (see Experimental Treatment Areas below) to allow them to adapt to the change in environment. Tiles were glued as weights to the base of the mini-cups to prevent floating along the artificial stream. Five holes approximately one cm in diameter were also drilled into the sides of the mini-cups at equal intervals to allow flow of stream water in and out.

Experimental Design

Growth treatments took place at the University of Michigan Biological Station stream lab facility in Pellston, MI (45°33’ N, 84°45’ W). Juvenile crayfish were exposed to one of six treatments:

Refuge (Control) N = 42

Fish Gelatin (Control) N = 39

River Detritus(Control) N = 39

SmartStax N = 42

VT Triple Pro N = 42

Roundup Ready 2 N = 42

Four of the treatments were diets consisting of different varieties of GM corn obtained from

Rupp Seeds (Wauseon, Ohio): SmartStax (OECD Unique Identifer: MON-89Ø34-3 x DAS-

Ø15Ø7-1 x MON-88Ø17 x DAS-59122-7), SmartStax refuge, VT Triple Pro (OECD Unique

Identifier: MON-89Ø34 x MON-88Ø17-3), and Roundup Ready 2 (OECD Unique Identifier:

MON-ØØ6Ø3-6). SmartStax refuge is a non-Bt variety of corn seed that is packaged with the

SmartStaxseeds and contains the two HT transgenes within SmartStax. Refuge seeds were dyed a different color than SmartStax seeds and were therefore easy to separate out. The other two experimental treatments were diets consisting of fish gelatin or in situ river detritus.

Diets

GM Corn

All maize was planted in the Bowling Green State University (BGSU) greenhouse

(Bowling Green, OH) from December 17th, 2014 to May 5th, 2015 in Metromix 852 topsoil using a fertilizer containing 10% each of phosphorus, nitrogen and potassium. All corn was grown using a 16-hour natural photoperiod (16 hr L: 6 hr D). After maturation, the maize was given time to senesce until the silk on the plant started to brown (~ 120 days), which is when corn is typically harvested from agricultural fields. The stalks and leaves from each variety were dried separately piled into an 80°C oven for 18-24 hours to mimic the drying that would occur for plants left on the field after harvest (Linn & Moore, 2014). The oven was wiped clean with a wet cloth after each drying session to eliminate the possibility of cross-contamination between corn varieties. Dried corn was stored in paper grocery bags until experimental use.

Before experimental use, 453.6 g of stalks and leaves from all four strains of maize were analyzed by Dairy One Forage Testing Laboratory (Ithaca, NY) to assess carbon, nitrogen and lignin content in plant parts across the four varieties. Only leaves from each variety were used in feeding experiments because of their similar C:N and lignin content (Table II).

For seven days prior to growth experiments, the dried corn tissue was preconditioned in mesh bags in the Maple River (Pellston, MI) to simulate breakdown in the natural environment and allow for microbial growth. The corn leaves were then cut into squares before being distributed to the crayfish. Each corn treatment was carried out for seven weeks. The fish gelatin and naturally-occurring detritus treatments were carried out for four weeks.

Naturally-Occurring Detritus

Naturally-occurring detritus was obtained by finding decomposed leaf litter along the stream bed of the east branch of the Maple River (45°33’N, 84°45’W) each week and using a blender to process the material into small pieces. Common leaf types found in the detritus were red oak (Quercus rubra), beech (Fagus grandifolia), red maple (Acer rubrum) and big-tooth aspen (Populus gradidentata). Estimates of relative densities of intact leaf types were approximately equal across these species. The remaining 20% of detritus was too decomposed to identify by species type.

Fish Gelatin

Fish gelatin was made by homogenizing approximately 46 g of Ocean Prince canned sardines in a blender and mixing with 28 g of Knox unflavored gelatin and 600 ml of boiling water (Wolf et al., 2004; Lahman et al., 2015). Gelatin was refrigerated in 3 cm x 3 cm circular caps wrapped with Parafilm® for at least 12 hours before being cut into 0.1 g cubes for use.

Experimental Treatment Areas

Eight flow-through stream treatments were made of rain gutters (200 x 13 x 8.9 cm) raised on a single layer of cinder blocks (40.6 x 20.3 x 20.3 cm). All streams received water via

12.7 cm long garden hose spouts from a 208.2 L head tank with water input directly from the

Maple River. Incoming detritus from the river was filtered out with nylon (0.01 cm2 holes) covering the inflow pipe, which was switched out as needed. The streams had an average discharge rate of 0.04 L/s ± 0.012.

At the start of each week, the crayfish were weighed on a Sartorius g scale (model BA

310 P) to the third decimal place. Their right chelae and intraorbital carapace length were measured by photographing each juvenile on a laminated grid with a millimeter scale. The length was determined by digitizing the photo and measuring each line distance with TPS morphometric software (F. J. Rohlf, State University of New York Stony Brook, USA; http://life.bio.sunysb.edu/morph/index.html) using the millimeter graphing paper as a reference with a 5-10% error. Old food was removed from the juvenile’s container at the start of each week, and a fresh 0.1 g of maize detritus, river detritus or fish gelatin was added each week according to the juvenile’s assigned treatment.

Data Analysis

Specific growth rate (Holdich, 2002) of each crayfish was calculated to account for differential experimental duration and starting weights among crayfish throughout treatments:

푙푛(푊푓– 푊푖 ) ∙ 100 Specific Growth Rate = 푡 where Wf = final weight, Wi = initial weight and t = days of survival. Differences in average crayfish growth rate among the treatments were then analyzed using an ANOVA in Statistica version 13.3 (TIBCO) and a post-hoc Tukey-HSD test to determine which treatments were

10 significantly different. Differential survival rates were analyzed with a Kaplan-Meier log rank test using the survdiff function (Harrington & Fleming, 1982) in R statistical software (version

3.5.1 GUI1.70 El Capitan build) in the Survival package (Therneau, 2018). A post hoc pairwise log rank test using the Survminer package (Kassambara et al., 2018) was also utilized to compare survivorship within treatments in pairs (Tripathi & Pandey, 2017).

RESULTS

Survival

Crayfish diet significantly altered the mortality of juvenile crayfish (X2 = 110, p < 0.01;

Figure II). A post-hoc pairwise log rank test showed that both the river detritus and fish gelatin diets had significantly lower mortality than all the corn treatments (p < 0.01). There were no significant differences in mortality among the four corn treatments (p > 0.05). Approximately

88% of the juveniles in the Triple Pro and 90% in the Roundup Ready treatments died, while

100% of juveniles died in the Smartstax and refuge treatments. Only 41% and 30% of the juveniles died in the Maple River detritus and fish gelatin control treatments respectively.

Growth

Specific growth rate varied among the six treatments (ANOVA; F(5, 202, 0.05) = 8.06; p <

0.01; Figure III). The river detritus and fish gelatin treatments exhibited significantly higher specific growth rate than the Smartstax, Triple Pro and refuge corn treatments (Tukey-HSD post-hoc analysis; p < 0.01) but comparable specific growth rate to one another. The Roundup

Ready corn treatments grew at a significantly higher rate than the Triple Pro corn treatment (p

< 0.01). Refuge, Smartstax and Triple Pro treatments all grew at a similar rate (p > 0.01;

Figure III).

DISCUSSION

The results of this study demonstrate that juvenile crayfish fed both GM and non-GM corn had significantly lower survivorship compared to those fed more natural diets consisting of river detritus or fish gelatin (Figure II; p < 0.01). However, there were no survivorship differences between juveniles fed GM corn and those fed the non-Bt control corn (Figure II; p >

0.05). River detritus and fish gelatin diets resulted in significantly higher specific growth rates than juveniles fed Triple Pro and Smartstax corn (Figure III; p < 0.05). There were no significant differences in specific growth rate of juveniles fed river detritus, fish gelatin and

Roundup Ready corn (Figure III; p > 0.05). Lastly, juveniles fed Roundup Ready corn grew at a significantly higher rate than juveniles fed Triple Pro corn (Figure III; p < 0.05) and trended toward a significantly higher rate than juveniles fed Smartstax corn (Figure III; p = 0.09). The remainder of the corn diets showed no significant specific growth rate differences. Overall, a pure corn diet had detrimental effects on crayfish growth and survivorship with one Bt corn variety significantly amplifying the negative effect on growth.

The entry of Bt corn into streams and lakes near agricultural fields is detrimental to the growth and survivorship of juvenile F. rusticus, with certain Bt varieties exacerbating the negative growth effect (Figures 2 & 3). A decrease in growth rates of juveniles will contribute to a higher rate of mortality in natural populations because smaller crayfish are more vulnerable to predators than larger crayfish (DiDonato & Lodge, 1993; Elvira et al., 1996). Also, since body size is associated with reproductive maturity, decreased growth rates would cause crayfish to reach reproductive size later (Alcorlo et al., 2008). Even when crayfish molt into reproductive form at smaller sizes due to environmental factors, these small crayfish are not very fecund

(Holdich, 2002). Moreover, smaller female crayfish carry significantly fewer eggs (Maguire et

13 al., 2005; Tropea et al., 2015), and female crayfish generally produce fewer eggs for smaller males (Galeotti et al., 2006). Hypothetically, fewer young could survive to adulthood due to predation and poor diet in areas where corn inputs are persistent and substantial. The individuals that do survive would reproduce later, less often and produce fewer eggs. The interaction of these factors could lead to decreases in natural crayfish populations located near agricultural fields with corn monocultures.

Decreases in crayfish sizes and abundance would have cascading effects through aquatic ecosystems in agricultural landscapes. Crayfish are keystone species based on their domination of energy transfers between trophic levels (Reynolds et al., 2013). At a microscopic level, crayfish densities have a large impact on periphyton abundance and quality (Creed, 1994; Dorn

& Wojdak, 2004; Lodge et al., 1994). The influence of crayfish on detrital processing rates and the distribution of fine particulate matter qualify them as ecosystem engineers (Creed & Reed,

2004). The role of crayfish as bioturbators means that they affect sediment accumulation and therefore habitat quality and resource abundance (Jones et al., 1994; Statzner et al., 2003). At a macroscopic level, bioturbation further affects invertebrate distribution (Usio & Townsend,

2004), and crayfish densities impact invertebrates further via size-selective predation (Creed &

Reed, 2004; Kreps et al., 2012). Crayfish determine the abundance and distribution of macrophytes as well (Carreira et al, 2014; Chambers et al., 1990; Peters et al., 2008; Roessink et al., 2017). A loss in crayfish biomass via population and body size decreases may lead to increases of periphyton and macrophytes, causing bottom-up effects on stream ecosystems which would vary with existing food web structure (Creed & Reed, 2004; Roth et al., 2007; Usio et al.,

2009). Since crayfish are consumed by for over 200 predatory species, a decrease in crayfish

14 biomass would affect species abundance and diversity of higher order predators as well

(Reynolds et al., 2013; Tablado et al., 2010).

Crayfish may have survived better on river detritus and fish gelatin over corn for a variety of reasons. Juvenile crayfish may not recognize corn leaf litter as a food source like their adult counterparts, or if they do, they may not find the corn litter appealing enough to consume.

Furthermore, juveniles may not have been capable of digesting the corn in the whole leaf form that was offered, as studies have found that zooplankton was a suitable food source for juveniles but that free-floating plants were not (Jones, 1995). Moreover, juveniles feed heavily on macroinvertebrates as compared to adult crayfish, with an ontogenetic shift occurring with growth from highly carnivorous to omnivorous (Momot, 1995). Juvenile crayfish may be more reliant on animal food sources for survival than adult crayfish, due to the higher protein content of these sources (Celada et al., 2013; Whitledge & Rabeni, 1997). Since the leaf material in the corn contained a maximum of 5.5% crude protein (Mosse, 1990), diets of solely corn would not fulfill the crude protein needs of the crayfish. Moreover, the lack of diversity in the corn diets could have led to a vitamin deficiency, as juveniles require, for example, 5000 IU/kg of vitamin

A (Shah et al., 2016).

Despite the significant differences in survivorship between crayfish fed corn and those fed river detritus and fish gelatin, there were no significant differences in survivorship among the corn treatments themselves. Our hypothesis was not fully supported by the GM/non-GM feeding trials, despite the findings of Linn & Moore (2014) that found 31% lower survivorship in adult F. rusticus fed GM corn than adults fed non-GM corn. This difference could be due to physiological differences between juvenile and adult crayfish. Juvenile crustaceans have significantly higher respiration rates, lower immune function and lower metabolic energy than

15 adults (Dissanayake et al., 2008). Moreover, ontogenetic shifts in digestive proteases and carbohydrases have been observed in C. quadricarinatus (Figeuiredo & Anderson, 2003), which are enzymes involved in the cytotoxic mechanism of Cry1AB. If these shifts exist for F. rusticus, these physiological differences may have affected the juvenile response to Bt and subsequently their survivorship.

The significant deficits in growth rates of crayfish fed Triple Pro corn when compared to growth rates of crayfish fed Roundup Ready corn contribute to the existing evidence for a possible detrimental mechanism involving Bt proteins and crayfish. Crayfish and target insects of Bt corn, such as Lepidoptera, have common Bt toxin receptors in their midguts, such as aminopeptidase and alkaline phosphatase (Holdich, 2002; Jurat-Fuentes et al., 2011). Despite these commonalities, researchers may still dismiss the possibility of a functional mechanism in non-target organisms because of the established specificity of some Bt proteins (Vachon et al.,

2012). For example, cadherin has been posited as essential to the mechanism that makes Cry1Aa proteins toxic (Nagamatsu et al., 1999), which crayfish are not known to contain. Regardless of this missing link, Smartstax and Triple Pro corn varieties contain other transgenic, pesticidal proteins, such as Cry3Bb and Cry34Ab, whose mechanisms are much less understood. These proteins may be active in the crayfish midgut after all, leading to decreased survivorship in adult

F. rusticus (Linn & Moore, 2014) and the differences in growth rates seen within our study.

Ongoing research has yet to determine what the synergistic effects of multiple Bt proteins might mean for non-target organisms.

Regardless of the mechanisms involved, our study shows that corn inputs to streams may be harmful to crayfish populations as a result of reduced survivorship and growth. Subsequently, the substantial conversion of corn byproducts to stream detritus will have cascading effects at all

16 levels of the stream food web by altering the abundance and distribution of algae, macrophytes and macroinvertebrates and decreasing the total biomass of animals at higher trophic levels.

Given the negative effects on growth rates seen in juvenile crayfish fed Triple Pro corn, more research is needed regarding the non-target effects of various Bt proteins on crayfish and other aquatic organisms.

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APPENDIX A – TABLES

Table I Active protein products in each GM corn variety

Name Protein Products

SmartStax Cry1A.105, Cry2Ab2, Cry1F, Cry3Bb1,

Cry34Ab1, Cry35Ab1, CP4 EPSPS, PAT

VT Triple Pro Cry1A.105, Cry1Ab2, Cry3Bb1, CP4 EPSPS

Roundup Ready 2 CP4 EPSPS

Smartstax Refuge CP4 EPSPS, PAT

Table II Nutritional quality (stalks and leaves) of each corn variety (Dairy One Forage Testing

Laboratory, Ithaca, NY

% % Nitrogen % Carbon C : N

Lignin

SmartStax Leaves 4.0 0.88 46.5 52.7

SmartStax Stalks 7.6 0.61 46.8 76.5

VT Triple Pro Leaves 3.6 0.75 45.8 61.5

VT Triple Pro Stalks 8.7 0.49 47.2 97.2

Roundup Ready 2 Leaves 3.6 0.83 46.6 55.9

Roundup Ready 2 Stalks 8.2 0.65 47.6 72.7

Refuge Leaves 4.0 0.92 46.4 50.5

Refuge Stalks 7.3 0.52 47.9 91.5

APPENDIX B – FIGURES

Figure I Flow through artificial stream setup. Rectangles represent rain gutters elevated on cinder blocks. Water was drawn from the Maple River in Pellston, MI into the head tank and distributed to rain gutters via garden hose spouts

Figure II Kaplain-Meier survival curve for Smartstax, Triple Pro, Roundup Ready, refuge, river detritus and fish gelatin diets. The control treatments (river detritus and fish gelatin) had significantly greater survival (log rank test) than the experimental treatments (Smartstax, Triple

Pro, Roundup Ready and refuge) (X2 = 110; p < 0.01). Survivorship did not differ between experimental treatments (p > 0.05)

Figure III Specific growth rate of crayfish in each of six diet treatments. Fish gelatin, river detritus and Roundup Ready corn treatments exhibited the highest specific growth rate, whereas

Smartstax and Triple Pro corn treatments experienced negative growth overall. Roundup Ready and Triple Pro corn had significantly different growth rates from one another (F(5, 202, 0.05) = 8.06, p < 0.01)