Testing the Effects of Glyphosate and a Possible Tradeoff with Immunity on Native And

Home , Gryllus lineaticeps, Gryllus pennsylvanicus, Velarifictorus, Velarifictorus micado

Testing the effects of glyphosate and a possible tradeoff with immunity on native and

non-native species of crickets

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Lydia Rose Mullins

Graduate Program in Evolution, Ecology and Organismal Biology

The Ohio State University

2020

Thesis Committee

Dr. Susan Gershman, Advisor

Dr. Ian Hamilton

Dr. Roman Lanno

Copyrighted by

Lydia Rose Mullins

2020

Abstract

Introduced insects can compete with native species and alter population and community dynamics. To minimize these effects and potential threats to biodiversity, it is necessary to understand the mechanisms that lie behind successful colonization of novel environments by introduced species, including anthropogenic factors such as herbicides.

Glyphosate, the active ingredient of Roundup, is a broad-spectrum herbicide that is commonly applied to various types of land across the world. Its application increased dramatically after the introduction of glyphosate-resistant crops, leading glyphosate to become the top-selling herbicide worldwide. Although it is so widely used, its effects on wildlife are extremely under-studied. Of the few studies examining glyphosate’s effects on non-target organisms, it has been shown to affect locomotion, reproduction, memory and learning of various species of arthropods. It is necessary to understand how glyphosate may be impacting invasion success of non-native insects. The present study examines these effects on native and non-native species of crickets. Further, competition with invasive species and exposure to herbicides may reveal or emphasize existing tradeoffs between traits. For example, many crickets trade off effort devoted to reproduction and immunity. Crickets commonly face immune challenges in the wild, so it is possible that a trade-off may lie in managing exposure to agrochemicals like herbicides and investment into immune function. If present, this trade-off may be contributing to the ii success of non-native species competing with native species and colonizing novel environments. Therefore, in the present study, I examine the effects of glyphosate and a possible trade-off with immunity on lifespan, calling effort and fecundity in the native fall field cricket, Gryllus pennsylvanicus and the non-native Japanese burrowing cricket

Velarifictorus micado. G. pennsylvanicus and V. micado occupy a similar niche and therefore are expected to be in competition with each other for resources. V. micado has also been observed in higher abundance than G. pennsylvanicus in the field, suggesting higher success in competition for resources. In this study, we found that glyphosate decreases survival of juvenile Gryllus vocalis and has no effect on adult lifespan of

Gryllus pennsylvanicus or Velarifictorus micado. Both G. pennsylvanicus and V. micado females produce more eggs when sprayed with glyphosate. G. pennsylvanicus females also show an interaction between glyphosate and immune challenge; those sprayed with glyphosate and provided with an induced immune challenge produce fewer eggs than females sprayed with glyphosate that are not immune challenged. Because this interaction is not present in V. micado females, this could potentially explain the successful establishment of V. micado. Glyphosate does not affect male calling effort of either G. pennsylvanicus or V. micado. These results suggest that glyphosate may impact survival and reproduction of non-target organisms and has varying effects based on species and may contribute to colonization by non-native species. The survival effect that we observed may be due to additional ingredients in commercial formulations of glyphosate such as surfactants, rather than pure glyphosate. In order to minimize the potential contribution of glyphosate to colonization of novel habitats by non-native species, further

iii studies are needed to better understand the potential consequences of this extensively used herbicide and to disentangle whether these effects are actually caused by glyphosate or other ingredients commonly found in Roundup.

Dedicated to my Papaw, my Mamaw, and my Poppy –

You are so missed

Acknowledgments

I would like to thank my advisor, Susan Gershman, for her continued support and guidance. Without her assistance, this work would not have been possible. I would also like to thank the other members of my committee, Ian Hamilton and Roman Lanno, for their valuable help throughout this process.

This work would also not have been completed without the help of Salvatore Sidoti,

Bridget Brown, Jaret Cingel, Leigh Carrabia, Erin Schuster, Marisa Nicol, Malek

Soumakieh, Nihit Tyagi and Haley Ries.

To my parents, my brother and my grandma Carolyn – I would be lost without your unwavering love and support.

Thank you to the faculty and my fellow graduate students in EEOB for being so welcoming and encouraging.

To my friends Jimmy Chiucchi, Chloe Flanigan, Drew Spacht, and Kunal Chatterjee – thank you for your endless support and helping me grow as a teacher, a graduate student, and most of all as a person along the way. vi

Vita

May 2018…………………………………………………………………….B.S. Biology, The Ohio State University 2018-2020………………...…………….…………………..Graduate Teaching Associate, The Ohio State University

Fields of Study

Major Field: Evolution, Ecology and Organismal Biology

vii

Table of Contents

Abstract ...... ii Dedication ...... v Acknowledgments ...... vi Vita ...... vii List of Tables ...... ix List of Figures ...... x Introduction ...... 1 Materials and Methods ...... 8 Results ...... 16 Discussion ...... 20 Conclusions ...... 26 Bibliography ...... 40

viii

List of Tables

Table 1. Summary of studies examining the effects of glyphosate on non-target terrestrial arthropods ...... 28

List of Figures

Figure 1. Recruitment of crickets for treatments ...... 29 Figure 2. Effect of glyphosate on juvenile G. vocalis survival ...... 30 Figure 3. Effect of glyphosate on water consumption ...... 31 Figure 4. Effect of glyphosate on food consumption ...... 32 Figure 5. Effect of spray and injection on G. pennsylvanicus fecundity ...... 33 Figure 6. Effect of spray and injection on V. micado fecundity ...... 34 Figure 7. Effect of species on fecundity ...... 35 Figure 8. Effect of species on adult lifespan ...... 36 Figure 9. Effect of spray and injection on lifespan of G. pennsylvanicus adults ...... 37 Figure 10. Effect of spray and injection on lifespan of V. micado adults ...... 38 Figure 11. Effect of species, injection and spray on calling effort ...... 39

Introduction

Introduced insect species are highly successful at becoming established in novel environments (Brockerhoff and Liebhold 2017). They play influential roles in ecosystems, including various roles in food web interactions and contributions to numerous ecosystem processes such as nutrient cycling and pollination. Therefore, they may have drastic impacts on recipient habitats, including altering community dynamics and threatening biodiversity (Brockerhoff and Liebhold 2017; Fahrner and Aukema

2018). To become established, invasive species must compete with native species that occupy similar niches. This could potentially lead to niche displacement or competitive exclusion of native species (Mooney and Cleland 2001). Considering the various impacts non-native insects may have on natural systems, it is necessary to understand the mechanisms that lie behind their successful establishment. In addition to competition with introduced species, insects face other challenges in the wild such as immune challenges and negative effects caused by anthropogenic factors like herbicides. The present study examines potential differences in life-history traits between the native fall field cricket, Gryllus pennsylvanicus, and the invasive Japanese burrowing cricket,

Velarifictorus micado, after exposure to a glyphosate-based herbicide and an immune challenge.

The fall field cricket, G. pennsylvanicus, can be found throughout most of the eastern and midwestern United States. Its habitat includes fields, edges of forests, and lawns. Adults emerge each summer around August and they remain active through October. Males signal reproductive availability to females through singing (Zuk 1988). Development of eggs or nymphs pauses during the winter and resumes in the summer (Carmona 1999).

The Japanese burrowing cricket, V. micado, originated in Asia and was introduced to the

United States in the 1950s (Bowles 2018). While the mechanism of introduction isn’t known, it is speculated that it was brought to the United States either as fishing bait or inadvertently transported as eggs in the soil of ornamental plants (Bowles 2018). It overwinters by diapausing in the egg stage (Zeng et al. 2014).

When introduced species occupy a similar niche as a native species, as is the case with G. pennsylvanicus and V. micado, competition for resources can increase and may emphasize or reveal tradeoffs. In many species, crickets exhibit trade-offs and increase investment of effort into one trait and less into another if resources are limited. For example, crickets trade off effort between immunity and reproduction. Gryllodes sigillatus invest reproductive effort in producing large nutritive courtship feedings

(spermatophylaxes) for females (Kerr et al. 2010). When provided with an immune challenge, they produce smaller spermatophylaxes than unchallenged individuals (Kerr et al. 2010). Additionally, males induced to produce more spermatophylaxes exhibit less ability to mount an immune response (Kerr et al. 2010). Similarly, Gryllus campestris

2 males show reduced daily calling song when immunologically challenged (Jacot et al.

2004). Environmental stress, such as low-quality diet, also leads to tradeoffs with immunity in crickets. For example, low-protein diets cause reduced levels of enzymes associated with immune activity in the Mormon cricket Anabrus simplex (Srygley et al.

2009). Similarly, female Teleogryllus commodus produce weaker encapsulation, an immune response to foreign particles, when provided with limited food (Zuk et al. 2004).

As competition increases after introduction of an invasive species and more effort is required to acquire resources, crickets may end up reducing investment into other traits like immunity. Additionally, increased time spent searching for limited resources like food may reduce time devoted to other tasks such as searching for mates. Other influences, such as anthropogenic factors, in addition to the presence of non-native species, may also cause tradeoffs. For example, exposure to agrochemicals like herbicides may require investment to manage the effects of the presence of these chemicals, potentially reducing effort invested elsewhere. Herbicides are commonly applied to various types of land, including areas occupied by crickets, so frequent exposure of crickets to these chemicals is expected.

Use of glyphosate-based herbicides has grown by almost 100 times between the years of

1974 and 2014, leading to them to become the top-selling herbicides in the world

(Vandenberg et al. 2017; Levya-Soto et al. 2018). Because glyphosate is a broad- spectrum herbicide that kills any plant it contacts, its use was initially limited. Originally, farmers applied glyphosate before any crops were planted (Young 2006). However, after

3 the introduction of glyphosate-resistant crops in 1996, labor and time required for weed control was reduced, and its use increased dramatically (Benbrook 2016). This led to an increase in use of glyphosate from almost 27 million kg/yr just in soybean between the years of 1996 and 2002 (Young 2006) to 826 million kg in 2014 (Benbrook 2016).

Today, 56% of the total glyphosate application worldwide is on these crops (Benbrook

2016; Young 2006). Because of the vast amount of these agrochemicals being applied, it is necessary to understand the potential impacts on wildlife that inhabit both application sites and adjacent areas, as drift from target sites commonly occurs. There is a major lack of understanding regarding the impact of herbicides on terrestrial species. It is necessary to examine these potential effects of herbicides on non-target terrestrial organisms, as exposure to these chemicals may influence population dynamics, predator-prey interactions, and competition within communities and may lead to tradeoffs, specifically in combination with competition between native and non-native species.

Although there is limited research on glyphosate’s effects on terrestrial invertebrates, prior research shows that glyphosate negatively affects honeybee learning and appetitive behavior. It also reduces abundance of prominent bacteria species in the gut microbiota of honeybees, leading to increased mortality of honeybees after exposure to pathogens

(Motta et al. 2018). Glyphosate also negatively impacts reproduction and locomotion in spiders. Female wolf spiders (Pardosa milvina) sprayed with glyphosate are selected as mates fewer times than females sprayed with water (Griesinger et al. 2011). When both female pheromones and glyphosate are present in the air, male wolf spiders show

4 increased locomotion compared to when glyphosate is absent (Griesinger et al. 2011).

Glyphosate also affects foraging behavior of multiple species of wolf spiders. Tigrosa helluo are able to capture prey faster using the same number of lunges when exposed to glyphosate, while P. milvina take the same amount of time but use more lunges to capture prey (Rittman et al. 2013). The mechanism by which glyphosate causes these effects found on non-target invertebrates in the previously mentioned studies is not well understood. However, it is speculated that glyphosate may interfere with chemical cues commonly used by arthropods (Rittman et al. 2013). Crickets rely on chemical cues for a range of tasks, such as predator recognition and mate choice (Tyler et al. 2015; Storm and

Lima 2008). The presence of glyphosate may interfere with the ability of crickets to avoid predators or successfully identify and choose mates. If differences in these effects exist between native and non-native crickets, they may impact successful competition by either species. For example, interference by herbicides with either the production of daily calling song by males or the interpretation of songs by females could impact reproductive success. Additionally, this could also impact species-specific recognition of male songs by females of either species, leading them to respond to songs produced by the other species and possibly incurring fitness costs.

It has been suggested that some herbicides may mimic specific hormones found in arthropods, potentially interfering with their endocrine systems (Benamú et al. 2010).

Griesinger et al. (2011) hypothesized that glyphosate interferes with the release or perception of pheromones produced by female P. milvina. If glyphosate interacts with

5 endocrine systems of terrestrial invertebrates, it could result in changes in reproduction.

Herbicides may have an effect on how individuals allocate resources to life history traits.

The terminal investment hypothesis states that individuals should invest more in current reproduction if chances of future reproductive events are low (Creighton et al. 2009).

Evidence of this hypothesis is found in numerous species of insects, including crickets. In

Texas field crickets (Gryllus texensis), females who have received an immune challenge produce more eggs than healthy females do (Shoemaker et al. 2006). In southern ground crickets (Allonemobius socius) when males were subjected to an immune challenge, younger males reduced effort put into calling song, while older males increased their reproductive effort (Copeland and Fedorka 2012).

If glyphosate reduces chances of future reproduction, it could result in an increase in current reproductive effort. Additionally, the pesticide-induced homeostatic modulation hypothesis posits that chemicals that are inhibitory at high doses may be stimulatory at low doses (Cohen 2006). Under this hypothesis, although glyphosate may negatively impact non-target organisms at high doses, it may lead to increased investment in reproduction at low doses.

It is important to note that within the currently limited work on glyphosate’s impacts on terrestrial invertebrates, there is a major lack of consistency in route of exposure and exposure doses of glyphosate on study organisms, making comparisons of results among studies difficult (see Table 1). In the present study, we used a residual exposure of

6 glyphosate most similar to methods in the previously mentioned work by Rittman et al.

(2013) and Griesinger et al. (2011), who found effects of glyphosate on reproduction and locomotion.

Because glyphosate’s impacts on most terrestrial species are not well explored, I aimed to examine its effects on native and non-native species of crickets. I also aimed to investigate how glyphosate may affect a possible trade-off in crickets between managing the presence of an environmental toxicant like glyphosate and investment into immune responses. If a trade-off between immunity and exposure to glyphosate exists in a native species but not in a non-native species, it may explain the successful colonization of the non-native species. Because V. micado has been observed in the field in higher abundance than G. pennsylvanicus and they occupy the same habitat and are using the same resources, I aimed to examine whether the presence of glyphosate and a potential trade-off with immunity may be contributing to the success of V. micado in the historic range of G. pennsylvanicus. I hypothesized that glyphosate will have an effect on cricket reproduction and lifespan and that a trade-off between immunity and exposure to glyphosate exists. Specifically, I predicted that glyphosate will affect male calling effort and female fecundity of both invasive V. micado and native G. pennsylvanicus and that this effect will change in combination with an immune challenge. I also predicted that these effects differ between the two species.

Materials and Methods

Preliminary experiments

Because G. pennsylvanicus and V. micado are diapausing species that are challenging to maintain in large numbers in colony, prior to our main experiment, we performed preliminary experiments on the non-diapausing vocal field cricket, (Gryllus vocalis), which is maintained year-round in colony. In our preliminary experiments, we determined whether glyphosate applied as a spray to substrate, food, and water would affect cricket mortality or consumption of food and water.

Gryllus vocalis rearing

G. vocalis individuals were collected from a lab colony in Riverside, California in 2015.

This colony is kept in overlapping generations, with 100 to 1,000 crickets separated by size cohort per housing box (59.7 cm x 42.9 cm x 31.1 cm), on a light cycle of 12 h day:

12 h night at 25°C. Housing boxes contain egg cartons for shelter and ad libitum food and tap water: two 7 cm diameter petri dishes of ground rabbit food (Ranch Pro rabbit pellets) and two 35 mL vials of water that are refreshed twice per week. Adult females are provided ad libitum with moist cotton for oviposition. Eggs are incubated for 10 days at 25°C before hatching.

Effect of Glyphosate on Gryllus vocalis Juvenile Survival

To determine at which life stage we should spray G. pennsylvanicus and V. micado with glyphosate, we tested whether glyphosate would cause substantial mortality in juvenile

G. vocalis. Five juvenile G. vocalis individuals ranging between 0.25 g and 0.40 g in weight were placed in each of 40 1.04 L plastic home containers. Twenty containers were randomly assigned to the glyphosate spray treatment (GLY), and twenty to the control spray treatment (CTRL). Glyphosate spray treatment containers were sprayed with 3.5 mL of 1.5% Roundup ProMax (Monsanto Company, St. Louis, MO), active ingredient glyphosate (Glyphosate, N-(phosphonomethyl)glycine potassium salt 48.7%, Other ingredients 51.3%, CAS # 1071-83-6), diluted with deionized (DI) water. In all experiments, a manufacturer-recommended concentration of 1.5% Roundup was used.

Control spray treatment containers were sprayed with 3.5 mL of DI water. Each home container had a small condiment cup (22.2 mL) of ground rabbit food. Glyphosate treatment containers included a 35 mL plastic vial of 1.5% Roundup plugged with cotton; control treatment containers included a 35 mL plastic vial of water with cotton. After the crickets were placed in the containers, each container was sprayed with its assigned treatment. Food and water were changed weekly. In the GLY treatment, we alternated between 1.5% Roundup and tap water in the water vials every other week, so those in the

GLY treatment were both sprayed with 1.5% Roundup and provided with 1.5% Roundup as a water source. Because glyphosate may also be sprayed on crickets’ water sources in the field, 1.5% Roundup was provided as a water source for one week, then tap water was

9 provided for one week. Spraying occurred once at the beginning of the experiment. The number of surviving crickets per container was counted weekly for two weeks.

Effect of Glyphosate on Food and Water Consumption by Gryllus vocalis

a. Water

The following experiment assessed whether the presence of glyphosate would affect water consumption by crickets. Groups of ten juvenile (0.25 g – 0.40 g) G. vocalis were housed in three (42.9 cm x 29.2 cm 23.8 cm) boxes. Each box contained two 35 mL plastic water vials plugged with cotton, one filled with tap water and one with a 1.5%

Roundup solution. The initial masses of each water vial were measured. The final mass of each vial was recorded after ten days. In addition, a control box containing two water vials (GLY and DI water) without any crickets was used to determine the baseline change in water vial weight due to evaporation rather than consumption by crickets.

b. Food

This experiment aimed to determine if the presence of glyphosate would affect consumption of food by crickets. Groups of ten juvenile (0.25 g – 0.40 g) G. vocalis were housed in three (42.9 cm x 29.2 cm x 23.8 cm) plastic boxes. Each box contained two small plastic petri dishes (7 cm diameter), one containing ground rabbit food sprayed with 3.5 mL of tap water and the other containing ground rabbit food sprayed with 3.5

10 mL of 1.5% Roundup. Each box also contained one 35 mL plastic water vial plugged with cotton. The initial mass of each petri dish was measured. Final masses of each petri dish were recorded after seven days. A control box containing two petri dishes of food

(sprayed with DI water and GLY) without any crickets was also included to determine whether there was a baseline change in food weight not due to consumption by crickets.

The effect of glyphosate and immune challenge on Gryllus pennsylvanicus and

Velarifictorus micado

Cricket collection

G. pennsylvanicus and V. micado adults were collected from the field during the summer of 2018 from Columbus and Marion, Ohio. It is not possible to know whether or not these areas had been previously treated with herbicides. Around 60 G. pennsylvanicus adults and 250 V. micado adults were collected. Females of each species were allowed to oviposit into soil and moistened cotton, which were then stored for 6 – 7 months at 4° C to allow eggs to diapause, then incubated at 25°C to initiate hatching. To ensure uniformity in environments for both species, lab-hatched nymphs of each species were housed in (59.7 cm x 42.9 cm x 31.1 cm) plastic boxes with ad libitum ground rabbit food, two 35 mL vials of water plugged with cotton, and leaf litter for shelter. Leaf litter was collected from a single location known to have no previous herbicide treatment in

Autumn 2018 and was stored outside, exposed to various weather conditions, bacteria and visiting insects, to naturally decompose for around five months. At the start of the experiment, leaf litter was brought inside. Leaves varied in size but were large enough to provide shelter for crickets. There was evidence that crickets visibly consumed some of the leaves during the experiment.

Recruiting crickets for experimental treatments

Because glyphosate increased mortality of juvenile G. vocalis, we determined that mortality would be best minimized by spraying penultimate instar G. pennsylvanicus and

V. micado individuals. Penultimate instar individuals of each species could be identified by their fleshy wing buds. Penultimate instar individuals were randomly assigned to spray treatments and were moved to designated spray boxes (GLY or CTRL). Spray boxes (42.9 cm x 29.2 cm x 23.8 cm) contained a petri dish (7 cm diameter) of ground rabbit food, two vials of water (35 mL) plugged with cotton and leaf litter as shelter.

Crickets in the glyphosate spray box were sprayed once with 10.5 mL of 1.5% Roundup

ProMax. Crickets in the control spray box were sprayed once with 10.5 mL of DI water.

To control the duration of exposure to the treatments, we moved crickets to designated clean boxes 24 hours after spraying occurred (Figure 1). These clean boxes contained food and water vials, but egg cartons were provided for shelter instead of leaf litter. We checked clean boxes three times a week for newly-eclosed adults. When adults were found, they were housed individually in 1.04 L plastic home containers with a small

12 condiment cup (22.2 mL) containing rabbit food, a vial of water (40 mL) plugged with cotton, and a piece of egg carton for shelter. Adults were housed individually to ensure that mating did not occur. The mortality of crickets prior to the isolation of adults was not recorded. Adults were randomly assigned to receive either control injections or immune injections (see Figure 1). All of the procedures described above were duplicated for each species of cricket. The two species were never exposed to each other. Spray boxes and clean boxes were duplicated to prevent any contact between species.

Immune challenge (G. pennsylvanicus and V. micado)

Individuals from treatment groups of both species received an injection of either lipopolysaccharides (LPS), known to induce an immune response in crickets, or Grace’s insect medium as a control. Crickets were injected between their lower abdominal segments on their ventral side with 5 μL of either LPS or control solution. The LPS stock solution consisted of 25 μg of LPS per 100 μL Grace’s insect medium (50-70% sucrose,

1-5% calcium chloride, L-malic acid and glutamic acid). Individuals assigned to receive control injections were injected with only Grace’s insect medium. Crickets were restrained by hand to avoid unnecessary morphological damage. Injections were performed rapidly without anesthesia. Crickets were then placed back in their original storage containers.

Measuring fecundity (G. pennsylvanicus and V. micado)

To measure fecundity, females were given the opportunity to mate with two different males. A male was added to each female’s home container; if she did not mate with the male within 30 minutes, the first male was removed and replaced by a second male for up to 30 additional minutes. If the female did not mate with either male, the same procedure was repeated 48 hours later with two different males. The males used for mating were not otherwise used in this experiment. Most males were lab-reared but several field-caught adult males were used to supplement the need for more males later in the experiment.

Once mated, the water vial in the female’s container was removed and replaced with moistened cheesecloth as both a source for water and as oviposition substrate. The cheesecloth was removed every other day and eggs were counted.

Measuring calling effort (G. pennsylvanicus and V. micado)

To measure male calling effort, 48 hours after males received injections, males were observed by scan sampling. Scan sampling entailed observing each male every five minutes for a total of four hours and recording whether or not males were actively calling during each observation period. We observed males at the start of their dark period in blocks of two hours, 11:30 am to 1:30 pm and 3:00 pm to 5:00 pm. We observed males nine days after eclosion and kept them in their home containers during observation.

Observations occurred during the dark period of their cycle, when males normally call in the field.

Statistical analysis

For the preliminary tests on G. vocalis, I used a t-test to analyze the effect of spray on juvenile survival. To examine the effect of glyphosate on food and water consumption, each box contained both glyphosate and water treatments. Thus, the masses of paired vials and dishes were not independent of each other. So, a paired t-test was used to analyze the effect of spray on food and water consumption by juvenile G. vocalis.

To analyze the effects of species, spray and injection on fecundity and calling effort on

G. pennsylvanicus and V. micado, a generalized linear model specifying a Poisson distribution was used. Because G. pennsylvanicus fecundity data and calling effort for both species contained excess zeroes, I used a zero-inflated Poisson regression. A Cox proportional hazards model was used to analyze the effects of species, spray and injection on adult lifespan. No effect of species on calling effort was found, so data for both species was pooled for the analysis. All analyses were performed in R (R Core Team, version: Version 1.1.463) and a significance level of α = 0.05 was used for all hypothesis testing.

Results

Effect of Glyphosate on Juvenile Survival on G. vocalis

More juvenile G. vocalis per container survived spraying with DI water (N = 20) than survived spraying with glyphosate (N = 20) (t = 3.0861, df = 37.16, P = 0.00382; Figure

2).

Effect of Glyphosate on G. vocalis Food and Water Consumption

a. Water

There was not a statistically significant difference detected between the mean weight loss of DI water vials and glyphosate water vials (N = 3, paired t = -3.5828, df = 2, P =

0.06984; Figure 3).

b. Food

There was not a statistically significant difference between the mean weight loss of food sprayed by glyphosate and food sprayed by water (N = 3, paired t = 1.3029, df =

2, P = 0.3224; Figure 4).

G. pennsylvanicus fecundity

A significant interaction between spray and injection was found (zero-inflated

Poisson regression, model: fecundity predicted by spray and injection and the interaction of spray and injection, z value = -14.133, P < 0.001, Figure 5). Females sprayed with glyphosate and injected with Grace’s insect medium (N = 17) produced the most eggs while those sprayed with DI water and injected with Grace’s insect medium (N = 17) produced the fewest eggs. Both spray and injection treatments had effects on fecundity of

G. pennsylvanicus. Females sprayed with glyphosate (N = 18) produced more eggs than those sprayed with water (N = 25, zero-inflated Poisson regression, z value = 19.900, p <

0.001; Figure 5). Females injected with Grace’s insect medium (N = 22) produced more eggs than those injected with LPS (N = 21, zero-inflated Poisson regression, model: fecundity predicted by spray and injection and the interaction of spray and injection, z value = 3.218, p < 0.01).

V. micado fecundity

Both spray and injection had a statistically significant effect on V. micado fecundity.

Individuals sprayed with glyphosate (N = 9) produced more total eggs than those sprayed with DI water (N = 13, GLM, model: fecundity predicted by spray and injection and the interaction of spray and injection, z value = 3.116, p < 0.01; Figure 6). Injection also affected the total eggs produced by V. micado; females injected with LPS (N = 12)

17 produced fewer eggs than those injected with Grace’s insect medium (N = 10) GLM, z value = -4.714, p < 0.001). There was not a statistically significant interaction between spray and injection (GLM, z value = 1.679, P = 0.093).

V. micado females (N = 22) produced more eggs than G. pennsylvanicus (N = 43) females (zero-inflated Poisson regression, model: fecundity predicted by species, z value

= 21.15, p < 0.001; Figure 7).

Adult lifespan

G. pennsylvanicus adults (N = 116) survived longer than V. micado adults (N = 68) (Cox proportional hazards model, model: adult lifespan predicted by species, p = 0.013; Figure

8). Because of the difference in survival between species, I analyzed the effects of injection and spray on adult lifespan separately by species. Neither spray nor injection had an effect on adult lifespan of G. pennsylvanicus (spray: N = 61 for GLY, N = 55, injection: N = 60 for LPS, N = 56 for CTRL) or V. micado (spray: N = 32 for GLY, N =

36 for CTRL, injection: N = 31 for LPS, N = 37 for CTRL, Figures 9 and 10).

Calling Effort

Species did not have a significant effect on calling effort, so data for both species was pooled. Neither spray (z value = 0.917, p = 0.656, N = 8 for GLY, N = 10 for CTRL) nor

18 injection (z value = -1.524, p = 0.128, N = 7 for LPS, N = 11 for CTRL) had a significant effect on total calls observed (zero-inflated Poisson regression, model: total calls predicted by spray and injection and the interaction of spray and injection; Figure 11).

Additionally, I did not find a statistically significant interaction between spray and injection (z value = 0.371, p = 0.711).

Discussion

In this study, I evaluated the effects of glyphosate sprayed at manufacturer- recommended concentrations, in combination with an induced immune challenge, on fecundity, calling effort, and adult lifespan of native and non-native species of crickets.

Our results show that a single application of glyphosate, sprayed at a concentration recommended by the manufacturer (1.5%) leads to increased fecundity in both G. pennsylvanicus and V. micado, but may not impact male calling effort or adult lifespan.

Here, I will discuss the implications for each variable studied in this experiment.

Fecundity

Females of both G. pennsylvanicus and V. micado sprayed with glyphosate produced more eggs, while females of both species induced to produce an immune response laid fewer eggs. A significant interaction between spray and injection treatments was found on G. pennsylvanicus fecundity. G. pennsylvanicus females injected with lipopolysaccharides (LPS) and sprayed with glyphosate produced fewer eggs than those sprayed with glyphosate that received injections of Grace’s insect medium. These results contradict those from other studies of glyphosate’s impacts on arthropod reproduction, which found that glyphosate negatively affects fecundity. After consuming prey exposed to glyphosate, the spider Alpaida veniliae showed impaired ovary development, reduced egg production, and reduced number of hatchlings from those eggs (Benamú et al. 2010).

Chrysoperla externa, a species of green lacewings, also experience reduced fertility and fecundity after exposure to glyphosate (Schneider et al. 2009). C. externa produced abnormal eggs and pupae showed increased mortality after treatment by glyphosate

(Schneider et al. 2009). Glyphosate also reduced the reproductive period of female C. externa (Schneider et al. 2009). The mechanism by which glyphosate affects reproduction in arthropods is not well understood, but some have speculated that glyphosate may mimic hormones found in arthropods and interfere with endocrine systems, leading to changes in reproduction (Benamú et al. 2010).

In contrast, other herbicides, such as atrazine, can positively affect reproduction, as found with glyphosate in the current study. Exposure of P. milvina females to atrazine leads to higher probability of producing a second egg sac after a single mating (Godfrey and

Rypstra 2018). Females also produce similar numbers of eggs after treatment by atrazine, but these eggs are larger in mass (Godfrey and Rypstra 2018). One possible explanation for our results is the concept of pesticide-induced homeostatic modulation, a term coined to include hormesis and the stimulatory effects of pesticides on non-target pests (Cohen

2006). This term was suggested to be used in place of hormesis when specifically discussing stimulatory effects of pesticides on non-target organisms, as Cohen (2006) argues that pesticides can’t be referred to as stressors on non-target organisms as it can be difficult to measure mortality and or inhibition at high doses of these pesticides. This hypothesis suggests that chemicals that may be stressful and inhibitory to organisms at high doses actually show stimulatory effects at low doses and could explain why low,

21 manufacturer-recommended doses of herbicides can lead to various stimulatory effects on arthropods (Cohen 2006). This has been shown to be the case with various herbicides leading to stimulatory effects on reproduction in numerous species of arthropods.

Another alternative explanation for this increase in egg production by females sprayed with glyphosate is the terminal investment hypothesis. The terminal investment hypothesis states that the trade-off between current and future reproduction declines as females age and investment in current reproduction should therefore increase with age

(Creighton et al. 2009). Although we did not find actual reductions in lifespan, females sprayed with glyphosate may invest more into current reproduction if a reduction in lifespan is anticipated, supporting our results that females sprayed with glyphosate produced more eggs. This effect is seen in other species of crickets. Upon infection by bacteria, both female Texas field crickets (G. texensis) and house crickets (Acheta domesticus) increase egg production when a favorable oviposition substrate is present

(Shoemaker et al. 2006; Copeland and Fedorka 2012). There is limited evidence of terminal investment after exposure to herbicides in crickets, but exposure of male mealworm beetles (Tenebrio molitor) to increased concentrations of atrazine leads to increased preference by females, indicating terminal investment through dishonest signaling by the males (McCallum et al. 2013). Support for glyphosate causing this effect in insects has not previously been documented. These results suggest that individual effects of glyphosate and immune challenges on female fecundity may not be contributing to the successful colonization of V. micado, as both species experienced increases in fecundity after exposure to glyphosate and reductions in fecundity when

22 immune challenged. However, we found a significant interaction between glyphosate exposure and induced immune challenge on G. pennsylvanicus fecundity. G. pennsylvanicus females produced fewer eggs when sprayed with glyphosate and also provided with an induced immune challenge and this interaction was not present in V. micado females. Because V. micado females don’t experience this interaction, they may have increased fecundity compared to G. pennsylvanicus females when both glyphosate and immune challenges are present, which could contribute to their establishment success.

Calling effort

There was no effect detected of species, injection, or spray treatments on male calling effort. Production of calls by male crickets is known to have a large metabolic cost

(Hoback 1997). We hypothesized that it may be costly to respond to the effects of an environmental toxicant such as glyphosate and that this investment may result in a trade off with calling effort. Additionally, it has been shown that investment into immune responses may also incur an energetic cost resulting in reduced reproductive output. This led us to hypothesize that inducing an immune response in males would impact calling effort. However, neither glyphosate nor LPS injection affected male calling effort. One explanation for these results is the immunocompetence handicap hypothesis, which posits that sexually selected male traits should be positively correlated with immunocompetence, or strong immune function (Drayton et al. 2012). This hypothesis

23 states that males that are of “higher quality” possess more resources to invest into both sexual displays and immune responses. This may have been the case as lower fitness males may have died before reaching adulthood, so only those of highest quality survived to observations and were able to invest sufficiently into both calling effort and immune responses.

Our results show that glyphosate does not affect male calling effort or the lifespan of crickets that survive exposure during penultimate instar. It also suggests that an immune challenge has no negative effect on male calling. As males call to attract females for mating, this implies that neither exposure to glyphosate nor immune challenges impacts potential reproductive success as related to female attraction through calling.

Adult lifespan

We found that G. pennsylvanicus adults had longer lifespans than V. micado adults.

However, neither glyphosate nor immune challenge had an effect on adult lifespan of either species. These results contradict our findings with exposure of juvenile G. vocalis to glyphosate. Mortality of penultimate individuals after spraying was not recorded during this experiment. However, juvenile G. vocalis sprayed with glyphosate showed reduced survival compared to those sprayed with DI water. If glyphosate also affects survival of penultimate individuals, it is possible that our measurements were taken on higher fitness individuals that were able to sustain spraying by glyphosate and reach

24 adulthood. This could have led to lack of differences in adult lifespan and male calling effort. Few studies on glyphosate’s effect on lifespan of arthropods exist, but our results agree with those found by Galin et al. (2019): D. melanogaster adult males showed reduced lifespan after exposure to glyphosate (Galin et al. 2019). Alternatively, in another study, D. melanogaster females did not show a difference in lifespan after exposure to pure glyphosate (Bednarova et al. 2020). Additionally, route of exposure may affect survival. Wolf spiders (P. milvina) exposed to glyphosate topically survive the same amount of time as those exposed to water and survive longer than those exposed to glyphosate residually (Evans et al. 2010). Residual exposure entailed spraying half of a piece of filter paper with glyphosate and allowing spiders to walk within an arena on both pieces of filter paper (Evans et al. 2010). This method of exposure is most similar to the application method used in this project. Topical exposure entailed direct application of glyphosate or water to spiders’ abdomens (Evans et al. 2010). It is evident that further work on glyphosate’s effect on lifespan of arthropods is needed. Determining the mechanisms by which glyphosate could impact lifespan of arthropods may explain the range of results found in both the current study and previous studies.

Conclusions

Non-native species can have substantial impacts on competition with native species and may affect community dynamics and threaten biodiversity. Understanding the mechanisms that lie behind successful colonization of non-native species is imperative to conserving biodiversity. This includes understanding how factors such as anthropogenic influences like herbicide application can impact establishment of non-native species. Use of herbicides has grown increasingly common in recent decades, and glyphosate is by far the most common. As of 2018, it was the top-selling herbicide worldwide (Levya-Soto et al. 2018). Yet, the impact of glyphosate on non-target species is still widely under- studied. Of the limited studies on glyphosate, most focus on its potential lethal effects on non-target organisms, and a major lack of information regarding its non-lethal effects still exists. Additionally, Bednarova et al. (2020) found that, while pure glyphosate did not affect lifespan, geotaxis, or fecundity of D. melanogaster females, Roundup Concentrate

Plus and polyethoxylated tallowamine (POEA), a surfactant commonly used in Roundup, both negatively impacted female fecundity, geotaxis, and lifespan. These results suggest that potential effects of Roundup on non-target organisms may be due to ingredients other than glyphosate: further studies are needed to determine whether the negative effects of Roundup are caused by its non-glyphosate ingredients. It is also important to note that there is no standard exposure dose or route of exposure across studies examining glyphosate’s impacts on arthropods. Routes of exposure range from residual or topical to ingestion of glyphosate. Most studies remain within manufacturer-

26 recommended ranges of doses, but these ranges still vary considerably. Only a fraction of previous studies have quantified the exposure of study organisms to glyphosate and these quantities also vary considerably (see Table 1). The present study used a residual route of exposure most similar to those used by Gaupp-Berghausen, et al. (2015), Behrend and

Rypstra (2017) and Rittman et al. (2013). We found that glyphosate sprayed at manufacturer-recommended rates increases egg production in G. pennsylvanicus and V. micado. We also found that V. micado females produce more eggs than G. pennsylvanicus females and that an interaction between glyphosate and investment into immunity exists only in G. pennsylvanicus females, suggesting a tradeoff. These results could explain why V. micado has been observed in higher numbers in the field.

Glyphosate did not have an effect on male calling effort or adult lifespan. Our results suggest that glyphosate may not influence competition between invasive V. micado and native G. pennsylvanicus, at least regarding adult lifespan and calling by males to attract females for mating. Limited studies of the potential effects of glyphosate on non-target terrestrial organisms exist, so further work is needed to understand its impacts on other arthropods and the impacts it may have on invasion success of introduced species. Due to the current lack of consistency across studies, future studies should also aim for increased standardization of exposure doses of glyphosate. This would include methods that allow for the quantification of glyphosate exposure dose, allowing comparison among studies.

Addressing each of these questions could potentially lead to an effective herbicide that lacks the negative effects on non-target organisms and minimizes contributions to successful invasion of novel habitats by non-native species.

Table 1. Summary of studies examining the effects of glyphosate on non-target terrestrial arthropods. Included in the table are species, mass of glyphosate in the dose applied, form of glyphosate used in the study, dosage of glyphosate used in the study, the route of exposure of study organisms to glyphosate, and author names and year article publication.

DI water

LPS

Grace’s insect medium

1.5% Roundup

LPS

Penultimate Penultimate Grace’s Adults are isolated insect Juvenile individuals moved individuals moved medium V. micado to be sprayed to a clean box 24 hours after spraying Penultimate individuals sprayed Figure 1. Juveniles of each species were housed separately in large plastic boxes. Three times a week, penultimate instar individuals were identified and transferred to small plastic boxes to be sprayed with glyphosate or DI water. Twenty-four hours after spraying, crickets were moved to clean boxes, remaining separated by treatment. Three times a week, clean boxes were checked for newly-eclosed adults, which were then transferred to individual home containers. One week later, adults were assigned injected with either LPS or Grace’s insect medium. Forty-eight hours after injections, male calling effort was measured and females were presented with mating opportunities. For females, eggs were collected for 2 weeks after mating. The date of natural death for each adult was also collected.

Effect of glyphosate on G. vocalis juvenile survival

Figure 2. Average number of surviving juvenile (± 1 SE) G. vocalis individuals (out of 5) per container sprayed with water (CTRL, N=20) or glyphosate (GLY, N=20). Fewer juveniles sprayed with glyphosate survived than those sprayed with DI water (* denotes statistical significance at a level of p<0.01).

Change in water weight - GLY vs CTRL 50

30 Weight (g) 25 NS 20

15 Start End

#1 #2 #3 #1 #2 #3

Figure 3. Average change in weight of water vials containing DI water (dotted lines) or 1.5% Roundup (solid lines) between the first and last measurements recorded. The presence of glyphosate did not affect consumption of water by G. vocalis. “NS” denotes p > 0.05.

Change in food weight - GLY vs CTRL 12 11 10 9 NS

Weight (g) 8 7 6 Start End

#1 #2 #3 #1 #2 #3

Figure 4. Average change in weight of petri dishes containing ground rabbit food sprayed once with either DI water (dotted lines) or 1.5% Roundup (solid lines) between the first and last measurements recorded. The presence of glyphosate did not affect food consumption by G. vocalis. “NS” denotes p > 0.05.

A B Effect of spray on fecundity (GP) Effect of injection on fecundity (GP) *

Figure 5. A. Average (± 1 SE) fecundity in G. pennsylvanicus females sprayed with DI water (CTRL) or glyphosate (GLY). Females sprayed with glyphosate (N = 18) produced more eggs than those sprayed with DI water. B. Average fecundity (± 1 SE) in G. pennsylvanicus injected with LPS or Grace’s insect medium (CTRL). Females injected with LPS produced fewer eggs than those injected with Grace’s insect medium. C. Interaction plot of spray and injection treatments. Females sprayed with glyphosate and injected with Grace’s insect medium produced the most eggs while females sprayed with DI water and injected with Grace’s insect medium produced the fewest eggs. Statistical significance at a level of p<0.01 is indicated by *; p<0.001 is indicated by **.

A Effect of spray on fecundity (VM) B Effect of injection on fecundity (VM) * **

Figure 6. A. Average (± 1 SE) fecundity in V. micado females sprayed with DI water (CTRL) or glyphosate (GLY). Females sprayed with glyphosate produced more eggs than those sprayed with DI water. B. Average fecundity (± 1 SE) in V. micado females injected with LPS or Grace’s insect medium (CTRL). Females injected with LPS produced fewer eggs than those injected with Grace’s insect medium. Statistical significance at a level of p<0.01 is indicated by *; p<0.001 is indicated by **.

Effect of species on fecundity

Figure 7. Average (± 1 SE) fecundity in V. micado and G. pennsylvanicus females. V. micado females produced more eggs than G. pennsylvanicus females. Statistical significance at a level of p<0.001 is indicated by **.

A B Effect of species on adult lifespan Effect of species on adult lifespan *

Time (days)

Figure 8. A. Average lifespan of G. pennsylvanicus (GP) and V. micado (VM) individuals. G. pennsylvanicus had a longer lifespan than V. micado adults. B. Average (± 1 SE) lifespan in G. pennsylvanicus (GP) and V. micado (VM) adults. Statistical significance at a level of p<0.01 is indicated by *.

A B

Effect of spray on adult lifespan (GP) Effect of injection on adult lifespan (GP)

NS NS

Time (days) Time (days)

C D Effect of spray on adult lifespan (GP) Effect of injection on adult lifespan (GP) NS NS

Figure 9. A and C. Average (± 1 SE) lifespan in G. pennsylvanicus adults sprayed with glyphosate (GLY) or DI water (CTRL). There was not a difference in lifespan between those sprayed with glyphosate or DI water. B and D. Average (± 1 SE) lifespan in G. pennsylvanicus adults injected with LPS or Grace’s insect medium (CTRL). There was not a difference in adult lifespan between those injected with LPS or Grace’s insect medium. Lack of a statistically significant difference (p>0.01) is indicated by “NS.”

A B Effect of injection on adult lifespan (VM) Effect of spray on adult lifespan (VM)

NS NS

Time (days) Time (days)

C D Effect of injection on adult lifespan (VM) Effect of spray on adult lifespan (VM) NS NS

Figure 10. A and C. Average (± 1 SE) lifespan in V. micado adults injected with LPS or Grace’s insect medium (CTRL). There was not a difference in adult lifespan of those injected with LPS or Grace’s insect medium (CTRL). B and D. Average (± 1 SE) lifespan in V. micado adults sprayed with glyphosate (GLY) or DI water (CTRL). There was not a difference in lifespan of those sprayed with glyphosate or DI water (CTRL). Lack of a statistically significant difference (p>0.01) is indicated by “NS.”

A B Effect of species on calling effort Effect of spray on calling effort NS NS

C Effect of injection on calling effort NS

Figure 11. A. Average (± 1 SE) calling effort of G. pennsylvanicus (GP) and V. micado (VM) males. There was no difference in calling effort between the G. pennsylvanicus and V. micado. B. Average (± 1 SE) calling effort of males sprayed with glyphosate (GLY) or DI water (CTRL). There was not a difference in calling effort of those sprayed with glyphosate or DI water. C. Average (± 1 SE) calling effort of males injected with LPS or Grace’s insect medium (CTRL). There was not a difference in calling effort of individuals injected with LPS or Grace’s insect medium. Lack of a statistically significant difference (p>0.01) is indicated by “NS.”

Bibliography

Bednářová, A., Kropf, M., & Krishnan, N. (2020). The surfactant polyethoxylated tallowamine (POEA) reduces lifespan and inhibits fecundity in Drosophila melanogaster-In vivo and in vitro study. Ecotoxicology and Environmental Safety, 188, 109883.

Benamú, M. A., Schneider, M. I., & Sánchez, N. E. (2010). Effects of the herbicide glyphosate on biological attributes of Alpaida veniliae (Araneae, Araneidae), in laboratory. Chemosphere, 78(7), 871-876.

Benbrook, C. M. (2016). Trends in glyphosate herbicide use in the United States and globally. Environmental Sciences Europe, 28(1), 3.

Bowles, D. E. (2018). Introduced Japanese burrowing cricket (Orthoptera: Gryllidae: Velarifictorus (Velarifictorus) micado) range continues to expand in North America. Journal of Orthoptera Research, 27, 177.

Brockerhoff, E. G., & Liebhold, A. M. (2017). Ecology of forest insect invasions. Biological Invasions, 19(11), 3141-3159.

Carmona, D. M., Menalled, F. D., & Landis, D. A. (1999). Gryllus pennsylvanicus (Orthoptera: Gryllidae): laboratory weed seed predation and within field activity- density. Journal of Economic Entomology, 92(4), 825-829.

Clark, R. M., McConnell, A., Zera, A. J., & Behmer, S. T. (2013). Nutrient regulation strategies differ between cricket morphs that trade off dispersal and reproduction. Functional Ecology, 27(5), 1126-1133.

Cohen, E. (2006). Pesticide-mediated homeostatic modulation in arthropods. Pesticide Biochemistry and Physiology, 85(1), 21-27.

Copeland, E. K., & Fedorka, K. M. (2012). The influence of male age and simulated pathogenic infection on producing a dishonest sexual signal. Proceedings of the Royal Society B: Biological Sciences, 279(1748), 4740-4746.

Creighton, J. C., Heflin, N. D., & Belk, M. C. (2009). Cost of reproduction, resource quality, and terminal investment in a burying beetle. The American Naturalist, 174(5), 673-684.

Drayton, J. M., Hall, M. D., Hunt, J., & Jennions, M. D. (2012). Sexual signaling and immune function in the black field cricket Teleogryllus commodus. PloS one, 7(7).

Evans, S. C., Shaw, E. M., & Rypstra, A. L. (2010). Exposure to a glyphosate-based herbicide affects agrobiont predatory arthropod behaviour and long-term survival. Ecotoxicology, 19(7), 1249-1257.

Fahrner, S., & Aukema, B. H. (2018). Correlates of spread rates for introduced insects. Global Ecology and Biogeography, 27(6), 734-743.

Galin, R. R., Akhtyamova, I. F., & Pastukhova, E. I. (2019). Effect of herbicide glyphosate on Drosophila melanogaster fertility and lifespan. Bulletin of Experimental Biology and Medicine, 167(5), 663-666.

Godfrey, J. A., & Rypstra, A. L. (2018). Impact of an atrazine-based herbicide on an agrobiont wolf spider. Chemosphere, 201, 459-465.

Griesinger, L. M., Evans, S. C., & Rypstra, A. L. (2011). Effects of a glyphosate-based herbicide on mate location in a wolf spider that inhabits agroecosystems. Chemosphere, 84(10), 1461-1466.

Hoback, W. W., & Wagner Jr., W.E. (1997). The energetic cost of calling in the variable field cricket, Gryllus lineaticeps. Physiological Entomology, 22(3), 286-290.

Jacot, A., Scheuber, H., & Brinkhof, M. W. (2004). Costs of an induced immune response on sexual display and longevity in field crickets. Evolution, 58(10), 2280-2286.

Kerr, A. M., Gershman, S. N., & Sakaluk, S. K. (2010). Experimentally induced spermatophore production and immune responses reveal a trade-off in crickets. Behavioral Ecology, 21(3), 647-654.

Leyva-Soto, L. A., Balderrama-Carmona, A. P., Moran-Palacio, E. F., Diaz-Tenorio, L. M., & Gortares-Moroyoqui, P. (2018). Glyphosate and aminomethylphosphonic acid in population of agricultural fields: Health risk assessment overview. Applied Ecology and Environmental Research, 16, 5127-5140.

McCallum, M. L., Matlock, M., Treas, J., Safi, B., Sanson, W., & McCallum, J. L. (2013). Endocrine disruption of sexual selection by an estrogenic herbicide in the mealworm beetle (Tenebrio molitor). Ecotoxicology, 22(10), 1461-1466.

Mole, S., & Zera, A. J. (1994). Differential resource consumption obviates a potential flight–fecundity trade-off in the sand cricket (Gryllus firmus). Functional Ecology, 573-580.

Mooney, H. A., & Cleland, E. E. (2001). The evolutionary impact of invasive species. Proceedings of the National Academy of Sciences, 98(10), 5446-5451.

Motta, E. V., Raymann, K., & Moran, N. A. (2018). Glyphosate perturbs the gut microbiota of honey bees. Proceedings of the National Academy of Sciences, 115(41), 10305-10310.

Woodburn, A. T. (2000). Glyphosate: production, pricing and use worldwide. Pest Management Science: formerly Pesticide Science, 56(4), 309-312.

Pimentel, D., & Levitan, L. (1986). Pesticides: amounts applied and amounts reaching pests. Bioscience, 36(2), 86-91.

Rittman, S., Wrinn, K. M., Evans, S. C., Webb, A. W., & Rypstra, A. L. (2013). Glyphosate-based herbicide has contrasting effects on prey capture by two co- occurring wolf spider species. Journal of chemical ecology, 39(10), 1247-1253.

Schneider, M. I., Sanchez, N., Pineda, S., Chi, H., & Ronco, A. (2009). Impact of glyphosate on the development, fertility and demography of Chrysoperla externa (Neuroptera: Chrysopidae): Ecological approach. Chemosphere, 76(10), 1451- 1455.

Shoemaker, K. L., Parsons, N. M., & Adamo, S. A. (2006). Egg-laying behaviour following infection in the cricket Gryllus texensis. Canadian journal of zoology, 84(3), 412-418.

Srygley, R. B., Lorch, P. D., Simpson, S. J., & Sword, G. A. (2009). Immediate protein dietary effects on movement and the generalised immunocompetence of migrating Mormon crickets Anabrus simplex (Orthoptera: Tettigoniidae). Ecological Entomology, 34(5), 663-668.

Storm, J. J., & Lima, S. L. (2008). Predator-naïve fall field crickets respond to the chemical cues of wolf spiders. Canadian Journal of Zoology, 86(11), 1259-1263.

Tyler, F., Fisher, D., d'Ettorre, P., Rodríguez-Muñoz, R., & Tregenza, T. (2015). Chemical cues mediate species recognition in field crickets. Frontiers in Ecology and Evolution, 3, 48.

Vandenberg, L. N., Blumberg, B., Antoniou, M. N., Benbrook, C. M., Carroll, L., Colborn, T., & Mesnage, R. (2017). Is it time to reassess current safety standards for glyphosate-based herbicides?. Journal of Epidemiological Community Health, 71(6), 613-618.

Young, B. G. (2006). Changes in herbicide use patterns and production practices resulting from glyphosate-resistant crops. Weed Technology, 20(2), 301-307.

Zeng, Y., & Zhu, D. H. (2014). Geographical variation in body size, development time, and wing dimorphism in the cricket Velarifictorus micado (Orthoptera: Gryllidae). Annals of the Entomological Society of America, 107(6), 1066-1071.

Zuk, M. (1988). Parasite load, body size, and age of wild-caught male field crickets (Orthoptera: Gryllidae): Effects on sexual selection. Evolution, 42(5), 969-976.

Zuk, M., Simmons, L. W., Rotenberry, J. T., & Stoehr, A. M. (2004). Sex differences in immunity in two species of field crickets. Canadian Journal of Zoology, 82(4), 627-634.