Roundup and Glyphosate
In all studies, we used 1.5% Roundup ProMax (Active ingredient: glyphosate, N-(phosphonomethyl) glycine 48.7%; Other ingredients: 51.3%) rather than pure glyphosate solution. Roundup is the most commonly available form of glyphosate. The non-active ingredients of Roundup, including surfactants to penetrate plant cuticle, have the potential to affect insects or to mediate the effects of glyphosate on insects (Straw et al. 2022). Because the manufacturer of Roundup will not provide information about these non-active ingredients, the most biologically realistic test of the effects of glyphosate requires Roundup in its commercially available form. The manufacturer-recommended concentration of Roundup is 1.5%, so this is what we used throughout the study.
In our preliminary experiments, we also tested the effects of Roundup Natural Weedkiller (without glyphosate) (Active ingredient: acetic acid 5–10%; Other ingredients: 90–95%). We intended to include Roundup Natural Weedkiller as a control for Roundup’s proprietary non-active ingredients. However, unlike 1.5% Roundup ProMax, which has a mild odor, no color, and foams only slightly when sprayed, Roundup Natural Weedkiller has a strong vinegary odor, and a foamy, milky appearance. Although the odor of Roundup Natural Weedkiller can be attributed to the active ingredient (acetic acid), the color and foam cannot. Thus, it is likely that the two formulations have different non-active ingredients.
Preliminary experiments to determine mode of delivery
Prior to conducting experiments to determine the effect of glyphosate on G. pennsylvanicus and V. micado, we conducted experiments to determine an appropriate mode of delivery for Roundup in our experiments. The goal was to find a biologically realistic route of delivery that would allow us to measure the effect of Roundup on reproductive traits, without bias due to immediate behavioral changes (avoidance) induced by the Roundup or mode of delivery. We used Gryllus vocalis the vocal field cricket as our model cricket for these preliminary tests. Unlike G. pennsylvanicus and V. micado, G. vocalis is a non-diapausing cricket, and was available year-round for experiments. Our preliminary tests indicated that juvenile crickets do not avoid food or water dosed with 1.5% Roundup solution and juvenile cricket sprayed directly with 1.5% Roundup solution have slightly reduced survival as compared to juveniles sprayed with water (S1).
We then tested the effects on direct versus indirect (substrate) Roundup spray on juvenile survival in crickets. In the field, crickets are rarely out in the open during the day. Instead, crickets are hidden in crevices and under grass thatch. Thus, Roundup exposure may occur when crickets contact substrates treated with Roundup spray. For this experiment, we housed lab-reared juvenile (0.25 g – 0.40 g) G. pennsylvanicus individually in 1L plastic tubs with field-collected dried leaves for shelter and ad libitum water (wet cotton ball) and food (ground rabbit pellets). Crickets were sprayed with either deionized water, Roundup Pro Max with glyphosate or Roundup Natural Weedkiller (without glyphosate). Crickets were either directly misted with spray, or dried leaves were misted with spray, “tossed” to ensure even coating (covered with droplets but not soaked), and then immediately used as substrate in cricket containers. Crickets were monitored for mortality for 10 days. Ideally, crickets would have been observed until adult eclosion, however, the experiment was prematurely ended due to pandemic shutdowns. Notably, all mortality observed in the experiment occurred within the first 24 hours after spraying. The pandemic shutdown also prevented us from replicating this experiment using V. micado.
Cricket maintenance
The parents of experimental G. pennsylvanicus and V. micado were field-collected from the same sites in central Ohio. In 2018, 60 G. pennsylvanicus adults and 250 V. micado adults were collected. Females of each species were allowed to oviposit into soil which was stored for 6–7 months at 4°C to allow eggs to diapause, then incubated at 25°C to initiate hatching. Crickets were housed in a growth chamber at 28°C with 12h light: 12h dark per 24-hour cycle. Lab-raised hatchlings of each species were housed in (59.7 cm x 42.9 cm x 31.1 cm) plastic boxes with dried leaves for shelter and ad libitum ground rabbit food and vials of water plugged with cotton. Leaves were collected in 2018 from a single location known to be free of herbicide treatment. Leaves were allowed to decompose outdoors until the start of the experiment. We used only whole leaves that were large enough to provide shelter for crickets. Although crickets preferentially consumed rabbit food, crickets sometimes also consumed small quantities of dried leaves.
Part 1: The effect of Roundup and immune challenge on life history traits
The general experimental design was to expose juvenile G. pennsylvanicus and V. micado crickets to substrate sprayed with either water or 1.5% Roundup Pro Max, then expose crickets to either an immune challenge or a sham injection and determine the effect on female fecundity, male calling effort and survival (Fig. 1). Crickets of each species were recruited into the study as penultimate instar nymphs and randomly assigned to either a 1.5% Roundup Pro Max treatment (GLY) spray box or a water (H2O) spray box. We did not mix species within spray boxes, so we used four spray boxes in total. Spray boxes (42.9 cm x 29.2 cm x 23.8 cm) contained ad libitum ground rabbit food and water in vials plugged with cotton. In both spray treatments, a solid layer of leaf litter was laid over the contents of the box. When the boxes were opened or disturbed, crickets hid beneath the leaves and were not visible on the top surface of the leaf litter. In each box, the top surface of the leaf litter was misted once with 10.5 mL of spray. The GLY treatment box was sprayed with 1.5% Roundup Pro Max and the H2O box was sprayed with deionized water. To control the duration of exposure to the treatments, 24 hours after spraying, we moved crickets to clean boxes containing egg carton for shelter and ad libitum food and water vials. Boxes were checked three times per week for newly-eclosed adults. At eclosion, adults were housed individually in 1 L plastic tubs with egg carton for shelter, rabbit food and a vial of water and randomly assigned to receive either control injections or immune injections.
One week after adult eclosion, each cricket received an injection of either lipopolysaccharides (LPS), known to induce an immune response in crickets, or Grace’s insect medium (SHAM) as a control for the effects of handling stress and injection. Crickets were injected between their 5th and 6th ventral abdominal segments with 5 µL of 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 the SHAM injection treatment were injected with Grace’s insect medium. To minimize stress to animals, crickets were briefly restrained by hand and injections were performed rapidly without anesthesia. Crickets were then returned to their home containers.
To measure fecundity, females were given the opportunity to mate with two different conspecific 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. Males used for mating were not otherwise used in this experiment. Most non-focal males were lab-reared but several field-caught adult males were used to supplement the need for additional 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. Females were checked daily to determine date of death.
To measure male calling effort, 48 hours after injection, males’ calling song behavior was quantified. Every five minutes for four hours, observers recorded whether or not each male was singing. Each male remained in his home container, but the egg carton shelter was removed for unobscured observation. Observations took place during the dark portion of the light cycle. After recording, males were checked daily to determine date of death.
To analyze the effects of species, spray treatment and injection treatment on female fecundity and male calling effort, a generalized linear model specifying a Poisson distribution was used. Because G. pennsylvanicus fecundity data and calling effort for both species contained excess zeroes, we used zero-inflated Poisson regressions. A Cox proportional hazards model was used to analyze the effects of species, spray treatment and injection treatment on adult lifespan. No effect of species on calling effort was found, so data for both species was pooled for this analysis. A significance level of α = 0.05 was used for all hypothesis testing.
Part 2: The effect of Roundup on song characteristics
To determine how adult Roundup exposure affects components of male calling song, we field-caught G. pennsylvanicus and V. micado adult males, recorded them, sprayed them with either Roundup or water, then recorded them again. Adult crickets were captured from sites in central Ohio that were, to the best of our information, not recently treated with herbicides. It is not possible to test for trace amounts of glyphosate byproducts in the environment, so we could not eliminate the possibility that unauthorized spraying or carry-over from other spray sites occurred. However, the presence of live vegetation on cricket collection sites suggests that manufacturer-recommended doses of glyphosate-based herbicides had not been applied this season. Each collection site contained both species of cricket. For 24–48 hours after capture, adult crickets were housed in group boxes by species with ad libitum water and food (alfalfa pellets, dried oatmeal and dried dog food) at 25–27°C in a lab room with natural light. This allowed crickets to feed, rehydrate and acclimate to the lab before recording. On day 1 of the experiment, male crickets were housed individually in 1 L plastic tubs with leaves for shelter and ad libitum water and alfalfa pellets. On day 2, each cricket was recorded for 4 hours between 12-5pm. Each recording took place in a (interior dimensions: 16 x 18 x 20 cm) Styrofoam box containing a button microphone connected to an iPod Touch. Up to eight crickets could be recorded simultaneously. After recording, each cricket was returned to its home container. At 8 am on day 3, the leaf shelters in each home container were sprayed with 3.5 mL of either de-ionized water or 1.5% Roundup Pro Max. On day 4 between 12-5pm, each cricket was recorded for 4 hours.
Cricket calling song is composed of pulses of sound (Fig. 2). G. pennsylvanicus chirps are comprised of four pulses. V. micado chirps are comprised of 6–8 pulses. For each 4-hour recording of cricket song, we used Audacity software (www.audacityteam.org) file labels to identify and mark the start and end of end of each chirp. For each species, we calculated the number of chirps per four-hour recording, average chirp duration (the time from the beginning of a chirp’s first pulse to the end of its last pulse), the number of trains (bouts of chirps), the average duration of trains (length of time spent in bouts of chirps), and total duration of song (total length of time spent chirping). To calculate average dominant frequency of chirps we used Audacity to select 10 consecutive chirps from each cricket song, then used the dfreq function of Seewave (https://rug.mnhn.fr/seewave/). The parameters for the dfreq function included a threshold percentage of 10, as well as a 3-6kHz bandpass filter to eliminate background noise. Researchers analyzing song characteristics did not know the spray treatment (water or Roundup) of the crickets that they analyzed. The final sample size for each treatment was 5–12 males (see S2 for details about sample sizes). To account for individual variation in call characteristics, we calculated the residual of the regression of the second (post-treatment) recording on the first (pre-treatment) recording for each cricket, by species. We used ANOVAs to determine the effect of spray treatment, species, and the interaction between treatment and species on the residuals of each song characteristic.