Roundup and immune challenge have different effects on a native field cricket and its introduced competitor

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We examine recently introduced Velafictorus micado Japanese burrowing crickets which share a microhabitat and season with native Gryllus pennsylvanicus field crickets. In this study, we assess the combined effects of Roundup (glyphosate-based herbicide) and a lipopolysaccharide (LPS) immune challenge on both crickets. In both species, an immune challenge reduced the numbers of eggs that female laid, however, this effect was much larger in G. pennsylvanicus . Conversely, Roundup caused both species to increase egg production, potentially representing a terminal investment strategy. The combined effect of immune challenge and herbicide harmed G. pennsylvanicus fecundity more than V. micado fecundity. Further, V. micado females laid significantly more eggs than G. pennsylvanicus , suggesting that introduced V. micado may have a competitive edge in fecundity over native G. pennsylvanicus . LPS and Roundup each had differing effects on male G. pennsylvanicus and V. micado calling effort. Overall, introduced male V. micado spent significantly more time calling than native G. pennsylvanicus , which could potentially interfere with G. pennsylvanicus mate-location behavior in their shared natural habitat. Despite the population-level spread of introduced V. micad o, in our study, this species did not outperform native G. pennsylvanicus in tolerating immune and chemical challenge. Although V. micado appears to possess traits that make this introduced species successful in colonizing new habitats, it may be less successful in traits that would allow it to outcompete a native species. Roundup glyphosate life history traits insect immunity immune challenge cricket song Gryllus pennsylvanicus Velafictorus micado fecundity survival Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Biotic and abiotic factors can work additively or synergistically to cause population decline and extinction in native species. Human movement can facilitate the introduction of new species (Brockerhoff and Liebhold 2017 ) and human land use alters habitat quality and introduces novel stressors like herbicides (Sánchez-Bayo and Wyckhuys 2019 ). For species that live in or near residential, commercial and agricultural areas, pesticides can be a part of their abiotic environment. Glyphosate, the world’s top-selling pesticide, is the active ingredient in the herbicide Roundup and other glyphosate-based herbicides (GBHs). Glyphosate works by inhibiting the enzymatic activity of EPSP synthase in plants, preventing the synthesis of three aromatic amino acids and causing plant death (Costas-Ferreira et al. 2022 ). Because animals lack this metabolic pathway, glyphosate has been regarded as relatively safe to animals (Duke and Powles 2008 ). Further, although glyphosate was initially thought to have low to moderate persistence in the environment (Duke and Powles 2008 ), there is evidence that glyphosate can remain in soils for months or years (Battaglin et al. 2014 ; Singh et al. 2020 ). In most commercial applications, GBHs contain not only glyphosate but also unknown inactive ingredients, including surfactants that allow the glyphosate to penetrate plant cuticle (Mullin 2015 ). Surfactants can also damage arthropods’ protective waxy cuticle, facilitating drowning and allowing chemicals to penetrate. Moreover, the “inactive” ingredients in commercial pesticides can be highly toxic to insects, either alone or in combination with active ingredients (Mullin 2015 , Straw et al. 2022 , Stahlschmidt et al. 2022 ). There is now a substantial body of data demonstrating that glyphosate and GBH exposure can negatively affect animals. Glyphosate has potential neurotoxic effects in mammals, fish, and invertebrates (Costas-Ferreira et al. 2022 ). Many studies have demonstrated that glyphosate and GBHs are toxic to bees (Battisti et al. 2021 ): GBHs impair development, learning, feeding, digestion, and survival in honey bees (Battisti et al. 2021 ), increase mortality in bumble bees (Straw et al. 2020), and reduce reproduction in solitary bees (Graffigna et al. 2021 ). GBHs also have negative effects on other terrestrial insects. In wolf spiders, GBHs affect predatory and reproductive behaviors (Rittman et al. 2013 ; Behrend and Rypstra 2018 ; Lacava et al. 2021 ). In Madagascar hissing cockroaches, GBHs can reduce locomotion and slow neural activity (Kanabar et al. 2021 ). The majority of studies of the effects of glyphosate in insects focus on species that feed on parts of living plants (e.g. bees, ants) or forage for prey in living plants (e.g. wolf spiders). Relatively few studies on terrestrial insects focus on detritivores (Kanabar et al. 2021 ; Stahlschmidt et al. 2022 ). However, detritivores like field crickets that live adjacent to humans may experience multiple routes of exposure to GBHs: eggs and hatchlings live within soils that may have been treated with GBHs, crickets ingest decomposing plant matter that may have been sprayed with GBHs, and older nymphs and adult crickets shelter in live weeds and tall grass that may be directly sprayed with GBHs. In addition to abiotic environmental stressors, pathogens and parasites can also contribute to population declines and local extinctions (de Castro and Bolker 2005 ). Because each individual has access to a finite pool of resources, effort devoted to fighting an immune challenge can come at the cost of other life history traits like growth and reproduction (Roff 1992 ). In crickets, an immune challenge can slow growth (Simmons 2012 ), decrease survival (Limberger et al. 2022 ), prevent males from being able to mate (Leman et al. 2009 ), cause males to allocate less effort to calling (Jacot et al. 2004 ) or reproduction (Kerr et al. 2010 ; Simmons 2012 ), reduce female fecundity (Stahlschmidt et al. 2013 ; Limberger et al. 2022 ) or damage the viability of sperm stored by females (McNamara et al. 2014 ). Alternatively, an immune challenge can elicit a terminal investment, meaning that when faced with a threat to survival, an individual maximizes reproductive success by diverting resources away from long-term reproduction and survival and towards current reproduction (Clutton-Brock 1984 ). Female crickets that have experienced an immune challenge may respond by increasing egg size (Bascuñá-García et al. 2010 ) or daily egg output (Adamo 1999 ). In this study, we focus on the effects of glyphosate and immune challenge on two crickets: the fall field cricket Gryllus pennsylvanicus and the Japanese burrowing cricket Velafictorus micado. G. pennsylvanicus is endemic to the mid- and northern United States and southern Canada. The Japanese burrowing cricket Velafictorus micado was first observed in North America in the 1950s (Alexander and Walker 1962 ) and has rapidly spread across the U.S. (Bowles 2018 ). The two species have synchronous reproductive seasons (Alexander and Bigelow 1960 ; Alexander and Walker 1962 ) and can be commonly found co-occupying the same microhabitats (SNG unpublished obs ). In some urban and suburban areas, populations of G. pennsylvanicus are becoming scarce, while populations of V. micado are becoming increasingly common (SNG unpublished obs ). Previous studies demonstrate that the addition of non-native species into a community can contribute to declines in native species (Sánchez-Bayo and Wyckhuys 2019 ). Further, biotic and abiotic factors can contribute to the competitive ability of introduced species relative to native species (Oduor 2013 ; Shivega and Aldrich-Wolf 2017; Wiśniewski et al. 2020 ). When a non-native competitor is introduced, potential outcomes include local extinction of the native species (Freed et al. 2008 ; Catford et al. 2018 ), niche displacement of the native species (Race 1982 ; Shucksmith et al. 2009 ; Silva et al. 2021 ), coexistence with a cost to the native species (Flanagan et al. 2010 ; Jessop et al. 2015 ; Putnam and Peckol 2018 ), or local extinction of the introduced species (Simberloff and Gibbons 2004 ; García-Ramos et al. 2015 ). In some instances, exposure to introduced competitors can promote adaptations in native species that allow them a competitive advantage in future introductions (Leger and Espeland 2010 ; Oduor 2013 ; Dostál 2022 ). In our study, we compare how herbicide exposure and immune challenge affect behavioral and life history traits in both a native species and its introduced competitor. By doing so, we hope to gain insight into whether the decline of a native insect and the spread of an introduced species are driven by differences in their responses to immune challenges and anthropogenic chemicals. To determine the combined effect of glyphosate exposure and an immune challenge on cricket behavior and life history traits, in Part 1 of this study, we sprayed penultimate instar G. pennsylvanicus and V. micado crickets with either Roundup or water, and later injected adults with either an immune challenge (LPS) or a sham injection. We assayed the effect of herbicide and immune treatment on male calling effort, female egg-laying and adult survival. We predicted that individually, LPS and Roundup would harm male calling effort, female egg-laying and adult survival. Further, individuals who received both an LPS immune challenge and Roundup would fare worse than individuals who received either LPS or Roundup. In Part 2 of this study, we examined the effects of Roundup on male calling song components. In many crickets, males in better condition produce songs with specific characteristics and songs with these characteristics are preferred by females (Wagner 1996 ; Holzer et al. 2003 ; Scheuber et al. 2003 ). As an environmental stressor, glyphosate could potentially alter characteristics of male calling song. Further, glyphosate exposure may occur when individuals are adults, and it is possible that adult exposure to glyphosate may have a stronger effect on behavior and traits than juvenile exposure (Stahlschmidt et al. 2022 ). Here, we sprayed field-caught G. pennsylvanicus and V. micado adults with either glyphosate or water to determine how adult glyphosate exposure affected components of male calling song. Methods 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 (H 2 O) 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 H 2 O 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. Results Preliminary experiments: The effects of direct versus indirect spraying on juvenile survival The mode of administration (direct spraying vs. substrate spraying) had an effect on the survival of juvenile G. pennsylvanicus (Likelihood ratio χ 2 = 25.7, P < 0.0001), as did the substance sprayed (water, Roundup Pro Max, or Roundup Natural Week Killer) (Likelihood ratio χ 2 = 23.6, P < 0.0001). Regardless of what was sprayed, there was no immediate mortality in crickets who were exposed to sprayed leaves but not directly sprayed. However, when crickets were directly sprayed, Roundup Pro Max (with glyphosate) slightly decreased survival, and Roundup Natural Weed Killer (without glyphosate) dramatically decreased survival (Water: 15/15 survived; Roundup Pro Max (with glyphosate): 12/15 survived; Roundup Natural Week Killer (without glyphosate): 2/15 survived). Part 1: The effect of Roundup and immune challenge on life history traits In both V. micado and G. pennsylvanicus , females injected with LPS laid fewer eggs than sham-injected females (ZI Poisson Wald V. micado 𝞆 2 = 4.48, P = 0.034; G. pennsylvanicus 𝞆 2 = 297.6, P < 0.0001; Fig. 4 ). Surprisingly, female V. micado sprayed with Roundup laid more eggs than those sprayed with water (ZI Poisson Wald V. micado 𝞆 2 = 7.5, P = 0.0061); we found the same non-significant trend in G. pennsylvanicus (ZI Poisson Wald G. pennsylvanicus 𝞆 2 = 2.30, P = 0.13). In G. pennsylvanicus there was an interaction between immune and spray treatments (ZI Poisson Wald G. pennsylvanicus 𝞆 2 = 199.7, P < 0.001) with LPS-injected females laying relatively few eggs, regardless of spray treatment, whilst sham-injected females who were sprayed with Roundup laid more eggs. In V. micado , there was not a significant interaction between immune treatment and spray treatment (ZI Poisson Wald V. micado 𝞆 2 = 0.07, P = 0.78). Further, V. micado females laid more eggs than G. pennsylvanicus females (ZI Poisson Wald 𝞆 2 447.5, P < 0.0001). We found different effects of immune challenge on each species: In V. micado , males injected with LPS sang less (ZI Poisson Wald 𝞆 2 = 7.72, P = 0.0055; Fig. 5 ), but in male G. pennsylvanicus , LPS injection did not affect singing (ZI Poisson Wald 𝞆 2 = 0.16, P = 0.69). We also found opposite effects of herbicide on each species: V. micado males sprayed with Roundup sang less than individuals sprayed with water, but G. pennsylvanicus males sprayed with Roundup sang more (ZI Poisson Wald V. micado 𝞆 2 = 6.93, P = 0.0085; G. pennsylvanicus 𝞆 2 = 7.87, P = 0.005). There was a significant interaction between immune challenge and herbicide in each species. In LPS-injected V. micado , Roundup decreased calling effort, but in sham-injected V. micado , there was relatively little effect of Roundup (ZI Poisson Wald 𝞆 2 = 11.2, P = 0.0008). In G. pennsylvanicus , Roundup increased male singing in both LPS-injected and sham-injected males, but there was less of an increase in singing in LPS-injected males than sham-injected males (ZI Poisson Wald 𝞆 2 = 11.0, P = 0.0009). We found effects of both species and sex on survival: G. pennsylvanicus survived longer than V. micado (Cox proportional hazard 𝛘 2 = 5.53, P = 0.019; Fig. 6 ), and in each species, females survived longer than males (Cox proportional hazard V. micado 𝛘 2 = 12.3, P = 0.0005; G. pennsylvanicus 𝛘 2 = 25.1, P < 0.0001). Analyzing each species by sex, there was not a statistically significant effect of Roundup, LPS, or their interaction on survival (Cox proportional hazard V. micado females 𝛘 2 3 = 1.26, P = 0.74; V. micado males 𝛘 2 3 = 8.34, P = 0.08; G. pennsylvanicus females 𝛘 2 3 = 3.66, P = 0.30; G. pennsylvanicus males 𝛘 2 3 = 0.94, P = 0.81). Part 3: Effect of glyphosate on male song characteristics Prior to spray treatment, we found differences between G. pennsylvanicus and V. micado in call characteristics. The dominant frequencies of the two species differed. V. micado produced more chirps and trains of chirps and chirped for longer periods of time than G. pennsylvanicus . G. pennsylvanicus produced longer trains than V. micado . Although V. micado spent more time singing than G. pennsylvanicus , this difference was not statistically significant (Table 1 ). Table 1 Differences in song characteristics between G. pennsylvanicus and V. micado. An asterisk indicates a p-value < 0.05 Song characteristic G. pennsylvanicus (mean ± SE) V. micado (mean ± SE) t Ratio p Number of chirps 1791.2 ± 2312.5 10601.8 ± 1656.0 -3.10 0.0030* Duration of chirps 0.106 ± 0.010 0.141 ± 0.008 -2.68 0.0096* Number of trains 34.9 ± 97.3 530.0 ± 69.7 -4.14 0.0001* Duration of trains 30.3 ± 7.81 4.2 ± 5.59 2.72 0.0087* Duration of song 1277.1 ± 680.9 2728.6 ± 487.6 -1.73 0.0885 Dominant frequency 4.33 ± 0.073 3.74 ± 0.068 5.61 < 0.0001* Roundup did not have an effect on song characteristics. There was not a statistically significant effect of spray treatment, species or the interaction between spray treatment and species on the residuals of any song characteristics (number of chirps ANOVA F 3,25 = 1.14, p = 0.35; duration of chirps ANOVA F 3,25 = 0.70, p = 0.56; number of trains ANOVA F 3,25 = 0.18, p = 0.91; duration of trains ANOVA F 3,25 = 0.15, p = 0.93; duration of song ANOVA F 3,25 = 1.21, p = 0.33; average dominant frequency ANOVA F 3,15 = 0.39, p = 0.77). Discussion In this study, we found sub-lethal effects of Roundup on both native and introduced species of cricket. We found that female fecundity was affected by both Roundup and immune challenge. In both V. micado and G. pennsylvanicus , females who had experienced an immune challenge (LPS) laid fewer eggs than females injected with a sham control. This result is consistent with previous studies that found a trade-off between immunity and reproduction in female crickets (Stahlschmidt et al. 2013 ; Limberger et al. 2022 ). Because we used non-pathogenic LPS to stimulate an immune challenge, we can deduce that the reduction in fecundity was not due to somatic damage caused by infection. When females faced an immune challenge, they devoted fewer resources towards reproduction. In this study we found that female V. micado crickets exposed to Roundup laid more eggs than V. micado sprayed with water. We found a similar trend (not statistically significant) in G. pennsylvanicus females. The mechanism behind this pattern is not clear, but we can propose three possibilities. First, this pattern could be explained by an unmeasured trade-off (Strobl et al. 2020). For example, egg size and number are negatively correlated in many species (Roff 1992 ), including field crickets (Carrière and Roff 1995 ; Stahlschmidt et al. 2013 ). If Roundup reduced egg size, we might expect to see the observed increase in numbers of eggs. Smaller eggs may have lower survival and fitness (Stahlschmidt et al. 2013 ). Consequently, in future studies, it would be valuable to explore the effects of Roundup on egg size and offspring survival to determine the total effect of Roundup on female fitness. A second explanation for why Roundup increased female fecundity is terminal investment (Clutton-Brock 1984 ): the presence of Roundup could be perceived by female crickets as a survival threat, causing females to lay all of their eggs immediately, rather than withholding eggs for later. Although no previous studies have looked for an effect of Roundup on cricket fecundity, we know that female crickets faced with other threats may respond by increasing daily egg output (Adamo 1999 ). In other arthropod taxa in which researchers have looked for an effect of Roundup, glyphosate or GBHs on insect fecundity, herbicide reduced fecundity (Mirande et al. 2010; Schneider et al. 2009; de Cuhra et al. 2013; Lares et al. 2022) or did not affect female fecundity (Saska et al. 2017; Bednarova et al. 2020). As none of these previous studies focused on orthopterans or detritovores, it is unclear whether our results are contrary to previous results or whether they reflect taxonomic and ecological differences between our study species and previously studied systems. Either way, our results highlight the need for additional studies on the effects of Roundup on diverse taxa. A third possibility for why females sprayed with Roundup laid more eggs is that Roundup (glyphosate or inactive ingredients) directly chemically stimulates female crickets to lay eggs. In field crickets, males produce prostaglandin synthetase that are transferred to females during mating and stimulate females to lay eggs (Destephano and Brady 1977 ; Loher et al. 1981 ; Stanley-Samuelson et al. 1987 ). These chemicals represent a form of sexual conflict: a male benefits from inducing a female to use all of her mature eggs to produce his offspring. Conversely, a female may gain genetic benefits from retaining some mature eggs for a later mating with a different male (Tregenza and Wedell 1998 ). It is possible that either glyphosate or an inactive ingredient in Roundup can mimic the effect of male prostaglandins on female crickets. If Roundup induces short-term egg release because of similarity to prostaglandins, it would be valuable to find out whether other insects (e.g. 28-spotted potato ladybird, rice brown planthopper, silk moth) that are similarly affected by prostaglandins (Stanley and Kim 2011) also increase oviposition in response to Roundup. Further, prostaglandins also affect insect immunity (Stanley and Kim 2011), so if Roundup chemical compounds bind with receptors that respond to prostaglandins, Roundup could potentially affect insect immunity. We did not find a statistically significant interaction between the effects of Roundup and immune challenge on V. micado female fecundity: both LPS and sham-injected females responded similarly to Roundup by laying more eggs. However, in G. pennsylvanicus , there was a statistically significant interaction between the effects of Roundup and immune challenge on female fecundity: G. pennsylvanicus females who had experienced an immune challenge laid relatively few eggs, regardless of whether they were sprayed with Roundup. However, female G. pennsylvanicus who had not received an immune challenge (sham injection) laid more eggs when exposed to Roundup. So, a previous immune challenge can mediate the effects of Roundup. Although studies have examined the effect of glyphosate on insect immunity (Smith et al. 2021 ), no previous studies have examined the combined effect of an immune challenge and Roundup exposure on other life history traits. As insects are actually exposed to many challenges in nature (e.g. pathogen exposure, low-quality diet, desiccation, predation stress), it is necessary to examine the interactions between these variables to fully assess the effects of glyphosate and GBHs on animals. Because we found that an immune challenge had a negative effect on fecundity in both species, and Roundup did not have a negative effect on fecundity for either species, the ability of introduced V. micado to thrive in the field relative to native G. pennsylvanicus cannot be attributed to either differences in immunity or response to herbicide. However, we did find that V. micado females lay significantly more eggs than native G. pennsylvanicus females. This difference could allow V. micado a strong competitive edge over G. pennsylvanicus in the field. Although we found differences in survival among sexes and species, we found no effect of Roundup or LPS on survival in either species. Under equivalent lab conditions, we found that G. pennsylvanicus survived longer than V. micado. It is unknown whether the increased survival of G. pennsylvanicus results in additional reproductive success. We found that in both species, females survived longer than males. This pattern is consistent with both previous published studies on crickets (Kelly et al. 2018 ) and life history theory that because males need only live long enough to mate, but females must live long enough to mate and lay eggs, females should allocate more resources to somatic maintenance and longevity than males (Rolff 2002 ). We did not find an effect of Roundup or LPS on survival in either species. We did not expect to find an effect of Roundup on survival, as we chose our mode of delivery and dosage to be sub-lethal. Further, as LPS is immunogenic not pathogenic, we did not expect that LPS-injected individuals would have reduced lifespan. We predicted that survival could be affected by the interaction between Roundup and LPS, with doubly challenged individuals suffering a reduced lifespan as compared to individuals who received only Roundup, only LPS, or control, but we did not find a difference in survival among treatment groups. In our study, we found that V. micado males that had been injected with an immune challenge sang less. Male calling song effort is considered a condition-dependent trait in many crickets: females prefer males that sing more (Wagner 1996 ; Holzer et al. 2003 ; Zeng et al. 2022 ) and males in better condition sing more (Wagner and Hoback 1999 ; Holzer et al. 2003 ; Scheuber et al. 2003 ; Bertram and Rook 2012 ). So, our result is congruent with previous studies, as well as studies that find a relationship between immunity and male cricket song (Ryder and Siva-Jothy 2000 ; Simmons et al. 2005 ). However, we did not find an effect of LPS immune challenge on calling effort in G. pennsylvanicus males. This difference in response to immune challenge among the two species could potentially reflect differences in how resources are re-allocated after an immune challenge, with V. micado trading off effort devoted to immunity with effort devoted to song, and G. pennsylvanicus preserving calling effort at the expense of another unmeasured trait. We found opposite effects of Roundup on male calling effort in each species: V. micado males sprayed with Roundup sang less, while G. pennsylvanicus sprayed with Roundup sang more. It is notable that for V. micado , both challenges (LPS and Roundup) caused males to reduce calling effort. This solution to harm or re-allocation of resources may be related to V. micado ’s population structure: introduced V. micado is found at much higher densities in the field than native G. pennsylvanicus (SNG unpublished data). Consequently, a small decrease in long-distance calling song effort may have smaller impact on male V. micado reproductive success than it would on male G. pennsylvanicus reproductive success. Conversely, the response of male G. pennsylvanicus to Roundup (increasing calling effort) is congruent to the response of female G. pennsylvanicus to Roundup (increasing egg production). It seems possible that in this species, both males and females respond to Roundup as a survival threat and employ a terminal investment strategy, allocating more resources to current reproduction at the expense of future reproduction. In each species, there was a statistically significant interaction between the effects of LPS and Roundup on male calling effort. When V. micado males were injected with LPS immune challenge, Roundup caused males to decrease in calling effort, but in sham-injected V. micado , there was relatively little effect of Roundup on calling effort. In G. pennsylvanicus , Roundup caused males to increase calling effort in both LPS-injected and sham-injected males, but there was less of an increase in calling effort in LPS-injected males than in sham-injected males. We did not find an effect of adult exposure to Roundup on male calling song characteristics. Predictably, we found differences among species in song characteristics. In addition to the song characteristics that distinguish one species from the other, V. micado spent much more time chirping than G. pennsylvanicus did: V. micado produced more chirps and trains of chirps and chirped for longer periods of time than G. pennsylvanicus . When G. pennsylvanicus did sing, they tended to chirp more continuously than V. micado , produced longer trains of chirps. Although G. pennsylvanicus song structure has been extensively studied (e.g. Ciceran et al. 1994 ; Judge 2011 ; Harrison et al. 2013 ), this paper is the first to examine the effect of male condition on V. micado song. Nothing is known about female preference for male song characteristics in V. micado ; we intend to pursue this issue in future studies. Given that male V. micado call from similar (or the same) microhabitats as G. pennsylvanicus , future studies will also be necessary to determine the impact of V. micado song on G. pennsylvanicus behavior. We also highlight an incidental finding from our preliminary experiments: Roundup Natural Weedkiller, the glyphosate-free formulation of Roundup, is overwhelmingly lethal to crickets when crickets are directly exposed to it. It is likely that this product has a similar effect on other insects. As previously noted, the surfactants and other inactive ingredients included in herbicides may be substantially more lethal than the active ingredients (Mullin 2015 ; Bednarova et al. 2020; Stahlschmidt et al. 2022 ; Straw et al. 2022 ). We urge others to avoid this product and discourage its sale and use. In sum, we found effects of both immune challenge and Roundup on life history traits and behavior in native G. pennsylvanicus and introduced V. micado crickets. Exposure to immune challenges and Roundup in the field cause individuals to alter traits important to both sexual selection and natural selection. However, the ability of V. micado to thrive and spread across the U.S. while native G. pennsylvanicus become patchy and locally extinct cannot be explained by differences in the two species in their ability to tolerate an immune challenge or withstand Roundup. Instead, the ability of female V. micado to produce more eggs than G. pennsylvanicus females and the ability of male V. micado to chirp more than male G. pennsylvanicus may give this introduced species a competitive edge over native G. pennsylvanicus . The impacts of introduced competitors on native species are unpredictable (Davis 2003 ; Wittmann et al. 2013 ). Factors like rate of introduction and growth of the new species and the degree of interspecific competition can influence whether an introduced species is capable of supplanting a native competitor (Wittmann et al. 2013 ). Further, biotic and abiotic factors can contribute to the competitive ability of introduced species relative to native species (Oduor 2013 ; Shivega and Aldrich-Wolf 2017; Wiśniewski et al. 2020 ). In our study, we found that Roundup and immune challenge have different effects on introduced V. micado as compared to native G. pennsylvanicus . Despite the population-level spread of introduced V. micad o, in our study, this species does not appear to outperform the native G. pennsylvanicus in tolerating immune and chemical challenge. However, the ability to colonize a new area may trade off with the ability to compete once there (Catford et al. 2018 ). In V. micado , increased egg production and long-range calling ability are likely to facilitate the spread of this species into new habitats. However, once in this new habitat, V. micado may not outperform G. pennsylvanicus in biotic and abiotic challenges. Other studies have shown that the ability of an introduced competitor to supplant native species can be affected by biotic factors like pathogens (Pizzatto & Shine 2011 ; Vilscinskas et al. 2013; Wiśniewski et al. 2020 ), and abiotic environmental factors (Shivega and Aldrich-Wolf 2017; Wiśniewski et al. 2020 ). Future studies should examine the abilities of competing native and introduced species to tolerate herbicides, insecticides and other anthropogenic chemicals, as these have the potential to influence whether native species are able to persist, co-exist or face local extinction. Declarations Availability of Data and Materials Raw data for this study can be accessed through OSU Knowledge Bank (url will be provided later) Funding This work was funded by a Fred E. Obey Scholarship, an OSU Marion Research Development Grant, and a Regional Campus Faculty Research/Creative Activity grant from The OSU College of Arts & Sciences. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Acknowledgements We thank Jaret Cingel, Leigh Carabbia, and Erin Schuster who assisted in the laboratory work for this project. We thank committee members Roman Lanno and Ian Hamilton for their constructive feedback on this project and its analysis. Author Contributions LRM and SNG designed the preliminary studies and main experiment. LRM performed the main experiment. DJB, SRL, TAB and SNG designed and performed the song analysis. LRM and SNG analyzed the results. SNG wrote the paper. Ethical Approval Not applicable Consent to participate Not applicable Consent to Publish Not applicable Conflict of interest The authors declare no competing interests. References Adamo SA (1999) Evidence for adaptive changes in egg laying in crickets exposed to bacteria and parasites. Anim Behav 57:117–124. doi: 10.1006/anbe.1998.0999 Alexander RD, Bigelow RS (1960) Allochronic speciation in field crickets, and a new species, Acheta veletis . Evolution 14:334–346. doi: 10.2307/2405976 Alexander RD, Walker TJ (1962) Two introduced field crickets new to eastern United States (Orthoptera: Gryllidae). 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Ecol Entomol 47:1–7. doi: 10.1111/een.13193 Supplementary Files MullinsetalSupplementalmaterials.docx Cite Share Download PDF Status: Published Journal Publication published 07 Jun, 2023 Read the published version in Environmental Science and Pollution Research → Version 1 posted Editorial decision: Major Revision 18 Mar, 2023 Reviewers agreed at journal 09 Feb, 2023 Editor invited by journal 08 Feb, 2023 Editor assigned by journal 29 Jan, 2023 First submitted to journal 05 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2440526","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":174914556,"identity":"f855b321-7dcd-4844-a157-e4f097a3ceed","order_by":0,"name":"Lydia R Mullins","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Lydia","middleName":"R","lastName":"Mullins","suffix":""},{"id":174914557,"identity":"9d84785a-30bb-44b7-b6eb-7fc1a11eb24a","order_by":1,"name":"Dylan J Brown","email":"","orcid":"","institution":"The Ohio State University - Marion Campus","correspondingAuthor":false,"prefix":"","firstName":"Dylan","middleName":"J","lastName":"Brown","suffix":""},{"id":174914558,"identity":"ed7bbcac-71da-4981-9335-96d1e569fb44","order_by":2,"name":"Shelly R Lovsey","email":"","orcid":"","institution":"The Ohio State University - Marion Campus","correspondingAuthor":false,"prefix":"","firstName":"Shelly","middleName":"R","lastName":"Lovsey","suffix":""},{"id":174914559,"identity":"ac96a1fa-5ff5-46e9-84de-2f068b298f9d","order_by":3,"name":"Troy A Bowers","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Troy","middleName":"A","lastName":"Bowers","suffix":""},{"id":174914560,"identity":"803080dd-c3b2-4566-804b-1d766edae95f","order_by":4,"name":"Susan N Gershman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYJACZjDJ3nwAwj1AtBaeYwmkapHwMSBOi7x77+PPhTl2eQwSPB8f/mxjkOO7kYBfi+GZ42bSM7clFzNI92425m1jMJYkqGVGGhsz7zbmxAaZs9ukGdsYEjcQ1DL/GfNn3m31iQ0SOc8kgQ6rJ6hFXoKNQZp322GQFjYJoMMSDAhpMeBJYwNqOZ7YxnPM2JjnnIThzDMPCNjSfgzksOrEfvbmhw9/lNnI8x0nZMsBKIMNQkngVw62pYGwmlEwCkbBKBjpAADy70Ec0PdFygAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4451-820X","institution":"The Ohio State University - Marion Campus","correspondingAuthor":true,"prefix":"","firstName":"Susan","middleName":"N","lastName":"Gershman","suffix":""}],"badges":[],"createdAt":"2023-01-03 21:49:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2440526/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2440526/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-023-27866-6","type":"published","date":"2023-06-07T21:05:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":32943154,"identity":"e93a5f47-f8a6-4b6f-8dd7-c9f0a5ddaac9","added_by":"auto","created_at":"2023-02-14 21:57:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109456,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design for Part 1: The effect of glyphosate and immune challenge on life history traits\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/8046728da004fa96d9037aad.png"},{"id":32942804,"identity":"713dfe21-9af0-4f6d-8cbc-1f2893c84a5c","added_by":"auto","created_at":"2023-02-14 21:49:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62293,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental design of Part 2: The effect of Roundup on song characteristics.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/a60b494e8e703086476e7701.png"},{"id":32942112,"identity":"56e99a9e-fc83-421a-88e7-6ccb7cf5c6a4","added_by":"auto","created_at":"2023-02-14 21:41:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":19909,"visible":true,"origin":"","legend":"\u003cp\u003eMode of delivery and Roundup formulation affect juvenile mortality in \u003cem\u003eG. pennsylvanicus\u003c/em\u003e. Late instar crickets were either directly sprayed (Direct spray) or dried leaves sheltering crickets were sprayed (Leaf spray). Individuals were sprayed with either deionized water (white bar), Roundup Natural Weedkiller without glyphosate (grey bar) or Roundup ProMax with glyphosate (black bar).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/646082a0b89c9d986e517dbd.png"},{"id":32943153,"identity":"9da43760-a5d9-4ac6-ad1f-ff87850815b7","added_by":"auto","created_at":"2023-02-14 21:57:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57092,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of spray treatment (water or Roundup) and immune treatment (sham injection or lipopolysaccharide (LPS) injection) on mean (± 1 SE) number of eggs laid by (a) \u003cem\u003eV. micado\u003c/em\u003e and (b) \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/480a50306e595b82cf1fddc6.png"},{"id":32942806,"identity":"966a0901-c303-4bc7-85af-fc339289c004","added_by":"auto","created_at":"2023-02-14 21:49:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56036,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of spray treatment (water or Roundup) and immune treatment (sham injection or lipopolysaccharide (LPS) injection on mean (± 1 SE) calling effort (number times observed singing over four hours) by (a) \u003cem\u003eV. micado\u003c/em\u003e and (b) \u003cem\u003eG. pennsylvanicus\u003c/em\u003e males.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/e34ad32df3ee42febf8aa802.png"},{"id":32942117,"identity":"0d583c2f-ed95-4253-a96d-0132a10cade7","added_by":"auto","created_at":"2023-02-14 21:41:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":25648,"visible":true,"origin":"","legend":"\u003cp\u003eDaily survival of \u003cem\u003eV. micado\u003c/em\u003e (Vm) males (dashed grey), \u003cem\u003eV. micado\u003c/em\u003e females (solid grey), \u003cem\u003eG. pennsylvanicus\u003c/em\u003e (Gp) males (dashed black) and \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females (solid black). Within each species and sex, daily survival from all individuals from the four herbicide and immune challenge treatment are pooled.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/69e908f42cda2836c1d4636d.png"},{"id":44730609,"identity":"8f20d941-01ba-4862-873f-6bac6142d032","added_by":"auto","created_at":"2023-10-16 21:32:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":571402,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/4b9b9c7b-829a-40da-a20f-1924fca20352.pdf"},{"id":32942808,"identity":"bbf992ac-429f-47a1-80b3-a35d4ef6c88a","added_by":"auto","created_at":"2023-02-14 21:49:14","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":26481,"visible":true,"origin":"","legend":"","description":"","filename":"MullinsetalSupplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-2440526/v1/4f693ea727e368f2448f5096.docx"}],"financialInterests":"","formattedTitle":"Roundup and immune challenge have different effects on a native field cricket and its introduced competitor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiotic and abiotic factors can work additively or synergistically to cause population decline and extinction in native species. Human movement can facilitate the introduction of new species (Brockerhoff and Liebhold \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and human land use alters habitat quality and introduces novel stressors like herbicides (S\u0026aacute;nchez-Bayo and Wyckhuys \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For species that live in or near residential, commercial and agricultural areas, pesticides can be a part of their abiotic environment. Glyphosate, the world\u0026rsquo;s top-selling pesticide, is the active ingredient in the herbicide Roundup and other glyphosate-based herbicides (GBHs). Glyphosate works by inhibiting the enzymatic activity of EPSP synthase in plants, preventing the synthesis of three aromatic amino acids and causing plant death (Costas-Ferreira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Because animals lack this metabolic pathway, glyphosate has been regarded as relatively safe to animals (Duke and Powles \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Further, although glyphosate was initially thought to have low to moderate persistence in the environment (Duke and Powles \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), there is evidence that glyphosate can remain in soils for months or years (Battaglin et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In most commercial applications, GBHs contain not only glyphosate but also unknown inactive ingredients, including surfactants that allow the glyphosate to penetrate plant cuticle (Mullin \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Surfactants can also damage arthropods\u0026rsquo; protective waxy cuticle, facilitating drowning and allowing chemicals to penetrate. Moreover, the \u0026ldquo;inactive\u0026rdquo; ingredients in commercial pesticides can be highly toxic to insects, either alone or in combination with active ingredients (Mullin \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Straw et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Stahlschmidt et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere is now a substantial body of data demonstrating that glyphosate and GBH exposure can negatively affect animals. Glyphosate has potential neurotoxic effects in mammals, fish, and invertebrates (Costas-Ferreira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Many studies have demonstrated that glyphosate and GBHs are toxic to bees (Battisti et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e): GBHs impair development, learning, feeding, digestion, and survival in honey bees (Battisti et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), increase mortality in bumble bees (Straw et al. 2020), and reduce reproduction in solitary bees (Graffigna et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). GBHs also have negative effects on other terrestrial insects. In wolf spiders, GBHs affect predatory and reproductive behaviors (Rittman et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Behrend and Rypstra \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lacava et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In Madagascar hissing cockroaches, GBHs can reduce locomotion and slow neural activity (Kanabar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The majority of studies of the effects of glyphosate in insects focus on species that feed on parts of living plants (e.g. bees, ants) or forage for prey in living plants (e.g. wolf spiders). Relatively few studies on terrestrial insects focus on detritivores (Kanabar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Stahlschmidt et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, detritivores like field crickets that live adjacent to humans may experience multiple routes of exposure to GBHs: eggs and hatchlings live within soils that may have been treated with GBHs, crickets ingest decomposing plant matter that may have been sprayed with GBHs, and older nymphs and adult crickets shelter in live weeds and tall grass that may be directly sprayed with GBHs.\u003c/p\u003e \u003cp\u003eIn addition to abiotic environmental stressors, pathogens and parasites can also contribute to population declines and local extinctions (de Castro and Bolker \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Because each individual has access to a finite pool of resources, effort devoted to fighting an immune challenge can come at the cost of other life history traits like growth and reproduction (Roff \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). In crickets, an immune challenge can slow growth (Simmons \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), decrease survival (Limberger et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), prevent males from being able to mate (Leman et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), cause males to allocate less effort to calling (Jacot et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) or reproduction (Kerr et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Simmons \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), reduce female fecundity (Stahlschmidt et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Limberger et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) or damage the viability of sperm stored by females (McNamara et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Alternatively, an immune challenge can elicit a terminal investment, meaning that when faced with a threat to survival, an individual maximizes reproductive success by diverting resources away from long-term reproduction and survival and towards current reproduction (Clutton-Brock \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Female crickets that have experienced an immune challenge may respond by increasing egg size (Bascu\u0026ntilde;\u0026aacute;-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) or daily egg output (Adamo \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we focus on the effects of glyphosate and immune challenge on two crickets: the fall field cricket \u003cem\u003eGryllus pennsylvanicus\u003c/em\u003e and the Japanese burrowing cricket \u003cem\u003eVelafictorus micado. G. pennsylvanicus\u003c/em\u003e is endemic to the mid- and northern United States and southern Canada. The Japanese burrowing cricket \u003cem\u003eVelafictorus micado\u003c/em\u003e was first observed in North America in the 1950s (Alexander and Walker \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) and has rapidly spread across the U.S. (Bowles \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The two species have synchronous reproductive seasons (Alexander and Bigelow \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1960\u003c/span\u003e; Alexander and Walker \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) and can be commonly found co-occupying the same microhabitats (SNG \u003cem\u003eunpublished obs\u003c/em\u003e). In some urban and suburban areas, populations of \u003cem\u003eG. pennsylvanicus\u003c/em\u003e are becoming scarce, while populations of \u003cem\u003eV. micado\u003c/em\u003e are becoming increasingly common (SNG \u003cem\u003eunpublished obs\u003c/em\u003e). Previous studies demonstrate that the addition of non-native species into a community can contribute to declines in native species (S\u0026aacute;nchez-Bayo and Wyckhuys \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Further, biotic and abiotic factors can contribute to the competitive ability of introduced species relative to native species (Oduor \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shivega and Aldrich-Wolf 2017; Wiśniewski et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). When a non-native competitor is introduced, potential outcomes include local extinction of the native species (Freed et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Catford et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), niche displacement of the native species (Race \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Shucksmith et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), coexistence with a cost to the native species (Flanagan et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Jessop et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Putnam and Peckol \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), or local extinction of the introduced species (Simberloff and Gibbons \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Garc\u0026iacute;a-Ramos et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In some instances, exposure to introduced competitors can promote adaptations in native species that allow them a competitive advantage in future introductions (Leger and Espeland \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Oduor \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dost\u0026aacute;l \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, we compare how herbicide exposure and immune challenge affect behavioral and life history traits in both a native species and its introduced competitor. By doing so, we hope to gain insight into whether the decline of a native insect and the spread of an introduced species are driven by differences in their responses to immune challenges and anthropogenic chemicals. To determine the combined effect of glyphosate exposure and an immune challenge on cricket behavior and life history traits, in Part 1 of this study, we sprayed penultimate instar \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e crickets with either Roundup or water, and later injected adults with either an immune challenge (LPS) or a sham injection. We assayed the effect of herbicide and immune treatment on male calling effort, female egg-laying and adult survival. We predicted that individually, LPS and Roundup would harm male calling effort, female egg-laying and adult survival. Further, individuals who received both an LPS immune challenge and Roundup would fare worse than individuals who received either LPS or Roundup. In Part 2 of this study, we examined the effects of Roundup on male calling song components. In many crickets, males in better condition produce songs with specific characteristics and songs with these characteristics are preferred by females (Wagner \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Holzer et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Scheuber et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). As an environmental stressor, glyphosate could potentially alter characteristics of male calling song. Further, glyphosate exposure may occur when individuals are adults, and it is possible that adult exposure to glyphosate may have a stronger effect on behavior and traits than juvenile exposure (Stahlschmidt et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Here, we sprayed field-caught \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e adults with either glyphosate or water to determine how adult glyphosate exposure affected components of male calling song.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eRoundup and Glyphosate\u003c/p\u003e \u003cp\u003eIn 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. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eIn our preliminary experiments, we also tested the effects of Roundup Natural Weedkiller (without glyphosate) (Active ingredient: acetic acid 5\u0026ndash;10%; Other ingredients: 90\u0026ndash;95%). We intended to include Roundup Natural Weedkiller as a control for Roundup\u0026rsquo;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.\u003c/p\u003e \u003cp\u003ePreliminary experiments to determine mode of delivery\u003c/p\u003e \u003cp\u003ePrior to conducting experiments to determine the effect of glyphosate on \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e, 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 \u003cem\u003eGryllus vocalis\u003c/em\u003e the vocal field cricket as our model cricket for these preliminary tests. Unlike \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e, \u003cem\u003eG. vocalis\u003c/em\u003e 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).\u003c/p\u003e \u003cp\u003eWe 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 \u0026ndash; 0.40 g) \u003cem\u003eG. pennsylvanicus\u003c/em\u003e individually in 1L plastic tubs with field-collected dried leaves for shelter and \u003cem\u003ead libitum\u003c/em\u003e 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, \u0026ldquo;tossed\u0026rdquo; 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 \u003cem\u003eV. micado\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCricket maintenance\u003c/p\u003e \u003cp\u003eThe parents of experimental \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e were field-collected from the same sites in central Ohio. In 2018, 60 \u003cem\u003eG. pennsylvanicus\u003c/em\u003e adults and 250 \u003cem\u003eV. micado\u003c/em\u003e adults were collected. Females of each species were allowed to oviposit into soil which was stored for 6\u0026ndash;7 months at 4\u0026deg;C to allow eggs to diapause, then incubated at 25\u0026deg;C to initiate hatching. Crickets were housed in a growth chamber at 28\u0026deg;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 \u003cem\u003ead libitum\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003ePart 1: The effect of Roundup and immune challenge on life history traits\u003c/p\u003e \u003cp\u003eThe general experimental design was to expose juvenile \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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 (H\u003csub\u003e2\u003c/sub\u003eO) 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 \u003cem\u003ead libitum\u003c/em\u003e 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 H\u003csub\u003e2\u003c/sub\u003eO 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 \u003cem\u003ead libitum\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOne week after adult eclosion, each cricket received an injection of either lipopolysaccharides (LPS), known to induce an immune response in crickets, or Grace\u0026rsquo;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 \u0026micro;L of solution. The LPS stock solution consisted of 25 \u0026micro;g of LPS per 100 \u0026micro;L Grace\u0026rsquo;s insect medium (50\u0026ndash;70% sucrose, 1\u0026ndash;5% calcium chloride, L-malic acid and glutamic acid). Individuals assigned to the SHAM injection treatment were injected with Grace\u0026rsquo;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.\u003c/p\u003e \u003cp\u003eTo measure fecundity, females were given the opportunity to mate with two different conspecific males. A male was added to each female\u0026rsquo;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\u0026rsquo;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.\u003c/p\u003e \u003cp\u003eTo measure male calling effort, 48 hours after injection, males\u0026rsquo; 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.\u003c/p\u003e \u003cp\u003eTo 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 \u003cem\u003eG. pennsylvanicus\u003c/em\u003e 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\u0026thinsp;=\u0026thinsp;0.05 was used for all hypothesis testing.\u003c/p\u003e \u003cp\u003ePart 2: The effect of Roundup on song characteristics\u003c/p\u003e \u003cp\u003eTo determine how adult Roundup exposure affects components of male calling song, we field-caught \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e 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\u0026ndash;48 hours after capture, adult crickets were housed in group boxes by species with \u003cem\u003ead libitum\u003c/em\u003e water and food (alfalfa pellets, dried oatmeal and dried dog food) at 25\u0026ndash;27\u0026deg;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 \u003cem\u003ead libitum\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eCricket calling song is composed of pulses of sound (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eG. pennsylvanicus\u003c/em\u003e chirps are comprised of four pulses. \u003cem\u003eV. micado\u003c/em\u003e chirps are comprised of 6\u0026ndash;8 pulses. For each 4-hour recording of cricket song, we used Audacity software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.audacityteam.org\" target=\"_blank\"\u003ewww.audacityteam.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.audacityteam.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) 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\u0026rsquo;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 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rug.mnhn.fr/seewave/\u003c/span\u003e\u003cspan address=\"https://rug.mnhn.fr/seewave/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). 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\u0026ndash;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePreliminary experiments: The effects of direct versus indirect spraying on juvenile survival\u003c/p\u003e\n\u003cp\u003eThe mode of administration (direct spraying vs. substrate spraying) had an effect on the survival of juvenile \u003cem\u003eG. pennsylvanicus\u003c/em\u003e (Likelihood ratio \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;25.7, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as did the substance sprayed (water, Roundup Pro Max, or Roundup Natural Week Killer) (Likelihood ratio \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;23.6, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Regardless of what was sprayed, there was no immediate mortality in crickets who were exposed to sprayed leaves but not directly sprayed. However, when crickets were directly sprayed, Roundup Pro Max (with glyphosate) slightly decreased survival, and Roundup Natural Weed Killer (without glyphosate) dramatically decreased survival (Water: 15/15 survived; Roundup Pro Max (with glyphosate): 12/15 survived; Roundup Natural Week Killer (without glyphosate): 2/15 survived).\u003c/p\u003e\n\u003cp\u003ePart 1: The effect of Roundup and immune challenge on life history traits\u003c/p\u003e\n\u003cp\u003eIn both \u003cem\u003eV. micado\u003c/em\u003e and \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, females injected with LPS laid fewer eggs than sham-injected females (ZI Poisson Wald \u003cem\u003eV. micado\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;4.48, P\u0026thinsp;=\u0026thinsp;0.034; \u003cem\u003eG. pennsylvanicus\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;297.6, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Surprisingly, female \u003cem\u003eV. micado\u003c/em\u003e sprayed with Roundup laid more eggs than those sprayed with water (ZI Poisson Wald \u003cem\u003eV. micado\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;7.5, P\u0026thinsp;=\u0026thinsp;0.0061); we found the same non-significant trend in \u003cem\u003eG. pennsylvanicus\u003c/em\u003e (ZI Poisson Wald \u003cem\u003eG. pennsylvanicus\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;2.30, P\u0026thinsp;=\u0026thinsp;0.13). In \u003cem\u003eG. pennsylvanicus\u003c/em\u003e there was an interaction between immune and spray treatments (ZI Poisson Wald \u003cem\u003eG. pennsylvanicus\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;199.7, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with LPS-injected females laying relatively few eggs, regardless of spray treatment, whilst sham-injected females who were sprayed with Roundup laid more eggs. In \u003cem\u003eV. micado\u003c/em\u003e, there was not a significant interaction between immune treatment and spray treatment (ZI Poisson Wald \u003cem\u003eV. micado\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.07, P\u0026thinsp;=\u0026thinsp;0.78). Further, \u003cem\u003eV. micado\u003c/em\u003e females laid more eggs than \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females (ZI Poisson Wald 𝞆\u003csup\u003e2\u003c/sup\u003e 447.5, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\n\u003cp\u003eWe found different effects of immune challenge on each species: In \u003cem\u003eV. micado\u003c/em\u003e, males injected with LPS sang less (ZI Poisson Wald 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;7.72, P\u0026thinsp;=\u0026thinsp;0.0055; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), but in male \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, LPS injection did not affect singing (ZI Poisson Wald 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.16, P\u0026thinsp;=\u0026thinsp;0.69). We also found opposite effects of herbicide on each species: \u003cem\u003eV. micado\u003c/em\u003e males sprayed with Roundup sang less than individuals sprayed with water, but \u003cem\u003eG. pennsylvanicus\u003c/em\u003e males sprayed with Roundup sang more (ZI Poisson Wald \u003cem\u003eV. micado\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;6.93, P\u0026thinsp;=\u0026thinsp;0.0085; \u003cem\u003eG. pennsylvanicus\u003c/em\u003e 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;7.87, P\u0026thinsp;=\u0026thinsp;0.005). There was a significant interaction between immune challenge and herbicide in each species. In LPS-injected \u003cem\u003eV. micado\u003c/em\u003e, Roundup decreased calling effort, but in sham-injected \u003cem\u003eV. micado\u003c/em\u003e, there was relatively little effect of Roundup (ZI Poisson Wald 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;11.2, P\u0026thinsp;=\u0026thinsp;0.0008). In \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, Roundup increased male singing in both LPS-injected and sham-injected males, but there was less of an increase in singing in LPS-injected males than sham-injected males (ZI Poisson Wald 𝞆\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;11.0, P\u0026thinsp;=\u0026thinsp;0.0009).\u003c/p\u003e\n\u003cp\u003eWe found effects of both species and sex on survival: \u003cem\u003eG. pennsylvanicus\u003c/em\u003e survived longer than \u003cem\u003eV. micado\u003c/em\u003e (Cox proportional hazard 𝛘\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;5.53, P\u0026thinsp;=\u0026thinsp;0.019; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), and in each species, females survived longer than males (Cox proportional hazard \u003cem\u003eV. micado\u003c/em\u003e 𝛘\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;12.3, P\u0026thinsp;=\u0026thinsp;0.0005; \u003cem\u003eG. pennsylvanicus\u003c/em\u003e 𝛘\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;25.1, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Analyzing each species by sex, there was not a statistically significant effect of Roundup, LPS, or their interaction on survival (Cox proportional hazard \u003cem\u003eV. micado\u003c/em\u003e females 𝛘\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.26, P\u0026thinsp;=\u0026thinsp;0.74; \u003cem\u003eV. micado\u003c/em\u003e males 𝛘\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.34, P\u0026thinsp;=\u0026thinsp;0.08; \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females 𝛘\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.66, P\u0026thinsp;=\u0026thinsp;0.30; \u003cem\u003eG. pennsylvanicus\u003c/em\u003e males 𝛘\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.94, P\u0026thinsp;=\u0026thinsp;0.81).\u003c/p\u003e\n\u003cp\u003ePart 3: Effect of glyphosate on male song characteristics\u003c/p\u003e\n\u003cp\u003ePrior to spray treatment, we found differences between \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e in call characteristics. The dominant frequencies of the two species differed. \u003cem\u003eV. micado\u003c/em\u003e produced more chirps and trains of chirps and chirped for longer periods of time than \u003cem\u003eG. pennsylvanicus\u003c/em\u003e. \u003cem\u003eG. pennsylvanicus\u003c/em\u003e produced longer trains than \u003cem\u003eV. micado\u003c/em\u003e. Although \u003cem\u003eV. micado\u003c/em\u003e spent more time singing than \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, this difference was not statistically significant (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" style=\"width: 1025px;\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eDifferences in song characteristics between \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado.\u003c/em\u003e An asterisk indicates a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr style=\"height: 59px;\"\u003e\n\u003cth style=\"height: 59px; width: 240.594px;\" align=\"left\"\u003e\n\u003cp\u003eSong characteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px; width: 352.406px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. pennsylvanicus\u003c/em\u003e (mean \u0026plusmn; SE)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px; width: 184px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eV. micado\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(mean \u0026plusmn; SE)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px; width: 88px;\" align=\"left\"\u003e\n\u003cp\u003et Ratio\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px; width: 122px;\" align=\"left\"\u003e\n\u003cp\u003ep\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px; width: 240.594px;\" align=\"left\"\u003e\n\u003cp\u003eNumber of chirps\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 352.406px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1791.2 \u0026plusmn; 2312.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 184px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e10601.8 \u0026plusmn; 1656.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 88px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e-3.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 122px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0030*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px; width: 240.594px;\" align=\"left\"\u003e\n\u003cp\u003eDuration of chirps\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 352.406px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.106 \u0026plusmn; 0.010\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 184px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.141 \u0026plusmn; 0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 88px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e-2.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 122px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0096*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px; width: 240.594px;\" align=\"left\"\u003e\n\u003cp\u003eNumber of trains\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 352.406px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e34.9 \u0026plusmn; 97.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 184px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e530.0 \u0026plusmn; 69.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 88px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e-4.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 122px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0001*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px; width: 240.594px;\" align=\"left\"\u003e\n\u003cp\u003eDuration of trains\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 352.406px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e30.3 \u0026plusmn; 7.81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 184px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.2 \u0026plusmn; 5.59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 88px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 122px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0087*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px; width: 240.594px;\" align=\"left\"\u003e\n\u003cp\u003eDuration of song\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 352.406px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1277.1 \u0026plusmn; 680.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 184px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e2728.6 \u0026plusmn; 487.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 88px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e-1.73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 122px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0885\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px; width: 240.594px;\" align=\"left\"\u003e\n\u003cp\u003eDominant frequency\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 352.406px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.33 \u0026plusmn; 0.073\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 184px;\" align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e3.74 \u0026plusmn; 0.068\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 88px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px; width: 122px;\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.0001*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eRoundup did not have an effect on song characteristics. There was not a statistically significant effect of spray treatment, species or the interaction between spray treatment and species on the residuals of any song characteristics (number of chirps ANOVA F\u003csub\u003e3,25\u003c/sub\u003e = 1.14, p\u0026thinsp;=\u0026thinsp;0.35; duration of chirps ANOVA F\u003csub\u003e3,25\u003c/sub\u003e = 0.70, p\u0026thinsp;=\u0026thinsp;0.56; number of trains ANOVA F\u003csub\u003e3,25\u003c/sub\u003e = 0.18, p\u0026thinsp;=\u0026thinsp;0.91; duration of trains ANOVA F\u003csub\u003e3,25\u003c/sub\u003e = 0.15, p\u0026thinsp;=\u0026thinsp;0.93; duration of song ANOVA F\u003csub\u003e3,25\u003c/sub\u003e = 1.21, p\u0026thinsp;=\u0026thinsp;0.33; average dominant frequency ANOVA F\u003csub\u003e3,15\u003c/sub\u003e = 0.39, p\u0026thinsp;=\u0026thinsp;0.77).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found sub-lethal effects of Roundup on both native and introduced species of cricket. We found that female fecundity was affected by both Roundup and immune challenge. In both \u003cem\u003eV. micado\u003c/em\u003e and \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, females who had experienced an immune challenge (LPS) laid fewer eggs than females injected with a sham control. This result is consistent with previous studies that found a trade-off between immunity and reproduction in female crickets (Stahlschmidt et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Limberger et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Because we used non-pathogenic LPS to stimulate an immune challenge, we can deduce that the reduction in fecundity was not due to somatic damage caused by infection. When females faced an immune challenge, they devoted fewer resources towards reproduction.\u003c/p\u003e \u003cp\u003eIn this study we found that female \u003cem\u003eV. micado\u003c/em\u003e crickets exposed to Roundup laid \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emore\u003c/span\u003e eggs than \u003cem\u003eV. micado\u003c/em\u003e sprayed with water. We found a similar trend (not statistically significant) in \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females. The mechanism behind this pattern is not clear, but we can propose three possibilities. First, this pattern could be explained by an unmeasured trade-off (Strobl et al. 2020). For example, egg size and number are negatively correlated in many species (Roff \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), including field crickets (Carri\u0026egrave;re and Roff \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Stahlschmidt et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). If Roundup reduced egg size, we might expect to see the observed increase in numbers of eggs. Smaller eggs may have lower survival and fitness (Stahlschmidt et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Consequently, in future studies, it would be valuable to explore the effects of Roundup on egg size and offspring survival to determine the total effect of Roundup on female fitness.\u003c/p\u003e \u003cp\u003eA second explanation for why Roundup increased female fecundity is terminal investment (Clutton-Brock \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1984\u003c/span\u003e): the presence of Roundup could be perceived by female crickets as a survival threat, causing females to lay all of their eggs immediately, rather than withholding eggs for later. Although no previous studies have looked for an effect of Roundup on cricket fecundity, we know that female crickets faced with other threats may respond by increasing daily egg output (Adamo \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In other arthropod taxa in which researchers have looked for an effect of Roundup, glyphosate or GBHs on insect fecundity, herbicide reduced fecundity (Mirande et al. 2010; Schneider et al. 2009; de Cuhra et al. 2013; Lares et al. 2022) or did not affect female fecundity (Saska et al. 2017; Bednarova et al. 2020). As none of these previous studies focused on orthopterans or detritovores, it is unclear whether our results are contrary to previous results or whether they reflect taxonomic and ecological differences between our study species and previously studied systems. Either way, our results highlight the need for additional studies on the effects of Roundup on diverse taxa.\u003c/p\u003e \u003cp\u003eA third possibility for why females sprayed with Roundup laid more eggs is that Roundup (glyphosate or inactive ingredients) directly chemically stimulates female crickets to lay eggs. In field crickets, males produce prostaglandin synthetase that are transferred to females during mating and stimulate females to lay eggs (Destephano and Brady \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Loher et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Stanley-Samuelson et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). These chemicals represent a form of sexual conflict: a male benefits from inducing a female to use all of her mature eggs to produce his offspring. Conversely, a female may gain genetic benefits from retaining some mature eggs for a later mating with a different male (Tregenza and Wedell \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). It is possible that either glyphosate or an inactive ingredient in Roundup can mimic the effect of male prostaglandins on female crickets. If Roundup induces short-term egg release because of similarity to prostaglandins, it would be valuable to find out whether other insects (e.g. 28-spotted potato ladybird, rice brown planthopper, silk moth) that are similarly affected by prostaglandins (Stanley and Kim 2011) also increase oviposition in response to Roundup. Further, prostaglandins also affect insect immunity (Stanley and Kim 2011), so if Roundup chemical compounds bind with receptors that respond to prostaglandins, Roundup could potentially affect insect immunity.\u003c/p\u003e \u003cp\u003eWe did not find a statistically significant interaction between the effects of Roundup and immune challenge on \u003cem\u003eV. micado\u003c/em\u003e female fecundity: both LPS and sham-injected females responded similarly to Roundup by laying more eggs. However, in \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, there was a statistically significant interaction between the effects of Roundup and immune challenge on female fecundity: \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females who had experienced an immune challenge laid relatively few eggs, regardless of whether they were sprayed with Roundup. However, female \u003cem\u003eG. pennsylvanicus\u003c/em\u003e who had not received an immune challenge (sham injection) laid more eggs when exposed to Roundup. So, a previous immune challenge can mediate the effects of Roundup. Although studies have examined the effect of glyphosate on insect immunity (Smith et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), no previous studies have examined the combined effect of an immune challenge and Roundup exposure on other life history traits. As insects are actually exposed to many challenges in nature (e.g. pathogen exposure, low-quality diet, desiccation, predation stress), it is necessary to examine the interactions between these variables to fully assess the effects of glyphosate and GBHs on animals. Because we found that an immune challenge had a negative effect on fecundity in both species, and Roundup did not have a negative effect on fecundity for either species, the ability of introduced \u003cem\u003eV. micado\u003c/em\u003e to thrive in the field relative to native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e cannot be attributed to either differences in immunity or response to herbicide. However, we did find that \u003cem\u003eV. micado\u003c/em\u003e females lay significantly more eggs than native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females. This difference could allow \u003cem\u003eV. micado\u003c/em\u003e a strong competitive edge over \u003cem\u003eG. pennsylvanicus\u003c/em\u003e in the field.\u003c/p\u003e \u003cp\u003eAlthough we found differences in survival among sexes and species, we found no effect of Roundup or LPS on survival in either species. Under equivalent lab conditions, we found that \u003cem\u003eG. pennsylvanicus\u003c/em\u003e survived longer than \u003cem\u003eV. micado.\u003c/em\u003e It is unknown whether the increased survival of \u003cem\u003eG. pennsylvanicus\u003c/em\u003e results in additional reproductive success. We found that in both species, females survived longer than males. This pattern is consistent with both previous published studies on crickets (Kelly et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and life history theory that because males need only live long enough to mate, but females must live long enough to mate and lay eggs, females should allocate more resources to somatic maintenance and longevity than males (Rolff \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). We did not find an effect of Roundup or LPS on survival in either species. We did not expect to find an effect of Roundup on survival, as we chose our mode of delivery and dosage to be sub-lethal. Further, as LPS is immunogenic not pathogenic, we did not expect that LPS-injected individuals would have reduced lifespan. We predicted that survival could be affected by the interaction between Roundup and LPS, with doubly challenged individuals suffering a reduced lifespan as compared to individuals who received only Roundup, only LPS, or control, but we did not find a difference in survival among treatment groups.\u003c/p\u003e \u003cp\u003eIn our study, we found that \u003cem\u003eV. micado\u003c/em\u003e males that had been injected with an immune challenge sang less. Male calling song effort is considered a condition-dependent trait in many crickets: females prefer males that sing more (Wagner \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Holzer et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zeng et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and males in better condition sing more (Wagner and Hoback \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Holzer et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Scheuber et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Bertram and Rook \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). So, our result is congruent with previous studies, as well as studies that find a relationship between immunity and male cricket song (Ryder and Siva-Jothy \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Simmons et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, we did not find an effect of LPS immune challenge on calling effort in \u003cem\u003eG. pennsylvanicus\u003c/em\u003e males. This difference in response to immune challenge among the two species could potentially reflect differences in how resources are re-allocated after an immune challenge, with \u003cem\u003eV. micado\u003c/em\u003e trading off effort devoted to immunity with effort devoted to song, and \u003cem\u003eG. pennsylvanicus\u003c/em\u003e preserving calling effort at the expense of another unmeasured trait.\u003c/p\u003e \u003cp\u003eWe found opposite effects of Roundup on male calling effort in each species: \u003cem\u003eV. micado\u003c/em\u003e males sprayed with Roundup sang less, while \u003cem\u003eG. pennsylvanicus\u003c/em\u003e sprayed with Roundup sang more. It is notable that for \u003cem\u003eV. micado\u003c/em\u003e, both challenges (LPS and Roundup) caused males to reduce calling effort. This solution to harm or re-allocation of resources may be related to \u003cem\u003eV. micado\u003c/em\u003e\u0026rsquo;s population structure: introduced \u003cem\u003eV. micado\u003c/em\u003e is found at much higher densities in the field than native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e (SNG unpublished data). Consequently, a small decrease in long-distance calling song effort may have smaller impact on male \u003cem\u003eV. micado\u003c/em\u003e reproductive success than it would on male \u003cem\u003eG. pennsylvanicus\u003c/em\u003e reproductive success. Conversely, the response of male \u003cem\u003eG. pennsylvanicus\u003c/em\u003e to Roundup (increasing calling effort) is congruent to the response of female \u003cem\u003eG. pennsylvanicus\u003c/em\u003e to Roundup (increasing egg production). It seems possible that in this species, both males and females respond to Roundup as a survival threat and employ a terminal investment strategy, allocating more resources to current reproduction at the expense of future reproduction. In each species, there was a statistically significant interaction between the effects of LPS and Roundup on male calling effort. When \u003cem\u003eV. micado\u003c/em\u003e males were injected with LPS immune challenge, Roundup caused males to decrease in calling effort, but in sham-injected \u003cem\u003eV. micado\u003c/em\u003e, there was relatively little effect of Roundup on calling effort. In \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, Roundup caused males to increase calling effort in both LPS-injected and sham-injected males, but there was less of an increase in calling effort in LPS-injected males than in sham-injected males.\u003c/p\u003e \u003cp\u003eWe did not find an effect of adult exposure to Roundup on male calling song characteristics. Predictably, we found differences among species in song characteristics. In addition to the song characteristics that distinguish one species from the other, \u003cem\u003eV. micado\u003c/em\u003e spent much more time chirping than \u003cem\u003eG. pennsylvanicus\u003c/em\u003e did: \u003cem\u003eV. micado\u003c/em\u003e produced more chirps and trains of chirps and chirped for longer periods of time than \u003cem\u003eG. pennsylvanicus\u003c/em\u003e. When \u003cem\u003eG. pennsylvanicus\u003c/em\u003e did sing, they tended to chirp more continuously than \u003cem\u003eV. micado\u003c/em\u003e, produced longer trains of chirps. Although \u003cem\u003eG. pennsylvanicus\u003c/em\u003e song structure has been extensively studied (e.g. Ciceran et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Judge \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Harrison et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), this paper is the first to examine the effect of male condition on \u003cem\u003eV. micado\u003c/em\u003e song. Nothing is known about female preference for male song characteristics in \u003cem\u003eV. micado\u003c/em\u003e; we intend to pursue this issue in future studies. Given that male \u003cem\u003eV. micado\u003c/em\u003e call from similar (or the same) microhabitats as \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, future studies will also be necessary to determine the impact of \u003cem\u003eV. micado\u003c/em\u003e song on \u003cem\u003eG. pennsylvanicus\u003c/em\u003e behavior.\u003c/p\u003e \u003cp\u003eWe also highlight an incidental finding from our preliminary experiments: Roundup Natural Weedkiller, the glyphosate-free formulation of Roundup, is overwhelmingly lethal to crickets when crickets are directly exposed to it. It is likely that this product has a similar effect on other insects. As previously noted, the surfactants and other inactive ingredients included in herbicides may be substantially more lethal than the active ingredients (Mullin \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bednarova et al. 2020; Stahlschmidt et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Straw et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We urge others to avoid this product and discourage its sale and use.\u003c/p\u003e \u003cp\u003eIn sum, we found effects of both immune challenge and Roundup on life history traits and behavior in native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and introduced \u003cem\u003eV. micado\u003c/em\u003e crickets. Exposure to immune challenges and Roundup in the field cause individuals to alter traits important to both sexual selection and natural selection. However, the ability of \u003cem\u003eV. micado\u003c/em\u003e to thrive and spread across the U.S. while native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e become patchy and locally extinct cannot be explained by differences in the two species in their ability to tolerate an immune challenge or withstand Roundup. Instead, the ability of female \u003cem\u003eV. micado\u003c/em\u003e to produce more eggs than \u003cem\u003eG. pennsylvanicus\u003c/em\u003e females and the ability of male \u003cem\u003eV. micado\u003c/em\u003e to chirp more than male \u003cem\u003eG. pennsylvanicus\u003c/em\u003e may give this introduced species a competitive edge over native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe impacts of introduced competitors on native species are unpredictable (Davis \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Wittmann et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Factors like rate of introduction and growth of the new species and the degree of interspecific competition can influence whether an introduced species is capable of supplanting a native competitor (Wittmann et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Further, biotic and abiotic factors can contribute to the competitive ability of introduced species relative to native species (Oduor \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shivega and Aldrich-Wolf 2017; Wiśniewski et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study, we found that Roundup and immune challenge have different effects on introduced \u003cem\u003eV. micado\u003c/em\u003e as compared to native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e. Despite the population-level spread of introduced \u003cem\u003eV. micad\u003c/em\u003eo, in our study, this species does not appear to outperform the native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e in tolerating immune and chemical challenge. However, the ability to colonize a new area may trade off with the ability to compete once there (Catford et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In \u003cem\u003eV. micado\u003c/em\u003e, increased egg production and long-range calling ability are likely to facilitate the spread of this species into new habitats. However, once in this new habitat, \u003cem\u003eV. micado\u003c/em\u003e may not outperform \u003cem\u003eG. pennsylvanicus\u003c/em\u003e in biotic and abiotic challenges. Other studies have shown that the ability of an introduced competitor to supplant native species can be affected by biotic factors like pathogens (Pizzatto \u0026amp; Shine \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Vilscinskas et al. 2013; Wiśniewski et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and abiotic environmental factors (Shivega and Aldrich-Wolf 2017; Wiśniewski et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Future studies should examine the abilities of competing native and introduced species to tolerate herbicides, insecticides and other anthropogenic chemicals, as these have the potential to influence whether native species are able to persist, co-exist or face local extinction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eRaw data for this study can be accessed through OSU Knowledge Bank (url will be provided later)\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThis work was funded by a Fred E. Obey Scholarship, an OSU Marion Research Development Grant, and a Regional Campus Faculty Research/Creative Activity grant from The OSU College of Arts \u0026amp; Sciences.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWe thank Jaret Cingel, Leigh Carabbia, and Erin Schuster who assisted in the laboratory work for this project. We thank committee members Roman Lanno and Ian Hamilton for their constructive feedback on this project and its analysis.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eLRM and SNG designed the preliminary studies and main experiment. LRM performed the main experiment. DJB, SRL, TAB and SNG designed and performed the song analysis. LRM and SNG analyzed the results. SNG wrote the paper.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdamo SA (1999) Evidence for adaptive changes in egg laying in crickets exposed to bacteria and parasites. 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Ecol Entomol 47:1\u0026ndash;7. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/een.13193\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Roundup, glyphosate, life history traits, insect immunity, immune challenge, cricket song, Gryllus pennsylvanicus, Velafictorus micado, fecundity, survival","lastPublishedDoi":"10.21203/rs.3.rs-2440526/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2440526/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCrickets face many natural selection pressures, and humans have added to this burden by applying potentially harmful herbicides and unintentionally introducing competitors. We examine recently introduced \u003cem\u003eVelafictorus micado\u003c/em\u003e Japanese burrowing crickets which share a microhabitat and season with native \u003cem\u003eGryllus pennsylvanicus\u003c/em\u003e field crickets. In this study, we assess the combined effects of Roundup (glyphosate-based herbicide) and a lipopolysaccharide (LPS) immune challenge on both crickets. In both species, an immune challenge reduced the numbers of eggs that female laid, however, this effect was much larger in \u003cem\u003eG. pennsylvanicus\u003c/em\u003e. Conversely, Roundup caused both species to increase egg production, potentially representing a terminal investment strategy. The combined effect of immune challenge and herbicide harmed \u003cem\u003eG. pennsylvanicus\u003c/em\u003e fecundity more than \u003cem\u003eV. micado\u003c/em\u003e fecundity. Further, \u003cem\u003eV. micado\u003c/em\u003e females laid significantly more eggs than G. \u003cem\u003epennsylvanicus\u003c/em\u003e, suggesting that introduced \u003cem\u003eV. micado\u003c/em\u003e may have a competitive edge in fecundity over native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e. LPS and Roundup each had differing effects on male \u003cem\u003eG. pennsylvanicus\u003c/em\u003e and \u003cem\u003eV. micado\u003c/em\u003e calling effort. Overall, introduced male \u003cem\u003eV. micado\u003c/em\u003e spent significantly more time calling than native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e, which could potentially interfere with \u003cem\u003eG. pennsylvanicus\u003c/em\u003e mate-location behavior in their shared natural habitat. Despite the population-level spread of introduced \u003cem\u003eV. micad\u003c/em\u003eo, in our study, this species did not outperform native \u003cem\u003eG. pennsylvanicus\u003c/em\u003e in tolerating immune and chemical challenge. Although \u003cem\u003eV. micado\u003c/em\u003e appears to possess traits that make this introduced species successful in colonizing new habitats, it may be less successful in traits that would allow it to outcompete a native species.\u003c/p\u003e","manuscriptTitle":"Roundup and immune challenge have different effects on a native field cricket and its introduced competitor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-14 21:41:08","doi":"10.21203/rs.3.rs-2440526/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2023-03-18T07:11:42+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-02-10T00:43:38+00:00","index":0,"fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2023-02-08T18:56:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-01-30T04:52:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2023-01-05T09:57:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4f2e5943-60b8-460c-b06f-bcdf4020180c","owner":[],"postedDate":"February 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:15:26+00:00","versionOfRecord":{"articleIdentity":"rs-2440526","link":"https://doi.org/10.1007/s11356-023-27866-6","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2023-06-07 21:05:40","publishedOnDateReadable":"June 7th, 2023"},"versionCreatedAt":"2023-02-14 21:41:08","video":"","vorDoi":"10.1007/s11356-023-27866-6","vorDoiUrl":"https://doi.org/10.1007/s11356-023-27866-6","workflowStages":[]},"version":"v1","identity":"rs-2440526","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2440526","identity":"rs-2440526","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}