Exposure to sublethal concentration of flupyradifurone alters sexual behavior and cuticular hydrocarbon profile in Heriades truncorum, an oligolectic solitary bee

The aboveground oligolectic bee, Heriades truncorum, is a particularly good model for studying the impact of pesticides on sexual communication, since some aspects of its mating behavior have previously been described. We have tested (1) the interference of the pesticide flupyradifurone on male precopulatory behavior and male mating partner preferences, (2) the way that the pesticide interferes in male quality assessment by the female, and (3) the effects of the pesticide on the chemical compounds in the female cuticle. We exposed bees of both sexes to a sublethal concentration of flupyradifurone. Various behaviors were registered in a mating arena with two females (one unexposed and one exposed) and one male (either unexposed or exposed). Unexposed males were quicker to attempt to mate. Treatment also impacted precopulatory behavior and male quality assessment by females. Males approached unexposed females more quickly than insecticide‐exposed ones. Females exposed to insecticide produced lower amounts of some cuticular hydrocarbons (sex pheromone candidates) and appeared less choosy than unexposed females. Our findings suggest that insecticide exposure affects sexual communication, playing a role both in male preference and in male quality assessment by the female.


Introduction
In addition to the effects of the accumulated exploitation of natural resources and residues by humans, environmental stressors are leading to a dramatic biodiversity decline in the Anthropocene (Brooks et al., 2002;Foley et al., 2005;Fletcher et al., 2018).Land-use intensification in agricultural landscapes is a major threat for insects that provide essential pollination services (Potts et al., 2010;Kennedy et al., 2013;Goulson et al., 2015).The most important stressors are increases in habitat degradation (e.g., loss of food and nesting resources) and frag-Correspondence: Samuel Boff, Institute of Evolutionary Ecology and Conservation Genomics, Ulm University, Ulm, Germany.Email: samboff@gmail.commentation, climate change, and pesticide use (Godfray et al., 2015).
Moreover, environmental stressors, such as changes in temperature, drought, and ozone, have been found to impact insect reproduction.These stressors have been found to change the time needed for finding a partner, to alter courtship displays and mating duration, and to disrupt sex recognition (Candolin et al., 2007;Gefen & Gibbs, 2009;Conrad et al., 2017;Jiang et al., 2023).Given the ongoing environmental disturbance in agricultural fields with increased landscape alteration and use of pesticides, populations of solitary bees, which are among the most important group of pollinators worldwide (Klein et al., 2007;Giannini et al., 2015), have been demonstrated to be in decline (Potts et al., 2010;Raven & Wagner, 2021).Whereas most studies have indicated that populations of these insects are decreasing, probably because of lethal doses of pesticides increasing mortality (Siviter et al., 2021), less attention has been given to sublethal effects of pesticide on reproduction (Sandrock et al., 2014), which likely also contributes to population density decreases.
Sublethal effects of pesticides have been found to interfere in courtship and consequently in mating in bees (Boff et al., 2022;McAfee et al., 2022;Straub et al., 2022), providing an additional mechanism explaining wild bee decline in agricultural environments (Rundlöf et al., 2015;Woodcock et al., 2017;Willis Chan & Raine, 2021).Despite evidence of these impacts, only a few studies have demonstrated the mechanism in which pesticides affect mating behavior in wild bees (e.g.Boff et al., 2022).
Despite the use of specific behaviors and cuticular chemical compounds as inherent parts of mating behavior in solitary bees (Ayasse et al., 2001), their effectiveness has been determined to be influenced by environmental factors (Conrad et al., 2017;Boff et al., 2022).Horned mason bees (Osmia cornuta), for example, seem to be sensitive to pesticides (Boff et al., 2022).In this species, despite adult males exposed to a sublethal dose of a fungicide being as equally eager to copulate as untreated males, their ability to vibrate their thoracic muscles is affected, and they display a changed cuticular hydrocarbon composition, both of which result in a high rate of female rejections and in copulation rate decline (Boff et al., 2022).
Although these previous findings (Boff et al., 2022;Straub et al., 2022) have added important information related to bee reproduction deficits, they do not yet provide a complete explanation for wild bee population decline in agricultural environments attributable to the impact of pesticides on mating.For example, we know that male bees have a preference for a given mating partner (Bonduriansky, 2001) based on geographical context (Smith & Ayasse, 1987;Vereecken et al., 2007), but we lack other evidence, for example, whether they have mating preferences for females not exposed to pesticide.Moreover, the ability of females to choose high-quality males after exposure to pesticides targeting nicotinic acetylcholine receptors (e.g., flupyradifurone) or other receptors has not been investigated to date.
Flupyradifurone is a new butanolide insecticide widely used in multiple crops to control sucking insects such as aphids and whiteflies.In agricultural fields, bees and other insects can be exposed to this formulation while visiting crop flowers and ruderal plants affected by pesticide spraying and drift in neighboring meadows (Zioga et al., 2023).Despite being considered a promising alternative to neonicotinoids, concerns have been raised regarding the impact of this insecticide on bees.Whereas some studies have shown sublethal effects on honeybee learning abilities (Hesselbach & Scheiner, 2018), others describe abnormal movement behaviors (Tosi et al., 2021).Furthermore, nutritional stress has been found to exacerbate the impact of flupyradifurone on the survival, foraging behavior, and offspring production of the solitary bee Osmia bicornis (Knauer et al., 2022) and the fecundity of Trichogramma evanescens, an important wasp parasitoid used in biological control (Tabebordbar et al., 2020).These effects, particularly the observed impact on reproduction, highlight the necessity for further investigation into the sublethal effects of flupyradifurone on bees and other wild-oligolectic bee species, with specific attention be paid to reproductive-related factors.
Oligolectic bees are species specialized on a selective diet.Previous studies indicate that populations of oligolectic bee species are exposed to stronger environmental pressures than other generalist bees (Biesmeijer et al., 2006), because of their dependence on a few host plant species and their higher sensitivity to landscape change (see Wood et al., 2020) and supposedly to pesticides.The large-headed resin bee, Heriades truncorum (Megachilidae: Osmiini), is an oligolectic bee distributed across Europe and northern Africa.Females build nests by utilizing a variety of habitats, including hollow plant stems, cavities in wood, and various other structures, including human-made bee hotels.In Europe, these bees are active during May-July when they forage on Asteraceae flowers (De Almeida Correa, 1980;Konzmann et al., 2020).The males, which are chemically attracted by the females (Ganuza et al., 2022), display precopulatory behavior such as wing fanning, thoracic vibration, and a rocking motion movement, similar to a dance (Boff, 2022).Females actively perform male quality assessment by biting the male's abdomen when a male mounts the female (Boff, 2022;Ganuza et al., 2022).Males that remain attached to the females after quality assessment can than copulate.
Here, we have carried out mating experiments with the solitary oligolectic bee, H. truncorum, comparing behavioral outcomes for flupyradifurone-exposed and unexposed females and males.Since most solitary hymenopteran females copulate once and the males several times (Paxton, 2005), we have hypothesized that males, independent of pesticide exposure, show no particular preference for a given female, but rather copulate similarly with unexposed and exposed females, but that their preference may result in changes in the sequence of these events.Additionally, we hypothesize that flupyradifurone-exposed females display a different pattern of male quality assessment from that of unexposed females, leading to suboptimal choices and copulation with both flupyradifurone-exposed and unexposed males.Since pesticides can interfere with the reproduction of insects because of the effect of these stressors on the chemical profiles in the cuticle of treated individuals (Müller et al., 2017;Wang et al., 2018;Boff et al., 2022), we have further hypothesized that the effects of flupyradifurone on the composition and quantity of cuticle chemicals present on the surface of female H. truncorum is linked to their role as electrophysiologically active compounds detected by male antennae (Ganuza et al., 2022).

Materials and methods
Individuals of Heriades truncorum were obtained from colonized phragmite reeds set up in monitoring programs in Bavaria and Baden-Württemberg, Germany.Occupied nests with developing larvae were individually placed into transparent tubes, the end(s) of which were blocked with cotton to allow ventilation to the brood.These nests were left overwintering protected from snow, at ambient temperatures, until the natural emergence of the adults.Newly emerged males and females were immediately removed from the transparent tubes and transferred to Petri dishes (Ø 5 cm, 1.5 cm high) in which they were exposed to flupyradifurone.

Flupyradifurone exposure
Bees with different genetic backgrounds have been shown to display different sensitivities to flupyradifurone (Tan et al., 2017;Hesselbach et al., 2020;Tosi et al., 2021).In the current study, we tested the chronic effect of a concentration known to impact honey bees when mixed with a fungicide (Tosi & Nieh, 2019).We prepared a stock solution of flupyradifurone (3.8 mg/L) at room temperature by dissolving the active ingredient powder (flupyradifurone PESTANAL ® , analytical standard by Sigma Aldrich) in water.Later, the stock solution was diluted (1 : 10 4 ) in various sucrose solutions (30%) to produce a final flupyradifurone concentration of 0.38 μg/L.Females (n = 33) and males (n = 36) were placed individually into Petri dishes and fed ad libitum for a period of 120 h on sucrose solution containing flupyradifurone at a concentration of 0.38 μg/L or on sucrose solution only to account for survival.The presence of dead bees was recorded every 24 h.
A completely new set of bees was used in mating experiments.Since we allowed individuals to emerge under natural conditions, number of emerging sexes varied on a daily basis.The age of individuals were counted in days and was determined based on the instant that a newly emerged individual was transferred from its emergence tube to a Petri dish until the period it was placed in the mating arena.Exposed and unexposed bees were prepared as described above.Bees were allowed to feed ad libitum on 50% sucrose solution (unexposed group) or on 50% sucrose solution mixed with 0.38 μg/L flupyradifurone.

Behavioral analyses of unexposed and exposed bees
After treatment, an unexposed male was inserted into a mating arena (Ø 5 cm, 1.5 cm high) with a flupyradifurone-exposed female and a control female (n control males = 17, n exposed females = 17, n control females = 17).The same setting was repeated with exposed males (n exposed males = 16, n exposed females = 16, n control females = 16), giving a total of 33 males and 66 females used in the mating experiment.The behavioral interactions between individuals were video-recorded for 10 min for each group, totaling 330 min of observations.Each female and male used in the study was observed only once.We also checked the success rate of feeding by comparing the weight of individual feeders amongst unexposed (n = 5) and exposed (n = 5) males for 2 consecutive days.
During the courtship display, we recorded mating attempts (definition: male grabbing, trying (and eventually) mounting the female), sideways rocking motion movement (dance), and copulation, for both the flupyradifurone-exposed and unexposed control males.Moreover, male quality assessment by the female (female bites) was recorded for exposed and unexposed control females.To assess the size of individuals, we measured the intertegular distance in females (n = 49) and males (n = 22) by means of an Axiocam 105 color microscope camera (Zeiss, Germany) mounted on a Stemi 508 stereo microscope (Zeiss, Germany) and the analytical software ZEN 3.2 (blue edition, Zeiss).Despite females being significantly larger than males, an intrasex comparison between females and between males under the different treatment conditions revealed no differences in size (Fig. S1) indicating that size did not bias the results.Experiments were performed in the laboratory under room temperature at 25.5 °C and 50%−52% relative humidity.

Chemical analyses
We sought to determine the effect of pesticide exposure on the production of relevant electrophysiologically active compounds (termed here: "sex pheromone candidates").Individuals were freeze-killed at the end of each mating behavior observation.Unexposed and exposed female bodies were placed in 1.5 mL glass vials.Prior to extraction, 10 μL tridecane solution (100 ng/μL in n-pentane) was applied to the thorax of each female as an internal standard for quantitative analysis.Subsequently, the females were washed with 300 μL pentane for 3 min on a shaker (Vortex Gene 2) with the speed set to 2 880 r/min in order to extract cuticular chemicals from their cuticle surface.Solvent extracts were concentrated to a final volume of 100 μL by using a gentle stream of nitrogen.All chemical analyses were performed on a gas chromatograph (Agilent 7890A, AgilentTechnologies, Waldbronn, Germany) with a DB-5 capillary column (30 m × 0.25 mm inner diameter, J&W) and a flame ionization detector (FID).Hydrogen at a constant flow of 2.0 mL/min was used as the carrier gas.One microliter of the respective extract was injected splitless into the gas chromatograph at an injector port temperature of 300 °C.After an initial time of 1 min at 60 °C, the oven temperature increased continuously by 5 °C/min to a final temperature of 310 °C and held at that temperature for 10 min resulting in a total working time of 59 min.For the identification of the GC-EAD (gas-chromatographicelectroantennographic detection)-active chemical compounds in female extracts, we used gas chromatography/mass spectrometry (Agilent 7890B GC plus 5977A MSD, Agilent Technologies, Santa Clara, CA, USA; method as described above for GC, carrier gas: helium) and compared mass spectra by using references from the NIST11 library and GC retention times with those of authentic reference samples (Chemical Ecology Group of the Institute of Evolutionary Ecology and Conservation Genomics, Ulm University).Furthermore, we used reference mixtures containing synthetic alkanes and alkenes and compared compounds with those from our female extracts.Double-bond positions of alkenes were identified by the co-injection of samples with GC-EADactive compounds with reference mixtures of different alkene isomers.We named nine sex pheromone candidates present on the surface of virgin females of H. truncorum.Based on electroantennography, these nine sex pheromone candidates had been observed to influence male antennae responses in virgin females extracts (Ganuza et al., 2022).The sex pheromone candidates of interest were: (1) (Z)-7-tricosene, (2) tricosane, (3) (Z)-7pentacosene, (4) pentacosane, (5) (Z)-7-heptacosene, (6) heptacosane, (7) (Z)-7-nonacosene, (8) nonacosane, and (9) hentriacontane.

Statistics
The analysis of survival was conducted using the Kaplan-Meier method from the survival package (Therneau, 2020) and surv-miner package (Kassambara & Kosinski, 2020).Pairwise comparisons were performed among the survival groups by using the pair-wise_survdiff function (Therneau, 2020) with P values adjusted for multiple comparisons and the log-rank test.To explore whether flupyradifurone-exposed individuals were affected during the courtship display and male quality assessment by the female, we used generalized linear models (GLM) with insecticide-exposed female treatment and insecticide-exposed male treatments as fixed factors.The time that males needed to perform the first mating attempt, the number of mating attempts, the number of sideways rocking motion movements (dances), and male quality assessment by the female were used as response variables with a Poisson distribution.In the model (GLM) with copulation (yes/no) events as a response variable, we used a binomial distribution.We also considered female age as an interaction factor with insecticide female treatment in our response variables.The fit of the models and the violation of model assumptions were assessed with the package DHARMa (Hartig, 2020).We performed a nonmetric multidimensional scaling analysis (NMDS) with Bray-Curtis distances with respect to treatment groups and their cuticular hydrocarbons and tested discrepancy between observed values and the values expected with goodness of fit model.A linear model was used to compare the total amount of sex pheromone candidates and of individual candidates, with flupyradifurone-exposed female treatment and age as fixed factors.The analyses were performed in R 1.4.1106(R Core Team, 2018) with the package lme4.We utilized a 95% confidence interval to interpret statistical significances.

Behavioral responses-males
Male insecticide treatment had a significant effect on the time for the first mating attempt, sideways rocking motion, and male quality assessment by the female but did not differ in the number of mating attempts and copulations (Table 1).
The age of the females had no effect on mating attempts by males and increased with male quality assessment by the female (Fig. S2).Moreover, it interacted with female insecticide treatment regarding the time for first mating attempt by males (χ 2 = 308.4,df = 1, P < 0.0001) and sideways rocking motion movement (χ 2 = 5.22, df = 1, P = 0.02, Fig. S3).

Discussion
We show that, in Heriades truncorum, an oligolectic solitary bee species, mating behavior and chemical profiles can be impaired following chronic exposure to a sublethal concentration of an insecticide (flupyradifurone).Males, either exposed or unexposed to the insecticide, performed similar mating attempts/copulation with females independent of whether they had been exposed to flupyradifurone, but unexposed males were quicker to approach unexposed females.Unexposed males dedicated less effort in courtship with flupyradifurone-exposed females, which presented lower amounts of chemical profiles, called here sex pheromone candidates.Male quality assessment by the female was performed more quickly with the control males, and control females performed this assessment for longer.
Although flupyradifurone has been developed as a beesafer pesticide, sublethal doses have been found to impact the life of bees in diverse ways.Similar to results described for bumblebees (Zhang et al., 2022), H. truncorum exposed to flupyradifurone presented in a sucrose solution consume as much food solution as the control group indicating these bees are not averse to the compound.Further, honey bees exposed to field realistic doses of flupyradifurone have been shown to have memory and learning problems (Tan et al., 2017;Hesselbach & Scheiner, 2018) and poor coordination (Tosi & Nieh, 2019) probably decreasing flight ability (Tong et al., 2019) and leading to the premature onset of foraging (Hesselbach et al., 2020).Moreover, the pesticide has been found to cause negative effect on olfactory and visual modalities in bumble bees (Siviter & Muth, 2020) and an associated impact on the immunity of honey bees (Al Naggar & Baer, 2019;Harwood et al., 2022).It has been found negatively to impact the reproductive rate of the egg parasitoid wasp, Trichogramma evanescens (Tabebordbar et al., 2020).
Heriades truncorum, which is primarily dependent on flowers of ruderal plants from the Asteraceae family (Konzmann et al., 2020), might also face reproductive issues when exposed to flupyradifurone.With the presence of Asteraceae species surrounding crop fields, in meadows, and in flower strips that are important food sources for wild bee sustenance (Kuppler et al., 2023), H. truncorum is probably exposed to flupyradifurone while foraging for nectar or collecting pollen from nontarget Asteraceae plants along field edges (Zioga et al., 2023).Moreover, given the limited dispersal capacity of less than 100 meters of H. truncorum (Hofmann et al., 2020), populations of this species are especially vulnerable to local pesticide exposure, especially in view of the higher concentrations to which they may be subjected in the field (Carleton, 2014).The potential amplifying effects of elevated flupyradifurone concentrations on sex communication and reproduction should not be disregarded as a potential mechanistic factor contributing to declines in flower-specialized bee populations, such as H. truncorum, in agricultural environments (Biesmeijer et al., 2006;Peters et al., 2022).

Impacts of pesticides on precopulatory behavior
Our findings corroborate impact of pesticides on mating behavior, even though the effects can vary across individuals.A previous study found that imidacloprid (a neonicotinoid) disrupted the sexual communication of parasitoid wasps (Tappert et al., 2017): female wasps treated with imidacloprid had difficulty detecting male pheromones and showed delayed receptivity during courtship, and male wasps treated with imidacloprid had trouble recognizing females and engaging in courtship, resulting in decreased mating rates for treated pairs.In another study, but with mason bees, males exposed to a fungicide changed their precopulatory behavior and scent bouquet, leading to a high rate of female mating rejection (Boff et al., 2022).Our current study involving the use of a different pesticide class and route of exposure has revealed slightly different negative effects.Flupyradifurone-exposed males are slower to attempt to copulate.Moreover, the negative effect on dancing possibly highlights the effect of flupyradifurone on motor ability (Hesselbach & Scheiner, 2019) or motivation.However, in a limited size arena, exposed males are similarly engaged in copulating regardless of their insecticide treatment, as has been found with Trichograma chilonis (Hymenoptera: Trichogrammatidae) (Wang et al., 2018).
Unlike the pronounced quick interaction of unexposed males with unexposed females, individual copulation was 43% lower between flupyradifurone-exposed bees.However, mating among pesticide-free individuals is probably rare, because bees in the field are probably exposed to high doses of several pesticides, including flupyradifurone (Carleton, 2014).This scenario becomes even more dramatic if changes in the odor of individuals make the finding of a mating partner a challenge (Tappert et al., 2017) and cause an increase in female rejection (Boff et al., 2022).Thus, based on chemical communication and precopulatory behavior being estimators of reproductive output and on the evidence of bee population decline in agricultural environments (Sandrock et al., 2014;Rundlöf et al., 2015;Woodcock et al., 2017;Stuligross & Williams, 2020;Willis Chan & Raine, 2021), we need to acknowledge the genuine concerns being raised that effects on mating behavior lead to reductions in bee populations.Although our overall knowledge of the impact of pesticides on mating is limited at present, it is supported for at least three major crop pollinators with differing sociality levels: (1) eusocial honey bees, Apis mellifera (McAfee et al., 2022), (2) primitively eusocial bees, Bombus terrestris (Straub et al., 2022), and (3) the solitary bee Osmia cornuta (Boff et al., 2022), in addition to the oligolectic bee H. truncorum (this study).

Impact on male quality assessment by females
Females of H. truncorum actively participate in the mating behavior process and normally respond mechanically to male mounting by performing male quality assessment (Boff, 2022), as is commonly observed in other solitary bee species (Ayasse et al., 2001;Conrad et al., 2017;Boff et al., 2022).Here, we have been able to compare this behavior between insecticide-exposed and control females.We have found that flupyradifuroneexposed females are less keen to invest in male quality assessment.They spend less time on male quality assessment, potentially impacting the selection for mating partners.Age also has an interaction effect with treatment.Whereas unexposed females seem to be attractive to males depending on age, this effect is less pronounced in the females exposed to insecticide.

Chemical profile of females
Males, irrespective of treatment, were rapidly able to distinguish between females, since mating attempts were significantly quicker toward unexposed females.We found that exposed females presented the same nine studied sex pheromones candidates as those of the controls, but that the amounts of six of these compounds were significantly lower in exposed females.Here, the significant reduction, especially in alkenes and alkanes, was linked to increased mating latency, but not to mating avoidance.In this scenario, unexposed females were contacted before females with lower amounts of these chemicals.Thus, either males first detected the volatilizing odors of unexposed females and consequently tried initially to copulate with them, or the lower amounts of pheromone in insecticide-exposed females led to their having a lower attraction for the males.Our analysis does not allow conclusions to be made regarding changes in the chemical composition of the whole scent bouquet, because we focused our research to sex pheromone candidates.Nevertheless, the impact shown here on reproduction might be a consequence of the impact of stressors on sex pheromones and odors (Müller et al., 2017;Tappert et al., 2017;Wang et al., 2018;Boff et al., 2022).
The changes in chemical composition of cuticular odors, especially those with sexual functions, in insects affected by environmental stressors is probably causing alterations in mating behavior, as shown in several species exposed to detrimental conditions resulting from climate change (see Pilakouta & Ålund, 2021).The alkanes and alkenes studied here have been shown to be electrophysiologically active compounds that also stimulate mating behavior in male bees (Schiestl et al., 1999;Cuervo et al., 2017).Moreover, in malaria mosquitoes, males resistant to insecticide produce higher amounts of cuticular hydrocarbon sex pheromones and are more successful in mating (Adams et al., 2021) suggesting that the abundance of cuticular hydrocarbon sex pheromones is driven by pesticide pressure in insects.Additionally, the sex pheromone candidates studied here have also been found to influence sexual behavior in other insect species.
For example, (Z)-7-tricosene acts as a sex chemical stimulant in Drosophila melanogaster (Grillet et al., 2006), and when present together with (Z)-7-pentacosene, both occur in different relative amounts when the fruit flies are exposed to ozone (Jiang et al., 2023).Furthermore, (Z)-7-heptacosene and (Z)-7-nanocosene seem to act as sex pheromones in wood wasps (Böröczky et al., 2009).The chemical profile of H. truncorum females studied here is common in females of other Megachilidae bees species (Buckner et al., 2009) and, thus, probably in other bee species.However, whether the profile changes when these other bees are exposed to pesticides, impacting their sex communication, still needs to be discovered.Finally, although bee age may have an effect on sex-stimulating cuticular hydrocarbons (Polerstock et al., 2002), the few days' difference in age among females in the current study is probably too short for the detection of differences in chemical composition.Nonetheless, insecticide coupled to female age might influence male behavioral responses.
Wild solitary bees are among the most important insect pollinators on earth.Their crucial services in plant reproduction might be at risk because of the sublethal effects of pesticide on various aspects of their reproduction.More studies are necessary if a global assessment of the effects of pesticides on sexual communication and reproduction is to be obtained for bees.The inclusion of reproductive aspects, including mating behavior and chemical communication, is required as a further step (Sgolastra et al., 2020) during pesticide risk assessment in (wild) bees.

Fig. 1
Fig. 1 Effect of a sublethal concentration of flupyradifurone on male and female mating behavior in H. truncorum.(A) Effect on mating attempts by males.(B) Time span from releasing bees into the arena until first male mating attempt.(C) Time spent by male in performing sideways rocking motion.(D) Time females continue to bite males.Box portion of the plot represents quartiles; line inside the box represents the median.ns = nonsignificant, **P < 0.01, ***P < 0.001.

Fig. 2
Fig. 2 Effect of flupyradifurone on the total amount production of nine sex pheromone candidates between females exposed to a sublethal concentration (0.38 μg/L) of flupyradifurone and unexposed (control) females.Box portion of the plot represents quartiles; line inside the box represents the median.*P value < 0.05.

Fig. 3
Fig. 3 Comparison of the amount (μg) of nine sex pheromone candidates in the cuticle surface extracts of pesticide-exposed and unexposed females of H. truncorum.Box portion of the plot represents quartiles; line inside the box represents the median.*P < 0.05, **P < 0.01, ns = nonsignificantly different.

Table 1
Measured effect of flupyradifurone in precopulatory behaviors and copulation in male Heriades truncorum.