Interspecific foraging response to the thiacloprid treatment of co-existing top spider predators

Interspecific foraging response to the thiacloprid treatment of co-existing top spider predators | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Interspecific foraging response to the thiacloprid treatment of co-existing top spider predators Anděla Šimečková, Filip Solar, Michaela Kolářová, Eva Líznarová, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8060416/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Neonicotinoids are nicotine-based synthetic insecticides used in agriculture to control plant pests. They are neurotoxic substances that attack the nervous system of insects and can cause paralysis or death. These selective insecticides should have a negligible effect on non-target organisms, including spiders, which are one of the most abundant and diverse natural predators that contribute to the control of pests. Current studies show that selective insecticides such as neonicotinoids have negative effects on non-target invertebrates. They can have both lethal effects resulting in mortality, and sublethal effects involving various aspects of their lives, e.g. breeding, movement, hunting, the ability to defend against predators, and predatory activity. We studied the species-specific responses to neonicotinoid treatments with the active ingredient thiacloprid of two top spider predators coexisting in tree crowns in Europe—respectively, spiders of the genus Philodromus ( aureoles group, Philodromidae) and species Anyphaena accentuata (Walckenaer) (Anyphaenidae). Spiders were exposed to field-realistic concentrations of the tested substance, while the control group was treated with distilled water. We compared the species-specific responses of three components of spider predatory activity: functional response, prey consumption, and overkilling. Further, we observed the long-term survival and paralysis of treated individuals compared to control, and the effect of insecticide residues on predation activity 14 days after insecticide application. We found that an hour-long tarsal contact with the active ingredient thiacloprid reduced predatory activity in both Anyphaena and Philodromus spiders, but the effect was species-specific in many aspects. Feeding was affected by fresh residua in Anyphaena , but not in Philodromus . Furthermore, 14 days after treatment, there were differences in the rates of predation, feeding, and overkilling between species. The treatment caused paralysis, but no mortality in Philodromus . In contrast, the treatment caused significant mortality, but no paralysis in Anyphaena . Further, after 14 days, we found that the insecticide had no significant effect on predation activity. Overall, the study revealed a species-specific response to the given pesticide of top pest predators sharing the same ecological niche in orchards. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Zoology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Many pesticides and active substances that were considered safe when first introduced commercially were later found to have negative effects on certain groups of organisms [ 1 , 2 , 3 ]. These effects are often sublethal, meaning that they do not lead to immediate mortality, but may cause other harmful effects such as reduced fitness, changes in behaviour, or impaired development [ 4 , 5 , 6 , 7 ]. Such sublethal effects often go undetected during the registration process because standard testing focuses primarily on lethal effects [ 8 ]. As a result, the full range of ecological impacts, especially those occurring at sub-lethal levels, are often overlooked. This highlights the need for testing protocols that are more rigorous and comprehensive than EU Commission Regulation 2013 (No 283/2013) in order to account for such potential non-lethal impacts on non-target species. Furthermore, there is a lack of consideration of the specificity of the response to pesticide applications across taxa, as the results come from a commonly tested model organism and may not represent taxa that are actually affected in nature. Neonicotinoids were commercialised in the 80s and widely used in agriculture to protect crops against insect pests; however, research later revealed their significant negative impacts on various important groups of organisms [ 9 , 10 ]. For example, studies have linked neonicotinoid exposure to widespread losses of honeybee colonies in the UK, raising concerns about their wider ecological consequences [ 11 , 12 , 13 ]. These insecticides have been shown to affect not only pollinators but also other non-target organisms, including beneficial arthropods, aquatic invertebrates, and birds, leading to declines in populations over large areas [ 14 , 15 , 16 , 17 ]. Although, spiders are less sensitive to neonicotinoids compared to insects because of the different structure of their acetylcholine receptors, the binding targets of neonicotinoids [ 18 ], several sublethal effects of neonicotinoids have been documented in spiders that affect their physiology and behaviour. These include temporary paralysis [ 19 ], changes in silk production [ 20 ], disruption of chemoreception [ 21 ], and a decrease in predation rate [ 19 ]. All these together cause a significant reduction in spiders’ potential for biological pest control and can damage their local populations in agroecosystems. There are restrictions on neonicotinoid use to mitigate their adverse effects on ecosystems. For example, most neonicotinoid active substances have been banned in the European Union since 2020, except for acetamiprid, which is considered to be less of a risk to bees [ 22 ]. However, these substances are still actively used in various parts of the world, including the United States, particularly on cotton and fruit [ 23 ]. Pesticide sensitivity can vary significantly even among ecologically similar species. For example, Jütte et al. [ 24 ] exposed seven bee species, including Apis mellifera (Linné 1758), Bombus terrestris (Linné 1758) and several solitary species, to field‑realistic levels of the pyrethroid insecticide lambda‑cyhalothrin. They observed marked species-specific differences in both mortality and sublethal behavioural effects. This challenges the adequacy of using A. mellifera as a sole surrogate in risk assessments. Short et al. [ 25 ] found over 30-fold differences in imidacloprid sensitivity among five earthworm species, explained by the species-specific expression of nonclassical acetylcholine-binding proteins. Henriques Martins et al. [ 26 ] demonstrated that different dipteran pollinators exhibit species-specific sensitivity to imidacloprid, with LD₅₀ values varying by nearly a factor of two, and showed that sublethal endpoints such as fecundity can be highly sensitive indicators of pesticide effects. Specific responses to neoticotinoids including thiacloprid have already been documented in spiders by [ 19 ], although they studied three different spiders from three different foraging guilds and three different microhabitats—respectively, Pardosa lugubris (Walckenaer 1802) (Lycosidae), a dominant ground dwelling spider; Philodromus cespitum (Walckenaer 1802) (Philodromidae), inhabiting tree crowns; and sheet-weaving spiders from Linyphiidae inhabiting vegetation. Documented different responses to neonicotinoid treatment were expected because of differences in ecology and in adaptations to different microhabitats. In order to reveal the functional impact of pesticide application on predation potential in pest control in a specific habitat, it is necessary to know the impact of application on a representative proportion of the predator community in the given habitat. We focused on understanding the species specificity of co-existing species from the same foraging guild, sharing the same microhabitat, and playing a similar role in the biological control of herbivorous insects in tree crowns in orchards. As models for testing the thiacloprid effect, we used two common tree crown and shrub-dwelling spiders—respectively, Anyphaena accentuata (Walckenaer 1802) (Anyphaenidae) and Philodromus spp. ( aureolus group) (Philodromidae). Both taxa are top predators of insect pests in European orchards [ 27 ] and their ecological niches and prey spectra overlap considerably [ 28 ]. Both are known as winter-active spiders, which are important in reducing hibernating insect pests during the winter season [ 29 , 30 ]. These two species differ in their main predation activity during the day period. Anyphaena is a nocturnal hunter, which effectively preys on non-flying aphids and lepidopteran larvae, with recent studies confirming a strong preference for hemipterans, especially pear psyllids, during winter [ 28 , 31 , 32 ]. In contrast, Philodromus are diurnal hunters of hymenopterans and lepidopterans [ 31 ], but also frequently prey on other spiders, dipterans, and hemipterans, indicating a broad and opportunistic diet [ 28 ]. Spiders help regulate prey populations not only through consumption but also by overkilling – killing more prey than they consume [ 33 ]. Overkilling is common in many species, especially active hunters, and tends to increase with prey density [ 34 , 35 ]. It may help spiders to avoid ingesting unsuitable or toxic prey [ 36 ], although some interpret it as merely a by-product of increased aggression [ 37 , 38 ]. In the present study, we focused on the active substance thiacloprid from the chloronicotinoid group. It acts as a contact and ingestive poison, has systemic effects, and inhibits the transfer of impulses within the insect nervous system. The mechanism of effect is similar to that of acetylcholinesterase inhibitors, but thiacloprid is inactivated only slowly. Its persistent effect leads to general dysfunction of the nervous system and subsequently to the death of the affected target organism [ 23 ]. Thiacloprid is the active ingredient in formulations such as Biscaya, Calypso, Proteus, and Sonido ((Bayer CropScience). We investigated the effect of thiacloprid treatment (fresh and 14-day-old residues) on the predation activity of spiders—specifically, on functional response, feeding and overkilling. Further effects of thiacloprid treatment, including mortality and paralysis, were documented for four weeks after treatment. We tested the hypothesis that treatment (fresh and 14-day-old residues) with the active ingredient thiacloprid negatively affects the traits of the predation activity of Anyphaena and Philodromus spiders. Further, we tested the hypothesis that the response to thiacloprid treatment differs between those two coexisting top spider predators. Material and methods Individuals of two top spider predators, Anyphaena accentuata and Philodromus spp. ( Philodromus : aureolus -group), with an above 70% dominance of P. cespitum (Walckenaer 1802) in the spider community were collected from apple orchards and their surroundings in Prague–Sedlec, Czech Republic (50.13°N, 14.39°E) in September and October 2023. We kept spiders individually in 10 ml plastic tubes (diameter – 15 mm; length – 50 mm) with pierced lids and a layer of gypsum plaster at the bottom. We moisturized the plaster every week with few drops of water to retain humidity. We kept the spiders in controlled conditions under a natural photoperiod and temperature for 14 days to acclimatise and fed them once a week. As prey, we used wingless Drosophila melanogaster (Meigen 1830) cultivated on the commercial medium NEKTON. We tested the effect of neonicotinoid insecticide on the predation rate of two top spider predators in orchards. We used neonicotinoid insecticide with the active substance thiacloprid which was formulated as Biscaya 240 OD (Bayer CropScience, Monheim, Germany). The insecticide was applied in the dilution recommended by the manufacturer for crop spraying, with a thiacloprid concentration of 23.1% and at a suggested application rate of 250 ml ha − 1 . We applied the insecticide on filter paper sheets rolled into tubes 5.5 cm in diameter (0.737 ml each paper). The treated filter papers were left to dry for 30 minutes and then placed inside plastic tubes into which spiders were later transferred individually. To ensure tarsal contact with the substance, the filter papers covered the whole inner surface of the tubes. The spiders remained inside the tubes for 60 minutes. To test the effect of thiacloprid on predation activity, we transferred the spiders individually to Petri dishes (height – 1 cm; diameter – 5.5 cm) and allowed them 10 minutes to acclimatize to their new environment. We examined 150 individuals of Philodromus spp. and 150 individuals of A. accentuata in total; half of each species was treated with neonicotinoids and half was treated with distilled water as a control. There were no significant differences in body length between the control groups and the treatened Anyphaena (Wilcoxon test, W = 2738, p = 0.8867) and Philodromus groups (Wilcoxon test, W = 2741.5, p = 0.2562). We divided the spiders into groups based on the numbers of offered prey: 1, 3, 6, 9, 12 (15 spiders in each group). As prey, we provided living wingless D. melanogaster , which we kept at a constant density according to the group. We replaced all killed or eaten prey every 30 minutes for 4 hours. We observed two prey capture behaviours each round – feeding (at least 30% of the prey was eaten) and killing (more than 70% of the prey remained). Spiders were measured during the experiment to observe the effect of body size on predation activity. The effect of neonicotinoid residues on predation activity was tested 14 days after exposure, which was the expected period in which the effect of the treatment could disappear. Spiders were treated with thiacloprid only once during the fresh treatment experiment and were not fed in the following 14 days, as they had been allowed to feed to satiation before. The effect of 14- day-old residues was tested on spider groups at a prey density of 6, as predation activity was generally highest at this density. In total, 30 Anyphaena and 30 Philodromus spiders (15 treated and 15 control individuals per species) were tested using the same design as in the fresh treatment. A constant density of 6 D. melanogaster was maintained, with all killed or consumed prey replaced every 30 minutes for a period of 4 hours. Additionally, we monitored spider mortality and visible signs of paralysis both during the experiment and at specific times following thiacloprid exposure. Mortality was recorded 24 hours, 1 week, 2 weeks, 3 weeks and 4 weeks after the treatment to assess delayed effects. Paralysis was recognized when spiders showed markedly reduced mobility and appeared unusually limp, but without the characteristic stiff and curled posture of dead individuals. The paralysis was further characterized by its reversibility, with the spiders recovering normal activity. Statistical analyses Predation activity in response to prey density (number of killed prey, overkilling rate, and consumption rate) was analyzed using a generalized linear model (GLM) with Poisson distribution. These data were fitted with a Type II functional response curve following the Holling [ 39 ] disc equation. To assess whether the treatment effect differed between species, we used post hoc pairwise interaction contrasts based on the GLM using the emmeans package (estimated marginal means). The effect of neonicotinoid residues on predation activity after 14 days was tested using a GLM with Poisson distribution. We used a generalized linear model (GLM) with binomial distribution to test the effect of treatment on spider survival rate. We performed all statistical analyses in the R environment [ 40 ]. Results Species specific response to thiacloprid treatment The functional response (predation activity response to prey density) of untreated spiders was significantly higher in Philodromus than in Anyphaena spiders (GLM-p, χ 2 1 = 10.535, p = 0.001, Fig. 1 ). Treatment with fresh thiacloprid significantly decreased the functional response of both Philodromus (GLM-p, χ 2 1 = 4.534, p = 0.033, Fig. 1 ) and Anyphaena (GLM-p, χ 2 1 = 59.137, p < 0.001, Fig. 1 ). Anyphaena exhibited a significantly stronger reduction in functional response under the treatment compared to Philodromus (interaction contrast = 0.88, z = 7.15, p < 0.0001). The predation activity (number of captured prey) of spiders affected by 14-day-old thiacloprid residues differed significantly among Philodromus and Anyphaena spiders. Whereas Philodromus spiders exhibiting a higher predation rate than Anyphaena spiders (GLM-p, χ 2 1 = 154.703, p < 0.001, Fig. 2 ). Anyphaena spiders under treatment killed non-significantly more flies when compared to the control group (GLM-p, χ 2 1 = 0.22, p = 0.633), Philodromus spiders under treatment killed significantly fewer flies than the control group (GLM-p, χ 2 1 = 6.083 p = 0.014). The effect of thiacloprid treatment on feeding and overkilling differed significantly between Philodromus and Anyphaena spiders (Table 1 ). The feeding (number of consumed prey) of Anyphaena decreased significantly under treatment with fresh thiacloprid (GLM-p, χ 2 1 = 34.564, p < 0.001, Fig. 3 A). In contrast, the feeding of Philodromus did not decrease significantly under treatment with fresh thiacloprid (GLM-p, χ 2 1 = 1.0601, p = 0.303, Fig. 3 B). Table 1. Response of different traits to thiacloprid treatment in Philodromus and Anyphaena spiders. “-” means no data collected. The feeding of spiders 14 days after treatment differed significantly between Anyphaena and Philodromus spiders; the feeding rate was overall higher in Philodromus (GLM-p, χ 2 1 = 154.703, p < 0.001, Fig. 4 ). The feeding rate did not differ significantly between spiders 14 days after treatment and the control group in either Anyphaena (GLM-p, χ 2 1 = 2.789, p = 0.095) or Philodromus (GLM-p, χ 2 1 = 1.0085, p = 0.315). The rate of overkilling significantly decreased under fresh residue treatment in both species; in Anyphaena (GLM-p, χ 2 1 = 25.1767, p < 0.001, Fig. 5 A) and in Philodromus (GLM-p, χ 2 1 = 3.741, p = 0.016, Fig. 5 B). The rate of overkilling by spiders 14 days after treatment was significantly higher in Philodromus spiders than in Anyphaena (GLM-p, χ 2 1 = 75.314, p < 0.001, Fig. 6 ). The overkilling rate of Anyphaena did not differ significantly between spiders 14 days after treatment and control (GLM-p, χ 2 1 = 0.516, p = 0.472), but it was significantly lower in Philodromus spiders 14 days after treatment than in the control (GLM-p, χ 2 1 = 5.899, p = 0.015). Mortality Thiacloprid treatment did not cause significant mortality in Philodromus spiders (GLM-b, χ 2 1 = 1.88711, p = 0.1695); in contrast, mortality in Anyphaena spiders was significantly higher in treated individuals (GLM-b, χ 2 1 = 36.238, p < 0.001). During the whole experiment, 30 Anyphaena spiders died in the treated group, corresponding to 20% of the total number, whereas only two individuals died in the control group, corresponding to about 1% of the total number. Of the total mortality in treated Anyphaena spiders, 7% occurred immediately after treatment, 13% within one week, 7% within two weeks, 20% within three weeks, and 53% within four weeks (Fig. 7 ). Both individuals (100%) that died in the control group died within one week. Paralysis Fresh Thiacloprid treatment induced paralysis in 13% of Philodromus spiders, which was significantly higher than in the control group (GLM-b, χ 2 1 = 40.203, p < 0.001, Fig. 8 A), where no individual was paralysed after the experiment. Paralysis occurred during the experiment or shortly after the end. One week after treatment, 19% of the paralyzed spiders began to move again, and after two weeks, 100% of the paralyzed spiders returned to normal (Fig. 8 B). In Anyphaena spiders, paralysis did not appear, either after fresh or 14-day-old residue treatment. Discussion Predation The ability of predators to suppress pests is determined by the rate of predation on pests, which varies depending on prey density and can be expressed as a functional response [ 39 ]. A high increase in the number of prey is typical when the pest population is overpopulated, and therefore a positive functional response of the predator is a strong predictor of its effectiveness in crop pest suppression. Both studied species, Anyphaena and Philodromus , are dominant active predators on fruit trees [ 27 ]. Our results confirm a strong functional response already observed by Řezáč et al. [ 41 ] in Philodromus and also revealed it in Anyphaena spiders for the first time. The absolute number of prey caught under laboratory conditions does not fully reflect the situation in nature because a saturated predator may avoid prey, or prey may escape. Predation measured in the laboratory differs from predation in nature, but laboratory data can approximate our knowledge of the situation in nature and indicate the predatory potential of the studied species. Such ecotoxicological laboratory data are valuable for assessing the effects of agrochemical treatments on predators. Effect of thiacloprid treatment We report for the first time a species-specific effect of neonicotinoid treatment with the active ingredient thiacloprid on the foraging ecology of two top spider predators co-occurring in tree crowns in Central Europe, respectively Anyphaena and Philodromus spiders. Both species belong to the same foraging guild of active hunters [ 42 ] and represent some of the most important natural predators in fruit orchards, where they share the prey and habitat [ 30 , 43 ] even during the winter [ 29 , 30 ]and also engage in intraguild predation [ 44 ]. In both hunter spiders, thiacloprid treatment significantly decreased predation activity at all tested prey densities. The functional response of treated spiders correlated with the functional response of the control group, but the predation rate was significantly lower in thiacloprid treatment groups. The predation rate of treated spiders decreased in both species at all prey densities: by more than half for Anyphaena and by more than a quarter for Philodromus . The greater negative effect of thiacloprid treatment on the predation rate of Anyphaena may be related to the physiological adaptation of nocturnal Anyphaena to lower temperatures than those experienced by diurnal Philodromus . At temperatures below freezing, the lower limit for predatory activity was set at − 3.73°C for Anyphaena and − 1.2°C for Philodromus . At increasing temperatures, the predatory activity of Anyphaena was highest at 15°C and then declined. In contrast, the predation rate of Philodromus increased monotonically with temperature and reached a maximum at 30°C, which was the highest temperature tested [ 29 ]. The link between differences in the effect of thiacloprid on the two spider hunters studied and the differences in their temperature adaptations is only a hypothesis and requires further research. However, it has already been documented that higher temperatures have a synergistic effect on the toxicity of thiacloprid, as observed in crayfish [ 45 ]. A decrease in predation was also documented in other spiders, e.g. in Pardosa pseudoannulata (Araneae: Lycosidae) Widiarta et al . [ 46 ] after imidacloprid exposure, and in Pardosa agrestis (Araneae: Lycosidae) after thiacloprid exposure [ 47 ]. In addition, a reduction in predation after neonicotinoid treatment with thiacloprid was found by [ 19 ], who also observed that dorsal application had a higher impact on spiders than tarsal application, the latter producing only moderate effects. In the present study, we found a significantly negative effect of tarsal exposure to thiacloprid on the predation rate of both Anyphaena and Philodromus spiders. Řezáč et al. [ 41 ] also found that neonicotinoid (acetamiprid) reduced the predation due to prolonged prey handling. In contrast, no effect of neonicotinoids on feeding was observed in Philodromus cespitum (Araneae: Philodromidae) after exposure to acetamiprid [ 41 ]. Predation, feeding, overkilling The predation rate expresses the number of prey caught, which includes two different components: feeding, i.e., prey caught and completely or partially consumed, and so-called overkilling, i.e., prey caught but not consumed by the predator [ 35 ]. Feeding is important for the predator itself, as it provides a source of nutrients [ 48 ]. Prey overkilling is not directly beneficial to the predator, but it is important in terms of protecting crops from pests. The ground dwelling spider Pardosa agrestis fed less on thiacloprid-treated prey than on control prey, but the rate of overkilling increased, especially among female spiders, from just 2.6% of control flies to 44.7% of thiacloprid-treated flies [ 47 ]. Similar compensation of low feeding by overkilling was also found in our results, but overkilling also decreased in comparison with control. At first sight, it seems that treatment with thiacloprid does not have a significant negative effect on pest population control, because low feeding is complemented by overkilling, and hypothetically the pressure on the pest population remains high. However, conditions in the field may differ from those in the laboratory (as already mentioned), and, together with other sublethal effects of thiacloprid treatment on the life history of predators, may reduce their long-term impact on pest populations in orchards. Due to all the above-mentioned factors, it is important to evaluate the obtained knowledge correctly and to correctly implement it when assessing the impact of thiacloprid treatment on beneficial arthropods. Paralysis and Mortality The toxicity of neonicotinoids to spiders is lower than to insects, most likely because the structure of acetylcholine receptors, which mediate the action of neonicotinoids in arthropods, differs between insects and spiders. In spiders, acetylcholine receptors are present [ 18 , 49 ], but their sensitivity to neonicotinoids is lower than that of insect receptors [ 18 ]. We found significant mortality under thiacloprid treatment only in Anyphaena spiders, not in Philodromus . This indicates that nocturnal Anyphaena seems to be more sensitive to thiacloprid treatment than diurnal Philodromus . On the other hand, Philodromus suffered significant temporary paralysis caused by tarsal contact with thiacloprid. Although we observed the recovery of all affected Philodromus individuals within 14 days under laboratory conditions, such paralysis could be fatal in natural conditions due to the increased risk of dehydration or predation by other predators. Paralysis was observed in spiders of the family Linyphiidae under acetamiprid and thiacloprid treatment, and in Philodromus cespitum under treatment with acetamiprid [ 19 ]. Sýkora [ 50 ] reported that tarsal contact with neonicotinoids (actemapirid, imidacloprid, thiacloprid and thiamethoxam) did not cause significant mortality in Phylloneta impresa (Koch 1881) and that no paralysis was observed. However, Řezáč et al. [ 19 ] found that the dorsal application of thiacloprid to spiders of the family Linyphiidae caused paralysis and mortality, which was up to 57% in males and 29% in females. Several studies have reported the paralysing effects of neonicotinoids on other non-target invertebrates, particularly honeybees. Following neonicotinoid application, bees are often found lying motionless around the hive [ 51 ]. Bumblebees showed significant growth inhibition following imidacloprid application, due to disorientation and the inability to search for food [ 52 ]. The paralyzing effects of neonicotinoids have also been found in predatory insects. For example, in the beetle Harmonia axyridis (Pallas 1773), 72% of larvae treated with thiamethoxam or clothianidin developed neurotoxic symptoms, including paralysis [ 53 ]. Other sub-lethal effects of neonicotinoids There may be other sublethal effects of neonicotinoids which should be considered. For example, Korenko et al. [ 21 ] found that after treatment with thiacloprid, male Pardosa agrestis (Westring 1861) are unable to complete the mating dance due to the disruption of chemical communication, which may reduce the chances of reproduction. Neonicotinoids also have a significant effect on locomotion. According to Řezáč et al. [ 54 ], they reduce the speed of Pardosa lugubris (Walckenaer 1802), which may affect predation success or escape. Further research is needed to better understand the long-term effects of neonicotinoids on spider populations and other non-target organisms. Research should focus on their persistence in soil and plants, their accumulation after repeated applications, and their interactions with other pesticides. In addition, research should go beyond predation to include sub-lethal effects on locomotion, food intake, and reproduction, as these factors are key to assessing the wider ecological consequences of pesticide exposure. A comprehensive examination of these parameters could provide crucial insights into the risks associated with neonicotinoid use. Regulatory frameworks should ensure that the approval of neonicotinoid-based pesticides is conditional on thorough research into their impacts on a wider range of organisms, including both lethal and sub-lethal effects. Prioritizing such studies over commercial considerations could help to minimise unintended ecological disturbances while maintaining effective pest management strategies. Declarations Competing interests The authors declare that they have no competing interests. Funding The study was supported by Czech University of Life Sciences Prague (Student Grant Competition 2025 provided by Faculty of Agrobiology, Food and Natural Resources). Author Contribution AŠ, FS and SK and conceived, designed the research and conducted laboratory experiments; AŠ, SK, MK and EL analyzed the data; all authors wrote the manuscript; all authors read and approved the manuscript. Acknowledgement The study was supported by the Student Grant Competition 2025 provided by Faculty of Agrobiology, Food and Natural Resources. Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files. References Alkassab, A. T. & Kirchner, W. H. Sublethal exposure to neonicotinoids and related side effects on insect pollinators: honeybees, bumblebees, and solitary bees. J. Plant. Dis. Prot. 124 , 1–30 (2017). Ward, W. et al. 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07:20:15","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":751,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/4e05f42f0af361a5f38839f6.png"},{"id":96710397,"identity":"c9e5ebf0-15aa-4cce-a80a-3ffc037d8c5c","added_by":"auto","created_at":"2025-11-25 10:10:35","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":751,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/333992201a56fd9b6233691a.png"},{"id":96694551,"identity":"0a26b1a0-f52e-4b21-876b-fd6c48df2220","added_by":"auto","created_at":"2025-11-25 07:20:15","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":751,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/0c8999f8ecf20f9bc5dede9f.png"},{"id":96710842,"identity":"a4c24066-3663-46e6-92f4-e6df5288cd0b","added_by":"auto","created_at":"2025-11-25 10:11:16","extension":"xml","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121542,"visible":true,"origin":"","legend":"","description":"","filename":"6b3df10eb33041f08bcd8056616634031structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/76b4d909802afad0a50053ca.xml"},{"id":96694550,"identity":"d6020300-0284-428c-8568-e7962d2cb873","added_by":"auto","created_at":"2025-11-25 07:20:15","extension":"html","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134131,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/acbea01eb8fda8e22de25128.html"},{"id":96710555,"identity":"38bc8999-2898-4866-8768-5ac0da5a4c44","added_by":"auto","created_at":"2025-11-25 10:10:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75605,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional response of fresh-thiacloprid-treated \u003cem\u003ePhilodromus\u003c/em\u003e and \u003cem\u003eAnyphaena \u003c/em\u003espiders compared to the control groups.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/ed54534c60c7739c9a762feb.jpg"},{"id":96694512,"identity":"bf673ac2-6606-4537-94c3-71aa2910ca40","added_by":"auto","created_at":"2025-11-25 07:20:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59644,"visible":true,"origin":"","legend":"\u003cp\u003ePredation activity of 14-day-thiacloprid-residua-treated \u003cem\u003ePhilodromus\u003c/em\u003eand \u003cem\u003eAnyphaena \u003c/em\u003espiders compared to the control groups.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/b6d737bdbb9d1d82135baddb.jpg"},{"id":96710587,"identity":"82ed97ea-c713-492e-8a36-7c8fd822fb52","added_by":"auto","created_at":"2025-11-25 10:10:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71393,"visible":true,"origin":"","legend":"\u003cp\u003eFeeding of the fresh thiacloprid treated \u003cem\u003eAnyphaena\u003c/em\u003e (A) and \u003cem\u003ePhilodromus \u003c/em\u003e(B) spiders compared to the control group at different prey densities. Points show the average number of eaten prey at each density.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/3fe6903f0b8093a7e63e3887.jpg"},{"id":96694513,"identity":"83a538f7-2278-4407-9c3d-196d4ae1ca48","added_by":"auto","created_at":"2025-11-25 07:20:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52604,"visible":true,"origin":"","legend":"\u003cp\u003eFeeding of 14-day-thiacloprid-residua-treated \u003cem\u003ePhilodromus\u003c/em\u003eand \u003cem\u003eAnyphaena \u003c/em\u003espiders compared to the control groups.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/6970fed067330771b706fcd1.jpg"},{"id":96710971,"identity":"682488b6-f1d4-4937-812f-3890bb2d5bc2","added_by":"auto","created_at":"2025-11-25 10:11:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66849,"visible":true,"origin":"","legend":"\u003cp\u003eOverkilling rate of fresh-thiacloprid-treated \u003cem\u003eAnyphaena\u003c/em\u003e (A) and \u003cem\u003ePhilodromus\u003c/em\u003e (B) spiders compared to the control group at different prey densities. Points show the average number of eaten prey at each density.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/4da1b9f00003116d60fa9a6d.jpg"},{"id":96694522,"identity":"c31b8609-013a-4fc1-9f2c-56bba8f8f2e8","added_by":"auto","created_at":"2025-11-25 07:20:14","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":55055,"visible":true,"origin":"","legend":"\u003cp\u003eOverkilling rate of 14-day-thiacloprid-residua-treated \u003cem\u003ePhilodromus\u003c/em\u003e and \u003cem\u003eAnyphaena \u003c/em\u003espiders compared to the control groups.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/436b93feeb1f2b9385e635b9.jpg"},{"id":96711267,"identity":"1ab40deb-fa7b-40da-811d-306dee8e566f","added_by":"auto","created_at":"2025-11-25 10:11:49","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52134,"visible":true,"origin":"","legend":"\u003cp\u003eTime of mortality of \u003cem\u003eAnyphaena\u003c/em\u003e spiders within 4 weeks of treatment - fresh treatment compared to the control group.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/a291b6a9f2c459a7b482756b.jpg"},{"id":96694530,"identity":"b9cfd597-5033-400d-a482-ce3e6ca65172","added_by":"auto","created_at":"2025-11-25 07:20:15","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":66182,"visible":true,"origin":"","legend":"\u003cp\u003eOccurrence (A) and duration (B) of paralysis (%) of \u003cem\u003ePhilodromus \u003c/em\u003espiders in treated and in the control group.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/f8a9828bf4290463dbd32f76.jpg"},{"id":96913009,"identity":"99829776-0efe-413a-8ba8-80bc9ea19638","added_by":"auto","created_at":"2025-11-27 13:49:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1242939,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8060416/v1/146860e3-a675-40ca-8b03-0d6374e74eec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interspecific foraging response to the thiacloprid treatment of co-existing top spider predators","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMany pesticides and active substances that were considered safe when first introduced commercially were later found to have negative effects on certain groups of organisms [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These effects are often sublethal, meaning that they do not lead to immediate mortality, but may cause other harmful effects such as reduced fitness, changes in behaviour, or impaired development [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Such sublethal effects often go undetected during the registration process because standard testing focuses primarily on lethal effects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As a result, the full range of ecological impacts, especially those occurring at sub-lethal levels, are often overlooked. This highlights the need for testing protocols that are more rigorous and comprehensive than EU Commission Regulation 2013 (No 283/2013) in order to account for such potential non-lethal impacts on non-target species. Furthermore, there is a lack of consideration of the specificity of the response to pesticide applications across taxa, as the results come from a commonly tested model organism and may not represent taxa that are actually affected in nature. Neonicotinoids were commercialised in the 80s and widely used in agriculture to protect crops against insect pests; however, research later revealed their significant negative impacts on various important groups of organisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For example, studies have linked neonicotinoid exposure to widespread losses of honeybee colonies in the UK, raising concerns about their wider ecological consequences [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These insecticides have been shown to affect not only pollinators but also other non-target organisms, including beneficial arthropods, aquatic invertebrates, and birds, leading to declines in populations over large areas [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although, spiders are less sensitive to neonicotinoids compared to insects because of the different structure of their acetylcholine receptors, the binding targets of neonicotinoids [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], several sublethal effects of neonicotinoids have been documented in spiders that affect their physiology and behaviour. These include temporary paralysis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], changes in silk production [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], disruption of chemoreception [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and a decrease in predation rate [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All these together cause a significant reduction in spiders\u0026rsquo; potential for biological pest control and can damage their local populations in agroecosystems.\u003c/p\u003e\u003cp\u003eThere are restrictions on neonicotinoid use to mitigate their adverse effects on ecosystems. For example, most neonicotinoid active substances have been banned in the European Union since 2020, except for acetamiprid, which is considered to be less of a risk to bees [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, these substances are still actively used in various parts of the world, including the United States, particularly on cotton and fruit [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePesticide sensitivity can vary significantly even among ecologically similar species. For example, J\u0026uuml;tte et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] exposed seven bee species, including \u003cem\u003eApis mellifera\u003c/em\u003e (Linn\u0026eacute; 1758), \u003cem\u003eBombus terrestris\u003c/em\u003e (Linn\u0026eacute; 1758) and several solitary species, to field‑realistic levels of the pyrethroid insecticide lambda‑cyhalothrin. They observed marked species-specific differences in both mortality and sublethal behavioural effects. This challenges the adequacy of using \u003cem\u003eA. mellifera\u003c/em\u003e as a sole surrogate in risk assessments. Short et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] found over 30-fold differences in imidacloprid sensitivity among five earthworm species, explained by the species-specific expression of nonclassical acetylcholine-binding proteins. Henriques Martins et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] demonstrated that different dipteran pollinators exhibit species-specific sensitivity to imidacloprid, with LD₅₀ values varying by nearly a factor of two, and showed that sublethal endpoints such as fecundity can be highly sensitive indicators of pesticide effects. Specific responses to neoticotinoids including thiacloprid have already been documented in spiders by [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], although they studied three different spiders from three different foraging guilds and three different microhabitats\u0026mdash;respectively, \u003cem\u003ePardosa lugubris\u003c/em\u003e (Walckenaer 1802) (Lycosidae), a dominant ground dwelling spider; \u003cem\u003ePhilodromus cespitum\u003c/em\u003e (Walckenaer 1802) (Philodromidae), inhabiting tree crowns; and sheet-weaving spiders from Linyphiidae inhabiting vegetation. Documented different responses to neonicotinoid treatment were expected because of differences in ecology and in adaptations to different microhabitats. In order to reveal the functional impact of pesticide application on predation potential in pest control in a specific habitat, it is necessary to know the impact of application on a representative proportion of the predator community in the given habitat.\u003c/p\u003e\u003cp\u003eWe focused on understanding the species specificity of co-existing species from the same foraging guild, sharing the same microhabitat, and playing a similar role in the biological control of herbivorous insects in tree crowns in orchards. As models for testing the thiacloprid effect, we used two common tree crown and shrub-dwelling spiders\u0026mdash;respectively, \u003cem\u003eAnyphaena accentuata\u003c/em\u003e (Walckenaer 1802) (Anyphaenidae) and \u003cem\u003ePhilodromus\u003c/em\u003e spp. (\u003cem\u003eaureolus\u003c/em\u003e group) (Philodromidae). Both taxa are top predators of insect pests in European orchards [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and their ecological niches and prey spectra overlap considerably [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Both are known as winter-active spiders, which are important in reducing hibernating insect pests during the winter season [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These two species differ in their main predation activity during the day period. \u003cem\u003eAnyphaena\u003c/em\u003e is a nocturnal hunter, which effectively preys on non-flying aphids and lepidopteran larvae, with recent studies confirming a strong preference for hemipterans, especially pear psyllids, during winter [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In contrast, \u003cem\u003ePhilodromus\u003c/em\u003e are diurnal hunters of hymenopterans and lepidopterans [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], but also frequently prey on other spiders, dipterans, and hemipterans, indicating a broad and opportunistic diet [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Spiders help regulate prey populations not only through consumption but also by overkilling \u0026ndash; killing more prey than they consume [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Overkilling is common in many species, especially active hunters, and tends to increase with prey density [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It may help spiders to avoid ingesting unsuitable or toxic prey [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], although some interpret it as merely a by-product of increased aggression [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, we focused on the active substance thiacloprid from the chloronicotinoid group. It acts as a contact and ingestive poison, has systemic effects, and inhibits the transfer of impulses within the insect nervous system. The mechanism of effect is similar to that of acetylcholinesterase inhibitors, but thiacloprid is inactivated only slowly. Its persistent effect leads to general dysfunction of the nervous system and subsequently to the death of the affected target organism [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thiacloprid is the active ingredient in formulations such as Biscaya, Calypso, Proteus, and Sonido ((Bayer CropScience).\u003c/p\u003e\u003cp\u003eWe investigated the effect of thiacloprid treatment (fresh and 14-day-old residues) on the predation activity of spiders\u0026mdash;specifically, on functional response, feeding and overkilling. Further effects of thiacloprid treatment, including mortality and paralysis, were documented for four weeks after treatment. We tested the hypothesis that treatment (fresh and 14-day-old residues) with the active ingredient thiacloprid negatively affects the traits of the predation activity of \u003cem\u003eAnyphaena\u003c/em\u003e and \u003cem\u003ePhilodromus\u003c/em\u003e spiders. Further, we tested the hypothesis that the response to thiacloprid treatment differs between those two coexisting top spider predators.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eIndividuals of two top spider predators, \u003cem\u003eAnyphaena accentuata\u003c/em\u003e and \u003cem\u003ePhilodromus\u003c/em\u003e spp. (\u003cem\u003ePhilodromus\u003c/em\u003e: \u003cem\u003eaureolus\u003c/em\u003e-group), with an above 70% dominance of \u003cem\u003eP. cespitum\u003c/em\u003e (Walckenaer 1802) in the spider community were collected from apple orchards and their surroundings in Prague\u0026ndash;Sedlec, Czech Republic (50.13\u0026deg;N, 14.39\u0026deg;E) in September and October 2023. We kept spiders individually in 10 ml plastic tubes (diameter \u0026ndash; 15 mm; length \u0026ndash; 50 mm) with pierced lids and a layer of gypsum plaster at the bottom. We moisturized the plaster every week with few drops of water to retain humidity. We kept the spiders in controlled conditions under a natural photoperiod and temperature for 14 days to acclimatise and fed them once a week. As prey, we used wingless \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (Meigen 1830) cultivated on the commercial medium NEKTON. We tested the effect of neonicotinoid insecticide on the predation rate of two top spider predators in orchards. We used neonicotinoid insecticide with the active substance thiacloprid which was formulated as Biscaya 240 OD (Bayer CropScience, Monheim, Germany). The insecticide was applied in the dilution recommended by the manufacturer for crop spraying, with a thiacloprid concentration of 23.1% and at a suggested application rate of 250 ml ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. We applied the insecticide on filter paper sheets rolled into tubes 5.5 cm in diameter (0.737 ml each paper). The treated filter papers were left to dry for 30 minutes and then placed inside plastic tubes into which spiders were later transferred individually. To ensure tarsal contact with the substance, the filter papers covered the whole inner surface of the tubes. The spiders remained inside the tubes for 60 minutes.\u003c/p\u003e\u003cp\u003eTo test the effect of thiacloprid on predation activity, we transferred the spiders individually to Petri dishes (height \u0026ndash; 1 cm; diameter \u0026ndash; 5.5 cm) and allowed them 10 minutes to acclimatize to their new environment. We examined 150 individuals of \u003cem\u003ePhilodromus\u003c/em\u003e spp. and 150 individuals of \u003cem\u003eA. accentuata\u003c/em\u003e in total; half of each species was treated with neonicotinoids and half was treated with distilled water as a control. There were no significant differences in body length between the control groups and the treatened \u003cem\u003eAnyphaena\u003c/em\u003e (Wilcoxon test, W\u0026thinsp;=\u0026thinsp;2738, p\u0026thinsp;=\u0026thinsp;0.8867) and \u003cem\u003ePhilodromus\u003c/em\u003e groups (Wilcoxon test, W\u0026thinsp;=\u0026thinsp;2741.5, p\u0026thinsp;=\u0026thinsp;0.2562). We divided the spiders into groups based on the numbers of offered prey: 1, 3, 6, 9, 12 (15 spiders in each group). As prey, we provided living wingless \u003cem\u003eD. melanogaster\u003c/em\u003e, which we kept at a constant density according to the group. We replaced all killed or eaten prey every 30 minutes for 4 hours. We observed two prey capture behaviours each round \u0026ndash; feeding (at least 30% of the prey was eaten) and killing (more than 70% of the prey remained). Spiders were measured during the experiment to observe the effect of body size on predation activity.\u003c/p\u003e\u003cp\u003eThe effect of neonicotinoid residues on predation activity was tested 14 days after exposure, which was the expected period in which the effect of the treatment could disappear. Spiders were treated with thiacloprid only once during the fresh treatment experiment and were not fed in the following 14 days, as they had been allowed to feed to satiation before. The effect of 14- day-old residues was tested on spider groups at a prey density of 6, as predation activity was generally highest at this density. In total, 30 \u003cem\u003eAnyphaena\u003c/em\u003e and 30 \u003cem\u003ePhilodromus\u003c/em\u003e spiders (15 treated and 15 control individuals per species) were tested using the same design as in the fresh treatment. A constant density of 6 \u003cem\u003eD. melanogaster\u003c/em\u003e was maintained, with all killed or consumed prey replaced every 30 minutes for a period of 4 hours.\u003c/p\u003e\u003cp\u003eAdditionally, we monitored spider mortality and visible signs of paralysis both during the experiment and at specific times following thiacloprid exposure. Mortality was recorded 24 hours, 1 week, 2 weeks, 3 weeks and 4 weeks after the treatment to assess delayed effects. Paralysis was recognized when spiders showed markedly reduced mobility and appeared unusually limp, but without the characteristic stiff and curled posture of dead individuals. The paralysis was further characterized by its reversibility, with the spiders recovering normal activity.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003ePredation activity in response to prey density (number of killed prey, overkilling rate, and consumption rate) was analyzed using a generalized linear model (GLM) with Poisson distribution. These data were fitted with a Type II functional response curve following the Holling [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] disc equation. To assess whether the treatment effect differed between species, we used post hoc pairwise interaction contrasts based on the GLM using the emmeans package (estimated marginal means). The effect of neonicotinoid residues on predation activity after 14 days was tested using a GLM with Poisson distribution. We used a generalized linear model (GLM) with binomial distribution to test the effect of treatment on spider survival rate. We performed all statistical analyses in the R environment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eSpecies specific response to thiacloprid treatment\u003c/h2\u003e\n \u003cp\u003eThe functional response (predation activity response to prey density) of untreated spiders was significantly higher in \u003cem\u003ePhilodromus\u003c/em\u003e than in \u003cem\u003eAnyphaena\u003c/em\u003e spiders (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.535, p\u0026thinsp;=\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Treatment with fresh thiacloprid significantly decreased the functional response of both \u003cem\u003ePhilodromus\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.534, p\u0026thinsp;=\u0026thinsp;0.033, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and \u003cem\u003eAnyphaena\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;59.137, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eAnyphaena\u003c/em\u003e exhibited a significantly stronger reduction in functional response under the treatment compared to \u003cem\u003ePhilodromus\u003c/em\u003e (interaction contrast\u0026thinsp;=\u0026thinsp;0.88, z\u0026thinsp;=\u0026thinsp;7.15, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\n \u003cp\u003eThe predation activity (number of captured prey) of spiders affected by 14-day-old thiacloprid residues differed significantly among \u003cem\u003ePhilodromus\u003c/em\u003e and \u003cem\u003eAnyphaena\u003c/em\u003e spiders. Whereas \u003cem\u003ePhilodromus\u003c/em\u003e spiders exhibiting a higher predation rate than \u003cem\u003eAnyphaena\u003c/em\u003e spiders (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;154.703, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eAnyphaena\u003c/em\u003e spiders under treatment killed non-significantly more flies when compared to the control group (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.22, p\u0026thinsp;=\u0026thinsp;0.633), \u003cem\u003ePhilodromus\u003c/em\u003e spiders under treatment killed significantly fewer flies than the control group (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.083 p\u0026thinsp;=\u0026thinsp;0.014).\u003c/p\u003e\n \u003cp\u003eThe effect of thiacloprid treatment on feeding and overkilling differed significantly between \u003cem\u003ePhilodromus\u003c/em\u003e and \u003cem\u003eAnyphaena\u003c/em\u003e spiders (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The feeding (number of consumed prey) of \u003cem\u003eAnyphaena\u003c/em\u003e decreased significantly under treatment with fresh thiacloprid (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;34.564, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, the feeding of \u003cem\u003ePhilodromus\u003c/em\u003e did not decrease significantly under treatment with fresh thiacloprid (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0601, p\u0026thinsp;=\u0026thinsp;0.303, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cp\u003eTable 1. Response of different traits to thiacloprid treatment in \u003cem\u003ePhilodromus\u003c/em\u003e and \u003cem\u003eAnyphaena\u003c/em\u003e spiders. \u0026ldquo;-\u0026rdquo; means no data collected.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003cimg 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NY9KbY6L+LPPPjt1/E+fFh62bNlSx6qqbdu2VStWrEjhdAxT2yzlaQXSueiii+r/9rceUaYvDaJo2Svu6DVdePMYAfF4lIDzJtCiRJxr8ZgBcTnXCG969ODYY489II2Iz69HnE/8msR0pLl48eKx5csxDbeTL7jggjpk/zlKfDrGNyGtyANYPvIKlof/mW+OcaSZ5xmbNm2qx+7/1Yv8gzDixDzz5SecdEqRdsybNJiOYcKmS2w3Wv9ouqVP5Qvlr2/3339/6p9zzjmpTzrlfg8cE6wb+zPfXiF+eWt6DCC2XWwX+hwb3bAPYh8Rn2XLL/rY1rGfWObI8zkWOFbKMqDc78Rh2nI/sqxxTsQ2yLdLvtyER3r5/EiTtGMepZgmn3c+D8azvZkXyzMXxPk6W/lJvl84BmN/so1Jt2k7g2m4+xHHJfspjsNYpxLLyzyIE8ce8+00DWGxfnQMx75lueM4ZX1IK8IjTcLL4xCxjrG8pMF0DBM2W1gH8xvzmwPU7yr0VdPLzCVeimkdCCke8XmTno7/ebMe5YsmrYM3jSOMF6kD8WKe9EuE00mzrR/HIS+KcZzzkle8eFiea2WrDpxbID5xmTbOP+RxIyzHS2cRHudldJzLvMRGmiwbcenKl9DyF90YxzIxTKsWhJfrQBzWk7QiDyB+pMPy52LeTMMwIk+J/ILh6CKfIS7xyF8i7Tx/AXGIzzxjHMsU6TPddIllappH7ItyW7DtCI+XxmO/I9/vKPdn2coI6044+7lEumxz5sP2Q2yTpvgsF3Hzfcp0sZ9jPfg/XyYQP/6PadFp3xAv1h8RL0+DMPqxvVgGkA7h9PN04piMlzrpcuV2D8yXjvF0bKOYVy/lPPqNbcA6sVyx3cpzMbZDdCw/iE9cps2PqzxuhOVY9wgv0+Y4j/MxTzf2bYh8I44zpiOMaQhnnUpxLsUy0xEWxwX/h5g34+KcKLcPw9ERD2zPWP6YX7nsnY7ZOHeYbrbEMjctQ+QVLHcutnkc93FcID8uEGlEZ34zt/KbJu1ncB/ERo8DpZPYKKxA7HQ2ZAwTXm6YOMjKtCOtpsyB8DIdaTZM9TiMjCPOkU7nQ4jxZMZkRHQhMrFAmnHOcR7mGEf8XBQAZEK5OP8jEw2ReYFCiHUBy0T8fB0YR/osT7kskX5ekBGHuOWyxPrnyxJ5BdMzXZ5OFDp5/Eib5YllDsSlKwu7for9lBeELFMsa9NyxTaNwqfbfg9N+S3YPoTHMRdiHuU2B+Hl8dJtn8Z+yteR+IRRCLIOjGOeTB/r223fxHFSLndsN6aN7UKcWGbCY3/Sj2UAy8D4PH4utkm+HvnxFljWprKqCdNOl1iO2EaxH/JzMRfjpyM/ieOs3Jedlin2I2nFfkHsm3L7Rvx8mRHpM9/A/Pm/XJbYl/my5PuXZZjr+UkvsR/zY5hljnVpWu447umzrt2OixDHRsn8ZnbzmybTkgPFBitP7FKsUKcVYGfQ5WIHl2l3S4twOmm2TfU4JNPJC5HIVDn+m5DhxPgy4ye8PL/i3C3TI6PJM+jIuPJMNDSlEfFZXs7dPDONZcwLl07LgRjHOoD5sxxNBQVhZTjbIeaXZ6YgHcbFOkXaTesJ4jJuusR+Yh3YTrF8dGyHfJvl8sIpzyvz9HKsG+FRQOWIWx4n3fY/GEeX67ZP8+UNUWiyvzqVJSxbpzQ7zS/WJz8fosDleMi3aSxXHhdxDJXHD+kSnh/fvY6hXkhvusyV/ASd9mWnMj/2I8ufr0Psmzz9TmkgxuXrE8ud70fE+ufhMb9ByE96Mb8xv2kyLTlQbLBOGzuwQYlH/F7YoWzEOHDLtLulRTidNNumchw2FUTdMkNwvhAnLzQRFwRlhhThecELpm8qHJvO8chMm+JTMJRpl5laZKLlMgfSpwssA/HzDJd5k4mSbl4YIApA0s8z0igo8sKLOGXaIQqvTsvZD7FuE50H68c2KqfrtN9im5frGQV9edzFdmna/2xTxtGFmG+n9Sj3KfJ5NBV4scydyo8oRPNzI9aHfZwfn/xPeLmebEfCy/lH2vmxFWkzrhTrz7imdemG6aYD22+u5Cex7Zr2ZRwH+bGZxy+PwdiXsQ5xnpbHV4j0Y3niWiJfR/ZZ03JgkPKTXsxvzG+aTEsOFJlN007NxQnZacODOKwscdhRkfGUaXdLi3A6abZN9jgkU4hMpKnLM6EQBVXT+Mh4ynDOK8Lz84tzqzzfYvoyE4rCrsyEI35eaCKWMc/QmZawpnVqKkjYLnQsC+mRFv+TF5RpEIfp6cpCKvKWCO+0LiEy4/IiqJ9iu01kHrFN2QZ5oYVO+z0KrnJ/xjbJC6h8G5bxEfNnXqHbPo0CuCw0mYZ16LTu3bZNp2VsKj8iLvNqKojLYzaOi3z9ENuwPFdCXIiU6fXCNP021/KT2C9N+7Lp2In4jMv3b+zL/JyN7V6e74HtkO/L2I9sI5aVeZEeXXk+5cfZIOQnvXQ7pzqJ48L8pn0ZY30GKb9pMqcrCrGi+Y4hzaa0u6VFOJ002yZzHJKxNF30otuxHedPea50ynhQnl/Mu1PG1ZQBlYUjIj4ZY7kO5TkemWXTsiEyx4gf532kz3Zi2Zu2FaKQaMononCJjD4K7rJAAXHK+P2Wb7eJiG1aLnekV25blp/wpv3JOtLlYpt32kdsW8bHMdQtb2berB/jmwrNpmkC4+matn8cx+X0cTGQT8Ox1BS36VhGpM3xEUgjjs18PXLMk23WlGY3xO8nloN1bTpHmFen+U1XfoLYL6XYN8w7F+de5AOh6SIwjq+m46TpHOf/6NhOjOMYbjJI+Ukv5jfN0wTG0zXtnzjOy+lZl0HLb5pMy9VzueM66bZDY8eVGURstDLtbmkRTifNtskch5wDZYGIyHA7pUkGVWZSiAKrKfOI8yjOOzKvMiOKwrGcnoKB+ZUZesRvyg9iGUOn8xuRTh6/23kf8vWPzJXpck35TeRjZVxE4dhU2PVL7KcyD+ylqXBCpJcXOOi2PwmPC4BIr9s2j3H5/LvtU9JuujiIQrPpuA+MpysxX+bPuHzfxfqU2zP2ZTkvlonwcjuW4fRZN46Fcj3KbRTbZyLHDfH7ifWdS/lJp/0C8hLmSZwQ8ZuOv/LiqduxSpw4TvJl4n+6TvL1H6T8pBfzG/ObTmbtOwq9PPnkk6lftvsaH/sIZbu140GbstIgoO3lo48+eqxt6twrr7yS+q3MIfVztKPMly1bmVJbW9Tf+c53Up/2ozudC5x3tOV81FFHtbWlHe1llx/DiXaiI/0Q8S+99NLUD6Qfy8jydjuXGf/Tn/40DUc763QLFixIYZ2mZZniwzqID76VH4q8++67U5+PBfXKH/I2rmlLfNJtU/dw8803p/5EPmDEdmoVUF33+/nnn3/AOsb+Wbly5QHhsd3+zb/5N6kt8DfeeCP9f+SRR6Z+iX3w1a9+NQ3fcccdjW2g52gTnLRZ3lZBdsB2pB31ViHYeNwHxjfhw2EcV63C/4D9HOtTbk/WrZxXbEeW65133hlbNvoRzvqxzn/6p3+ajm2W+ayzzkphxCON+AhVmOgHSvttLuYnnfYL7dTzpd9bb731gO86RPwrrrgi9QPbPdrfX7JkSVrmTogb1xjse5Yp9nHroi/1iVPiWOH4CoOUn/RifmN+01FdYeibqMHQ8WtAWTvKRS2O2lhrY9Sh+0WNnK61kVINivQYJqy1oVPNLKaLWiTh5Tyjtse0dHmtT5pJHIfjwfEfv0A1/VLHMc7xz/im8yfOrfyXMhCPcNIm3XJ8nHecR6RRYr4xzziPSJP0CCvTi/icd6VYRn5ZiV9/Iv9g/qTL9PwixbLmv1wybZznsZ3YHhFGOoTnv2YxH+KVv3CB+TGO5Y/tHXkKy066LA/zJU7kQww3baepinnTTSS/im3aaT+U+z3CyVsJy7dNbFfilMsQ6x/xmZY0OAbKX8oYR9zYp3Tsq5gf42L/gvGEEaebmDb2e6RLWBxPuVi+XCxbuQ9j+9PPx8UxRHjMj3mz/ITnx2bEjTiI/TORfUr8qWI9Y3/G8Z1j+WLbzWR+AvYLcWL/sywxv6ZlbdqPyPcNHenQEZfw2OakyfhYNtabeceyM45w5hPbgT7HfOxbDFJ+0kssG535TbOYNs7nSJcwtl+p6TiNZSv3cWx/+vm42chvmvS1osACNXUcAKWmeOXGZiPESc6BEjudMA4CNjrKdOjyDRPp0DEszRaOzV4i88m7/LiNTKXs8vOM86PMpBAZFXHj/CkxnkwuMpscyxHjY77MK8+cchE/LxAC07CM5XkfmRvjiMM5j8ioo6ANDOfbjGUjzXJ5Ik7TejMv1iMvdJg+Ci/G5WmyDExTZvhTFetYdqzTeHTb74SX+z3PT8sCNwq6pjyT7cC6M57lK7dPiTSIS5cfK1G45dsxtkG5PE2Iy7yJH2nH8ZIjjDiMz3UqSGMZyv0b25EuX99YD7ZvzD/G58vINh3PeuWYbirycyO6uZKfxH6J9Bmmz/nVlF7EZ3wp8pr8+EIcezFd7OtYdpatnBfbJKZh2ZqOq9iuTcvJtEyX72uWaabzk17Mb8xvxuMg/rQSlDQD+KT6XD/l+OT7zp07G2/l8ujCbbfdlm6H5o8DSJoeg5BndNMtP+ExjIsuuqhqXdilRwolzT1z9h0FSTOP5zh5trTT854U7MuXL7eSIKmnXvlJp/edJM0dVhQkJRTq//7f//u2lw1DvHx8wQUX1CGS1KxXfsJLmLyQOTIy0rEiIWn2WVGQhhQtTixevDj1exXqtKpw/fXXp+ETTjgh9SUpTCQ/oZIQrQedfPLJqS9pbrKiIA2pd999N90h+Pa3v52aW+tUqPNewtKlS1NThTj33HPTBYEkhfHmJzy+eMQRR6R3nXDjjTem9zC6NWcqafb0fJmZE1iSJEnS/FZWC2z1SJpBg96CiaSZZZ4haTb56JEkSZKkNlYUJEmSJLWxoiBJkiSpjRUFSZIkSW2sKEiSJElq09eKAh9R2bJlS3Xsscemlhro8zXXQcBy0jb8hRdeWIfMPXz0iu0bH7WRBsVvfvOb6rzzzqvOP/98W3DRGI8LSZrb+lpROPvss1P/1VdfrbZv317t2bMnfZxpUCoLcxkfo7n99turq6++On3URhok9957b/WXf/mXqX/ffffVoRp2HheSNLf1raKwbdu29OVWvsgIfpmnsoCFCxem/lzRdNfgnHPOqfbt25e+GjkX8ZXLG264oRoZGalDJEmSpOnTt4pC/Bp0+OGHpz64IOd28mmnnVaHzD4qAm+//Xb9nyRJkqQmfaso7Nq1qx6au3iH4mtf+1r9nyRJkqRO+vqOwlx33XXXpfcmJEmSJHU35YoCrRvRhfifF5tjOP4PtNyTj8tb8OHF5/Xr16dwMG7FihXpf/q81NuER4oiXsTNX6Jm/rfddlsa5u5HxIt5Mz1x8uXMkVa+TqTf9D7DZJafOHnatBbFNpoq5rVp06axVpJYXoZJnxaUAuN4TCzmz7I0LWfZohXrmb/vwTSEMQ+wLfL4+f7IMR3pMF2+/twBypXrwzrEcjNNp/0R25/piM//pbLFLuL2Yx9IkiQNrNE+Iamm5LZu3ZrCR0ZG6pD99uzZM7po0aI0bvfu3Slsx44do2vWrBlLa/v27Wm6zZs3pz5hS5cuTXFz69atG12+fPlYOi+88MJY2qQZGE9YuSx5+uU4kD7pRVose8RnXJjM8scyRTp79+4dS4PpSpFOrGsnbIONGzemuHQM0zF//mfZQJ91i/To8z8daQSWhWlZdxCPbR7bi3XP58f/pMF4+nl4jvkTznHCutNFOqTP/yjXh/iMZ7nYdhEey4fYtrFujIttm2MepEU6sQyRZr5/+6Gc97C4++6707rT3XvvvXWohp3HRW/DmmdImhumvaIQF2txQZlruujlIi3SiovZEBe5+QVsXGjmaSAuKvM0ui1Lp3Fc2JbpgOUsL7ox0eWPC9d8+bstZ9M26ybisz3A8sW0XDg3pcXFdzl/hiONwHTlMjJdzI95gT4X4oSzDQLzpxLRdDEe26UcF+mU4XFhTwUisB7Ez7EszDPHtOV6RDzSHO+2Hg/SG0ZeEKqJx0Vvw5pnSJob5tw7CmWrSTkeC8F7772X+uDl5NbFYFvLSjQl2lq/tjQm6vrrr0/9M888M/UDy3nVVVel4fwF6Ykuf+vCuWpdkFZHHnlkHTI9PvvZz6Y+yxfbiu8yMP9y25166qmpzyNa+eM/PLqVP5LEdMccc0z934HY/rEt6N91111puFU5SI8NgfnzTYjPfe5z6f/ctddem/rMM39MKtK8+OKLUz8cffTRqf/OO++kfqDJ3vyRJKbP9wvrxzwuueSSOmQ/4q1cuTINP/jgg6kvSZI0TAb6ZWYu8rjwjIvHfiN9LjTRNI9TTjkl9VmG/GJ2Irig5vsNXHDHl5fLi9bp8txzz6Vl55n8vFu1alUdo6peeeWV1L/iiivSRf3v/d7vpfcQosJw6623pn4vrB8VOjzxxBOp/9BDD6V+03c2+G4EFSg8/fTTqT9RfO2VNC666KL03kW8I5Evc6wfccrtEC15sZ0kSZKGzUBXFOIib7r0Sp+L2fCzn/2sHpq4eCn3rLPOqg477LDqm9/8Zj1m+nHxzp2XTl3cbeCDdFQqRkZG0i/wVBi4+J5IBamsbEUlrJP4Rf+nP/1p6k8UlZOf/OQn1caNG9NFP18J565O3NHI7dixo3H96Xbu3FnHkiTNJvLv8kcdOsojqZum44ZO3Q10RSEe1ykfkemX/HGg/JGbJuXjO+PFYzE8/kP36quvVmvXrq2WLFlSj51+XKyPd9tx4c1F8+7du1OFge1ORWOi2/6EE05IfdYZL7/8cup3Eo9CTQaVE+7aUMlZt25d6nPHpKwsPP744/WQJGmuWrBgQSp/yu7kk0+uY0jNmo4bOnU30BUFLlzj8ZR77rkn9UtTaeKS9ONi9sknn0z9XPyaPpUDjcd4mAcXszON9QPfl2iybdu2sXWkWdJApYgKAxfePI7UaduXokLxmc98JvW5g4If/vCHqV+i4sT+Pf744+uQicmbWGVdeeRo+/bt6f9vf/vbqR+VwfL9i8D0NpMqSXMDd/Ipf8puNspQDZam44ZO3U17RSF+aec58/yXZ54X50Jwqq655prU5+XX/Fdi5sVF+FFHHVWHtCNOrzsF8RgQ6efLj3h2frJfe2beXGjzjkKe9htvvFEP7VfOt1/ihWAuknn0KbYF86OScN99941VJnhOv/wVvnyhOFc+kkTa3L2gchGPIP2X//JfUp8Xncu0mZ5f/9m/U3kHhRemc+UL5qwfFT32w+rVq9MdntjeLDO3s6dyR2PY3XLLLdWXv/zl6oMPPqhDqur9999PxwHvvWg4eVxI0oAY7QOaASUpurJJULQuxNI4mqqkyUqaviReHh7NWuZp5c2I0lzl0rp50byZzrzpzUiLdBc1NLtJehGPNIjH9IgmQZlHhAXSibRbF68pjCYzmUfZZOhEl580Im2WgWWK+UXc+PYA8474eTOgneTxy+UMhMe88o7p8uWPbRr7l/VhPxKWb6+YvtxW/E9XbtvYXqQTzZAyXcTPER7pl+sfzak27VP6ZVje5Gm+ncquPIamijSHCet7yCGHjC5btmxsm5544omjhx56aBpuXSjWMTVMPC7Gj+0hSbNlyjlQZPJlxwVZ4IKTCzjCuViOC1/CuMDL/y/TISwu7soucBFInLjYYx75/HNxYUycuBDO04wuv5AEF7RcuMb4fLnDZJafZYgKBOnHfBnOL8wJL6en66TXNsuxHuW6xUV+IKxcv6Z4MY4L+Vgv1oPtXlYSAutGWjEty1JWBDqtT6ftQjjTsMx5JSDfxjmWjUpBfgyNpzI2UaQ9TFasWDG6ePHi0YMPPnhsHzDMdl65cmUdS8PG42L8hi3PkDS3HMSfVkYk9UW0IOBh1YztM0zb5uc//3n6TkarQln98pe/TOv/8Y9/vDruuONSS1NHHHFEHVPDxONi/IYtz5A0t1hRUF9ZUehuGAv9X/ziF6lpWt5JYt0/9alPeTEoj4txsqIgaTZZUVBfWVHoblgLfS4K+To4L68+/PDDXgwq8bjozYqCpNlkRUF9Q8tF8VXn3bt3T/rbEvOZhb6kiTDPkDSbBvo7Cpo7aEY0Kglg2C9lSpIkDS7vKEgzyF8HJU2EeYak2eQdBUmSJEltrChIkiRJamNFQZIkSVIbKwqSJEmS2lhRkCRJktRmXlYUXnvttWrTpk3V4sWL65D+2rJlS2qJYtu2bXWIJEmaTXfeeWe1YsWKVD5T/lNW53qNl9Ru3lUUyAjWrFlT3XjjjdVbb71Vh0qaTb/5zW+q8847rzr//PNt6lFjPC7UL/w4eP/991c7d+6sXnjhhRR29dVXp3D0Gq/5z/xmkloba15i1aa6elu3bh3dvXt3/d/wGNb1ngnz+JTr6u677x47J++99946VMPO46K3Yc0zJqJ14Z+2044dO+qQ/WGLFi0a3bx5c8/x04H0pyttTY75zeT4jkIXN998cz00XIZ1vSVJg2fXrl2pv3DhwtTHsmXLqn379lUbNmzoOX46+Giy5gsrCh1wku/Zs6f+b3gM63pLkgbTI488Ug816zW+31588cXqtttuq/+TBpsVhQac5Ndee2393/AY1vWWJKkf3nzzzeqyyy6r/5MG35QrCg888EB14YUXVmeffXb6n1YEaE2AVgUI4+IzR/z169en8eBFooiby1snoGMaTsAmzPPYY49N8Xq1ZMAv5nm6DLNMgeHVq1ePvQi9atWqFC+Wj2UgDebXaT6E5/Ng+zz22GP12P1Ih3Uk3Ug70o1pOq1vrkyH/+mTRr58U13vMJH9IkkaTLQemJd15POU15SxdJ1eAiYe5UJeJvM/6U0E8ZkuridILy+zEGVdPFoU5RYdeo0PlM+UuTGO6cprF7BubItYNzqmi7gs82c+85nq2WefTf/zsnTEkwZW/a7CpPDS65o1a9KLISMjI6MbN24cXbp0aRomjI6Xhfbs2ZPi8yJRxKfjRR+mIQ7/R7x169aNLl++fOx/5sN4wvbu3ZvCAnGZPl6+pU+8mEeO+RG2ffv29D/ps7yElS/vxjrk4cQnjZimfFGJZWPedLzIBKaP+DFfsE6xnMyL9aDL0+f/Xjqlw/+kg6mudyBd5jWe/aJmbK9hdLcvkamBx0Vvs5FnkJ9TbkTZTDlN2UA/L7PzMg3xgjBlRV5OEEYX5WIvkQ7TgrSinCvniW7lFrqNJz3mFePoNy1vlO9cw8S6MS3pEjfCEGVueY2g2WV+MzlTzoE4qeJEiZMacaIzrrzgjR0VLRBwgsUJGSdteeEZJ3p+4sXJWJ78sUx0uVieXKcTulvG0mkaMlDC8wwDrBvh5TjWnzCWK8+QiBPh45Hvg0if9GK4H+s9kf2izsr9MCzMoNXE46K32cwzokwrfwyKC2QumnPEix+ocvGjUtO4JsQry5S8nOtUDjWV1+g0PsraMjzKR6YLXMc0zZt1JjwvwzuVr5pd5jeT07d3FFauXFmtXbu2/m9/iwK33nprGu70Us8555yT+sccc0yKj29961vpVt7hhx+e/g9nnHFG6n//+99Pfdx0001VK0OpTjvttDpkv/L/QNzWSV3/11/ckuTbDaTP+uRYN77tgFtuuSX1ES0wsO1i/RHTT/Q7EKQT05JeDPdjvSeyXyRJg++www5L/QsuuOCAvH/JkiWp//bbb6c+eCyIR26irMtxbdC6mE4NZfD4ajekQzzaus9FuU65+NRTT6Xhqbr99tsbryFOPfXU1OeRJcp2HiniOqZVWWgrA5955pnUelJehkvzybS+zHzmmWfWQ/ufARwPMhpOyHiuLzqe9YvxID0yDJ4VHC9OaDrwTCHPP1LZ6IfIuMpMJPD8Px566KHUn0n9WO/x7hdJ0vB5/PHHUz8qF6Wzzjor9R999NHU7+Sll15KfS7gy/ImRJypeu6551KlpJwP7zKEV155pfrxj3+chjutmzSfTWtFodNFcy8bN27kXmvHDk888UTqTxQVDF7G5QL39NNPT78Q9EOvjOvTn/506pMpzYZ+rPd49oskafhw0d3NSSedlPrjfal57969jeUMXT+/fcDd9qZ5RMfdhn5VTKRBNGPNox5//PH1UG/T9as7v6TzS8HXv/719Bl3HqXp1y8ERx11VOo//fTTqd/JyMhIPTRz+rXes3E3RJI098Wjrs8//3zqdxKPq/bSr8eLeuFuOI8XdRPlu4/YahhNa0UhTj5q7OO9u8DtRk7cTs8xRpNs+TOE4/mFgkdu4hnDeDein3g/ADwO1dSs2htvvJH6480k+6Vf6z3e/SJJGj7cqUanH5Ref/311I+yu5O4KL/++usbL+Ap03q95zBeUbm57rrrUr9E87BcX8QPnZSBTeU7y0lcaT7qW0Xh1VdfrYc+dM8996T+lVdemfrj8cUvfjH1L7rootRecVQC6PPLePwSzu1ALl7BIzGdREbz5JNPpn5ZqYjMK3T6ZaHXOxZkOPE4Tzy3n+O5TF7muvTSS+uQmdGv9R7vfpEkDR/uVFMm82NZ/g2fQAWCO+qdGhsJvNtIWclFOd8zKL/3c9555x3w/uNUXHzxxanPj2ksf1QC4sL/vvvuS2U7LyrH0wB8TC0vTxlmObstE+l2KmOlOW90iqLJMrrWhfJY02HRnCZhuWgSlC6aRy0tr9tLLjvC86bJaI6MeTCOZsxIjy7/VgPDLEveRClhNFtGehG3lcEd0PZzhBOHJuLyZs6YV6STY9li2fNtEU3DletLnJh3uV6EN03TJJqwY1vE8od+rnesW9kRni+/OmN7DZObb7559Etf+tJYc4p0d9111+jll18++pWvfKWOpWHjcTF+s5Vn5OVZWdZFmVaWOXmZHM2lkw5lTFP51AnlHmk0dRwzuXyezKfUa3yUn2XHNEwb8nTouA6Ia4FymfLtQxlabj/NLPObqelbRSEuLONE4gI0MooQJ1XeEdaEk4s0iEOapN10McrJGxe3dGQExIvhvH1kDpJYPqYh06IjjHmVmULMn3kjrxTlXY55l8vOcuRpo0yDjukmso1QxqXLL+4x1fXOjXe/qBnbbZiwvocccsjosmXL0jDdiSeeOHrooYem4Q8++KCOqWHicTF+bI+Z1qms6xSelzmULZR5UeZQXkymnKAsyst2rjHy8hxN5SVdlJm9xgcqJvkPYVFOlmLdIh7TNP2Yx7rGvOlbRs4u9oP5zeQdxJ/Whpo0Hk3hRdnWyZBelJXUGU3vTfGUGyi8u9MqXKt33323ev/991PYwQcfXC1YsKA67rjjZuyFRc0tHhfjN2x5htRv5jdTM2OtHkkaPj/4wQ/Sc8t8XPDQQw+tPvrRj45lzjt27Khjadh0Oi4+9alPeVxI6ivLoamxoiBp2nzyk59MGTGZ9G//9m9XH/vYx8Yy5yOOOKKOpWFTHhegkkCB7nEhqZ+a8hvLofGbckUhPnxGq0dlyzqS9M//+T9PGTKthxx99NFmzkry44LmJ60kSJouZX5jOTR+U3pHgWcnS5s3b+7rVxOl+cTnjSVNhHmGpNk05ZeZJY2fhb6kiTDPkDSbfEdBkiRJUhsrCpIkSZLaWFGQJEmS1MaKgiRJkqQ2VhQkSZIktbGiIEmSJKlNXysKb775ZrVly5bq2GOPTU260X/ggQfqsTPjzjvvrM4+++y0HDmWY/HixdWFF15Yh0iS5iLy8RUrVqRyhHy7zM97jddw8rjRZHjcdNfXigIX6OArzdu3b6/27NlTnXvuuTNSWeCr0OvXr0/drl276lBJc8FvfvOb6rzzzqvOP/9824RXV5s2baruv//+aufOndULL7yQwq6++uoUjl7jNZw8bjQZHjfjwAfX+mHr1q2U/qN79+6tQ0ZHW5WFFLZ79+46ZPpt3rw5zZO+NNf08ZQbKHfffXdad7p77723DpUO1CqI0zGyY8eOOmR/2KJFi1Ke3mv8dCD92SxPhjXPmAiPG02Gx8349O2Own333Zf6hx9+eOqDx3xa86hOO+20OkSSpGZxN3jhwoWpj2XLllX79u2rNmzY0HP8dNi2bVs9pLnK40aT4XEzPn2rKMQGlSRpMh555JF6qFmv8f324osvVrfddlv9n+YqjxtNhsfN+NjqkSRJBRrnuOyyy+r/pPHxuNFkzOnjZv8TSJNHEk3dyMhIHWM/3ldYvnz52Ph169Yd8D5DIIzns5YuXZridXsWjGe51qxZc8A84/9yGubP+HK5ePaMZWEa8D5FLCd95tGkXJ+ym2vPmGlu4NgYRr6jMHv27NmT3iEjTyVfIo/duHFjylvpGG5CPPLGPC/mf9KbCOIzHdOTDunlz/yCfDmOj7Ibz/hA/l2WCePNwxmO5WKZY73LbqbNxjzhcdN+3JTXJ3RMF3Hn0nEzWzxuBju/adK3pei2Uuw0Nkrs8HjxmTAOjsAwYcRnOA4c4tLPsTPiQIq4HJSxHAwHhmPH0w/spHwnR2Uij8/OK3GgM471QCwLYeVySjmOkWFkRWF2RL4Y+RN5F3kb/Si8CSfvy+X5a55vE0bXqTAsRTqRV5JWFJblPBH5bqcGMLqNJz3mFePod1pe1ovliLh5Hp5fVESZkpcnM435zzSPm/blZZuwDFwzxLoxLekSN8IwF46b2eBxM/j5TZNpryjExuQAysUOyDcIG5PwHNPFBo2NHGFlXDSlC6YlvCl9wunKAylqePlO58AjjJ2eY9pIR+pkWI8PKwqzi0KabU++lefFkW9x8ZMjXtOPJBTAxG8a14R4nfLibuVC5PWlTuMjXy7Do+DN8/1Y5zJubKO8HIjpy3WYScx/tnjcfHjccH3SNG/WmfD8OmEuHDezyeNmcPObJtP+jsK3vvWt1PpR3hoSzjjjjNT//ve/n/o8n8VLHJdcckn6PzDdypUr0/CDDz6Y+vfcc0/11ltvtcVFpDteZStNOT4Yh/feey/18fDDD6d+uT5nnnlmPSRJc8thhx2W+hdccMEBedeSJUtS/+2330598N2bZ599tmoV5nXIh9auXVu1Ctz0jRw+QtQN6RCPb2fkohU88vCnnnoqDU/V7bffXrUuEtpa2Dv11FNTn8Y2KGPwta99rWpdmLTFveGGG7gi96OcGY+b/ccN32ni+qRVWWgr+5955pnUCg6t4Wg/j5v5ld9Me0WBA4ATjC/a5R0frIjxeOWVV1L/oosuaosbLSo999xzqR9NscZBN5PiBCiVmYckDaLHH3889TvldWeddVbqP/roo6nfyUsvvZT6FKhlnh4izlRRNnCRUM5n1apVdYz9ZQyFN/HMr/tvPh83P/7xj9Nwp3XT5JnfzH0z0urRxo0bU82pU5fbsWNHYxw6voyH2WyKNe5uPP3002M1RvCLA5pqxZI0KOIHmU5OOumk1I88r5e9e/c25ud0/WyLnF/tmuYRHb/oxQ9S6r/5fNz06wJT7cxv5r4ZqSg89NBD9VBvUbucq4455phq+/bt6TbWddddlyoLdFSGuEV244031jElafCQx+H5559P/U7G+5hnv27398Ld6fzHmyZHHnlk6uePBqg/5vNxc9RRR6V+PCqt/jG/mfumvaLAbSA2aKfnyzZt2pT6sUF5TImPTpTYyFu2bEnDIyMjqf+9730v9Wcaz5Rx54Aa7hFHHJE6/PCHPxw76CVpEJ1++ump3+kHntdffz3143ncTuLi6vrrr28sJMnnez13PF6R7/LjTRO+dkp+TTx+0AHvujWJckYTM5+Pm+OPPz79z7VMp+sTv8Q8OeY3cz+/mfaKwhe/+MXU590DNkjcPqK/fv36sefS2KBUAPilfvXq1WmHxs5mB5999tljB0q8xNypUoF33nmnHuo/lptl5FGouM3E8voyk6RBxw8h/MBDXtxUiFGgk1eXL+eVaOCBQpKLK/JvXjYMDJ933nl9awTi4osvTn3KBJY/yoW4gOO9tijcr7nmmtS/9tprq8ceeywNg7jk7XHB0YR0my5CNL+PG8r2+IGSj2LFdQwYZjm7LZPHTWfmNwOQ37Qucqcsmn+iy5t6CsuXf/iRibwjPG+uas+ePakJq6a4NE2W43/CiU9TUjQ9Rb91wB0QTpqIZqcYn88zX/a8eTPiRFo0YxWIQxjLTprRkQ7LkKchlTh2hsnNN988+qUvfemA8+yuu+4avfzyy0e/8pWv1LE0ncjLIg9eUzRLGM0Pkl9GXgnysciLo01y0okmIvO43dBGeOz3sivLinyeZX6PXuOjucGyY5o8X863Bx3DrQuRFK9MN98+5PPl9psJzH82eNwceNzk6dBxzNAxXC7TXDhuZovHzWDnN02mnAPFypcdK5nj//wino3MBizlBwdxmSYOnFKeJn0Okghjmkg/linvuKiPkzzvCCONMpwO5U5v6khDasLxMUxY30MOOWR02bJlY+fHiSeeOHrooYem4Q8++KCOqelAPhfbPe86hef5NoVzmRd3yre7odCkwIt5kH8y/1xTXkwXeWmv8YEyIM+fmW/TRQbrwLrm65aveyBezJv+RNe9H5j3TPO4aT5uYt0iHtMwbWkuHDezweNm8PObJgfxp7VQmgBuF33uc5+rFi5cWL377rsHtIjAI0/cjrr11lvTLSkpRxNqw3TK0UpYK+NM58n777+fwg4++OBqwYIF1XHHHTdjL55Jg2rY8gxJc4sVhQniGToqA3wsoxPi8NyZFQWVhq3Q//nPf54q1a+++mr1q1/9Kq3/xz/+8VRJ2LFjx1hDAJKaWVGQNJtmpHnU+YIXS/hQHL+QdsKLJ4888kjfXpqRBtknP/nJVCHgK+dUEGAlQZKkweAdhQmgdYPly5ent/OXLl2amkjNvyZIM158zv073/mOLSCp0bD+OviLX/yi+uxnP1t98MEH1cMPP2wlQRon7yhImk1WFCaIOwa33357+vAKzXCFkZGR6g/+4A+q888/f+A/163pY6EvaSLMMyTNJisK0gyy0Jc0EeYZkmaT7yhIkiRJamNFQZIkSVIbKwqSJEmS2lhRkCRJktTGioIkSZKkNlYUJEmSJLUZqorCnXfeWZ199tmpGy/iLl68uHrsscfqkP0eeOCBxrT4zgLz4Uu0W7ZsqUM/1Ck9SZI0eZS9K1asSE3KUs6WZXCv8ZLaDU1FYdu2bdW3vvWtateuXXXI5JHW9ddf35ZWfIxt/fr11Z49e+pQSb/5zW+q8847L32Q0DbhFTwu1C+bNm2q7r///mrnzp3VCy+8kMKuvvrqFI5e4zX/md9MUmtjDY3du3dzZIyOjIzUIZPXLa3NmzencfSl3JCdcmPuvvvutO509957bx2qYedx0duw5hkT0brwT9tpx44ddcj+sEWLFqVyuNf46UD6XgPMLeY3k+M7CpIkaWDF3f2FCxemPpYtW1bt27ev2rBhQ8/x04EnD6T5wIqCJEkaWI888kg91KzX+H578cUXq9tuu63+TxpsVhQkSZL6gHcVL7vssvo/afD1raIQrQDRmgAdLQvQwkAuHx9d3vpPPr5sTShvrYCOF4Y5IUO0NsR0dPwf6XVr2YD5R5rRxTK99tpr6UWnfrVS1Ck9fn3Iw4l34YUXpmWh9aRyOwbiRrymrtyGkiSNB+UQj89EC36UqVFO0XV6CZh4lM9MRzlEXP4nvYkgPtMxPemQHtcZuSjj49GiVatWjZV/6DU+lGUp01Eul1g3tkWsGx3TRVyW+TOf+Uz17LPPpv95WTriSYOqLxUFTuY//MM/rK644or0Jjkt/hx++OHVRRddlMaF//2//3e1bt26+r+q2rt3b3XaaafV/1WpNYKNGzdWa9asSXEDadBi0V133ZXS37p1a7qtx8nMiYt77rnngFaNrrvuuuqYY45Jw3/+53+e+k2YP8u0dOnSavv27WPLRMZBmjfeeGP11ltv1bEnr1N6ZDB33333WPjLL7+c1v+kk05Ky8W2ZDuWmSwZJpkemSjLTLd8+fI0jnVhO7E9JUmaCMpVyqtrr702lUHvvPNO9YUvfCGNizKcMqv8EYvy7LjjjkvDDz30UCqH/uRP/iTFo3xquvhuQjzix3sELMOiRYuqc88994B5UsYxj5GRkfT/7t270/906DUepPf5z3+++vKXv5zCifP0009Xq1evPmB52SZcczz//PNj68Y1A9clxKWM5prj1VdfrTZv3pymoV/OTxo4rQN4SmhFgGRaJ0wdsl/rwnW0dcHaOC7CWyd/HfKhVuZwQDjT0jIB6eVobYg08lYFoiUi4kcatDwQw00tFZE+/5fph5gP0+aa0gosU7lsoVN6rDfhrUy4DtmP/wlvVY7qkP1iG+bLzXoS1pS+5gb2zTBqVYbHjk1bm1DwuOhtNvOMjRs3pvlTPuVlDeUm4WvWrKlD9iMeZVOJ8ov4TeOaEK8sP6PM7XY90Knc6zQ+yswyPMrwvHynLG6aN+tMONcaods1gGaP+c3kTPmOAt8TwJlnnpn6gTsKV111VRr+2te+lvrhi1/8YurfcsstqR/41b2VQYzdCQB3Cbi1R3q5M844I/W///3vp35u5cqVY2nwi0SeXo47FT/60Y/Srw5l+jMt5n/xxRenfjj66KNTn190Ar9ytDK4NJwvN+vZyrTq/yRJmrzDDjss9S+44IIDypolS5ak/ttvv5364C43j9xwR7y0du3adEeAcqvTo7SBdIhHW/e5ePqAO+9PPfVUGp4qvnvENUf+ZANOPfXU1OcJBe4kcLeApxi4m1JeKzzzzDPprgfXGtJ8NKWKAidQPIvXdKF9yimnpD4nff7ozKWXXpoyDU68PPx73/teuv2XI33ixXN+0fHsX4yfKJab9x24fXjDDTfUoYNjwYIF9VC7pv0gSdJ0evzxx1M/Khels846K/UfffTR1O/kpZdeSn0u4MtyP0ScqXruuefS9Uk5Hx7rDa+88kr14x//OA13WjdpPptSRYETqJu8hv2zn/2sHtp/MRvPOfIcJKgwUDMva/bgvYXR+jm/pm6i4mKaDCJ/h2JQcOeATBTchcnxfCSVsKbtKEnSdOCiuxveu0P+42A3e/fubSzv6fr57QPuwjfNIzrK0n5VTKRBNKWKwpFHHlkP7X8cppvywpW7CrjpppvSL/zcArzyyitTWIlf/vuNx4242OZuxSB+GOXee+9NFYKvfvWrYxkvrTFQ+bnjjjvS/5IkzYR4xJeXfbuJx4Z76dfjRb3wVALXIN0cddRRqd/0qLM0302popD/sv3kk0+mfi4uYEfqFgdyTMtdBZ43/NM//dNUGeBdhBLpcyJ3eq6xUxNtvXBXIS62L7/88p7PTc413K2hNQnwiwi3S8nEduzYUZ1zzjkpXJKkmXD66aenfqcf9l5//fXUj+f/O4mLct5/bLqA50fJfpXXUbmhlcQm/IjIdczxxx+f/udapOlHUZbTLzFrvpryy8zf/OY3U59m1MqTmibGUL7MHOLFXZpZ42WpJvHiM02E8ot5VD7o89jQVJ4Z5GL7r/7qr9IwafW6KzKXkFH+8Ic/HHuRilukDFtJkCTNNH7o44c9fvxr+nYRFQh+NOz1WCwNo/ADHhflNEeafzuB4fPOO6+t8ZTJimsQnizIv4cQF/733XdfqkxwrRA/ePIxtbgOAcMsZ7dlIt1edy2kOat1gTll0YQnzYTtyZoibZ3sqXm1bqLZsrLJsRzpEqfsCM+ni6bcmG8sRy7GtzKzA6aL5Wc6mnsNxCGMcWXzpJ3SYjjWqWw6rlN6LCthTfMhDcJJM58P6dDR/Fp0TMt279REnGYf+3KY3HzzzaNf+tKXxppTpLvrrrtGL7/88tGvfOUrdSwNG4+L8ZutPIPyJsresiyL5k4pg/KyliZCyzKOdChjy7jdRLPrTR3HTC6fJ/Mp9RofZXnZMU3e5GmeDh1lcpT15TLl24eyudx+mlnmN1PTtxyIHZBf0HNi5BfdnRCn6eQtcbJxUR4nHyd3fuEc8807pgmdxnNR3WlcGUamgMgc8i7mVYbTMY9O6TWF03VarqgEROWmU8e2Gm+mrJnDvhkmrO8hhxwyumzZsrFj88QTTxw99NBD0/AHH3xQx9Qw8bgYP7bHTOtW/jSFR/kHyp2oGDCOsqgsr8eDC/P4oYyO64so/0JTWUzXrazOxweuQ8rrl6byM9Yt4jFN03UO6xrzpj/RdVd/sR/MbybvIP60NpQGDI8e/fSnPx173vOJJ55I/cDXqGmO7tZbb61DNBfwLskwnXJ806RVuFbvvvtu9f7776ewgw8+ODXxyxdcZ+qFRc0tHhfjN2x5htRv5jdTY0VhANEkKq0d8U5CJ8ThuxRWFOaWYSv0f/7zn1ef+9znUib9y1/+Mq3/xz/+8ZQ579ixozriiCPqmBomHhfjZ0VBmppO+c2nPvWp6gc/+IH5TQ9TfplZM+/zn/98emGs28tRVBLKrzxLM+2Tn/xkuvBbunRp9du//dspzItBeVxImilN+Y2VhPHzjsIA4qvStAixaNGi1FLD0UcfXY+pqnfeeSe1LsE3KZqam9XsGtZfB3/xi19Un/3sZ6sPPvigevjhh82clXhc9OYdBak/zG8mx4rCgOIdhe9+97vVrl276pD931PgvQQ+ZhftQ2tusdCXNBHmGZJmkxUFaQZZ6EuaCPMMSbPJdxQkSZIktbGiIEmSJKmNFQVJkiRJbawoSJIkSWpjRUGSJElSGysKkiRJktpYUeiBrx/zzYJjjz222rJlSx36obPPPrtavHhx9dhjj9Uhkkr/9Nd/Xf3qD/6g+sf7769DJEnSXGdFoQsqCbfffnu1fv36as+ePXWopIn4h+9+t/rVOeekSgKVBf6XJElz31BUFC688MJ6aGIOP/zwasOGDdU111xTh7TbuXNntW/fvuq0006rQz60bds27zRoqFEp+HWroj3661/XIVX1d3/0R1YWJEkaAPO+osBjQ2+//Xb938y6+eab6yFp+DRVEgKVBR9DkiRpbpvXFQUeHfra175W/zezuJvg40oaZv/4v/5XWyXhoAUL6qGq+vVXvlIPSZKkuWheVxSuu+66WblYf/HFF6trr722/k8aTof8239b/daRR1Yf+//+v+ojv/u7KYyKwsf/4i+q31qypDr00ktTmCRJmpumXFHgGXxe9j3ooIPS/1wk0xIQ/9NS0AMPPJDC+XWfeLQQxDiGCWtC60IrVqxI8eh4x6B81j9aI2JedPwf82V6hm+77bYUd9euXWNpRTqvvfZaSjeWh363ZWpCGps2bTqg1SPWd/Xq1dVbb72V/l+1alVKP5YzliO6fL3y8QxLg+yf/af/VB32t3+b+rmPXnJJddjf/E31sf/23+oQzVfkh3n5QH4XeTt9yosmTJfnh8Qlv5ckzbDRKdi+ffvounXrRkmGjv+XL18+unnz5tE1a9aMhb/wwgujIyMjKe7GjRtHFy1alMIZzu3duzdNT8c02L179+jSpUvH0g9bt25N8QiPtGNZiA+mjfE55sMyMD3DYJmb4iLG0Q+kHeF0/J8jnTJ8z549Y8vMMsa8c2yTfLk0v7Dvh9E7v/u7o/ta6/72kUfWIZrvduzYcUA5QP5Nvki+Gflj5NU58nHyZ6YH+WbEZ9ywGdY8Q9Lc0JccKC8IcpG50yezD3EBXxYSXCQTnscFlYaYR1M6FCoRTtwYjvHMP0clg/D8wh8xj1JTRSHEOo6nogCWLSpKTZUBKglRQGr+aTq+hoEVheFEHscxT1eWD/EDUPwoBPK+prikE/HLcfMd6yxJs6Wv7yiUzZCeccYZY/1jjjkmDSOaEm1dNKc+eOTnxhtvrFoXygfExbJly6o1a9ak4VtuuSX1cytXrhybhrjl9KXf+Z3fSf0TTjgh9WcSyxbNrfKNhhy35Xlk6ZxzzqlDJGlw0cR0KMsHHk3Fe++9l/q4/vrrU//MM89M/UA6V111VRqerQYqJGkYzZmXmZ966qnUzwuWHM/946GHHkr9qeBCvFVJSv1414FnYGfKpZdeWi1atKi66aabDngn4nvf+95YYShJw4S88Nlnn03DTeXAKaeckvr8wMT7YZKk6TdnKgovvfRSPdTs05/+dOrndyGmgkKJF5E/85nPVD/60Y+q73znO/WY6UchyF0F7h7QMhMo+Hj5+vzzz0//S9IweeWVV+qhZtwtDj/72c/qIQ0b7rzHS+55ZwMg6qXpuKFTd3OmonDUUUel/tNPP536nYyMjNRDk0dLG8cdd1z13HPPpTsZN9xwwwGF0EyIuwpUDqgk3HPPPdW6des63lGRpPnsyCOPrIf259HdNH0JX8NhwYIF6Tqg7E4++eQ6htSs6bihU3dzpqLAewbgV/amQuKNN95I/XjvYSr+5E/+JM3n1ltvnbUL87irAN674DGktWvXpv8ladjw/tbSpUvT8JNPPpn6uXjcyIJ9uPGj3s6dO9s6fvCTumk6bujU3ZypKFBI8Is6rr766tTPPfroo+kXeH6JnyweN6ISEu855Levy2de83cH+iH/XkLI7ypQUZrpuxqSNJd885vfTH0+WFnmwXG32ZeZJWnmTLmiEB9UQ34ngEz++eefT8P080w/v2jOh7/xjW+kVo/4QFr+8bNt27ali+k77rjjgDsADz74YOpTgDS93MYtSpAe7yN84QtfqJYsWVKdddZZKfzzn/98+jgb89q4cWO6aAf/R4tELMMjjzyShmN9AuOi8Hr55ZdTP3ziE59I/a9+9atp3k888UT6P5ffVbjkkktSX5Lmi/wjaWX58Oqrr6bhyMdBy0j8YMQdX545j3ydciLyaR87kqQZtL+V1MkZqb8VkHeExfcLyo7wpmny7xPQXjb/R5vZrYv39JGdvK1tlGnQNX3nIL7NQHqRBvOI5SCc7yog4tIPkXbesR7xbYW8I83AvGId8vRKxGMdNRw4HoaR31EYPp3Kh6a8ky7HtxLi45R0fLhtWL8vU24bSZpJB/GnlRFplnC34bDDDqs2bNhQh2g+o4WFYTzl3v30p6sP/t//q37ryCOrw/72b+tQSb0Ma54haW6YM+8oDCNuv9skqiRJkuYiKwozjGd2470OvqHAM7m9viQtDbyPfCT1Dqrf3ZEkSXOfjx7NIF7IW7VqVf1flV6e/slPfuK3E4bIsD5G8E9//dfV37cqxh+98srq0N///TpUUi8+eiRpNllRmEE8akRLHs8++2y1Zs2a6sYbb/RuwpCx0Jc0EeYZkmaTFQVpBlnoS5oI8wxJs8l3FCRJkiS1saIgSZIkqY0VBUmSJEltrChIkiRJamNFQZIkSVIbKwqSJEmS2gxNRYGvIfMNA7qZwPwWL16cvrwsSZIkDZqhqChs27atuv7666tdu3bVIZIkSZK6GZoPrj322GPVqlWrqpGRkWrnzp11qDSz/HiSpIkwz5A0m3xHQZIkSVIbKwqSJEmS2lhRkCRJktRmyhUFWvehZZ9oTWjLli2ptR+eqyTsxRdfTOE53hdgHHHojj322DRd7s0336zuvPPOsZaK+D+myePyovKKFSvG0mKYZRqPfBmiY9lCPp7hwPxZZsLpr1+/vq11o3zZS+OZXhoU5bkKzss4xjm2idOEcyE/f4mbn4MaHO+tWlW91dqHefdPf/mX9dgDxzOc+4c/+7Pq3X/5L8fG/+rii6vfvPFGPXa/9x95pPrlOecckAb/5/OQJPUZLzNP1tatW0fXrFnDW1ajIyMjoxs3bhxdunRpGiaMbtGiRaN79uyppxgd3b17dwpft25d+n/v3r1jaWzevDmFgbSXL18+ljbx6fifeYD4/L99+/b0P/NhHGHMJxfzJa1A/JgH07EsJdaJODGOeRI31ol0GZ+nS5zYBnk4xjO95i+Oifmm07kaxzrhcb4HziemoXvhhRdSGOdCxI9zWoPj/eefH33nhBNG97X239u/8zujH/zN39RjPvSrtWtTnHzcLy+6KIUxPX79x3+c0sjj0X9r4cLRv7vqqrH/GSbeP957bwqbr+ZjniFpcEw5B4oLcCoEXDAECn/CyouEqBQwXYg0yovlPO24sCbdGI70c1F5oJ/rNA/SinSaKgpcyOzYsaP+bzRNT+UhR9qdlr0MH+/0mp/K43W+4Bxh3TiX4sIfnF8RnuMcIDzO5cC0hDeN09zHxT4X9FzAN1UUuPj/h7/4i/q/0dG//+//PcUv47572mkpjagYUBloSpN4VhQkafr07R2FlStXVmvXrq3/q6ply5ZVt956axq+7bbbUh9Lly6tWhcN1ZFHHlmH9EbaxxxzTBom3RgmrdaFfBqeLNK65ppr0vDtt9+e+oFHIN56663qnHPOqUP2Y33yR6pOO+20sWUaj6lOL801CxcuTH3OVc7REMc151HgMaQbb7wxnbvlcc+0a9asScO33HJL6mtwfORf/avqo3U5wONEOR4RGn3nnerQSy6pQ1px/uf/rA4599zqt5YsqUP2O/hf/+vU/6cHH0z98OsNG+qh/Q694IJ6SJI0Hab1ZeYzzzyzHtp/0Y0bbrih2rdvX7pAeO2119IzypdkBcdEPPPMM6kDF94863/TTTel/yfi0ksvTZUXps2fpf7e975XXXXVVfV/+11xxRXpouf3fu/30vzigj8qRb1MdXpp0D311FOpf/jhh6d+afXq1an/0EMPpb4Gy0dbedxBrYrjP2zbdsB7Bv94zz3VR//Df6j/2++Dl16q/nH79rH3DqL7+5tvHhuPQ/7dv6s+csIJKe47Rx2VKiGkzbwYJ0maHtNaUeh0IUAFgZcWzzrrrOqwww6rvvnNb9ZjJo4KCC9DXn311dXpp59erVu3rh4zfiwndxW4gL/uuutSGMvIL//nn39++j9wd2HPnj3pw22M54KfFziJPx5TnV4adC/VF3+dfPrTn059zhMNHu4OcFdh9N13x+4AfPB//2+6yD/0j/4o/Z8j7qLR0Y5dWPijH1Uf++M/Tncl/u4//sfq3dZx8vff+EY9VsOCMj8aP8i7poZDpFzTcUOn7masedTjjz8+9WkdhUeG6F599dX0uNKS4rbzePGLPF9b/vrXv56+tkzlg4rHZMRdBS7euWi/5557UqWjqbLD3RDmt3v37nTBv2vXrvQYRaeWXUpTnV4aZEcddVTqP/3006nfCeeGBlPcVaByMFZJuOiitkeM8P7jj9dDvf2z666rFr78cvXP6ju9v/6v/7XtcSTNbwsWLEh5Q9mdfPLJdQypWdNxQ6ce6ncVJq3TS7vg5WDG8UJw4KVGWjbJdUqjW9rx0mPZmspEX2bOxbSkyXLmL2WG8kVkEJ/p8pe5O81vvNNrfmI/z0fdzi/C8/XmJeUIazrHaPGIceU5rMESrRLRqhEvLP/T//k/9ZgP0ToScXipuQmtJIEXlvOXoEF6pEs3n+XnjiTNtL7dUeDuQIlf5XHllVemPs/j83gP7yjkv56/UbSXPZ5f1p988snULx/Zef311+uh/SbyK31+V6F8KTM899xzbe28X3zxxfVQb1OdXhp03FHjbh14ZLD06KOPpvOQ81GDK7+r8JETT6wOPuOMesyHDq2/H8OjRNwZ4O4D6PMtBaYP/1A0NkF6pCtJmj59qyjwPDGPAsWFOY8YXXvttemCID4mxoU3FwBUFniekBeZ6XNhAB7B2bRp09jLjg/WLV7wiEJZITjllFNSn2lIPz7cRCUEf/7nf56W55133kn/R1pUaDpVHuJdBXR7wfrzn/98Wj+Q1re//e20Xvn7DE888UTqN81vPNNLg4QX/1Ee73nrXvmHEL/xjW+kx+04f/N8gw+1UVG/4447Or7jpMEQ7yqgU+tEH2vl27ykDF5gfvekk9LLzPQ/+NGPUmUjvP/YY+kDa1GZoBUlwmIekqRpUN9ZmLR45IDHi3ispnXBm/7n8aKmR2l41IBxMQ3Tg2GmjQ8tMb7sykcRiBvz4/sMPNJARxjziMcaeByiV1qBaZi+E+ZTphfzDvm46GI9xzO95i/293yTH8vRcX41nXf5o0k8mki8yA8473gMr+lxJA2meDyoFx5TiseQiM8jR/k3E3j0iG8wRBw6hvk423zHuSFJs+Ug/rQyoknjMRpeKG5dAKQXdAcddzR4IXqDL8hpGtDCwhRPOWlg/N3ll6fHh7hzoMkxz5A0m2as1aNBwOMPPPbgI0CSNDV85yBaO5IkDaahryjwrkA8O803FHjfofxarCSpNz6E9o/f/W4a5uVkvrrM15olSYNpyo8e8RIxLZcsXbo0fUl1kC6y47GpsGjRouonP/mJL1Fq2vgYgeYrXi7+5Xnn1f+1jvWFC9M3D5q+naDxM8+QNJumdEeBDCyaN6TVIyoLVBwGBR+Bo+UVrFmzpnr22WetJEjSJHxk5cqxFowO+exnqwV//ddWEiRpwE35joIkSZKk+ceXmSVJkiS1saIgSZIkqY0VBUmSJEltrChIkiRJamNFQZIkSVIbKwqSJEmS2lhRkCRJktTGioIkSZKkNlYUJEmSJLWxoiBJkiSpjRUFSZIkSW2sKEiSJElqY0VBkiRJUhsrCpIkSZLaWFGQJEmS1MaKgiRJkqQ2VhQkSZIktbGiIEmSJKmNFQVJkiRJbawoSJIkSWpjRUGSJElSGysKkiRJktpYUZAkSZLUxoqCJEmSpDZWFCRJkiS1saIgSZIkqY0VBUmSJEltrChIkiRJamNFQZIkSVIbKwqSJEmS2lhRkCRJktTGioIkSZKkQlX9/3nKJvDwZmH7AAAAAElFTkSuQmCC\" style=\"width: 666px;\"\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eThe feeding of spiders 14 days after treatment differed significantly between \u003cem\u003eAnyphaena\u003c/em\u003e and \u003cem\u003ePhilodromus\u003c/em\u003e spiders; the feeding rate was overall higher in \u003cem\u003ePhilodromus\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;154.703, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The feeding rate did not differ significantly between spiders 14 days after treatment and the control group in either \u003cem\u003eAnyphaena\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.789, p\u0026thinsp;=\u0026thinsp;0.095) or \u003cem\u003ePhilodromus\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0085, p\u0026thinsp;=\u0026thinsp;0.315).\u003c/p\u003e\n \u003cp\u003eThe rate of overkilling significantly decreased under fresh residue treatment in both species; in \u003cem\u003eAnyphaena\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;25.1767, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) and in \u003cem\u003ePhilodromus\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.741, p\u0026thinsp;=\u0026thinsp;0.016, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). The rate of overkilling by spiders 14 days after treatment was significantly higher in \u003cem\u003ePhilodromus\u003c/em\u003e spiders than in \u003cem\u003eAnyphaena\u003c/em\u003e (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;75.314, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The overkilling rate of \u003cem\u003eAnyphaena\u003c/em\u003e did not differ significantly between spiders 14 days after treatment and control (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.516, p\u0026thinsp;=\u0026thinsp;0.472), but it was significantly lower in \u003cem\u003ePhilodromus\u003c/em\u003e spiders 14 days after treatment than in the control (GLM-p, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.899, p\u0026thinsp;=\u0026thinsp;0.015).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMortality\u003c/h3\u003e\n\u003cp\u003eThiacloprid treatment did not cause significant mortality in \u003cem\u003ePhilodromus\u003c/em\u003e spiders (GLM-b, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.88711, p\u0026thinsp;=\u0026thinsp;0.1695); in contrast, mortality in \u003cem\u003eAnyphaena\u003c/em\u003e spiders was significantly higher in treated individuals (GLM-b, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;36.238, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). During the whole experiment, 30 \u003cem\u003eAnyphaena\u003c/em\u003e spiders died in the treated group, corresponding to 20% of the total number, whereas only two individuals died in the control group, corresponding to about 1% of the total number. Of the total mortality in treated \u003cem\u003eAnyphaena\u003c/em\u003e spiders, 7% occurred immediately after treatment, 13% within one week, 7% within two weeks, 20% within three weeks, and 53% within four weeks (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Both individuals (100%) that died in the control group died within one week.\u003c/p\u003e\n\u003ch3\u003eParalysis\u003c/h3\u003e\n\u003cp\u003eFresh Thiacloprid treatment induced paralysis in 13% of \u003cem\u003ePhilodromus\u003c/em\u003e spiders, which was significantly higher than in the control group (GLM-b, \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;40.203, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA), where no individual was paralysed after the experiment. Paralysis occurred during the experiment or shortly after the end. One week after treatment, 19% of the paralyzed spiders began to move again, and after two weeks, 100% of the paralyzed spiders returned to normal (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). In \u003cem\u003eAnyphaena\u003c/em\u003e spiders, paralysis did not appear, either after fresh or 14-day-old residue treatment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003ePredation\u003c/h2\u003e\u003cp\u003eThe ability of predators to suppress pests is determined by the rate of predation on pests, which varies depending on prey density and can be expressed as a functional response [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. A high increase in the number of prey is typical when the pest population is overpopulated, and therefore a positive functional response of the predator is a strong predictor of its effectiveness in crop pest suppression. Both studied species, \u003cem\u003eAnyphaena\u003c/em\u003e and \u003cem\u003ePhilodromus\u003c/em\u003e, are dominant active predators on fruit trees [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our results confirm a strong functional response already observed by Řez\u0026aacute;č et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] in \u003cem\u003ePhilodromus\u003c/em\u003e and also revealed it in \u003cem\u003eAnyphaena\u003c/em\u003e spiders for the first time. The absolute number of prey caught under laboratory conditions does not fully reflect the situation in nature because a saturated predator may avoid prey, or prey may escape. Predation measured in the laboratory differs from predation in nature, but laboratory data can approximate our knowledge of the situation in nature and indicate the predatory potential of the studied species. Such ecotoxicological laboratory data are valuable for assessing the effects of agrochemical treatments on predators.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffect of thiacloprid treatment\u003c/h3\u003e\n\u003cp\u003eWe report for the first time a species-specific effect of neonicotinoid treatment with the active ingredient thiacloprid on the foraging ecology of two top spider predators co-occurring in tree crowns in Central Europe, respectively \u003cem\u003eAnyphaena\u003c/em\u003e and \u003cem\u003ePhilodromus\u003c/em\u003e spiders. Both species belong to the same foraging guild of active hunters [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and represent some of the most important natural predators in fruit orchards, where they share the prey and habitat [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] even during the winter [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]and also engage in intraguild predation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn both hunter spiders, thiacloprid treatment significantly decreased predation activity at all tested prey densities. The functional response of treated spiders correlated with the functional response of the control group, but the predation rate was significantly lower in thiacloprid treatment groups. The predation rate of treated spiders decreased in both species at all prey densities: by more than half for \u003cem\u003eAnyphaena\u003c/em\u003e and by more than a quarter for \u003cem\u003ePhilodromus\u003c/em\u003e. The greater negative effect of thiacloprid treatment on the predation rate of \u003cem\u003eAnyphaena\u003c/em\u003e may be related to the physiological adaptation of nocturnal \u003cem\u003eAnyphaena\u003c/em\u003e to lower temperatures than those experienced by diurnal \u003cem\u003ePhilodromus\u003c/em\u003e. At temperatures below freezing, the lower limit for predatory activity was set at \u0026minus;\u0026thinsp;3.73\u0026deg;C for \u003cem\u003eAnyphaena\u003c/em\u003e and \u0026minus;\u0026thinsp;1.2\u0026deg;C for \u003cem\u003ePhilodromus\u003c/em\u003e. At increasing temperatures, the predatory activity of \u003cem\u003eAnyphaena\u003c/em\u003e was highest at 15\u0026deg;C and then declined. In contrast, the predation rate of \u003cem\u003ePhilodromus\u003c/em\u003e increased monotonically with temperature and reached a maximum at 30\u0026deg;C, which was the highest temperature tested [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The link between differences in the effect of thiacloprid on the two spider hunters studied and the differences in their temperature adaptations is only a hypothesis and requires further research. However, it has already been documented that higher temperatures have a synergistic effect on the toxicity of thiacloprid, as observed in crayfish [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA decrease in predation was also documented in other spiders, e.g. in \u003cem\u003ePardosa pseudoannulata\u003c/em\u003e (Araneae: Lycosidae) Widiarta \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] after imidacloprid exposure, and in \u003cem\u003ePardosa agrestis\u003c/em\u003e (Araneae: Lycosidae) after thiacloprid exposure [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition, a reduction in predation after neonicotinoid treatment with thiacloprid was found by [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], who also observed that dorsal application had a higher impact on spiders than tarsal application, the latter producing only moderate effects. In the present study, we found a significantly negative effect of tarsal exposure to thiacloprid on the predation rate of both \u003cem\u003eAnyphaena\u003c/em\u003e and \u003cem\u003ePhilodromus\u003c/em\u003e spiders. Řez\u0026aacute;č et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] also found that neonicotinoid (acetamiprid) reduced the predation due to prolonged prey handling. In contrast, no effect of neonicotinoids on feeding was observed in \u003cem\u003ePhilodromus cespitum\u003c/em\u003e (Araneae: Philodromidae) after exposure to acetamiprid [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePredation, feeding, overkilling\u003c/h2\u003e\u003cp\u003eThe predation rate expresses the number of prey caught, which includes two different components: feeding, i.e., prey caught and completely or partially consumed, and so-called overkilling, i.e., prey caught but not consumed by the predator [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Feeding is important for the predator itself, as it provides a source of nutrients [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Prey overkilling is not directly beneficial to the predator, but it is important in terms of protecting crops from pests. The ground dwelling spider \u003cem\u003ePardosa agrestis\u003c/em\u003e fed less on thiacloprid-treated prey than on control prey, but the rate of overkilling increased, especially among female spiders, from just 2.6% of control flies to 44.7% of thiacloprid-treated flies [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Similar compensation of low feeding by overkilling was also found in our results, but overkilling also decreased in comparison with control. At first sight, it seems that treatment with thiacloprid does not have a significant negative effect on pest population control, because low feeding is complemented by overkilling, and hypothetically the pressure on the pest population remains high. However, conditions in the field may differ from those in the laboratory (as already mentioned), and, together with other sublethal effects of thiacloprid treatment on the life history of predators, may reduce their long-term impact on pest populations in orchards. Due to all the above-mentioned factors, it is important to evaluate the obtained knowledge correctly and to correctly implement it when assessing the impact of thiacloprid treatment on beneficial arthropods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eParalysis and Mortality\u003c/h2\u003e\u003cp\u003eThe toxicity of neonicotinoids to spiders is lower than to insects, most likely because the structure of acetylcholine receptors, which mediate the action of neonicotinoids in arthropods, differs between insects and spiders. In spiders, acetylcholine receptors are present [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], but their sensitivity to neonicotinoids is lower than that of insect receptors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We found significant mortality under thiacloprid treatment only in \u003cem\u003eAnyphaena\u003c/em\u003e spiders, not in \u003cem\u003ePhilodromus\u003c/em\u003e. This indicates that nocturnal \u003cem\u003eAnyphaena\u003c/em\u003e seems to be more sensitive to thiacloprid treatment than diurnal \u003cem\u003ePhilodromus\u003c/em\u003e. On the other hand, \u003cem\u003ePhilodromus\u003c/em\u003e suffered significant temporary paralysis caused by tarsal contact with thiacloprid. Although we observed the recovery of all affected \u003cem\u003ePhilodromus\u003c/em\u003e individuals within 14 days under laboratory conditions, such paralysis could be fatal in natural conditions due to the increased risk of dehydration or predation by other predators. Paralysis was observed in spiders of the family Linyphiidae under acetamiprid and thiacloprid treatment, and in \u003cem\u003ePhilodromus cespitum\u003c/em\u003e under treatment with acetamiprid [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. S\u0026yacute;kora [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] reported that tarsal contact with neonicotinoids (actemapirid, imidacloprid, thiacloprid and thiamethoxam) did not cause significant mortality in \u003cem\u003ePhylloneta impresa\u003c/em\u003e (Koch 1881) and that no paralysis was observed. However, Řez\u0026aacute;č et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] found that the dorsal application of thiacloprid to spiders of the family Linyphiidae caused paralysis and mortality, which was up to 57% in males and 29% in females.\u003c/p\u003e\u003cp\u003eSeveral studies have reported the paralysing effects of neonicotinoids on other non-target invertebrates, particularly honeybees. Following neonicotinoid application, bees are often found lying motionless around the hive [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Bumblebees showed significant growth inhibition following imidacloprid application, due to disorientation and the inability to search for food [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The paralyzing effects of neonicotinoids have also been found in predatory insects. For example, in the beetle \u003cem\u003eHarmonia axyridis\u003c/em\u003e (Pallas 1773), 72% of larvae treated with thiamethoxam or clothianidin developed neurotoxic symptoms, including paralysis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eOther sub-lethal effects of neonicotinoids\u003c/h2\u003e\u003cp\u003eThere may be other sublethal effects of neonicotinoids which should be considered. For example, Korenko et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] found that after treatment with thiacloprid, male \u003cem\u003ePardosa agrestis\u003c/em\u003e (Westring 1861) are unable to complete the mating dance due to the disruption of chemical communication, which may reduce the chances of reproduction. Neonicotinoids also have a significant effect on locomotion. According to Řez\u0026aacute;č et al. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], they reduce the speed of \u003cem\u003ePardosa lugubris\u003c/em\u003e (Walckenaer 1802), which may affect predation success or escape.\u003c/p\u003e\u003cp\u003eFurther research is needed to better understand the long-term effects of neonicotinoids on spider populations and other non-target organisms. Research should focus on their persistence in soil and plants, their accumulation after repeated applications, and their interactions with other pesticides. In addition, research should go beyond predation to include sub-lethal effects on locomotion, food intake, and reproduction, as these factors are key to assessing the wider ecological consequences of pesticide exposure. A comprehensive examination of these parameters could provide crucial insights into the risks associated with neonicotinoid use. Regulatory frameworks should ensure that the approval of neonicotinoid-based pesticides is conditional on thorough research into their impacts on a wider range of organisms, including both lethal and sub-lethal effects. Prioritizing such studies over commercial considerations could help to minimise unintended ecological disturbances while maintaining effective pest management strategies.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe study was supported by Czech University of Life Sciences Prague (Student Grant Competition 2025 provided by Faculty of Agrobiology, Food and Natural Resources).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAŠ, FS and SK and conceived, designed the research and conducted laboratory experiments; AŠ, SK, MK and EL analyzed the data; all authors wrote the manuscript; all authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe study was supported by the Student Grant Competition 2025 provided by Faculty of Agrobiology, Food and Natural Resources.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlkassab, A. 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Rep.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 14745 (2022).\u003c/span\u003e\u003c/li\u003e\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8060416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8060416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeonicotinoids are nicotine-based synthetic insecticides used in agriculture to control plant pests. They are neurotoxic substances that attack the nervous system of insects and can cause paralysis or death. These selective insecticides should have a negligible effect on non-target organisms, including spiders, which are one of the most abundant and diverse natural predators that contribute to the control of pests. Current studies show that selective insecticides such as neonicotinoids have negative effects on non-target invertebrates. They can have both lethal effects resulting in mortality, and sublethal effects involving various aspects of their lives, e.g. breeding, movement, hunting, the ability to defend against predators, and predatory activity.\u003c/p\u003e\u003cp\u003eWe studied the species-specific responses to neonicotinoid treatments with the active ingredient thiacloprid of two top spider predators coexisting in tree crowns in Europe\u0026mdash;respectively, spiders of the genus \u003cem\u003ePhilodromus\u003c/em\u003e (\u003cem\u003eaureoles\u003c/em\u003e group, Philodromidae) and species \u003cem\u003eAnyphaena accentuata\u003c/em\u003e (Walckenaer) (Anyphaenidae). Spiders were exposed to field-realistic concentrations of the tested substance, while the control group was treated with distilled water. We compared the species-specific responses of three components of spider predatory activity: functional response, prey consumption, and overkilling. Further, we observed the long-term survival and paralysis of treated individuals compared to control, and the effect of insecticide residues on predation activity 14 days after insecticide application.\u003c/p\u003e\u003cp\u003eWe found that an hour-long tarsal contact with the active ingredient thiacloprid reduced predatory activity in both \u003cem\u003eAnyphaena\u003c/em\u003e and \u003cem\u003ePhilodromus\u003c/em\u003e spiders, but the effect was species-specific in many aspects. Feeding was affected by fresh residua in \u003cem\u003eAnyphaena\u003c/em\u003e, but not in \u003cem\u003ePhilodromus\u003c/em\u003e. Furthermore, 14 days after treatment, there were differences in the rates of predation, feeding, and overkilling between species. The treatment caused paralysis, but no mortality in \u003cem\u003ePhilodromus\u003c/em\u003e. In contrast, the treatment caused significant mortality, but no paralysis in \u003cem\u003eAnyphaena\u003c/em\u003e. Further, after 14 days, we found that the insecticide had no significant effect on predation activity. Overall, the study revealed a species-specific response to the given pesticide of top pest predators sharing the same ecological niche in orchards.\u003c/p\u003e","manuscriptTitle":"Interspecific foraging response to the thiacloprid treatment of co-existing top spider predators","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 07:20:10","doi":"10.21203/rs.3.rs-8060416/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-23T16:04:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-22T07:38:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124438769430954741084551109889446789086","date":"2026-03-21T18:24:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T13:23:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208640229713184282657454714823005061776","date":"2025-12-01T20:11:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90728401231809119595318655333595249558","date":"2025-11-24T13:45:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T06:54:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-12T09:45:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-10T01:21:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-10T01:19:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-07T23:03:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cb7597e-54af-401a-9dce-eb788f0290f9","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58533634,"name":"Biological sciences/Ecology"},{"id":58533635,"name":"Earth and environmental sciences/Ecology"},{"id":58533636,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-05-04T17:39:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 07:20:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8060416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8060416","identity":"rs-8060416","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}