Repellent and acaricidal effects of the chlorfenapyr and acequinocyl on the predatory mites, Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae)

Repellent and acaricidal effects of the chlorfenapyr and acequinocyl on the predatory mites, Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae) | 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 Research Article Repellent and acaricidal effects of the chlorfenapyr and acequinocyl on the predatory mites, Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae) Navid Sehat-Niaki, Azadeh Zahedi Golpaygani, Ehssan Torabi, Behnam Amiri-Besheli, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4604689/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jan, 2025 Read the published version in Experimental and Applied Acarology → Version 1 posted You are reading this latest preprint version Abstract The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a major pest of various plants with a worldwide distribution. Extensive use of chemical pesticides has led to the development of resistance in this pest, making biological control agents a viable alternative for its management. The predatory mites, Neoseiulus californicus McGregor and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) are the most important predators of the two-spotted spider mites. In this study, the toxicity of two acaricides, chlorfenapyr and acequinocyl, on these predators was evaluated, and the walking behavior of predatory mites after exposure to residues of the pesticides was assessed using a video tracking system. While the LC 50 of both acaricides was estimated to be higher than the field concentration, chlorfenapyr was found to be five times more toxic than acequinocyl. In the behavioral assay, both acaricides significantly affected the distance and speed of walking, resting time, and frequency of stops of both predatory mites. In the escape assay, both compounds had an irritable effect on both predatory mites, as the mites avoided areas contaminated with pesticide residues and their presence in the untreated area was significantly longer than in the contaminated area ( P < 0.05). However, the study found no correlation between toxicity and repellency. According to the results of this study, N. californicus and P. persimilis possess the ability to detect the presence of pesticide residues in their environment and try to avoid them. Moreover, both compounds are at low risk to these mites, but acequinocyl is much safer and is a suitable option for using in integrated pest management. Predatory mites Bioassay Behavior Biological Control Repellency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a major pest of vegetable, orchard, ornamental, and field crops worldwide (Migeon and Dorkeld 2010). It rapidly forms colonies and has a short life cycle (Dermauw et al. 2013). It is a polyphagous species and damages the leaf surface by feeding on plant sap and destroying plant cells (Huffaker et al. 1970 ). Chemical acaricides have widely been used to control this pest (Van Leeuwen et al. 2010 ). However, one of the major challenges in controlling the process is its rapid development of resistance to these chemicals (Van Pottelberge et al. 2008 ). Therefore, researchers are trying to use alternative ways to manage this pest, including the use of biological control agents. The predatory mite, Neoseiulus californicus McGregor (Acari: Phytoseiidae) is known as an important component of biological control in integrated pest management programs (Canlas et al. 2006 ). This generalist predator (McMurtry et al. 2013 ) can feed on various sources, including the two-spotted spider mite, but it has a higher growth rate when feeding on this particular prey (Rhodes & Liburd 2009 ). Its rapid population growth, long-term establishment, and persistent populations in plant systems are the predator's valuable characteristics (Walzer et al. 2007 ). The predatory mite Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) is a key component in integrated pest management (IPM) programs worldwide, particularly for controlling tetranychid mites in greenhouses (Lee et al. 2002 ). This predatory mite possesses a high reproductive capacity, a short developmental period, and the ability to feed on all stages of the two-spotted spider mite, making it an important factor in IPM success (Moghadasi et al. 2016 ). It feeds exclusively on spider mites, primarily of the genus Tetranychus . Since most pesticides have had a broad spectrum of activity, they also eliminate non-target and beneficial species. The most important biological control agents of insect and mite pests are considered arthropod predators and parasitoids, which may be directly or indirectly affected by various pesticides through contact with pesticide residues or feeding on infested hosts (Croft 1990 ). Studies have shown that even selective pesticides can have negative effects on natural enemies (Rezac et al. 2010). Pesticides can cause mortality (lethal effects) or alter other biological traits of organisms (sublethal effects) (Rezac et al. 2010; Papachristos & Milonas 2010). Therefore, before incorporating any pesticide into a system, it is crucial to gather information on its lethal and sublethal effects on key predators in the system. Chlorfenapyr, marketed under the trade name Conqueror, is a systemic and contact insecticide-acaricide (Zhao et al. 2017 ) that is effective against various important pests, including tarsonemid and tetranychid mites, and whiteflies (Raghavendra et al. 2011 ). This pesticide belongs to the pyrrole chemical group and acts as an oxidative phosphorylation inhibitor by disrupting the proton concentration gradient (Dekeyser 2005 ), making it recommended against pests resistant to organophosphate, carbamate, and pyrethroid compounds (Sheppard & Joyce 1998 ; Zhao et al. 2017 ). Acequinocyl, marketed under the brand name Kanemite, is a naphthoquinone analog compound formulated as a suspension concentrate (SC). The U.S. Environmental Protection Agency (EPA) classifies it as a reduced-risk pesticide. Acequinocyl inhibits mitochondrial respiration and is used to control phytophagous mites (Yorulmaz Salman et al. 2015 ). Arthropods in agricultural fields are constantly exposed to sublethal doses of pesticide residues after application. To reduce contact with these compounds, predators may exhibit avoidance behaviors, which can alter their movement patterns and result in either irritability or repellency (Cordeiro et al. 2010 ). Irritability occurs when an insect is stimulated to move away from the pesticide after direct physical contact with the chemical residue, while repellency occurs from a distance, and the insect avoids the pesticide-treated area without direct physical contact (Roberts et al. 1997 ). Most laboratory bioassays in the past have focused on acute mortality, with sublethal effects being added to these studies more recently (Desneux et al. 2007 ). Sublethal effects typically involve estimates of reduced reproductive capacity, but these experiments are generally designed to expose insects to pesticides in a "worst-case" scenario, with no option to escape sprays or residues, likely overestimating the toxicity of pesticides (Gerson et al. 2003 ; Hassan 1989 ). This study aimed to determine the repellency and irritability properties of two acaricides for the predatory mites N. californicus and P. persimilis . The results of this study, along with other research on the sublethal effects of pesticides, will allow us to better determine which pesticides are suitable for integrated pest management programs. Materials and Methods Rearing of two-spotted spider mite ( T. urticae ) Common bean plants [[ Phaseolus vulgaris L. (Fabaceae) var. Red Alamouti] were grown in plastic pots in a greenhouse (soil: perlite; 50:50%) under controlled conditions (25 ± 5°C, 16L:8D photo-period, 65 ± 5% RH) at the Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran. Plants were daily irrigated with tap water and a fertilizer solution of NPK (20×20×20). Spider mites, Tetranychus urticae (green form; food source for the predatory mites) were reared on bean plants. Fresh bean plants were added to the rearing system regularly. Rearing of the predatory mites The predatory mites, Phytoseiulus persimilis , and Neoseiulus californicus species clarified before experiments, were reared on masses of detached bean leaves, infested with T. urticae , placed upside down on a plastic sheet on a water-saturated sponge. The plastic sheet was surrounded by napkin tapes which were put into the water from the otherwise that the predatory mites could drink water. Fresh T. urticae- infested leaves and fresh corn pollen (Zea mays) were added to the rearing system and the old predator-free leaves were removed regularly (Overmeer 1985 ). The cultures were kept in separate growth chambers under controlled conditions (25 ± 1°C, 16L:8D photoperiod, 65 ± 5% RH) in the Acarology laboratory at Jalal Afshar Zoological Museum, Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran. Toxicity bioassays of pesticides on predatory mites Bioassays were conducted using the leaf disc dipping method (Helle & Overmeers 1985 ). Preliminary tests were performed to determine the range of effective concentrations that caused 10–90% mortality in the predatory mites. Intermediate concentrations were determined using logarithmic intervals. Five concentrations determined in the preliminary tests were used in these experiments. For P. persimilis , the tested concentrations were 1500–6000 ppm for acequinocyl and 300–800 ppm for chlorfenapyr. Similarly, N. californicus was exposed to a range of 3000–9000 ppm for acequinocyl and 700–2000 ppm for chlorfenapyr. For each concentration, a 4 cm diameter leaf disc was excised from a bean plant. Each leaf disc was immersed in the toxic solution for 15 sec (the control leaf disc was immersed in distilled water). After drying the leaf for 2–3 h, it was placed on moistened cotton inside a 9 cm diameter Petri dish. The leaf disc was surrounded by a strip of moistened cotton. For each concentration, 20 same-age-matched adult mites (male and female) were randomly selected and placed on the leaf discs. Petri dishes were incubated in a growth chamber under controlled conditions (25 ± 5°C, 16L:8D photo-period, 70 ± 5% RH). After 24 h, mortality was assessed. Mites that were unable to move after stimulation with a brush were considered dead. Bioassays were performed with four replicates. Based on the results, three concentrations were selected for subsequent experiments, including the field-recommended rate, LC 30 and LC 50 . Assessment of predatory mites behavior after exposure to pesticides without escape chance The experiment was conducted following the method of Lima et al. ( 2012 ). Black polyvinyl chloride (PVC) discs (3 cm diameter, 1 mm thickness) were fully immersed in 40 mL of different concentrations of each pesticide and distilled water (Control) for 5 sec. Then the discs were air-dried under a fume hood for 20 min. A hole was punched in the center of the discs and they were attached to the center of Petri dishes (9 cm diameter, 1 cm height). Distilled water was added to each Petri dish up to half its height so that the disc would float on it. A single individual same-age-matched female was placed on each disc and the Petri dishes were transferred to a tracking system via a video camera connected to a computer. The assessment was performed for 10 min at 25–27°C. The parameters recorded for each mite were: distance traveled, average walking speed, resting time, and number of stops. Twenty replicates were performed for each pesticide, with each mite representing a replicate. The whole experiment was conducted in a completely randomized design. Assessment of predatory mites behavior after exposure to acaricides with an escape chance Similar to the previous step, PVC discs were attached to Petri dishes with a needle. A diameter was drawn inside the disc with a marker to divide it into two equal hemispheres (Fig. 1). One hemisphere was immersed in distilled water for 5 sec and then placed in a laminar flow hood for 20 min to dry. The other hemisphere was then immersed in 40 ml of the acaricide solution using the same method. This process was repeated for all treatments. A control treatment was also performed separately by immersing both hemispheres in distilled water. Finally, the dishes were placed in the tracking system to evaluate the time spent in each hemisphere of each disc. Acaricides were considered repellent if mites remained on the untreated hemisphere for the entire duration, and irritant if they entered the treated hemisphere but spent less than 50% of the time there. Data analysis Sublethal concentrations were determined using the probit method and SAS 9.4 software. Differences in the numbers of female predatory mites choosing the treated and untreated areas in the choice test were compared using independent F-tests and T-tests in SPSS 25. Total distance traveled, walking time, and walking speed in the observational tracking experiments were estimated following the methods described by Lima et al. ( 2012 ) and were analyzed using a multivariate analysis of variance (MANOVA) with pesticides as the independent variable. Mean comparisons were then performed using Tukey's HSD test. The results of repellency and irritability of acaricides were analyzed using the non-parametric Wilcoxon test (sum of ranks) and the UNIVARIATE procedure. In this study, the type of insecticide treatment was considered as the independent variable. Data were visualized with graphs created using Excel 2019 software. Results Bioassays experiments Table 1 presents the results of bioassays conducted with two acaricides, chlorfenapyr and acequinocyl, on the predatory mites, N. californicus and P. persimilis . Chlorfenapyr was about five times more toxic than acequinocyl for both species. The LC 50 values for N. californicus were significantly higher than those for P. persimilis , indicating that N. californicus can tolerate higher levels of pesticides than P. persimilis . Three concentrations (the field recommended rate, LC 30 , and LC 50 ) were used in the behavioral assay to investigate the relationship between concentration and behavioral changes. The field rate of chlorfenapyr was 0.4 ml/L and that of acequinocyl was 1 ml/L. Table 1 Bioassay of chlorfenapyr and acequinocyl acaricides on the predatory mites, Neoseiulus californicus and Phytoseiulus persimilis , and determination of sublethal concentrations. Mite Acaricide N * Hetero-geneity df P- value X 2 Slope ± SE LC 30 ** LC 50 N. californicus chlorfenapyr 480 0.382 4 0.67 1.52 5.54 ± 0.49 0.953 (0.882–1.017) 1.186 (1.118–1.258) acequinocyl 480 0.575 4 0.14 5.45 5.03 ± 0.47 4.711 (4.363–5.033) 5.987 (5.614–6.424) P. persimilis chlorfenapyr 480 0.532 4 0.15 5.18 5.42 ± 0.51 0.429 (0.398–0.456) 0.536 (0.505–0.571) acequinocyl 480 0.447 4 0.42 2.81 4.12 ± 0.37 2.558 (2.323–2.777) 3.428 (3.169–3.727) * Twenty individuals per replicate, four replicates per concentration, six concentrations per assay. ** Concentrations are reported in ml/L with 95% confidence intervals. Behavioral response of the predatory mites to acaricides without chance of escape The movement patterns of N. californicus and P. persimilis predatory mites on discs fully treated with acaricide residues are shown in Fig. 2 . In acaricide treatments, the mites walked shorter distances and at slower speeds, spent less time resting, and stopped more frequently. The longest distance traveled was observed when the discs were treated with distilled water, and the shortest distance traveled was observed for the LC 50 treatment of chlorfenapyr for both predatory mites. For both species the highest walking speed was observed in the control treatment and the field concentration of acequinocyl. This was determined by calculating the average speed, which is defined as the distance traveled in 10 minutes divided by the actual travel time excluding stops. The shortest resting time was observed in the control treatment and the longest resting time was observed in the LC 50 treatment of chlorfenapyr. Furthermore, the fewest stops were observed in both the control treatment and the field concentration of acequinocyl. It can be inferred that the field concentration of acequinocyl did not affect these mites, as it did not differ significantly from the control in all tests (Fig. 3 ). Moreover, the negative effect of chlorfenapyr was more pronounced than acequinocyl for all parameters. Behavioral responses of the predatory mites to acaricides with a chance of escape The movement patterns of N. californicus and P. persimilis mites on PVC discs are shown in Fig. 4 . One hemisphere of each disk was immersed in distilled water, while the other was immersed in an acaricide solution, as in the previous experiment, the longest distance traveled was observed in the control treatment. The average distance and percentage of time spent in each hemisphere are shown in Figs. 5 and 6 . In the control treatment, the mites showed no preference for either hemisphere, however, in the treated groups, it showed a lower tendency to stay in the hemisphere containing the acaricide. None of the acaricides had repellent properties since the mites also moved in the area treated with the acaricides, but they had irritant properties and the time spent by the mite in the contaminated area was significantly less than in the untreated area. This difference was observed in the treatment with the lowest concentration of acaricides and continued up to the highest concentration. These findings suggest that both predatory mites, N. californicus and P. persimilis can detect and avoid acaricide residues in their environment. Discussion Natural enemies may exhibit behavioral responses, such as avoidance of treated areas when exposed to certain pesticides. These behaviors, known as repellency and irritability effects, can influence the actual impact of pesticides in orchards and make their prediction more challenging (Beers and Schmidt-Jeffris 2015 ). Repellency can negatively affect pest population control since predatory mites will be less effective in controlling pest populations. However, it can potentially reduce the harmful effects of pesticides on natural enemies since they avoid contact with toxic substances by fleeing the treated area. This can help preserve natural enemy refuges and promote continued biological pest control (Hislop et al. 1981 ; Gerson et al. 2003 ). Of course, there is also the possibility that they may completely leave the treated area and not return. On the other hand, repellency can increase the dispersal of natural enemies in the agroecosystem, temporarily reducing their population in the treated area. This dispersal can reduce the probability of resistance evolution in natural enemies to pesticides as they migrate to areas with less selection pressure (Croft and Brown 1975 ). Therefore, it is difficult to determine whether repellency is beneficial or detrimental to agriculture. Bioassay results demonstrated that chlorfenapyr was about five times more toxic than acequinocyl to both predatory mites, N. californicus and P. persimilis . Acequinocyl acaricide exhibited low toxicity to this mite since the LC 50 value was estimated to be several-fold higher than the field concentration. The field concentration of acequinocyl is 2.5 times that of chlorfenapyr. This could mean that chlorfenapyr may be effective for pest control at lower concentrations, but it also poses a greater risk to natural enemies such as predatory mites. As shown in Fig. 2 , in the no-escape chance experiment, the mite tended to move across the whole area of the PVC disc. The distance traveled was significantly higher in the control treatment than in the pesticide treatments. In the no-escape chance experiment, the decrease in the distance and walking speed of the predatory mites, and conversely, the increase in resting time and the number of stops, could be due to the pesticide's effect on the motor system or substance's irritable effect that makes the mite lazier and more tired. In any case, these factors in turn reduce its efficiency in prey tracking and biological control. Although the LC 50 concentration of both acaricides did not cause mortality in the first 10 min, it showed that the mite tends to weaken significantly. If this experiment were evaluated for a longer period, the difference in these parameters would likely be more distinct than that of the control. Phytoseiulus persimilis was significantly more motile and faster than N. californicus , exhibiting a shorter resting time as well. Possible reasons for this phenomenon include: P. persimilis is smaller than N. californicus , which may give it an advantage in terms of agility and speed, also P. persimilis may have stronger muscles and longer legs allowing it to move faster. Finally, P. persimilis may have a faster metabolism, providing it with the energy needed to move more quickly. Lima et al. ( 2012 ) investigated the walking behavior of the predatory mite, Neoseiulus baraki exposed to the acaricides abamectin, fenpyroximate, azadarachtin, carbosulfan, chlorfenapyr, and chlorpyrifos. All of these compounds caused a significant reduction in the distance and speed of mite walking. The highest distance traveled was observed in the chlorfenapyr treatment and the lowest was for azadarachtin and carbosulfan. Resting time and stopping frequency was also significantly higher in the azadarachtin and carbosulfan treatments than in the other pesticides. Chlorfenapyr and chlorpyrifos had the least effect, but in our study, chlorfenapyr caused significant changes in these parameters. This is probably because they only tested one concentration of each pesticide, while we also tested higher concentrations. França et al. ( 2018 ) analyzed the walking behavior and oviposition rate of the mite Steneotarsonemus concavuscutum when exposed to products impregnated with the acaricides abamectin, azadarachtin, spiromesifen, fenproximate, and hexythiazox. They found that abamectin had the highest toxicity to this mite (LC 50 1.1 mg/L). The total distance traveled and walking speed was significantly lower when S. concavuscutum was exposed to abamectin compared to the other acaricides. The number of eggs laid on the treated area was also significantly lower for spiromesifen and abamectin compared to the other acaricides. In an experiment where escape from the treated area was possible, both predatory mites showed a preference for moving in the untreated area (Fig. 3 ). This suggests that the compounds had an irritable effect on the mites, as they did not avoid them completely but only spent less time on them. One possible reason for this is that the chemical residues are toxic to predatory mites and can harm them. Consequently, the predatory mites attempt to avoid the contaminated area to prevent exposure to the poison. Another possible reason is that the chemical residues may have a specific odor or scent that the predatory mites can sense. This odor or scent can be a warning signal for the predatory mites and keep them away from the contaminated area. Despite the advantages of P. persimilis in terms of speed and resting time, N. californicus showed a slightly greater tendency to stay in the untreated area, indicating a more cautious behavior. This could be attributed to N. californicus ' potentially greater caution to the chemicals in the pesticides, leading it to perceive the negative effects of the pesticides more quickly than P. persimilis and making it more likely to leave the contaminated area. According to Haynes ( 1988 ) and Soderlund and Bloomquist ( 1989 ), there is a relationship between repellent behavior and sensory perception in arthropods when they first come into contact with the treated area. Therefore, it is likely that predatory mites have mechanisms that allow it to minimize contact with acaricides. In the first experiment (with the possibility of escape), the parameters increased or decreased more or less regularly as the concentration of the pesticides increased, but in the second experiment, no regular pattern of changes was observed. In addition, the distance traveled by the mite in the treated and untreated areas (Fig. 5 ) did not correlate with the time spent by the mite in these two areas (Fig. 6 ), which is why we separated them. However, in the case of repellency, we use the time spent by the mite as the criterion because it is more logical. We tested two acaricides, but many other pesticides may not have this property. For example, Beers and Schmidt-Jeffries (2015) studied the repellent effect of several pesticides on the predatory mite Galendromus occidentalis and found that the compounds novaluron, carbaryl, mancozeb + copper hydroxide, and sulfur had the greatest repellent effect, as the mites consistently avoided the areas treated with these compounds. Spirotetramate and flubendiamide also had moderate repellency. In contrast, spinosad, spinetoram, imidacloprid, stamiprid, azinphos-methyl, and lambda-cyhalothrin had almost no repellent effect. The most acute toxicity was observed in the spinetoram and lambda-cyhalothrin treatments, which had no repellent effect. Novaluron, which was one of the most repellent substances, also had no significant acute toxicity. Therefore, strong repellents do not always lead to high mortality, and highly toxic substances may not be repellents. In our experiment, chlorfenapyr was also more toxic to predatory mites, but the results for the two acaricides were not very different. Therefore, repellency cannot be inferred from mortality assessments and must be evaluated separately for each compound. Lima et al. ( 2012 ) found that when the predatory mite Neoseiulus baraki was placed in Petri dishes with areas treated with the pesticides abamectin, fenproximate, azadarachtin, carbofuran, chlorfenapyr, and chlorpyrifos, the mites completely avoided the areas treated with chlorpyrifos and azadarachtin. However, they walked around in both the treated and untreated areas for the other pesticides (but mostly preferred the untreated area). This suggests that these pesticides have repellent properties for this mite. Saraiva et al. ( 2020 ) investigated the compatibility of babassu ( Attalea speciosa Mart.) and defatted soybean oils with the predatory mite Typhlodromus ornatus . They found that babaçu and defatted soybean oils were about 19 and 28 times less harmful to this predatory mite than to its prey ( Aceria guerreronis Keifer), respectively. Babaçu oil had no repellent effect on the predatory mite, but soybean oil showed repellent effects at different concentrations. Beers et al. ( 2009 ) studied the effect of three sulfur-based pesticides (lime sulfur, powdered sulfur, and ammonium thiosulfate, which is a type of plant fertilizer) on the predatory mite Galendromus occidentalis . They found that all three pesticides were highly repellent to adult predatory mites. Hislop et al. ( 1981 ) observed that residues of 4 out of 5 pesticides, namely phosmet, azinphos-methyl, captan, and dicofol, caused the predatory mite Amblyseius fallacis to move away and escape from the treated area. This avoidance caused these mites to feed less on eggs of the two-spotted spider mites, oviposit less, and be observed less frequently on treated sites. However, one insecticide, dodine, had no such effect. These pesticides were used as water-soluble powders at dosages specific for apple orchards. Qayyoum et al. ( 2021 ), in a study on the citrus red mite Panonychus citri under field conditions, found that the compounds SYP-9625, abamectin, vegetable oil, and EnSpray 99 all had repellent properties for this mite. The repellency of the chemicals varied depending on the release site of the mites and their preferred feeding location. Chen and Dai ( 2015 ) investigated the effects of two natural compounds, DTBP and EO, on the mortality, repellency, and oviposition deterrence of the red form of the two-spotted spider mite, T. urticae (also known as the carmine spider mite), Their results showed that after exposure to sublethal doses of DTBP and EO, mites exhibited increased repellency, moving away from sprayed bean leaves towards unsprayed surfaces. In both choice and no-choice tests, the number of eggs laid on bean leaves sprayed with DTBP and EO was significantly reduced compared to unsprayed leaves. Kumral et al. ( 2009 ) studied the effect of Datura stramonium plant extract on the two-spotted spider mite T urticae . They found that both leaf and seed extracts had high mortality effects on spider mites and caused them to repel, seeking refuge in untreated leaves. It also inhibited oviposition on leaves containing the extract, suggesting its potential as a natural weapon for controlling mites in gardens and fields. Freitas et al. ( 2018 ) found that coconut oil did not repel the predatory mite Typhlodromus ornatus nor did it interfere with its predation activity. Tak and Isman (2017) studied the insecticidal and repellent effects of 20 common plant essential oil compounds on the two-spotted spider mite ( T. urticae ). They found that combining some of these compounds could have synergistic or antagonistic effects on repellency, with vanillin significantly increasing the repellency of other compounds such as carvacrol, thymol, and alpha-terpineol. They also stated that there was a weak correlation between toxicity and repellency. Avoidance behavior has also been observed in insects. Kongmee et al. ( 2004 ) investigated the behavioral responses of Aedes aegypti (Diptera: Culicidae) to deltamethrin and found that these mosquitoes exhibited avoidance behavior when exposed to a standard concentration of deltamethrin. Two other researches reported the same results for permethrin, abamectin, and fipronil (Wang et al. 2004 ; Jallow and Hoy 2005 ). Relationship between repellency and excitability: Repellency and excitability are interrelated and can be difficult to distinguish, as both behaviors can lead to natural enemies fleeing from the treated area. Highly repellent materials are also somewhat irritable, but highly irritable materials may not be repellent at all. A material that causes high mortality may not have a repellent effect (Beers and Schmidt-Jeffris 2015 ). Role of repellency in explaining field results: Behavioral effects such as repellency or reduced prey consumption can be potential reasons for the poor fit between predator density and biological control, as these effects are rarely considered in other studies (Desneux et al. 2007 ). Some pesticides, instead of directly killing insect pest predators, may negatively affect them by repelling them or reducing their appetite. Or, mites that escape from the area may die later, resulting in an underestimate of the actual mortality rate. These behavioral effects are difficult to measure, but they can play an important role in the field. There are many field studies where different authors have found different results on the harmfulness of pesticides. For example, some reported that the pesticides imidacloprid and thiacloprid reduce predator populations, while others believe their impact is minimal (Martinez-Rocha et al. 2008 ; Duso et al. 2014). Spinosad also reduced phytoseiid mite populations in some studies (Miles & Dutton 2003 ) and had no effect due to its repellent properties in others (Holt et al. 2006 ). These differences may be due to environmental factors or the type of predator studied. Therefore, we conclude that the behavior of predators after spraying should not be ignored. These behaviors can significantly alter the actual results of pesticides in the field. According to the results of this study, the acaricide acequinocyl is a suitable compound for use in integrated pest control because it has low toxicity and irritable properties for N. californicus and P. persimilis . The ideal pesticide is not toxic or repellent, or if it is toxic, it directs natural enemies to safe havens (Beers and Schmidt-Jeffris 2015 ). Combining data on repellency and irritability with mortality and other sublethal effects can help fill gaps between natural enemies’ behavior in the field and bioassay results, as well as select appropriate pesticides for integrated pest management (IPM) programs. Declarations Consent to participate Informed consent was obtained from all individual participants included in the study. Funding The research was financially supported by the University of Tehran under Grant number 73149051.6.21 which is greatly appreciated. Author Contribution All authors contributed to the study conception and design. Also all authors read and approved the final manuscript. Acknowledgement The research was financially supported by the University of Tehran under Grant number 73149051.6.21 which is greatly appreciated. References Beers EH, Martinez-Rocha L, Talley RR, Dunley JE (2009) Lethal, Sublethal, and Behavioral Effects of Sulfur-Containing Products in Bioassays of Three Species of Orchard Mites. 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Journal of the Entomological Society of British Columbia 105: 1–12 McMurtry JA, Moraes GJD, Sourassou NF (2013) Revision of the lifestyles of phytoseiid mites (Acari: Phytoseiidae) and implications for biological control strategies. Systematic and Applied Acarology 18:297. https://doi.org/10.11158/saa.18.4.1 Migeon A, Nouguier E, Dorkeld F (2010) Spider Mites Web: A comprehensive database for the Tetranychidae. Trends in Acarology:557–560. http://dx.doi.org/10.1007/978-90-481-9837-5_96 Miles M, Dutton R (2003). Testing the effects of Spinosad to predatory mites in laboratory, extended laboratory, semi-field and field studies. Pesticides and Beneficial Organisms 26: 9–20 Moghadasi M, Allahyari H, Saboori A, Zahedi Golpayegani A. (2016) Life table and presation capacity of Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) feeding on Tetranychus urticae Koch (Acari: Tetranychidae) on Rose. Journal of Agricultural Science and Technology 18: 1279–1288 Overmeer WPJ (1985) Rearing and handling. In: Sabelis MW (Ed.) Spider mites, their biology, natural enemies and control. Elsevier Science, Amsterdam, the Netherlands, pp. 161–170 Papachristos, DP, Milonas PG (2008) Adverse effects of soil applied insecticides on the predatory coccinellid Hippodamia undecimnotata (Coleoptera: Coccinellidae). Biological Control 47:77–81. https://doi.org/10.1016/j.biocontrol.2008.06.009 Qayyoum MA, Song Z-W, Zhang B-X, Li D-S, Khan BS (2021) Behavioral response of Panonychus citri (McGregor) (Acari: Tetranychidae) to synthetic chemicals and oils. PeerJ 9:e10899. https://doi.org/10.7717/peerj.10899 Raghavendra K, Barik TK, Sharma P, Bhatt RM, Srivastava HC, Sreehari U, Dash AP (2011) Chlorfenapyr: a new insecticide with novel mode of action can control pyrethroid resistant malaria vectors. Malaria Journal 10. https://doi.org/10.1186/1475-2875-10-16 Řezáč M, Pekár S, Stará J (2010) The negative effect of some selective insecticides on the functional response of a potential biological control agent, the spider Philodromus cespitum . BioControl 55:503–510. http://dx.doi.org/10.1007/s10526-010-9272-3 Rhodes EM, Liburd OE (2009). Predatory mite, Neoseiulus californicus (McGregor) (Arachnida: Acari: Phytoseiidae). In EDIS 2005. University of Florida George A Smathers Libraries. http://dx.doi.org/10.32473/edis-in639-2005 Roberts DR, Chareonviriyaphap T, Harlan HH, Hshieh P (1997) Methods for testing and analyzing excito repellency responses of malaria vectors to pesticides. Journal of the American Mosquito Control Association 13:13–17 Saraiva WVA, Vieira IG, Galvão AS, Do Amaral EA, Rêgo AS, Teodoro AV, Dias-Pini NS (2020) Lethal and sublethal effects of babassu and degummed soybean oils on the predatory mite Typhlodromus ornatus (Acari: Phytoseiidae). International Journal of Acarology 46:180–184. https://doi.org/10.1080/01647954.2020.1734081 Sheppard DC, Joyce C (1998) Increased susceptibility of pyrethroid- resistant horn flies (Diptera: Muscidae) to chlorfenapyr, Journal of Economic Entomology 91: 398-400 Soderlund DM, Bloomquist JR (1989) Neurotoxic actions of pyrethroid pesticides. Annual Review of Entomology 34:77–96. https://doi.org/10.1146/annurev.en.34.010189.000453 Tak J-H, Isman MB (2017) Acaricidal and repellent activity of plant essential oil-derived terpenes and the effect of binary mixtures against Tetranychus urticae Koch (Acari: Tetranychidae). Industrial Crops and Products 108:786–792. http://dx.doi.org/10.1016/j.indcrop.2017.08.003 Van Leeuwen T, Vontas J, Tsagkarakou A, Dermauw W, Tirry L (2010) Acaricide resistance mechanisms in the two-spotted spider mite Tetranychus urticae and other important Acari: A review. Insect Biochemistry and Molecular Biology 40:563–572. https://doi.org/10.1016/j.ibmb.2010.05.008 Van Pottelberge S, Van Leeuwen T, Van Amermaet K, Tirry L (2008) Induction of cytochrome P450 monooxygenase activity in the two-spotted spider mite Tetranychus urticae and its influence on acaricide toxicity. Pesticide Biochemistry and Physiology 91:128–133. https://doi.org/10.1016/j.pestbp.2008.03.005 Walzer A, Castagnoli M, Simoni S, Liguori M, Palevsky E, Schausberger P (2007) Intraspecific variation in humidity susceptibility of the predatory mite Neoseiulus californicus : Survival, development and reproduction. Biological Control 41:42–52. https://doi.org/10.1016/j.biocontrol.2006.11.012 Wang C, Scharf ME, Bennett GW (2004) Behavioral and physiological resistance of the German cockroach to gel baits (Blattodea: Blattellidae). Journal of Economic Entomology 97: 2067–2072 https://doi.org/10.1093/jee/97.6.2067 Yorulmaz Salman S, Aydınlı F, Ay R (2015). Selection for resistance: Cross-resistance, inheritance, synergists and biochemical mechanisms of resistance to acequinocyl in Phytoseiulus persimilis A. H. (Acari: Phytoseiidae). Crop Protection 67: 109-115. https://doi.org/10.1016/j.cropro.2014.10.001 Zhao Y, Wang Q, Wang Y, Zhang Z, Wei Y, Liu F, Zhou C, Mu W (2017) Chlorfenapyr, a Potent Alternative Insecticide of Phoxim To Control Bradysia odoriphaga (Diptera: Sciaridae). Journal of Agricultural and Food Chemistry 65:5908–5915. https://doi.org/10.1021/acs.jafc.7b02098 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 17 Jan, 2025 Read the published version in Experimental and Applied Acarology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4604689","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323367124,"identity":"75006c2b-376e-4e02-8491-f552613c1f76","order_by":0,"name":"Navid Sehat-Niaki","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Navid","middleName":"","lastName":"Sehat-Niaki","suffix":""},{"id":323367125,"identity":"ec9d307b-1be0-4a58-a96d-19e57d782481","order_by":1,"name":"Azadeh Zahedi Golpaygani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYFACHiAyYJCD8iSI12LMwEaaFgaGxAY2Yp0l33724IM3BbXpG+43MH74wWCRT1ALY09esuEcg+O5G44xMEv2MEhYNhDSwsyQYybNY3AMpIVBGugXA4K2sPG/Mf8N1JJuALTlN1FaeCRyzJh5DGoSgFrYiLNFQuKNseQcgwOGM48ltln2GBChRb4/x/DDmz918nyHDx++8aOijrAWKDgMxIwNDAxEa2BgqCNe6SgYBaNgFIw8AAAjHDJPTasKowAAAABJRU5ErkJggg==","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Azadeh","middleName":"Zahedi","lastName":"Golpaygani","suffix":""},{"id":323367126,"identity":"cd167130-b6bd-478c-a410-f481073a31fc","order_by":2,"name":"Ehssan Torabi","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Ehssan","middleName":"","lastName":"Torabi","suffix":""},{"id":323367127,"identity":"78e8bde4-4a43-4976-bdc6-3c5219bdb500","order_by":3,"name":"Behnam Amiri-Besheli","email":"","orcid":"","institution":"Sari Agricultural Sciences and Natural Resources University","correspondingAuthor":false,"prefix":"","firstName":"Behnam","middleName":"","lastName":"Amiri-Besheli","suffix":""},{"id":323367128,"identity":"a4bc52d0-c02d-4c9f-ae0a-1fa2a9b6de13","order_by":4,"name":"Alireza Saboori","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Saboori","suffix":""}],"badges":[],"createdAt":"2024-06-19 09:12:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4604689/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4604689/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10493-024-00995-4","type":"published","date":"2025-01-17T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59906057,"identity":"9a19118b-d04f-4b54-aa4f-ed991e3a4f7c","added_by":"auto","created_at":"2024-07-09 06:55:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41860,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4604689/v1/52e577b99685f6a5a2c4c349.png"},{"id":59905564,"identity":"728c7ba1-5f86-4300-a43e-b262d24d1c58","added_by":"auto","created_at":"2024-07-09 06:47:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":166606,"visible":true,"origin":"","legend":"\u003cp\u003eTracks of predatory mites, \u003cem\u003eNeoseiulus californicus\u003c/em\u003e and \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e on a disc soaked with distilled water (control) and acaricides chlorfenapyr and acequinocyl for 10 min (a representative replicate is shown for each treatment).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4604689/v1/3b241c96e5a6aae27eef7b18.png"},{"id":59905560,"identity":"e16fae42-7acc-4861-8557-aac8f03b204c","added_by":"auto","created_at":"2024-07-09 06:47:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111065,"visible":true,"origin":"","legend":"\u003cp\u003eAverage values of walking distance, walking speed, resting time, and number of stops of the predatory mites, \u003cem\u003eNeoseiulus californicus\u003c/em\u003e and \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e in an environment treated with different concentrations of the acaricides chlorfenapyr and acequinocyl in 10 min. Lowercase letters indicate significant differences (P \u0026lt; 0.05) within each mite as determined by MANOVA and Tukey's HSD post-hoc test and are not related to another mite.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4604689/v1/5300baf16f578d3ba5b42f85.png"},{"id":59906058,"identity":"d971601c-3da8-447e-b62d-1b3058438660","added_by":"auto","created_at":"2024-07-09 06:55:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":154092,"visible":true,"origin":"","legend":"\u003cp\u003eTracks of \u003cem\u003eNeoseiulus californicus\u003c/em\u003eand \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e predatory mites on a disc soaked with distilled water (right hemisphere) and acaricide (left hemisphere) in 10 min. (a representative replicate is shown for each treatment).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4604689/v1/31936431924d7cb3f1a5dbf7.png"},{"id":59905561,"identity":"61905eb5-fff8-4b2c-baae-f058c84bf7f8","added_by":"auto","created_at":"2024-07-09 06:47:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61326,"visible":true,"origin":"","legend":"\u003cp\u003eAverage distance walked by the predatory mites \u003cem\u003eNeoseiulus californicus\u003c/em\u003eand \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e in each hemisphere of the disc soaked with distilled water (untreated area) and acaricide (treated area) with a chance of escape chance for 10 min. The asterisk (*) indicates a statistically significant difference (P \u0026lt; 0.05) between the two areas of each treatment as determined by MANOVA and Tukey’s HSD post-hoc test\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4604689/v1/61addd67489f8dc16f68ee5f.png"},{"id":59905563,"identity":"4ea245e4-4d87-4263-80a4-d1b3648328f1","added_by":"auto","created_at":"2024-07-09 06:47:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69866,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of time spent by the predatory mites, \u003cem\u003eNeoseiulus californicus\u003c/em\u003e and \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e in each hemisphere of the disc soaked with distilled water (untreated area) and acaricide (treated area) with a chance of escape for 10 min. The asterisk (*) indicates a statistically significant difference (P \u0026lt; 0.05) between the two areas of each treatment as determined by MANOVA and Tukey’s HSD post-hoc test\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4604689/v1/181069dfeb9f1e8585836bea.png"},{"id":74284560,"identity":"8b951695-a53d-45e0-b515-454860404b5c","added_by":"auto","created_at":"2025-01-20 16:08:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1316893,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4604689/v1/ff8ae1dd-8faf-46e2-bb30-2d2ae1c2dee8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Repellent and acaricidal effects of the chlorfenapyr and acequinocyl on the predatory mites, Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe two-spotted spider mite, \u003cem\u003eTetranychus urticae\u003c/em\u003e Koch (Acari: Tetranychidae), is a major pest of vegetable, orchard, ornamental, and field crops worldwide (Migeon and Dorkeld 2010). It rapidly forms colonies and has a short life cycle (Dermauw et al. 2013). It is a polyphagous species and damages the leaf surface by feeding on plant sap and destroying plant cells (Huffaker et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). Chemical acaricides have widely been used to control this pest (Van Leeuwen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, one of the major challenges in controlling the process is its rapid development of resistance to these chemicals (Van Pottelberge et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, researchers are trying to use alternative ways to manage this pest, including the use of biological control agents.\u003c/p\u003e \u003cp\u003eThe predatory mite, \u003cem\u003eNeoseiulus californicus\u003c/em\u003e McGregor (Acari: Phytoseiidae) is known as an important component of biological control in integrated pest management programs (Canlas et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This generalist predator (McMurtry et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) can feed on various sources, including the two-spotted spider mite, but it has a higher growth rate when feeding on this particular prey (Rhodes \u0026amp; Liburd \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Its rapid population growth, long-term establishment, and persistent populations in plant systems are the predator's valuable characteristics (Walzer et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The predatory mite \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e Athias-Henriot (Acari: Phytoseiidae) is a key component in integrated pest management (IPM) programs worldwide, particularly for controlling tetranychid mites in greenhouses (Lee et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). This predatory mite possesses a high reproductive capacity, a short developmental period, and the ability to feed on all stages of the two-spotted spider mite, making it an important factor in IPM success (Moghadasi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It feeds exclusively on spider mites, primarily of the genus \u003cem\u003eTetranychus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSince most pesticides have had a broad spectrum of activity, they also eliminate non-target and beneficial species. The most important biological control agents of insect and mite pests are considered arthropod predators and parasitoids, which may be directly or indirectly affected by various pesticides through contact with pesticide residues or feeding on infested hosts (Croft \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Studies have shown that even selective pesticides can have negative effects on natural enemies (Rezac et al. 2010). Pesticides can cause mortality (lethal effects) or alter other biological traits of organisms (sublethal effects) (Rezac et al. 2010; Papachristos \u0026amp; Milonas 2010). Therefore, before incorporating any pesticide into a system, it is crucial to gather information on its lethal and sublethal effects on key predators in the system.\u003c/p\u003e \u003cp\u003eChlorfenapyr, marketed under the trade name Conqueror, is a systemic and contact insecticide-acaricide (Zhao et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) that is effective against various important pests, including tarsonemid and tetranychid mites, and whiteflies (Raghavendra et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This pesticide belongs to the pyrrole chemical group and acts as an oxidative phosphorylation inhibitor by disrupting the proton concentration gradient (Dekeyser \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), making it recommended against pests resistant to organophosphate, carbamate, and pyrethroid compounds (Sheppard \u0026amp; Joyce \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Acequinocyl, marketed under the brand name Kanemite, is a naphthoquinone analog compound formulated as a suspension concentrate (SC). The U.S. Environmental Protection Agency (EPA) classifies it as a reduced-risk pesticide. Acequinocyl inhibits mitochondrial respiration and is used to control phytophagous mites (Yorulmaz Salman et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eArthropods in agricultural fields are constantly exposed to sublethal doses of pesticide residues after application. To reduce contact with these compounds, predators may exhibit avoidance behaviors, which can alter their movement patterns and result in either irritability or repellency (Cordeiro et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Irritability occurs when an insect is stimulated to move away from the pesticide after direct physical contact with the chemical residue, while repellency occurs from a distance, and the insect avoids the pesticide-treated area without direct physical contact (Roberts et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Most laboratory bioassays in the past have focused on acute mortality, with sublethal effects being added to these studies more recently (Desneux et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Sublethal effects typically involve estimates of reduced reproductive capacity, but these experiments are generally designed to expose insects to pesticides in a \"worst-case\" scenario, with no option to escape sprays or residues, likely overestimating the toxicity of pesticides (Gerson et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hassan \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aimed to determine the repellency and irritability properties of two acaricides for the predatory mites \u003cem\u003eN. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e. The results of this study, along with other research on the sublethal effects of pesticides, will allow us to better determine which pesticides are suitable for integrated pest management programs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eRearing of two-spotted spider mite (\u003cem\u003eT. urticae\u003c/em\u003e)\u003c/p\u003e \u003cp\u003eCommon bean plants [[\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L. (Fabaceae) var. Red Alamouti] were grown in plastic pots in a greenhouse (soil: perlite; 50:50%) under controlled conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C, 16L:8D photo-period, 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH) at the Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran. Plants were daily irrigated with tap water and a fertilizer solution of NPK (20\u0026times;20\u0026times;20). Spider mites, \u003cem\u003eTetranychus urticae\u003c/em\u003e (green form; food source for the predatory mites) were reared on bean plants. Fresh bean plants were added to the rearing system regularly.\u003c/p\u003e \u003cp\u003eRearing of the predatory mites\u003c/p\u003e \u003cp\u003eThe predatory mites, \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e, and \u003cem\u003eNeoseiulus californicus\u003c/em\u003e species clarified before experiments, were reared on masses of detached bean leaves, infested with \u003cem\u003eT. urticae\u003c/em\u003e, placed upside down on a plastic sheet on a water-saturated sponge. The plastic sheet was surrounded by napkin tapes which were put into the water from the otherwise that the predatory mites could drink water. Fresh \u003cem\u003eT. urticae-\u003c/em\u003einfested leaves and fresh corn pollen (Zea mays) were added to the rearing system and the old predator-free leaves were removed regularly (Overmeer \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The cultures were kept in separate growth chambers under controlled conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 16L:8D photoperiod, 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH) in the Acarology laboratory at Jalal Afshar Zoological Museum, Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran.\u003c/p\u003e \u003cp\u003eToxicity bioassays of pesticides on predatory mites\u003c/p\u003e \u003cp\u003eBioassays were conducted using the leaf disc dipping method (Helle \u0026amp; Overmeers \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Preliminary tests were performed to determine the range of effective concentrations that caused 10\u0026ndash;90% mortality in the predatory mites. Intermediate concentrations were determined using logarithmic intervals. Five concentrations determined in the preliminary tests were used in these experiments. For \u003cem\u003eP. persimilis\u003c/em\u003e, the tested concentrations were 1500\u0026ndash;6000 ppm for acequinocyl and 300\u0026ndash;800 ppm for chlorfenapyr. Similarly, \u003cem\u003eN. californicus\u003c/em\u003e was exposed to a range of 3000\u0026ndash;9000 ppm for acequinocyl and 700\u0026ndash;2000 ppm for chlorfenapyr. For each concentration, a 4 cm diameter leaf disc was excised from a bean plant. Each leaf disc was immersed in the toxic solution for 15 sec (the control leaf disc was immersed in distilled water). After drying the leaf for 2\u0026ndash;3 h, it was placed on moistened cotton inside a 9 cm diameter Petri dish. The leaf disc was surrounded by a strip of moistened cotton. For each concentration, 20 same-age-matched adult mites (male and female) were randomly selected and placed on the leaf discs. Petri dishes were incubated in a growth chamber under controlled conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C, 16L:8D photo-period, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH). After 24 h, mortality was assessed. Mites that were unable to move after stimulation with a brush were considered dead. Bioassays were performed with four replicates. Based on the results, three concentrations were selected for subsequent experiments, including the field-recommended rate, LC\u003csub\u003e30\u003c/sub\u003e and LC\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eAssessment of predatory mites behavior after exposure to pesticides without escape chance\u003c/p\u003e \u003cp\u003eThe experiment was conducted following the method of Lima et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Black polyvinyl chloride (PVC) discs (3 cm diameter, 1 mm thickness) were fully immersed in 40 mL of different concentrations of each pesticide and distilled water (Control) for 5 sec. Then the discs were air-dried under a fume hood for 20 min. A hole was punched in the center of the discs and they were attached to the center of Petri dishes (9 cm diameter, 1 cm height). Distilled water was added to each Petri dish up to half its height so that the disc would float on it. A single individual same-age-matched female was placed on each disc and the Petri dishes were transferred to a tracking system via a video camera connected to a computer. The assessment was performed for 10 min at 25\u0026ndash;27\u0026deg;C. The parameters recorded for each mite were: distance traveled, average walking speed, resting time, and number of stops. Twenty replicates were performed for each pesticide, with each mite representing a replicate. The whole experiment was conducted in a completely randomized design.\u003c/p\u003e \u003cp\u003eAssessment of predatory mites behavior after exposure to acaricides with an escape chance\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar to the previous step, PVC discs were attached to Petri dishes with a needle. A diameter was drawn inside the disc with a marker to divide it into two equal hemispheres (Fig.\u0026nbsp;1). One hemisphere was immersed in distilled water for 5 sec and then placed in a laminar flow hood for 20 min to dry. The other hemisphere was then immersed in 40 ml of the acaricide solution using the same method. This process was repeated for all treatments. A control treatment was also performed separately by immersing both hemispheres in distilled water. Finally, the dishes were placed in the tracking system to evaluate the time spent in each hemisphere of each disc. Acaricides were considered repellent if mites remained on the untreated hemisphere for the entire duration, and irritant if they entered the treated hemisphere but spent less than 50% of the time there.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eSublethal concentrations were determined using the probit method and SAS 9.4 software. Differences in the numbers of female predatory mites choosing the treated and untreated areas in the choice test were compared using independent F-tests and T-tests in SPSS 25. Total distance traveled, walking time, and walking speed in the observational tracking experiments were estimated following the methods described by Lima et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and were analyzed using a multivariate analysis of variance (MANOVA) with pesticides as the independent variable. Mean comparisons were then performed using Tukey's HSD test. The results of repellency and irritability of acaricides were analyzed using the non-parametric Wilcoxon test (sum of ranks) and the UNIVARIATE procedure. In this study, the type of insecticide treatment was considered as the independent variable. Data were visualized with graphs created using Excel 2019 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eBioassays experiments\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the results of bioassays conducted with two acaricides, chlorfenapyr and acequinocyl, on the predatory mites, \u003cem\u003eN. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e. Chlorfenapyr was about five times more toxic than acequinocyl for both species. The LC\u003csub\u003e50\u003c/sub\u003e values for \u003cem\u003eN. californicus\u003c/em\u003e were significantly higher than those for \u003cem\u003eP. persimilis\u003c/em\u003e, indicating that \u003cem\u003eN. californicus\u003c/em\u003e can tolerate higher levels of pesticides than \u003cem\u003eP. persimilis\u003c/em\u003e. Three concentrations (the field recommended rate, LC\u003csub\u003e30\u003c/sub\u003e, and LC\u003csub\u003e50\u003c/sub\u003e) were used in the behavioral assay to investigate the relationship between concentration and behavioral changes. The field rate of chlorfenapyr was 0.4 ml/L and that of acequinocyl was 1 ml/L.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBioassay of chlorfenapyr and acequinocyl acaricides on the predatory mites, \u003cem\u003eNeoseiulus californicus\u003c/em\u003e and \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e, and determination of sublethal concentrations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcaricide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHetero-geneity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP-\u003c/em\u003evalue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSlope\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLC\u003csub\u003e30\u003c/sub\u003e\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eN. californicus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echlorfenapyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.953\u003c/p\u003e \u003cp\u003e(0.882\u0026ndash;1.017)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.186\u003c/p\u003e \u003cp\u003e(1.118\u0026ndash;1.258)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eacequinocyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.711\u003c/p\u003e \u003cp\u003e(4.363\u0026ndash;5.033)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5.987\u003c/p\u003e \u003cp\u003e(5.614\u0026ndash;6.424)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP. persimilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echlorfenapyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e5.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.429\u003c/p\u003e \u003cp\u003e(0.398\u0026ndash;0.456)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.536\u003c/p\u003e \u003cp\u003e(0.505\u0026ndash;0.571)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eacequinocyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.558\u003c/p\u003e \u003cp\u003e(2.323\u0026ndash;2.777)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3.428\u003c/p\u003e \u003cp\u003e(3.169\u0026ndash;3.727)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e*\u003c/sup\u003e Twenty individuals per replicate, four replicates per concentration, six concentrations per assay.\u003c/p\u003e \u003cp\u003e \u003csup\u003e**\u003c/sup\u003e Concentrations are reported in ml/L with 95% confidence intervals.\u003c/p\u003e \u003cp\u003eBehavioral response of the predatory mites to acaricides without chance of escape\u003c/p\u003e \u003cp\u003eThe movement patterns \u003cem\u003eof N. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e predatory mites on discs fully treated with acaricide residues are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In acaricide treatments, the mites walked shorter distances and at slower speeds, spent less time resting, and stopped more frequently. The longest distance traveled was observed when the discs were treated with distilled water, and the shortest distance traveled was observed for the LC\u003csub\u003e50\u003c/sub\u003e treatment of chlorfenapyr for both predatory mites. For both species the highest walking speed was observed in the control treatment and the field concentration of acequinocyl. This was determined by calculating the average speed, which is defined as the distance traveled in 10 minutes divided by the actual travel time excluding stops. The shortest resting time was observed in the control treatment and the longest resting time was observed in the LC\u003csub\u003e50\u003c/sub\u003e treatment of chlorfenapyr. Furthermore, the fewest stops were observed in both the control treatment and the field concentration of acequinocyl. It can be inferred that the field concentration of acequinocyl did not affect these mites, as it did not differ significantly from the control in all tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, the negative effect of chlorfenapyr was more pronounced than acequinocyl for all parameters.\u003c/p\u003e \u003cp\u003eBehavioral responses of the predatory mites to acaricides with a chance of escape\u003c/p\u003e \u003cp\u003eThe movement patterns of \u003cem\u003eN. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e mites on PVC discs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e. One hemisphere of each disk was immersed in distilled water, while the other was immersed in an acaricide solution, as in the previous experiment, the longest distance traveled was observed in the control treatment. The average distance and percentage of time spent in each hemisphere are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e. In the control treatment, the mites showed no preference for either hemisphere, however, in the treated groups, it showed a lower tendency to stay in the hemisphere containing the acaricide. None of the acaricides had repellent properties since the mites also moved in the area treated with the acaricides, but they had irritant properties and the time spent by the mite in the contaminated area was significantly less than in the untreated area. This difference was observed in the treatment with the lowest concentration of acaricides and continued up to the highest concentration. These findings suggest that both predatory mites, \u003cem\u003eN. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e can detect and avoid acaricide residues in their environment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNatural enemies may exhibit behavioral responses, such as avoidance of treated areas when exposed to certain pesticides. These behaviors, known as repellency and irritability effects, can influence the actual impact of pesticides in orchards and make their prediction more challenging (Beers and Schmidt-Jeffris \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRepellency can negatively affect pest population control since predatory mites will be less effective in controlling pest populations. However, it can potentially reduce the harmful effects of pesticides on natural enemies since they avoid contact with toxic substances by fleeing the treated area. This can help preserve natural enemy refuges and promote continued biological pest control (Hislop et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Gerson et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Of course, there is also the possibility that they may completely leave the treated area and not return. On the other hand, repellency can increase the dispersal of natural enemies in the agroecosystem, temporarily reducing their population in the treated area. This dispersal can reduce the probability of resistance evolution in natural enemies to pesticides as they migrate to areas with less selection pressure (Croft and Brown \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). Therefore, it is difficult to determine whether repellency is beneficial or detrimental to agriculture.\u003c/p\u003e \u003cp\u003eBioassay results demonstrated that chlorfenapyr was about five times more toxic than acequinocyl to both predatory mites, \u003cem\u003eN. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e. Acequinocyl acaricide exhibited low toxicity to this mite since the LC\u003csub\u003e50\u003c/sub\u003e value was estimated to be several-fold higher than the field concentration. The field concentration of acequinocyl is 2.5 times that of chlorfenapyr. This could mean that chlorfenapyr may be effective for pest control at lower concentrations, but it also poses a greater risk to natural enemies such as predatory mites. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, in the no-escape chance experiment, the mite tended to move across the whole area of the PVC disc. The distance traveled was significantly higher in the control treatment than in the pesticide treatments. In the no-escape chance experiment, the decrease in the distance and walking speed of the predatory mites, and conversely, the increase in resting time and the number of stops, could be due to the pesticide's effect on the motor system or substance's irritable effect that makes the mite lazier and more tired. In any case, these factors in turn reduce its efficiency in prey tracking and biological control. Although the LC\u003csub\u003e50\u003c/sub\u003e concentration of both acaricides did not cause mortality in the first 10 min, it showed that the mite tends to weaken significantly. If this experiment were evaluated for a longer period, the difference in these parameters would likely be more distinct than that of the control.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e was significantly more motile and faster than \u003cem\u003eN. californicus\u003c/em\u003e, exhibiting a shorter resting time as well. Possible reasons for this phenomenon include: \u003cem\u003eP. persimilis\u003c/em\u003e is smaller than \u003cem\u003eN. californicus\u003c/em\u003e, which may give it an advantage in terms of agility and speed, also \u003cem\u003eP. persimilis\u003c/em\u003e may have stronger muscles and longer legs allowing it to move faster. Finally, \u003cem\u003eP. persimilis\u003c/em\u003e may have a faster metabolism, providing it with the energy needed to move more quickly.\u003c/p\u003e \u003cp\u003eLima et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) investigated the walking behavior of the predatory mite, \u003cem\u003eNeoseiulus baraki\u003c/em\u003e exposed to the acaricides abamectin, fenpyroximate, azadarachtin, carbosulfan, chlorfenapyr, and chlorpyrifos. All of these compounds caused a significant reduction in the distance and speed of mite walking. The highest distance traveled was observed in the chlorfenapyr treatment and the lowest was for azadarachtin and carbosulfan. Resting time and stopping frequency was also significantly higher in the azadarachtin and carbosulfan treatments than in the other pesticides. Chlorfenapyr and chlorpyrifos had the least effect, but in our study, chlorfenapyr caused significant changes in these parameters. This is probably because they only tested one concentration of each pesticide, while we also tested higher concentrations. Fran\u0026ccedil;a et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) analyzed the walking behavior and oviposition rate of the mite \u003cem\u003eSteneotarsonemus concavuscutum\u003c/em\u003e when exposed to products impregnated with the acaricides abamectin, azadarachtin, spiromesifen, fenproximate, and hexythiazox. They found that abamectin had the highest toxicity to this mite (LC\u003csub\u003e50\u003c/sub\u003e 1.1 mg/L). The total distance traveled and walking speed was significantly lower when \u003cem\u003eS. concavuscutum\u003c/em\u003e was exposed to abamectin compared to the other acaricides. The number of eggs laid on the treated area was also significantly lower for spiromesifen and abamectin compared to the other acaricides.\u003c/p\u003e \u003cp\u003eIn an experiment where escape from the treated area was possible, both predatory mites showed a preference for moving in the untreated area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This suggests that the compounds had an irritable effect on the mites, as they did not avoid them completely but only spent less time on them. One possible reason for this is that the chemical residues are toxic to predatory mites and can harm them. Consequently, the predatory mites attempt to avoid the contaminated area to prevent exposure to the poison. Another possible reason is that the chemical residues may have a specific odor or scent that the predatory mites can sense. This odor or scent can be a warning signal for the predatory mites and keep them away from the contaminated area.\u003c/p\u003e \u003cp\u003eDespite the advantages of \u003cem\u003eP. persimilis\u003c/em\u003e in terms of speed and resting time, \u003cem\u003eN. californicus\u003c/em\u003e showed a slightly greater tendency to stay in the untreated area, indicating a more cautious behavior. This could be attributed to \u003cem\u003eN. californicus\u003c/em\u003e' potentially greater caution to the chemicals in the pesticides, leading it to perceive the negative effects of the pesticides more quickly than \u003cem\u003eP. persimilis\u003c/em\u003e and making it more likely to leave the contaminated area. According to Haynes (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) and Soderlund and Bloomquist (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), there is a relationship between repellent behavior and sensory perception in arthropods when they first come into contact with the treated area. Therefore, it is likely that predatory mites have mechanisms that allow it to minimize contact with acaricides. In the first experiment (with the possibility of escape), the parameters increased or decreased more or less regularly as the concentration of the pesticides increased, but in the second experiment, no regular pattern of changes was observed. In addition, the distance traveled by the mite in the treated and untreated areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e) did not correlate with the time spent by the mite in these two areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e), which is why we separated them. However, in the case of repellency, we use the time spent by the mite as the criterion because it is more logical.\u003c/p\u003e \u003cp\u003eWe tested two acaricides, but many other pesticides may not have this property. For example, Beers and Schmidt-Jeffries (2015) studied the repellent effect of several pesticides on the predatory mite \u003cem\u003eGalendromus occidentalis\u003c/em\u003e and found that the compounds novaluron, carbaryl, mancozeb\u0026thinsp;+\u0026thinsp;copper hydroxide, and sulfur had the greatest repellent effect, as the mites consistently avoided the areas treated with these compounds. Spirotetramate and flubendiamide also had moderate repellency. In contrast, spinosad, spinetoram, imidacloprid, stamiprid, azinphos-methyl, and lambda-cyhalothrin had almost no repellent effect. The most acute toxicity was observed in the spinetoram and lambda-cyhalothrin treatments, which had no repellent effect. Novaluron, which was one of the most repellent substances, also had no significant acute toxicity. Therefore, strong repellents do not always lead to high mortality, and highly toxic substances may not be repellents. In our experiment, chlorfenapyr was also more toxic to predatory mites, but the results for the two acaricides were not very different. Therefore, repellency cannot be inferred from mortality assessments and must be evaluated separately for each compound.\u003c/p\u003e \u003cp\u003eLima et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) found that when the predatory mite \u003cem\u003eNeoseiulus baraki\u003c/em\u003e was placed in Petri dishes with areas treated with the pesticides abamectin, fenproximate, azadarachtin, carbofuran, chlorfenapyr, and chlorpyrifos, the mites completely avoided the areas treated with chlorpyrifos and azadarachtin. However, they walked around in both the treated and untreated areas for the other pesticides (but mostly preferred the untreated area). This suggests that these pesticides have repellent properties for this mite. Saraiva et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) investigated the compatibility of babassu (\u003cem\u003eAttalea speciosa\u003c/em\u003e Mart.) and defatted soybean oils with the predatory mite \u003cem\u003eTyphlodromus ornatus\u003c/em\u003e. They found that baba\u0026ccedil;u and defatted soybean oils were about 19 and 28 times less harmful to this predatory mite than to its prey (\u003cem\u003eAceria guerreronis\u003c/em\u003e Keifer), respectively. Baba\u0026ccedil;u oil had no repellent effect on the predatory mite, but soybean oil showed repellent effects at different concentrations. Beers et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) studied the effect of three sulfur-based pesticides (lime sulfur, powdered sulfur, and ammonium thiosulfate, which is a type of plant fertilizer) on the predatory mite \u003cem\u003eGalendromus occidentalis\u003c/em\u003e. They found that all three pesticides were highly repellent to adult predatory mites. Hislop et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) observed that residues of 4 out of 5 pesticides, namely phosmet, azinphos-methyl, captan, and dicofol, caused the predatory mite \u003cem\u003eAmblyseius fallacis\u003c/em\u003e to move away and escape from the treated area. This avoidance caused these mites to feed less on eggs of the two-spotted spider mites, oviposit less, and be observed less frequently on treated sites. However, one insecticide, dodine, had no such effect. These pesticides were used as water-soluble powders at dosages specific for apple orchards. Qayyoum et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), in a study on the citrus red mite \u003cem\u003ePanonychus citri\u003c/em\u003e under field conditions, found that the compounds SYP-9625, abamectin, vegetable oil, and EnSpray 99 all had repellent properties for this mite. The repellency of the chemicals varied depending on the release site of the mites and their preferred feeding location. Chen and Dai (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) investigated the effects of two natural compounds, DTBP and EO, on the mortality, repellency, and oviposition deterrence of the red form of the two-spotted spider mite, \u003cem\u003eT. urticae\u003c/em\u003e (also known as the carmine spider mite), Their results showed that after exposure to sublethal doses of DTBP and EO, mites exhibited increased repellency, moving away from sprayed bean leaves towards unsprayed surfaces. In both choice and no-choice tests, the number of eggs laid on bean leaves sprayed with DTBP and EO was significantly reduced compared to unsprayed leaves. Kumral et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) studied the effect of \u003cem\u003eDatura stramonium\u003c/em\u003e plant extract on the two-spotted spider mite \u003cem\u003eT urticae\u003c/em\u003e. They found that both leaf and seed extracts had high mortality effects on spider mites and caused them to repel, seeking refuge in untreated leaves. It also inhibited oviposition on leaves containing the extract, suggesting its potential as a natural weapon for controlling mites in gardens and fields. Freitas et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that coconut oil did not repel the predatory mite \u003cem\u003eTyphlodromus ornatus\u003c/em\u003e nor did it interfere with its predation activity. Tak and Isman (2017) studied the insecticidal and repellent effects of 20 common plant essential oil compounds on the two-spotted spider mite (\u003cem\u003eT. urticae\u003c/em\u003e). They found that combining some of these compounds could have synergistic or antagonistic effects on repellency, with vanillin significantly increasing the repellency of other compounds such as carvacrol, thymol, and alpha-terpineol. They also stated that there was a weak correlation between toxicity and repellency. Avoidance behavior has also been observed in insects. Kongmee et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) investigated the behavioral responses of \u003cem\u003eAedes aegypti\u003c/em\u003e (Diptera: Culicidae) to deltamethrin and found that these mosquitoes exhibited avoidance behavior when exposed to a standard concentration of deltamethrin. Two other researches reported the same results for permethrin, abamectin, and fipronil (Wang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Jallow and Hoy \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRelationship between repellency and excitability: Repellency and excitability are interrelated and can be difficult to distinguish, as both behaviors can lead to natural enemies fleeing from the treated area. Highly repellent materials are also somewhat irritable, but highly irritable materials may not be repellent at all. A material that causes high mortality may not have a repellent effect (Beers and Schmidt-Jeffris \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRole of repellency in explaining field results: Behavioral effects such as repellency or reduced prey consumption can be potential reasons for the poor fit between predator density and biological control, as these effects are rarely considered in other studies (Desneux et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Some pesticides, instead of directly killing insect pest predators, may negatively affect them by repelling them or reducing their appetite. Or, mites that escape from the area may die later, resulting in an underestimate of the actual mortality rate. These behavioral effects are difficult to measure, but they can play an important role in the field. There are many field studies where different authors have found different results on the harmfulness of pesticides. For example, some reported that the pesticides imidacloprid and thiacloprid reduce predator populations, while others believe their impact is minimal (Martinez-Rocha et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Duso et al. 2014). Spinosad also reduced phytoseiid mite populations in some studies (Miles \u0026amp; Dutton \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and had no effect due to its repellent properties in others (Holt et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These differences may be due to environmental factors or the type of predator studied. Therefore, we conclude that the behavior of predators after spraying should not be ignored. These behaviors can significantly alter the actual results of pesticides in the field.\u003c/p\u003e \u003cp\u003eAccording to the results of this study, the acaricide acequinocyl is a suitable compound for use in integrated pest control because it has low toxicity and irritable properties for \u003cem\u003eN. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e. The ideal pesticide is not toxic or repellent, or if it is toxic, it directs natural enemies to safe havens (Beers and Schmidt-Jeffris \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Combining data on repellency and irritability with mortality and other sublethal effects can help fill gaps between natural enemies\u0026rsquo; behavior in the field and bioassay results, as well as select appropriate pesticides for integrated pest management (IPM) programs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConsent to participate\u003c/h2\u003e \u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe research was financially supported by the University of Tehran under Grant number 73149051.6.21 which is greatly appreciated.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Also all authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe research was financially supported by the University of Tehran under Grant number 73149051.6.21 which is greatly appreciated.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBeers EH, Martinez-Rocha L, Talley RR, Dunley JE (2009) Lethal, Sublethal, and Behavioral Effects of Sulfur-Containing Products in Bioassays of Three Species of Orchard Mites. Journal of Economic Entomology 102:324\u0026ndash;335. https://doi.org/10.1603/029.102.0143\u003c/li\u003e\n\u003cli\u003eBeers EH, Schmidt-Jeffris RA (2015) Effects of Orchard Pesticides on \u003cem\u003eGalendromus occidentalis\u003c/em\u003e (Acari: Phytoseiidae): Repellency and Irritancy. 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Chemosphere 81:1352\u0026ndash;1357. https://doi.org/10.1016/j.chemosphere.2010.08.021\u003c/li\u003e\n\u003cli\u003eCroft B A (1990) Arthropod biological control agents and pesticides. John Wiley and Sons Inc, New York\u003c/li\u003e\n\u003cli\u003eCroft BA, Brown AWA (1975) Responses of Arthropod Natural Enemies to Insecticides. Annual Review of Entomology 20:285\u0026ndash;335. https://doi.org/10.1146/annurev.en.20.010175.001441\u003c/li\u003e\n\u003cli\u003eDekeyser MA (2005) Acaricide mode of action. Pest Management Science 61:103\u0026ndash;110. https://doi.org/10.1002/ps.994\u003c/li\u003e\n\u003cli\u003eDermauw W, Wybouw N, Rombauts S, Menten B, Vontas J, Grbić M, Clark RM, Feyereisen R, Van Leeuwen T (2012) A link between host plant adaptation and pesticide resistance in the polyphagous spider mite \u003cem\u003eTetranychus urticae\u003c/em\u003e. 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Extensive use of chemical pesticides has led to the development of resistance in this pest, making biological control agents a viable alternative for its management. The predatory mites, \u003cem\u003eNeoseiulus californicus\u003c/em\u003e McGregor and \u003cem\u003ePhytoseiulus persimilis\u003c/em\u003e Athias-Henriot (Acari: Phytoseiidae) are the most important predators of the two-spotted spider mites. In this study, the toxicity of two acaricides, chlorfenapyr and acequinocyl, on these predators was evaluated, and the walking behavior of predatory mites after exposure to residues of the pesticides was assessed using a video tracking system. While the LC\u003csub\u003e50\u003c/sub\u003e of both acaricides was estimated to be higher than the field concentration, chlorfenapyr was found to be five times more toxic than acequinocyl. In the behavioral assay, both acaricides significantly affected the distance and speed of walking, resting time, and frequency of stops of both predatory mites. In the escape assay, both compounds had an irritable effect on both predatory mites, as the mites avoided areas contaminated with pesticide residues and their presence in the untreated area was significantly longer than in the contaminated area (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the study found no correlation between toxicity and repellency. According to the results of this study, \u003cem\u003eN. californicus\u003c/em\u003e and \u003cem\u003eP. persimilis\u003c/em\u003e possess the ability to detect the presence of pesticide residues in their environment and try to avoid them. Moreover, both compounds are at low risk to these mites, but acequinocyl is much safer and is a suitable option for using in integrated pest management.\u003c/p\u003e","manuscriptTitle":"Repellent and acaricidal effects of the chlorfenapyr and acequinocyl on the predatory mites, Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-09 06:47:17","doi":"10.21203/rs.3.rs-4604689/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3f6ae87-f869-464c-b059-784da0bba816","owner":[],"postedDate":"July 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T16:01:10+00:00","versionOfRecord":{"articleIdentity":"rs-4604689","link":"https://doi.org/10.1007/s10493-024-00995-4","journal":{"identity":"experimental-and-applied-acarology","isVorOnly":false,"title":"Experimental and Applied Acarology"},"publishedOn":"2025-01-17 15:57:19","publishedOnDateReadable":"January 17th, 2025"},"versionCreatedAt":"2024-07-09 06:47:17","video":"","vorDoi":"10.1007/s10493-024-00995-4","vorDoiUrl":"https://doi.org/10.1007/s10493-024-00995-4","workflowStages":[]},"version":"v1","identity":"rs-4604689","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4604689","identity":"rs-4604689","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}