Susceptibility of Apis mellifera to Bacillus thuringiensis Berliner, nuclear multiple polyhedrosis virus, azadirachtin, pyrethrins, and abamectin

Susceptibility of Apis mellifera to Bacillus thuringiensis Berliner, nuclear multiple polyhedrosis virus, azadirachtin, pyrethrins, and abamectin | 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 Susceptibility of Apis mellifera to Bacillus thuringiensis Berliner, nuclear multiple polyhedrosis virus, azadirachtin, pyrethrins, and abamectin Michelle Alejandra Iubini-Aravena, Gonzalo Iván Silva, Marcela Rodríguez, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6421763/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In recent years, the density of Apis mellifera L. has declined due to a phenomenon known as Colony Collapse Disorder. The irrational use of pesticides is considered one of the causes. However, the primary focus in this area is on synthetic insecticides. Hence, this research aimed to assess the toxicity of commercial doses of Bacillus thuringiensis Berliner, nuclear multiple polyhedrosis virus (NPVs), azadirachtin, pyrethrins, and abamectin against A. mellifera . In contact and ingestion bioassays, the highest toxicity was achieved with abamectin (100% mortality). Pyrethrins exhibited a mortality rate of 53.2% whereas NPVs did not result in any mortality. Although untreated bees preferred control over treated diet in repellency bioassays, no significant differences between treatments were observed. We concluded that abamectin and pyrethrins are the most harmful to bees by contact and ingestion toxicity. Honeybee bioinsecticides abamectin pyrethrins Neem Bacillus thuringiensis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Honeybees ( Apis mellifera L.; Hymenoptera: Apidae) are essential pollinators for many economically important crops. However, due to pesticide exposure, a worldwide decline in population density has been observed in recent years, a phenomenon called Colony Collapse Disorder (CCD) (Suryanarayanan y Kleinman, 2013). Several factors have contributed to this decline, including changes in land use, loss of foraging habitat, parasites, diseases, and irrational use of insecticides (Yasuda et al. 2017 ). Therefore, a single cause has not been identified. It is suggested that multiple factors may contribute to cumulative adverse effects on honeybees (Grillone et al. 2017 ). However, the use of synthetic insecticides is one of the most relevant and studied factors (Del Sarto et al. 2014 ). Hardstone and Scott ( 2010 ) determined that A. mellifera , in comparison with other insect species, is more sensitive to insecticides such as organophosphates (Naggar et al. 2015a , 2015b and 2015c ; Wang et al. 2020 ; Delkash-Roudsari et al. 2022 ), pyrethroids (Hardstone and Scott, 2010 ; Wang et al. 2020 ), neonicotinoids (Blacquiere et al. 2012 ; Badawy et al. 2015 ; Lundin et al. 2015 ; Woodcock et al. 2017 ; Delkash-Roudsari et al. 2022 ), fipronil (Muñoz-Capponi et al. 2018 ; De Carvalho et al. 2024 ), Indoxacarb (Pashte and Patil, 2018 ), and some insect growth regulators (Delkash-Roudsari et al. 2022 ), among others. Neonicotinoids are restricted in the European Community due to their adverse effect on pollinators (Laycock et al. 2014 ). Insecticides of natural origin or bioinsecticides are alternatives to synthetic pesticides, but the amount of authorized commercial formulations is limited in countries with emerging economies (Isman, 2020 ). The most common options available on the market are Bacillus thuringiensis Berliner, pyrethrins from Tanacetum cinerariifolium (Trevir.) Sch.Bip. (Asteraceae), Neem from Azadiractha indica J. (Meliaceae), avermectins, spinosines, and some nucleopolyhedrovirus for Lepidoptera pest control. However, despite being insecticides of natural origin, they are not necessarily less toxic to non-target organisms or pollinators compared to synthetic ones. For example, Li et al. ( 2022 ) found that several gene clusters are negatively influenced by abamectin exposure in A. cerana cerana and A. mellifera lingustica . Steinigeweg et al. ( 2022 ) determined that B. thuringiensis subspecies aizawai (strain: ABTS-1857) inhibited brood development and reduced the percentage of emerged worker bees. In the case of Neem, Lopes-Amaral et al. ( 2015 ) documented that larvae and forager bees reduce their survival rate by ingesting food contaminated with this bioinsecticide. González-Gómez et al. ( 2016 ) determined that mortality and development of bee brood, queen oviposition, and colony performance increased when Neem oil concentrations increased. Hence, this research aimed to assess the toxicity of commercial formulations of Bacillus thuringiensis Berliner, nuclear multiple polyhedrosis virus (NPVs), azadirachtin, pyrethrins, and abamectin against Apis mellifera. Materials and methods Insects The bioassays were carried out with honeybee foragers (< 7 days old). The adult workers were obtained from the "El Nogal" Experimental Station at the Chillan Campus, Universidad de Concepcion, Chile. Bee colonies were maintained according to the standard rearing techniques, and seven days before bioassays, operculated brood frames were removed from the colony and transferred to our laboratory to obtain insects of known age. In the laboratory, the brood frame was kept in an entomological cage (60 x 60 x 30 cm) in a bioclimatic chamber (SHEL LAB, Model 2015-2E, Cornelius, Oregon, USA) at controlled environmental conditions [28 ± 5°C and 90 ± 5% of relative humidity (RH)] until adult emergence. Insecticides This assessment focused exclusively on commercial formulations of authorized and readily available bioinsecticides for agricultural pest control. The products evaluated included B. thuringiensis var. Kurstaki (DIPEL® WG), Nuclear Polyhedrosis Virus of Mamestra brassicae L. (NPVs) (En Vivo SC®), azadirachtin (Neem-X®), pyrethrins (Tec Fort®), and abamectin (Fast Plus®). These bioinsecticides were selected due to their prevalent use among fruit and vegetable growers in Chile (Table 1 ). Table 1 Insecticides assessed under laboratory conditions against Apis mellifera . Insecticide Active ingredient (a.i.) Recommended Dose IRAC Group* Company FAST PLUS Abamectin 18.0 g L − 1 6. Glutamate-gated chloride channel (GluCl) allosteric modulators ANASAC Chile S.A. NEEM-X® Azadirachtin 4.0 g L − 1 UN. Compounds of unknown or uncertain mode of action Marketing Arm International Inc. USA. DIPEL® WG Bacillus thuringiensis subsp. kurstaki 64 g Kg − 1 11. Microbial disruptors of insect midgut membranes Valent BioSciences, USA. EN VIVO SC® Nuclear multiple polyhedrosis virus of Mamestra brassicae (NPVs) 281.88 g L − 1 31. Baculoviruses. Host-specific occluded pathogenic viruses Point Chile S.A. TEC FORT® Pyrethrins 22.5 g L − 1 3. Sodium channel modulators MIP Agro Ltda. Chile. *IRAC= "The Insecticide Resistance Action Committee". The IRAC mode of action classification online (IRAC International, 2024) ( www.irac-online.org ) Bioassays In all bioassays, we initially determined the concentration range for each insecticide that caused from 0.0–100%. Then, three to four intermediate concentrations were evenly distributed between this range. Topical toxicity We used the methodology of Del Sarto et al. ( 2014 ) with slight modifications. To reduce bee's mobility, they were placed in a freezer at a temperature of -15 ± 2°C for 5 min (De Carvalho et al. 2024 ). Then, 2 µL of each insecticide concentration was applied in the ventral part of the ​​ abdomen with a micropipette, as Delkash-Roudsari et al. ( 2022 ) suggested. Each treatment had ten replications of 15 insects. The untreated control was similarly handled but exposed to distilled water. Mortality was evaluated 24 hours post-treatment, with a maximum accepted control mortality of 10%, adjusted using Abbott's equation (Abbott, 1925 ). A bee that did not react to an external stimulus consisting of touching with a brush and/or being found upside down was considered dead, as Del Sarto et al. ( 2014 ) suggested. Treated honeybees were transferred to 1 L plastic jars fitted with a lid with two holes for holding two 20 mL syringes containing a diet of 5 mL of honey (70%) and 5 mL of distilled water, respectively, both offered ad libitum . In addition, to facilitate the bee's mobility inside the jar, two wooden tongue depressors measuring 113 x 10 x 2 mm were placed, forming an "X" (Fig. 1 ). Ingestion Bioassay methodologies outlined by Del Sarto et al. ( 2014 ) and Phan et al. ( 2020 ) were utilized, incorporating minor modifications. This bioassay includes insecticides identified as the most or least toxic based on results from topical bioassays: Fast Plus (abamectin) and Neem-X® (azadirachtin) (Fig. 2 ). The methodology involved placing bees in 1 L plastic jars as previously done. Bees were starved for 4 h and then fed ad libitum with a 20 mL syringe containing 5 mL of a 70% honey mixture and the insecticide treatment. Then, the syringes were removed and replaced with a syringe with 5 mL of honey (70%). Mortality was assessed 24 h after treatment, and the mortality criterion was the same as in topical bioassay. The experimental unit consisted of 15 bees; each treatment was replicated ten times, and mortality was adjusted using Abbott's equation (Abbott, 1925 ). Residuality The residuality bioassays involving the most toxic compounds, Tec Fort® (pyrethrins) and Fast Plus (abamectin) (Fig. 2 ) in its commercial doses (22.5 g L − 1 and 18.0 g L − 1, respectively), were conducted using methodologies from Husain et al. ( 2014 ) and Abati et al. ( 2023 ), with minor modifications. The initial phase of the study was performed in an eight-year-old Fuji apple orchard at the "El Nogal" Experimental Station, located on the Chillan Campus of the Universidad de Concepcion, Chile. We randomly selected three central rows in each orchard containing 45 trees. Then, using a random block design, we had three treatments (each treatment had three trees) and five replicates; each block contained an untreated control. Treatments were sprayed with a two-wheel barrow sprayer of 50L capacity (Lerpain pulverizadores, Puente Alto, Chile) in a volume of 2.6 L per tree, using a hollow cone nozzle and 150 psi of working pressure. After 2, 24, and 48 h of treatment, five random leaves were collected from the central tree, identified, and stored in hermetically sealed bags; in less than 10 min, they were transferred to the laboratory. These leaves were placed in 1 L plastic jars to maintain leaf turgor; stems were kept in 1.5 mL Eppendorf tubes with a water solution and 2.0% agar. Then, each jar was inoculated with 15 honeybees previously cooled to -15 ± 2°C for 5 min to reduce mobility and aggressiveness. Bees were fed two syringes with 5 mL of honey (70%) and 5 mL of distilled water offered ad libitum . Mortality was assessed at 24, 48, 72, and 96 h until the untreated control showed a 50% mortality. Repellency This bioassay was performed via a double-choice method adapted from Signoretti et al. ( 2012 ), using the commercially recommended doses of each insecticide (Table 2 ). A Y-shaped glass olfactometer was used (two lateral arms 16.5 cm long and 3 cm in diameter were connected to a central tube with the same diameter and dimensions). Each arm was sealed with a rubber cap and attached to a glass flask containing the odor sources (treatment or control). The aroma source from the treatment included a cotton ball soaked with 20 mL of honey (70%) and mixed with the commercial dose of the respective insecticide. The untreated control contained a cotton ball soaked only with 20 mL of honey (70%). Each arm of the Y-shaped tube was connected to an aquarium air pump Super LB-1000 (Shanghai Luby Pet Industries Co., Shanghai, China) of 1.6 L air min − 1 flow. The airflow was filtered with an activated carbon filter and passed through a distilled flask to humidify the air. The airflow rate was controlled by SHLLJ LZQ-6 flow meters (Yuyao Shunhuan Instruments Co., Shanghai, China), which provided a constant rate of 1 L air min − 1 . Lines were traced outside each arm and at the base of the Y-shaped tube at 7 cm from the center. Then, groups of 75 adult honeybees were placed individually on the central tube for each concentration, and the repellent effect was evaluated. Repellency was monitored for five minutes after the insect crossed the threshold line of the central tube. Choices were recorded when an insect crossed the line of one of the two arms and stayed there for 20 seconds (Signoretti et al.). 2012). The bees that chose one of the two arms during five minutes were the only ones considered in the bioassay. Each insect was used a single time to prevent associative learning. The glass Y-shape tube was washed with neutral washing soap, distilled water, 90% ethanol (v/v), and 90% acetone (v/v) between replicates, while the connections with the odor sources were alternated to minimize bias. Table 2 Lethal Concentration 50% (LC 50 ) and 90% (LC 90 ) at 24 hours of abamectin, pyrethrins, nuclear multiple polyhedrosis virus of Mamestra brassicae (NPVs) and azadirachtin assessed by topical application against Apis mellifera . Treatment N a b ± EE b LC 50 (LC 95%) c (mg L − 1 ) LC 90 (LC 95%) d (mg L − 1 ) Pr > X 2e TR 50 f Abamectin 900 2.36 ± 0.38 0.221 (0.109–0.311) 0.77 (0.582–1.242) < 0.0001 1 Pyrethrins 900 1.41 ± 0.29 39.82 (8.85–67.05) 322.2 (156.1-7292.9) < 0.0001 180 NPVs g 1050 2.01 ± 0.24 2393.9 (1844.4-3088.5) 10386 (6983.9-20505) < 0.0001 10832 Azadirachtin 900 1.2 ± 0.21 2081 (14782 − 3983) 46847 (13148–670103) < 0.0001 9416 a Number of treated insects, b Slope value. c Lethal Concentration at 50% of effect with confidence limits at 95% probability. d Lethal Concentration at 90% of impact with confidence limits at 95% probability. e Model fit to a straight line. f Toxicity ratio 50% (RT 50 ) = CL 50 treatment/ CL 50 more toxic treatment. g Nuclear multiple polyhedrosis virus of Mamestra brassicae Table 3 Lethal Concentration 50% (LC 50 ) and 90% (LC 90 ) at 24 hours of abamectin and azadirachtin assessed by ingestion against Apis mellifera . a Number of treated insects, b Slope value. c Lethal Concentration at 50% of effect with confidence limits at 95% probability. d Lethal Concentration at 90% of effect with confidence limits at 95% probability. e Model fit to a straight line. f Toxicity ratio 50% (RT 50 ) = CL 50 treatment/ CL 50 more toxic treatment. Treatment N a b ± EE b LC 50 (LC 95%) c (mg L − 1 ) LC 90 (LC 95%) d (mg L − 1 ) Pr > X 2e TR 50 f Abamectin 1200 2.09 ± 0.20 0.233 (0.166-0.3) 0.949 (0.725-429) < 0.0001 1 Azadirachtin 750 2.09 ± 0.40 800.365 (631.88-1103.34) 3278.05 (1990.14-9370.63) < 0.0001 3621 a Number of treated insects, b Slope value. c Lethal Concentration at 50% of effect with confidence limits at 95% probability. d Lethal Concentration at 90% of effect with confidence limits at 95% probability. e Model fit to a straight line. f Toxicity ratio 50% (RT 50 ) = CL 50 treatment/ CL 50 more toxic treatment. Experimental design and statistical analysis The laboratory bioassays were performed with a completely randomized experimental design, while the residuality bioassay was a randomized block experimental design. The results of topical application and ingestion were adjusted to the Probit model (Finney, 1971 ) to estimate the lethal concentration of 50% (LC 50 ) and 90% (LC 90 ) mortality using the PoloPlus software (LeOra Software, 2022). Residuality analysis was performed with a Kaplan and Meier ( 1958 ) survival test using R software (R Core Team, 2020 ), and repellency data were analyzed with a Chi-square (X 2 ) test, also with R software. Results Topical application Abamectin showed the highest contact toxicity, with 100% mortality at its commercial dose and half and one-eighth of this (Fig. 2 A). Even this insecticide at the lowest dose evaluated, corresponding to 1/64 of commercial dose (0.28 mg L − 1 ), exceeded 50% of dead bees (57,89%). Pyrethrins at the commercial concentration of 45 mg L − 1 resulted in a mortality rate of 53.2%. NPVs did not cause mortality at the recommended dosage, so the concentration was increased by 64 times (3264 mg L − 1 ) to achieve a mortality rate of 90.7% (Fig. 2 D). Bacillus thuringiensis and azadirachtin, at 32 and 64 times the commercial dose, respectively, did not achieve 10% mortality, making Probit analysis unfeasible. The lowest LC 50 was obtained by abamectin with CL 50 = 0.221 mg L − 1, while NPVs and pyrethrins showed a CL 50 of 2393 mg L − 1 and 39.82 mg L − 1, respectively. All treatments differed significantly because the confidence limits did not overlap (Robertson et al. 2020 ). The TR 50 indicated that abamectin was 180 and 10832-fold more toxic to A. mellifera than NPVs and pyrethrins, respectively. Ingestion bioassays Abamectin was the more toxic insecticide, with a mortality of 100% at the commercial dosage (18 mg L − 1 ) (Fig. 3 ), while the lowest assessed concentration corresponding to 1/128 (0.14 mg L − 1 ) showed 20.55% of dead bees (LC 50 = 0.233 mg L − 1 ; LC 90 = 0.949 mg L − 1 ). Neem (azadirachtin) exhibited the lowest toxicity because it was necessary to increase to 64-fold the commercial dose to obtain a mortality of 1.43%. This insecticide had an LC 50 and LC 90 of 800.3 mg L − 1 and 3278.05 mg L − 1 , respectively. Furthermore, both treatments were significantly different because the confidence limits did not overlap (Robertson et al. 2020 ), and according to RT 50, abamectin is 3620-fold more toxic than Neem Residuality Bee longevity decreased in all treatments compared to the control, and shorter contact times with treated leaves resulted in lower survival rates (Fig. 4 ). Results from leaves collected two hours after the insecticide spray indicated that at 24 hours, 96% of the bees exposed to leaves sprayed with pyrethrins remained alive, while at 120 hours, 30.7% were still alive. These treatments did not show significant differences from the control sprayed only with water ( p = 0.0572). For abamectin, at 24 h, 8.0% of the bees were alive at 120 h, and none of the insects remained alive. This treatment significantly differed from the untreated control and pyrethrins ( p = 0.0025). In leaves collected 24 h after spraying with pyrethrins, 90.7% and 49.4% survival rates were observed at 24 and 120 h, without significant differences from the untreated control ( p = 0.9893). Abamectin caused 53.3% and 100% mortality at 24 and 96 h, respectively, registering statistical differences from the untreated control and pyrethrins ( p = 0.001). In leaves collected 48 h after spraying insecticides, those treated with pyrethrins showed 76% and 60.9% alive bees at 24 and 120 h, without significant differences from the untreated control ( p = 0.469). For abamectin, survival rates at 24 and 120 H were 92% and 69.3%, respectively. This treatment showed significant differences compared to the control (p = 0.0173) but not compared to pyrethrins ( p = 0.9185). Repellency NPVs was the only treatment that did not exhibit repellency. It attracted bees, with 55.26% choosing a diet containing insecticide and 44.74% opting for untreated control (Fig. 5 ). The highest repellent activity was observed in B. thuringiensis , with 63.16% control preference versus 36.84% of food mixed with the commercial dose. In bioassays with Neem, 51.32% of bees preferred the untreated control and 46.68% contaminated food. Abamectin and pyrethrins showed 53.95% preference for the untreated control and 46.05% for the insecticide treatment. The Chi-square test (χ 2 ) indicated no significant differences ( p = 0.1338) among treatments. Discussion Natural insecticides are considered less toxic to pollinators and non-target insects than synthetic ones. However, our results show that some of these are as toxic as the synthetic compounds. The contact and ingestion toxicity obtained for abamectin agrees with Del Sarto et al. ( 2014 ), who found that abamectin is moderately toxic to A. mellifera , and more harmful than deltamethrin, an insecticide of the pyrethroid family. Similarly, Costa et al. ( 2014 ) assessed, under laboratory conditions, the toxicity against A. mellifera of all insecticides used in Brazilian melon crops, concluding that abamectin, thiametoxam, and chlorfenapyr were the most toxic. Research indicates that pyrethroids, including deltamethrin (Besard et al. 2010 ; Del Sarto et al. 2014 ; Stanley et al. 2015 ), cypermethrin (Pashte and Patil, 2018 ), lambda-cyhalothrin, and bifenthrin (Wang et al. 2020 ), exhibit a high level of toxicity against A. mellifera . Thus, we expect the same harmful effects as pyrethrins but with a shorter duration since their residuality does not exceed 24 h (Feng et al. 2018 ). The toxicity of Neem (azadirachtin) in contact and ingestion bioassays did not exceed 2.0% mortality, coinciding with Melathopoulos et al. ( 2000 ), who found that an azadirachtin-rich extract (Neem-aza) and Neem oil did not exceed 10% mortality in bees infested with V. jacobsoni and those free of this pest. However, Peng et al. ( 2000 ) demonstrated that azadirachtin possesses the highest oral toxicity in A. mellifera , particularly when infested with V. jacobsoni. In addition, azadirachtin greatly reduces syrup consumption by worker bees, and over 90% of treated while prepupae and pupae exhibit abnormal mouthpart and appendage pigmentation. Kaur et al. ( 2022 ) also studied the effect of three concentrations (0.05%, 0.1%, and 1.0%) of Neem oil in A. mellifera larvae. They found that survival decreased as the dose increased. Emerged pupae reduction and early adult mortality showed the same trend. Our findings and additional research highlight the negative traits of certain botanical insecticides. Isman (2000) noted that the varied insecticidal activity of these insecticides is the reason they are not widely used. Bacillus thuringiensis was one of the less toxic insecticides. There are no previous studies about the toxicity of this insecticide in bees. However, in other pollinators such as Bombus terrestris L. (Hymenoptera: Apidae), Mommaerts et al. ( 2010 ) assessed Dipel ( B. thuringiensis subsp. kurstaki) and Xentari ( B. thuringiensis subsp. aizawai) against this species of insect, at the highest doses for field conditions, obtained a maximum mortality of 5.0%. Similarly, Libardoni et al. ( 2021 ), in a bioassay with a contaminated diet with Dipel, found that the survival rate of Africanized honey bees after 144 h of consuming the contaminated food was 80%. Most studies on the residual effects of insecticides against A. mellifera focus on synthetic insecticides, such as pyrethroids and neonicotinoids; there are nearly no studies on insecticides of natural origin. However, synthetic insecticides, such as pyrethroids, have been chemically improved to increase their field efficacy and residual effect under field conditions and are toxic to bees. Biopesticides do not have residue problems because they degrade fast in the environment (Kumar, 2012 ). However, novel formulations such as nano emulsions increase residuality (Ayilara et al. 2023 ). Repellency is one of the most studied properties of natural toxic compounds, mainly essential oils. In our research, although untreated bees exhibited higher preferences for control than the treated diet, no significant differences existed between these treatments. Thus, we infer that the studied insects made their choice randomly rather than being influenced by the type of diet. Naumann et al. ( 1994 ) demonstrated that bees can discriminate between untreated sugar syrup and syrup containing concentrations as low as 0.1 ppm of azadirachtin. Our research found azadirachtin to have a 51.3% repellency, contrary to Barbosa et al. ( 2015 ), who reported a maximum of 7.0%. Melathopoulos et al. ( 2000 ) assessed the toxicity of Neem against A. mellifera , concluding that Neem mixed with sucrose syrup in a concentration > 0.01 mg mL − 1 is a feeding deterrent to A. mellifera . Bacillus thuringiensis repelled bees by 63.2% (Fig. 5 ), whereas NPVs attracted them slightly, as bees favored the treated diet over the control. The low toxicity of these insecticides reduces this effect. Abamectin did not negatively affect bee behavior; treated and untreated diets exhibited similar repellent effects. Mubin et al. ( 2024 ) reached a similar conclusion in their study involving Tetragonula laeviceps Smith (Hymenoptera: Apidae: Meliponini). Their research indicated that the presence of abamectin did not influence the behavior of T. laeviceps , as evidenced by similar preferences between honey containing abamectin and honey alone. Natural insecticides can harm A. mellifera , similar to conventional ones. Abamectin and pyrethrins affect bees through contact and ingestion, even at commercial and sublethal doses. Farmers should protect bees when using these substances. Conversely, the nuclear polyhedrosis virus and B. thuringiensis are safe for honeybees. Declarations Acknowledgements Who would like to thank Dr. Selim Musleh of Núcleo Milenio de Salmónidos Invasores (INVASAL), Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Concepción. Chile, for his technical assistance in statistical analysis and R software management. Ethical Approval not applicable Competing interests: The authors declare no competing interests Financial interests: The authors declare they have no financial interests Author contribution Conceptualization, G.S.A., J.C.R., M.R., and J.S.B. Experiments M.I.A, B.C.M. Statistical Analysis M.I.A., G.S.A., M.R., writing-original draft preparation, M.I.A., writing-review and editing G.S.A., J.C.R. M.R. J.S.B., M.I.A. All authors approved the final manuscript. Data availability: No datasets were generated or analyzed during the current study References Abbott WS (1925) A method of computing the effectiveness of an insecticide. 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J Insect Physiol 82:85-91. https://doi.org/10.1016/j.jinsphys.2015.09.004 Naggar Y, Codling G, Vogt A, Naiem E, Mona M, Seif A, Giesy JP (2015b) Organophosphorus insecticides in honey, pollen and bees ( Apis mellifera L.) and their potential hazard to bee colonies in Egypt. Ecotoxicol Environ Saf 114:1-8. https://doi.org/10.1016/j.ecoenv.2014.12.039 Naggar Y, Vogt A, Codling G, Naiem E, Mona M, Seif A, Robertson SJ, Giesy JP (2015c) Exposure of honeybees ( Apis mellifera ) in Saskatchewan, Canada to organophosphorus insecticides. Apidologie 46:667-678. http://dx.doi.org/10.1007/s13592-015-0357-y Naumann K, Currie RW, Isman MB (1994) Evaluation of the repellent effects of a neem insecticide on foraging honey bees and other pollinators. Can Entomol 126:225-230. http://dx.doi.org/10.4039/Ent126225-2 Pashte VV, Patil SS (2018) Toxicity and poisoning symptoms of selected insecticides to honey bees ( Apis mellifera L.). Arch Biol Sci 70(1):5-12. http://dx.doi.org/10.2298/ABS170131020P Peng CYS, Trinh S, Lopez JE, Mussen EC, Hung A, Chuang R (2000) The effects of azadirachtin on the parasitic mite, Varroa jacobsoni and its host honey bee ( Apis mellifera ), J Apic Res 39(3-4):159-168. https://doi.org/10.1080/00218839.2000.11101037 Phan NT, Joshi NK, Rajotte EG, López-Uribe MM, Zhu F, Biddinger DJ (2020) A new ingestion bioassay protocol for assessing pesticide toxicity to the adult Japanese orchard bee ( Osmia cornifrons ). Scientific Reports 10(1): 9517. https://doi.org/10.1038/s41598-020-66118-2 R Core Team (2020) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/ Robertson M, Jones M, Olguin E, Alberts B (2020) Bioassays with arthropods. CRC Press Boca Raton. doi: 10.1201/9781420004045. Signoretti AGC, Peñaflor MFGV, Bento JMS (2012) Fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), female moths respond to herbivore-induced corn volatiles. Neotrop Entomol 41: 22-26. https://doi.org/ 10.1007/s13744-011-0003-y Stanley J, Sah K, Jain SK, Bhatt JC Sushil SN (2015) Evaluation of pesticide toxicity at their field recommended doses to honeybees, Apis cerana and A. mellifera through laboratory, semi-field and field studies. Chemosphere 119:668-674. https://doi.org/10.1016/j.chemosphere.2014.07.039 Steinigeweg C, Alkassab AT, Erler S, Beims H, Wirtz IP, Richter D, Pistorius J (2022) Impact of a microbial pest control product containing Bacillus thuringiensis on brood development and gut microbiota of Apis mellifera worker honey bees. Microb Ecol 85(4): 1300-1307. https://doi.org/10.1007/s00248-022-02004-w Suryanarayanan S, Kleinman DL (2013) Bee coming experts: The controversy over insecticides in the honey bee colony collapse disorder. Soc Stud Sci 43(2): https://doi.org/10.1177/0306312712466186 215-240. Wang Y, Zhu YC, Li W (2020) Comparative examination on synergistic toxicities of chlorpyrifos, acephate, or tetraconazole mixed with pyrethroid insecticides to honey bees ( Apis mellifera L.). Environ Sci Pollut Res 27:6971-6980. https://link.springer.com/article/10.1007/s11356-019-07214-3 Woodcock BA, Bullock JM, Shore RF, Heard MS, Pereira MG et al (2017) Country-specific effects of neonicotinoid pesticides on honey bees and wild bees. Science 356:1393-1395. https://doi.org/10.1126/science.aaa1190 Yasuda M, Sakamoto Y, Goka K, Nagamitsu T, Taki H (2017) Insecticide susceptibility in Asian honey bees ( Apis cerana (Hymenoptera: Apidae)) and implications for wild honey bees in Asia. J Econ Entomol. 110(2): 447-452. https://doi.org/10.1093/jee/tox032 Cite Share Download PDF Status: Posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6421763","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448133662,"identity":"bbf5c774-ebc5-41eb-94b1-3be7b56bfe71","order_by":0,"name":"Michelle Alejandra Iubini-Aravena","email":"","orcid":"","institution":"Universidad de Concepción Facultad de Agronomía: Universidad de Concepcion Facultad de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"Alejandra","lastName":"Iubini-Aravena","suffix":""},{"id":448133663,"identity":"724288dd-a4ae-4505-aafc-2ee1c3d2a44b","order_by":1,"name":"Gonzalo Iván Silva","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYJCCAw8YGOQYGBJAbAsitQAVG0O1SBBpDVBxYgPRWuTd2x8eSGyzS99wPPngB4YaIrQYnjljANSSnLvhzLNkCYZjxGiZkQP0yxnm3A03cswYGBuI0pL+AKilPt3gRv434rTISyQYHEioOJxgcCOHjTgtBjxnQFqOG84888xYIoEYv8i3tz/+8MGgWp7vePLDDx9qbIiw5QAyL4GwBqAtDcSoGgWjYBSMgpENAKYyPQaaJcSgAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2463-5540","institution":"Universidad de Concepcion Facultad de Agronomia","correspondingAuthor":true,"prefix":"","firstName":"Gonzalo","middleName":"Iván","lastName":"Silva","suffix":""},{"id":448133664,"identity":"c731ac37-d299-4161-91f2-9de1d3e842f1","order_by":2,"name":"Marcela Rodríguez","email":"","orcid":"","institution":"Universidad de Concepcion Facultad de Ciencias Naturales y Oceanograficas","correspondingAuthor":false,"prefix":"","firstName":"Marcela","middleName":"","lastName":"Rodríguez","suffix":""},{"id":448133665,"identity":"22c210f8-9756-41cd-8378-172b686be8dc","order_by":3,"name":"J. Concepción Rodríguez-Maciel","email":"","orcid":"","institution":"Colegio de Postgraduados Campus Montecillo: Colegio de Postgraduados","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"Concepción","lastName":"Rodríguez-Maciel","suffix":""},{"id":448133666,"identity":"a3176f95-93d0-4531-976c-037f858fd46c","order_by":4,"name":"Julio S. Bernal","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Julio","middleName":"S.","lastName":"Bernal","suffix":""},{"id":448133667,"identity":"1bfe4280-2bbb-4ecf-863d-8ca380f5cd8b","order_by":5,"name":"Bastián Campos-Monsalve","email":"","orcid":"","institution":"Universidad de Concepcion Facultad de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Bastián","middleName":"","lastName":"Campos-Monsalve","suffix":""}],"badges":[],"createdAt":"2025-04-10 15:55:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6421763/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6421763/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81627554,"identity":"fdb84f83-fce9-4312-85c7-257b3e3f8230","added_by":"auto","created_at":"2025-04-29 10:40:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":383282,"visible":true,"origin":"","legend":"\u003cp\u003eDevice for honeybee confinement after treated topically, including two wooden tongue depressors to facilitate the bee's mobility inside the jar and two 20 mL syringes containing a diet of 5 mL of honey (70%) and 5 mL of distilled water.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6421763/v1/ee750067bae3e13f4995c424.png"},{"id":81628116,"identity":"965c594a-427f-436f-8f9e-9bfcd94e1ba8","added_by":"auto","created_at":"2025-04-29 10:48:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21401,"visible":true,"origin":"","legend":"\u003cp\u003eMortality (%) by contact toxicity of azadirachtin (\u003cstrong\u003eA\u003c/strong\u003e), abamectin (\u003cstrong\u003eB\u003c/strong\u003e), pyrethrins (\u003cstrong\u003eC\u003c/strong\u003e), and nuclear multiple polyhedrosis virus of \u003cem\u003eMamestra brassicae\u003c/em\u003e (NPVs) (\u003cstrong\u003eD\u003c/strong\u003e) against \u003cem\u003eApis mellifera\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6421763/v1/78773b1132731bf740b6a19a.png"},{"id":81627552,"identity":"2d97a53a-3d6f-44d9-a1f9-a416b534f661","added_by":"auto","created_at":"2025-04-29 10:40:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16377,"visible":true,"origin":"","legend":"\u003cp\u003eMortality (%) by ingestion toxicity of azidarachtin (A) and abamectin (B) against \u003cem\u003eApis mellifera\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6421763/v1/fa7a4aa08d4a574105dc14e5.png"},{"id":81627555,"identity":"f99ba851-71e1-4370-8744-c46a315947ed","added_by":"auto","created_at":"2025-04-29 10:40:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59501,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival probability of\u003cem\u003e A. mellifera\u003c/em\u003e at 2, 24, and 48 hrs exposed to leaf treated with abamectin and pyrethrins.\u003c/p\u003e\n\u003cp\u003e*Values with different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e≤0.05) by chi-square (X\u003csup\u003e2\u003c/sup\u003e) test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6421763/v1/50a103256e368bfce4571fbb.png"},{"id":81628867,"identity":"1c3fa6ac-471d-427c-9538-231092496881","added_by":"auto","created_at":"2025-04-29 10:56:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9564,"visible":true,"origin":"","legend":"\u003cp\u003ePreference (%) of \u003cem\u003eA. mellifera\u003c/em\u003e for diet treated with insecticide or control. *NPVs= Nuclear multiple polyhedrosis virus of \u003cem\u003eMamestra brassicae\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6421763/v1/f67ea7e77532e15f2c649f74.png"},{"id":85404373,"identity":"90c58e19-ba9b-492c-99ca-30ae28ef8ad4","added_by":"auto","created_at":"2025-06-25 12:47:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1378781,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6421763/v1/b05ce5ec-ccaf-4489-aa9c-99d5bfbfa844.pdf"}],"financialInterests":"","formattedTitle":"Susceptibility of Apis mellifera to Bacillus thuringiensis Berliner, nuclear multiple polyhedrosis virus, azadirachtin, pyrethrins, and abamectin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHoneybees (\u003cem\u003eApis mellifera\u003c/em\u003e L.; Hymenoptera: Apidae) are essential pollinators for many economically important crops. However, due to pesticide exposure, a worldwide decline in population density has been observed in recent years, a phenomenon called Colony Collapse Disorder (CCD) (Suryanarayanan y Kleinman, 2013). Several factors have contributed to this decline, including changes in land use, loss of foraging habitat, parasites, diseases, and irrational use of insecticides (Yasuda et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, a single cause has not been identified. It is suggested that multiple factors may contribute to cumulative adverse effects on honeybees (Grillone et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the use of synthetic insecticides is one of the most relevant and studied factors (Del Sarto et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Hardstone and Scott (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) determined that \u003cem\u003eA. mellifera\u003c/em\u003e, in comparison with other insect species, is more sensitive to insecticides such as organophosphates (Naggar et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e and \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015c\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Delkash-Roudsari et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), pyrethroids (Hardstone and Scott, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), neonicotinoids (Blacquiere et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Badawy et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lundin et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Woodcock et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Delkash-Roudsari et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), fipronil (Mu\u0026ntilde;oz-Capponi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; De Carvalho et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), Indoxacarb (Pashte and Patil, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and some insect growth regulators (Delkash-Roudsari et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), among others. Neonicotinoids are restricted in the European Community due to their adverse effect on pollinators (Laycock et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsecticides of natural origin or bioinsecticides are alternatives to synthetic pesticides, but the amount of authorized commercial formulations is limited in countries with emerging economies (Isman, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The most common options available on the market are \u003cem\u003eBacillus thuringiensis\u003c/em\u003e Berliner, pyrethrins from \u003cem\u003eTanacetum cinerariifolium\u003c/em\u003e (Trevir.) Sch.Bip. (Asteraceae), Neem from \u003cem\u003eAzadiractha indica\u003c/em\u003e J. (Meliaceae), avermectins, spinosines, and some nucleopolyhedrovirus for Lepidoptera pest control. However, despite being insecticides of natural origin, they are not necessarily less toxic to non-target organisms or pollinators compared to synthetic ones. For example, Li et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) found that several gene clusters are negatively influenced by abamectin exposure in \u003cem\u003eA. cerana cerana\u003c/em\u003e and \u003cem\u003eA. mellifera lingustica\u003c/em\u003e. Steinigeweg et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) determined that \u003cem\u003eB. thuringiensis\u003c/em\u003e subspecies \u003cem\u003eaizawai\u003c/em\u003e (strain: ABTS-1857) inhibited brood development and reduced the percentage of emerged worker bees. In the case of Neem, Lopes-Amaral et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) documented that larvae and forager bees reduce their survival rate by ingesting food contaminated with this bioinsecticide. Gonz\u0026aacute;lez-G\u0026oacute;mez et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) determined that mortality and development of bee brood, queen oviposition, and colony performance increased when Neem oil concentrations increased. Hence, this research aimed to assess the toxicity of commercial formulations of \u003cem\u003eBacillus thuringiensis\u003c/em\u003e Berliner, nuclear multiple polyhedrosis virus (NPVs), azadirachtin, pyrethrins, and abamectin against \u003cem\u003eApis mellifera.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInsects\u003c/h2\u003e \u003cp\u003eThe bioassays were carried out with honeybee foragers (\u0026lt; 7 days old). The adult workers were obtained from the \"El Nogal\" Experimental Station at the Chillan Campus, Universidad de Concepcion, Chile. Bee colonies were maintained according to the standard rearing techniques, and seven days before bioassays, operculated brood frames were removed from the colony and transferred to our laboratory to obtain insects of known age. In the laboratory, the brood frame was kept in an entomological cage (60 x 60 x 30 cm) in a bioclimatic chamber (SHEL LAB, Model 2015-2E, Cornelius, Oregon, USA) at controlled environmental conditions [28\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C and 90\u0026thinsp;\u0026plusmn;\u0026thinsp;5% of relative humidity (RH)] until adult emergence.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInsecticides\u003c/h3\u003e\n\u003cp\u003eThis assessment focused exclusively on commercial formulations of authorized and readily available bioinsecticides for agricultural pest control. The products evaluated included \u003cem\u003eB. thuringiensis\u003c/em\u003e var. Kurstaki (DIPEL\u0026reg; WG), Nuclear Polyhedrosis Virus of \u003cem\u003eMamestra brassicae\u003c/em\u003e L. (NPVs) (En Vivo SC\u0026reg;), azadirachtin (Neem-X\u0026reg;), pyrethrins (Tec Fort\u0026reg;), and abamectin (Fast Plus\u0026reg;). These bioinsecticides were selected due to their prevalent use among fruit and vegetable growers in Chile (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eInsecticides assessed under laboratory conditions against \u003cem\u003eApis mellifera\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInsecticide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive ingredient\u003c/p\u003e \u003cp\u003e(a.i.)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRecommended Dose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIRAC Group*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCompany\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFAST PLUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbamectin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e6.\u003c/b\u003e Glutamate-gated chloride channel (GluCl) allosteric modulators\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eANASAC Chile S.A.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNEEM-X\u0026reg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAzadirachtin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eUN.\u003c/b\u003e Compounds of unknown or\u003c/p\u003e \u003cp\u003euncertain mode of action\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMarketing Arm International Inc. USA.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDIPEL\u0026reg; WG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus thuringiensis\u003c/em\u003e subsp. kurstaki\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64 g Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e11.\u003c/b\u003e Microbial disruptors of insect midgut membranes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eValent BioSciences, USA.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEN VIVO SC\u0026reg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNuclear multiple polyhedrosis virus of \u003cem\u003eMamestra brassicae\u003c/em\u003e (NPVs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e281.88 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e31.\u003c/b\u003e Baculoviruses. Host-specific occluded pathogenic viruses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePoint Chile S.A.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTEC FORT\u0026reg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyrethrins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e3.\u003c/b\u003e Sodium channel modulators\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMIP Agro Ltda. Chile.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*IRAC= \"The Insecticide Resistance Action Committee\". The IRAC mode of action classification online (IRAC International, 2024) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.irac-online.org\u003c/span\u003e\u003cspan address=\"http://www.irac-online.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eBioassays\u003c/h3\u003e\n\u003cp\u003eIn all bioassays, we initially determined the concentration range for each insecticide that caused from 0.0\u0026ndash;100%. Then, three to four intermediate concentrations were evenly distributed between this range.\u003c/p\u003e\n\u003ch3\u003eTopical toxicity\u003c/h3\u003e\n\u003cp\u003eWe used the methodology of Del Sarto et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with slight modifications. To reduce bee's mobility, they were placed in a freezer at a temperature of -15\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 5 min (De Carvalho et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Then, 2 \u0026micro;L of each insecticide concentration was applied in the ventral part of the ​​ abdomen with a micropipette, as Delkash-Roudsari et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) suggested. Each treatment had ten replications of 15 insects. The untreated control was similarly handled but exposed to distilled water. Mortality was evaluated 24 hours post-treatment, with a maximum accepted control mortality of 10%, adjusted using Abbott's equation (Abbott, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1925\u003c/span\u003e). A bee that did not react to an external stimulus consisting of touching with a brush and/or being found upside down was considered dead, as Del Sarto et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) suggested. Treated honeybees were transferred to 1 L plastic jars fitted with a lid with two holes for holding two 20 mL syringes containing a diet of 5 mL of honey (70%) and 5 mL of distilled water, respectively, both offered \u003cem\u003ead libitum\u003c/em\u003e. In addition, to facilitate the bee's mobility inside the jar, two wooden tongue depressors measuring 113 x 10 x 2 mm were placed, forming an \"X\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eIngestion\u003c/h3\u003e\n\u003cp\u003eBioassay methodologies outlined by Del Sarto et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Phan et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) were utilized, incorporating minor modifications. This bioassay includes insecticides identified as the most or least toxic based on results from topical bioassays: Fast Plus (abamectin) and Neem-X\u0026reg; (azadirachtin) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The methodology involved placing bees in 1 L plastic jars as previously done. Bees were starved for 4 h and then fed \u003cem\u003ead libitum\u003c/em\u003e with a 20 mL syringe containing 5 mL of a 70% honey mixture and the insecticide treatment. Then, the syringes were removed and replaced with a syringe with 5 mL of honey (70%). Mortality was assessed 24 h after treatment, and the mortality criterion was the same as in topical bioassay. The experimental unit consisted of 15 bees; each treatment was replicated ten times, and mortality was adjusted using Abbott's equation (Abbott, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1925\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eResiduality\u003c/h2\u003e \u003cp\u003eThe residuality bioassays involving the most toxic compounds, Tec Fort\u0026reg; (pyrethrins) and Fast Plus (abamectin) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in its commercial doses (22.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 18.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e respectively), were conducted using methodologies from Husain et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Abati et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with minor modifications. The initial phase of the study was performed in an eight-year-old Fuji apple orchard at the \"El Nogal\" Experimental Station, located on the Chillan Campus of the Universidad de Concepcion, Chile. We randomly selected three central rows in each orchard containing 45 trees. Then, using a random block design, we had three treatments (each treatment had three trees) and five replicates; each block contained an untreated control. Treatments were sprayed with a two-wheel barrow sprayer of 50L capacity (Lerpain pulverizadores, Puente Alto, Chile) in a volume of 2.6 L per tree, using a hollow cone nozzle and 150 psi of working pressure. After 2, 24, and 48 h of treatment, five random leaves were collected from the central tree, identified, and stored in hermetically sealed bags; in less than 10 min, they were transferred to the laboratory. These leaves were placed in 1 L plastic jars to maintain leaf turgor; stems were kept in 1.5 mL Eppendorf tubes with a water solution and 2.0% agar. Then, each jar was inoculated with 15 honeybees previously cooled to -15\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 5 min to reduce mobility and aggressiveness. Bees were fed two syringes with 5 mL of honey (70%) and 5 mL of distilled water offered \u003cem\u003ead libitum\u003c/em\u003e. Mortality was assessed at 24, 48, 72, and 96 h until the untreated control showed a 50% mortality.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRepellency\u003c/h3\u003e\n\u003cp\u003eThis bioassay was performed via a double-choice method adapted from Signoretti et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), using the commercially recommended doses of each insecticide (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A Y-shaped glass olfactometer was used (two lateral arms 16.5 cm long and 3 cm in diameter were connected to a central tube with the same diameter and dimensions). Each arm was sealed with a rubber cap and attached to a glass flask containing the odor sources (treatment or control). The aroma source from the treatment included a cotton ball soaked with 20 mL of honey (70%) and mixed with the commercial dose of the respective insecticide. The untreated control contained a cotton ball soaked only with 20 mL of honey (70%). Each arm of the Y-shaped tube was connected to an aquarium air pump Super LB-1000 (Shanghai Luby Pet Industries Co., Shanghai, China) of 1.6 L air min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e flow. The airflow was filtered with an activated carbon filter and passed through a distilled flask to humidify the air. The airflow rate was controlled by SHLLJ LZQ-6 flow meters (Yuyao Shunhuan Instruments Co., Shanghai, China), which provided a constant rate of 1 L air min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Lines were traced outside each arm and at the base of the Y-shaped tube at 7 cm from the center. Then, groups of 75 adult honeybees were placed individually on the central tube for each concentration, and the repellent effect was evaluated. Repellency was monitored for five minutes after the insect crossed the threshold line of the central tube. Choices were recorded when an insect crossed the line of one of the two arms and stayed there for 20 seconds (Signoretti et al.). 2012). The bees that chose one of the two arms during five minutes were the only ones considered in the bioassay. Each insect was used a single time to prevent associative learning. The glass Y-shape tube was washed with neutral washing soap, distilled water, 90% ethanol (v/v), and 90% acetone (v/v) between replicates, while the connections with the odor sources were alternated to minimize bias.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLethal Concentration 50% (LC\u003csub\u003e50\u003c/sub\u003e) and 90% (LC\u003csub\u003e90\u003c/sub\u003e) at 24 hours of abamectin, pyrethrins, nuclear multiple polyhedrosis virus of \u003cem\u003eMamestra brassicae\u003c/em\u003e (NPVs) and azadirachtin assessed by topical application against \u003cem\u003eApis mellifera\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u0026thinsp;\u0026plusmn;\u0026thinsp;EE\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(LC 95%)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(LC 95%)\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePr\u0026thinsp;\u0026gt;\u0026thinsp;X\u003csup\u003e2e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTR\u003csub\u003e50\u003c/sub\u003e\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbamectin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.221\u003c/p\u003e \u003cp\u003e(0.109\u0026ndash;0.311)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003cp\u003e(0.582\u0026ndash;1.242)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyrethrins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.82 \u003c/p\u003e \u003cp\u003e(8.85\u0026ndash;67.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e322.2 \u003c/p\u003e \u003cp\u003e(156.1-7292.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNPVs\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2393.9\u003c/p\u003e \u003cp\u003e(1844.4-3088.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10386 \u003c/p\u003e \u003cp\u003e(6983.9-20505)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10832\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzadirachtin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2081\u003c/p\u003e \u003cp\u003e(14782\u0026thinsp;\u0026minus;\u0026thinsp;3983)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46847\u003c/p\u003e \u003cp\u003e(13148\u0026ndash;670103)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9416\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003eNumber of treated insects, \u003csup\u003eb\u003c/sup\u003eSlope value. \u003csup\u003ec\u003c/sup\u003eLethal Concentration at 50% of effect with confidence limits at 95% probability. \u003csup\u003ed\u003c/sup\u003eLethal Concentration at 90% of impact with confidence limits at 95% probability. \u003csup\u003ee\u003c/sup\u003eModel fit to a straight line. \u003csup\u003ef\u003c/sup\u003eToxicity ratio 50% (RT\u003csub\u003e50\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;CL\u003csub\u003e50\u003c/sub\u003e treatment/ CL\u003csub\u003e50\u003c/sub\u003e more toxic treatment. \u003csup\u003eg\u003c/sup\u003eNuclear multiple polyhedrosis virus of \u003cem\u003eMamestra brassicae\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLethal Concentration 50% (LC\u003csub\u003e50\u003c/sub\u003e) and 90% (LC\u003csub\u003e90\u003c/sub\u003e) at 24 hours of abamectin and azadirachtin assessed by ingestion against \u003cem\u003eApis mellifera\u003c/em\u003e. \u003csup\u003ea\u003c/sup\u003eNumber of treated insects, \u003csup\u003eb\u003c/sup\u003eSlope value. \u003csup\u003ec\u003c/sup\u003eLethal Concentration at 50% of effect with confidence limits at 95% probability. \u003csup\u003ed\u003c/sup\u003eLethal Concentration at 90% of effect with confidence limits at 95% probability. \u003csup\u003ee\u003c/sup\u003eModel fit to a straight line. \u003csup\u003ef\u003c/sup\u003eToxicity ratio 50% (RT\u003csub\u003e50\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;CL\u003csub\u003e50\u003c/sub\u003e treatment/ CL\u003csub\u003e50\u003c/sub\u003e more toxic treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u0026thinsp;\u0026plusmn;\u0026thinsp;EE\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(LC 95%)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(LC 95%)\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePr\u0026thinsp;\u0026gt;\u0026thinsp;X\u003csup\u003e2e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTR\u003csub\u003e50\u003c/sub\u003e\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbamectin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e0.233\u003c/p\u003e \u003cp\u003e(0.166-0.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e0.949\u003c/p\u003e \u003cp\u003e(0.725-429)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzadirachtin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e800.365\u003c/p\u003e \u003cp\u003e(631.88-1103.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e3278.05\u003c/p\u003e \u003cp\u003e(1990.14-9370.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3621\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eNumber of treated insects, \u003csup\u003eb\u003c/sup\u003eSlope value. \u003csup\u003ec\u003c/sup\u003eLethal Concentration at 50% of effect with confidence limits at 95% probability. \u003csup\u003ed\u003c/sup\u003eLethal Concentration at 90% of effect with confidence limits at 95% probability. \u003csup\u003ee\u003c/sup\u003eModel fit to a straight line. \u003csup\u003ef\u003c/sup\u003eToxicity ratio 50% (RT\u003csub\u003e50\u003c/sub\u003e) = CL\u003csub\u003e50\u003c/sub\u003e treatment/ CL\u003csub\u003e50\u0026nbsp;\u003c/sub\u003emore toxic treatment.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eExperimental design and statistical analysis\u003c/h3\u003e\n\u003cp\u003eThe laboratory bioassays were performed with a completely randomized experimental design, while the residuality bioassay was a randomized block experimental design. The results of topical application and ingestion were adjusted to the Probit model (Finney, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1971\u003c/span\u003e) to estimate the lethal concentration of 50% (LC\u003csub\u003e50\u003c/sub\u003e) and 90% (LC\u003csub\u003e90\u003c/sub\u003e) mortality using the PoloPlus software (LeOra Software, 2022). Residuality analysis was performed with a Kaplan and Meier (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1958\u003c/span\u003e) survival test using R software (R Core Team, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and repellency data were analyzed with a Chi-square (X\u003csup\u003e2\u003c/sup\u003e) test, also with R software.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTopical application\u003c/h2\u003e \u003cp\u003eAbamectin showed the highest contact toxicity, with 100% mortality at its commercial dose and half and one-eighth of this (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Even this insecticide at the lowest dose evaluated, corresponding to 1/64 of commercial dose (0.28 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), exceeded 50% of dead bees (57,89%). Pyrethrins at the commercial concentration of 45 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e resulted in a mortality rate of 53.2%. NPVs did not cause mortality at the recommended dosage, so the concentration was increased by 64 times (3264 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) to achieve a mortality rate of 90.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). \u003cem\u003eBacillus thuringiensis\u003c/em\u003e and azadirachtin, at 32 and 64 times the commercial dose, respectively, did not achieve 10% mortality, making Probit analysis unfeasible. The lowest LC\u003csub\u003e50\u003c/sub\u003e was obtained by abamectin with CL\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.221 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e while NPVs and pyrethrins showed a CL\u003csub\u003e50\u003c/sub\u003e of 2393 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 39.82 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e respectively. All treatments differed significantly because the confidence limits did not overlap (Robertson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The TR\u003csub\u003e50\u003c/sub\u003e indicated that abamectin was 180 and 10832-fold more toxic to \u003cem\u003eA. mellifera\u003c/em\u003e than NPVs and pyrethrins, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIngestion bioassays\u003c/h2\u003e \u003cp\u003eAbamectin was the more toxic insecticide, with a mortality of 100% at the commercial dosage (18 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), while the lowest assessed concentration corresponding to 1/128 (0.14 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) showed 20.55% of dead bees (LC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.233 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; LC\u003csub\u003e90\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.949 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Neem (azadirachtin) exhibited the lowest toxicity because it was necessary to increase to 64-fold the commercial dose to obtain a mortality of 1.43%. This insecticide had an LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e of 800.3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3278.05 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Furthermore, both treatments were significantly different because the confidence limits did not overlap (Robertson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and according to RT\u003csub\u003e50,\u003c/sub\u003e abamectin is 3620-fold more toxic than Neem\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eResiduality\u003c/h2\u003e \u003cp\u003eBee longevity decreased in all treatments compared to the control, and shorter contact times with treated leaves resulted in lower survival rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Results from leaves collected two hours after the insecticide spray indicated that at 24 hours, 96% of the bees exposed to leaves sprayed with pyrethrins remained alive, while at 120 hours, 30.7% were still alive. These treatments did not show significant differences from the control sprayed only with water (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0572). For abamectin, at 24 h, 8.0% of the bees were alive at 120 h, and none of the insects remained alive. This treatment significantly differed from the untreated control and pyrethrins (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0025). In leaves collected 24 h after spraying with pyrethrins, 90.7% and 49.4% survival rates were observed at 24 and 120 h, without significant differences from the untreated control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9893). Abamectin caused 53.3% and 100% mortality at 24 and 96 h, respectively, registering statistical differences from the untreated control and pyrethrins (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). In leaves collected 48 h after spraying insecticides, those treated with pyrethrins showed 76% and 60.9% alive bees at 24 and 120 h, without significant differences from the untreated control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.469). For abamectin, survival rates at 24 and 120 H were 92% and 69.3%, respectively. This treatment showed significant differences compared to the control \u003cem\u003e(p\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0173) but not compared to pyrethrins (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9185).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRepellency\u003c/h2\u003e \u003cp\u003eNPVs was the only treatment that did not exhibit repellency. It attracted bees, with 55.26% choosing a diet containing insecticide and 44.74% opting for untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The highest repellent activity was observed in \u003cem\u003eB. thuringiensis\u003c/em\u003e, with 63.16% control preference versus 36.84% of food mixed with the commercial dose. In bioassays with Neem, 51.32% of bees preferred the untreated control and 46.68% contaminated food. Abamectin and pyrethrins showed 53.95% preference for the untreated control and 46.05% for the insecticide treatment. The Chi-square test (χ\u003csup\u003e2\u003c/sup\u003e) indicated no significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1338) among treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNatural insecticides are considered less toxic to pollinators and non-target insects than synthetic ones. However, our results show that some of these are as toxic as the synthetic compounds. The contact and ingestion toxicity obtained for abamectin agrees with Del Sarto et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), who found that abamectin is moderately toxic to \u003cem\u003eA. mellifera\u003c/em\u003e, and more harmful than deltamethrin, an insecticide of the pyrethroid family. Similarly, Costa et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) assessed, under laboratory conditions, the toxicity against \u003cem\u003eA. mellifera\u003c/em\u003e of all insecticides used in Brazilian melon crops, concluding that abamectin, thiametoxam, and chlorfenapyr were the most toxic. Research indicates that pyrethroids, including deltamethrin (Besard et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Del Sarto et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Stanley et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), cypermethrin (Pashte and Patil, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), lambda-cyhalothrin, and bifenthrin (Wang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), exhibit a high level of toxicity against \u003cem\u003eA. mellifera\u003c/em\u003e. Thus, we expect the same harmful effects as pyrethrins but with a shorter duration since their residuality does not exceed 24 h (Feng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The toxicity of Neem (azadirachtin) in contact and ingestion bioassays did not exceed 2.0% mortality, coinciding with Melathopoulos et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), who found that an azadirachtin-rich extract (Neem-aza) and Neem oil did not exceed 10% mortality in bees infested with \u003cem\u003eV. jacobsoni\u003c/em\u003e and those free of this pest. However, Peng et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) demonstrated that azadirachtin possesses the highest oral toxicity in \u003cem\u003eA. mellifera\u003c/em\u003e, particularly when infested with \u003cem\u003eV. jacobsoni.\u003c/em\u003e In addition, azadirachtin greatly reduces syrup consumption by worker bees, and over 90% of treated while prepupae and pupae exhibit abnormal mouthpart and appendage pigmentation.\u003c/p\u003e \u003cp\u003eKaur et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) also studied the effect of three concentrations (0.05%, 0.1%, and 1.0%) of Neem oil in \u003cem\u003eA. mellifera\u003c/em\u003e larvae. They found that survival decreased as the dose increased. Emerged pupae reduction and early adult mortality showed the same trend. Our findings and additional research highlight the negative traits of certain botanical insecticides. Isman (2000) noted that the varied insecticidal activity of these insecticides is the reason they are not widely used.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus thuringiensis\u003c/em\u003e was one of the less toxic insecticides. There are no previous studies about the toxicity of this insecticide in bees. However, in other pollinators such as \u003cem\u003eBombus terrestris L.\u003c/em\u003e (Hymenoptera: Apidae), Mommaerts et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) assessed Dipel (\u003cem\u003eB. thuringiensis\u003c/em\u003e subsp. kurstaki) and Xentari (\u003cem\u003eB. thuringiensis\u003c/em\u003e subsp. aizawai) against this species of insect, at the highest doses for field conditions, obtained a maximum mortality of 5.0%. Similarly, Libardoni et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), in a bioassay with a contaminated diet with Dipel, found that the survival rate of Africanized honey bees after 144 h of consuming the contaminated food was 80%.\u003c/p\u003e \u003cp\u003eMost studies on the residual effects of insecticides against \u003cem\u003eA. mellifera\u003c/em\u003e focus on synthetic insecticides, such as pyrethroids and neonicotinoids; there are nearly no studies on insecticides of natural origin. However, synthetic insecticides, such as pyrethroids, have been chemically improved to increase their field efficacy and residual effect under field conditions and are toxic to bees. Biopesticides do not have residue problems because they degrade fast in the environment (Kumar, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, novel formulations such as nano emulsions increase residuality (Ayilara et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRepellency is one of the most studied properties of natural toxic compounds, mainly essential oils. In our research, although untreated bees exhibited higher preferences for control than the treated diet, no significant differences existed between these treatments. Thus, we infer that the studied insects made their choice randomly rather than being influenced by the type of diet. Naumann et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) demonstrated that bees can discriminate between untreated sugar syrup and syrup containing concentrations as low as 0.1 ppm of azadirachtin. Our research found azadirachtin to have a 51.3% repellency, contrary to Barbosa et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), who reported a maximum of 7.0%. Melathopoulos et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) assessed the toxicity of Neem against \u003cem\u003eA. mellifera\u003c/em\u003e, concluding that Neem mixed with sucrose syrup in a concentration\u0026thinsp;\u0026gt;\u0026thinsp;0.01 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is a feeding deterrent to \u003cem\u003eA. mellifera\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus thuringiensis\u003c/em\u003e repelled bees by 63.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), whereas NPVs attracted them slightly, as bees favored the treated diet over the control. The low toxicity of these insecticides reduces this effect. Abamectin did not negatively affect bee behavior; treated and untreated diets exhibited similar repellent effects. Mubin et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reached a similar conclusion in their study involving \u003cem\u003eTetragonula laeviceps\u003c/em\u003e Smith (Hymenoptera: Apidae: Meliponini). Their research indicated that the presence of abamectin did not influence the behavior of \u003cem\u003eT. laeviceps\u003c/em\u003e, as evidenced by similar preferences between honey containing abamectin and honey alone.\u003c/p\u003e \u003cp\u003eNatural insecticides can harm \u003cem\u003eA. mellifera\u003c/em\u003e, similar to conventional ones. Abamectin and pyrethrins affect bees through contact and ingestion, even at commercial and sublethal doses. Farmers should protect bees when using these substances. Conversely, the nuclear polyhedrosis virus and \u003cem\u003eB. thuringiensis\u003c/em\u003e are safe for honeybees.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWho would like to thank Dr. Selim Musleh of N\u0026uacute;cleo Milenio de Salm\u0026oacute;nidos Invasores (INVASAL), Facultad de Ciencias Naturales y Oceanogr\u0026aacute;ficas, Universidad de Concepci\u0026oacute;n, Concepci\u0026oacute;n. Chile, for his technical assistance in statistical analysis and R software management.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial interests:\u0026nbsp;\u003c/strong\u003eThe authors declare they have no financial interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003eConceptualization, G.S.A., J.C.R., M.R., and J.S.B. Experiments M.I.A, B.C.M. Statistical Analysis M.I.A., G.S.A., M.R., writing-original draft preparation, M.I.A., writing-review and editing G.S.A., J.C.R. M.R. J.S.B., M.I.A. All authors approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eNo datasets were generated or analyzed during the current study\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol. 18: 265-267. https://doi.org/10.1093/jee/18.2.265a \u003c/li\u003e\n\u003cli\u003eAbati R, Libardoni G, Osowski G, de Souza E, Costa-Maia FM, Lozano ER, Adami PF, Potrich M (2023) Residual effect of imidacloprid and beta-cyfluthrin on Africanized \u003cem\u003eApis mellifera\u003c/em\u003e L. (Hymenoptera: Apidae) workers. 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The irrational use of pesticides is considered one of the causes. However, the primary focus in this area is on synthetic insecticides. Hence, this research aimed to assess the toxicity of commercial doses of \u003cem\u003eBacillus thuringiensis\u003c/em\u003e Berliner, nuclear multiple polyhedrosis virus (NPVs), azadirachtin, pyrethrins, and abamectin against \u003cem\u003eA. mellifera\u003c/em\u003e. In contact and ingestion bioassays, the highest toxicity was achieved with abamectin (100% mortality). Pyrethrins exhibited a mortality rate of 53.2% whereas NPVs did not result in any mortality. Although untreated bees preferred control over treated diet in repellency bioassays, no significant differences between treatments were observed. We concluded that abamectin and pyrethrins are the most harmful to bees by contact and ingestion toxicity.\u003c/p\u003e","manuscriptTitle":"Susceptibility of Apis mellifera to Bacillus thuringiensis Berliner, nuclear multiple polyhedrosis virus, azadirachtin, pyrethrins, and abamectin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 10:39:55","doi":"10.21203/rs.3.rs-6421763/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":"3e13a754-ef38-44fc-8dbf-3d78c0040145","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-25T12:39:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-29 10:39:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6421763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6421763","identity":"rs-6421763","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}