Genetic variability and pyrethroid susceptibility of the parasitic honey bee mite <Emphasis Type="Italic">Varroa destructor</Emphasis> (Acari: Varroidae) in Iran

The ectoparasitic honey bee mite Varroa destructor Anderson & Trueman (Acari: Varroidae) is one of the major concerns for worldwide beekeeping. The use of synthetic pyrethroids for controlling the mite was among the most popular treatments until resistance evolved in the mid 1990’s. In Iran, beekeepers are dealing with the parasite and they also used pyrethroids for controlling the mite for a long time. After the evolution of resistance to pyrethroids, they based mite control mostly on treatments with amitraz, organic acids and several management practices. Here we conducted a comprehensive characterization of V. destructor populations parasitizing Apis mellifera in Iran. We determined the genetic variability of mites collected from 28 localities distributed throughout the country. The haplotype of V. destructor was determined by PCR-RFLP, analyzing a fragment of the mitochondrial cox1 gene. It was found that only the Korean haplotype was present in samples from all localities. DNA fragments from cox1, atp6, cox3 and cytb mitochondrial genes were sequenced and the results showed that all samples were identical to the K1-1 or the K1-2 V. destructor haplotypes. Moreover, as it has been reported that resistance to pyrethroids in V. destructor is associated with mutations at position 925 of the voltage-gated sodium channel, a TaqMan^®-based allelic discrimination assay was conducted to genotype the mites collected. The results showed that all the mites tested were homozygous for the wild-type allele and, therefore, susceptible to treatment with pyrethroids.     

Mutations in the Bemisia tabaci para sodium channel gene associated with resistance to a pyrethroid plus organophosphate mixture

The voltage-gated sodium channel is the primary target site of pyrethroid insecticides. In some insects, super knockdown resistance (super-kdr) to pyrethroids is caused by point mutations in the linker fragment between transmembrane segments 4 and 5 of the para-type sodium channel protein domain II (IIS4-5). Here, we identify two mutations in the IIS4-5 linker of the para-type sodium channel of the whitefly, Bemisia tabaci: methionine to valine at position 918 (M918V) and leucine to isoleucine at position 925 (L925I). Although each mutation was isolated independently from strains >100-fold resistant to a pyrethroid (fenpropathrin) plus organophosphate (acephate) mixture, only L925I was associated with resistance in strains derived from the field in 2000 and 2001. The L925I mutation occurred in all individuals from nine different field collections that survived exposure to a discriminating concentration of fenpropathrin plus acephate. Linkage analysis of hemizygous male progeny of unmated heterozygous F1 females (L925I×wild-type) shows that the observed resistance is tightly linked to the voltage-gated sodium channel locus. The results provide a molecular tool for better understanding, monitoring and managing pyrethroid resistance in B. tabaci.     

钠离子通道与蜜蜂狄斯瓦螨对氟胺氰菊酯的抗性机理

狄斯瓦螨Varroadestructor是全世界蜜蜂最严重的寄生虫 ,目前 ,它对主要防治药物———拟除虫菊酯类的氟胺氰菊酯已产生明显抗性 ,严重影响其防治效果。近年来神经生理学研究结果证实 :电压门控的钠离子通道是拟除虫菊酯作用的位点。钠通道结构的改变 ,是拟除虫菊酯类杀虫剂毒理的主要基础 ,也是产生抗药性的基础。该文介绍了近年来国内外研究电压门控钠离子通道、拟除虫菊酯对钠通道的作用、钠通道与拟除虫菊酯的抗性和狄斯瓦螨对氟胺氰菊酯抗性机理研究的新进展     

Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases

Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.     

Effect of acaricide resistance on reproductive ability of the honey bee mite Varroa destructor

The reproduction of pyrethroid-resistant Varroa destructor mite, a brood parasite of honey bees, was observed in Weslaco, Texas, and the results compared with known susceptible mite populations from other studies. Seven Apis mellifera colonies that had mite populations resistant to the acaricide Apistan^™ were used. Pyrethroid-resistance was confirmed when only 17% rather than 90% of mites confined in dishes containing Apistan^™ died after 12 h of exposure. The average number of eggs laid by resistant mites invading worker and drone cells was 4.4 and 5.4 respectively. This is similar to the number of eggs laid by susceptible mites in worker (4.4–4.8) or drone (4.7–5.5) cells. Also the average number of fertilised V. destructor female mites produced by resistant mites in worker (1.0) and drone (2.1) cells were similar to the number produced by susceptible mites in worker (0.9) and drone (1.9–2.2) cells. In addition, no major differences between the resistant and susceptible mite populations were observed in either worker or drone cells when six different reproductive categories and offspring mortality rates were compared. Therefore, it appears that there is little or no reproductive fitness cost associated with pyrethroid resistance in V. destructor in Texas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spread and strain determination of Varroa destructor (Acari: Varroidae) in Madagascar since its first report in 2010

Varroa destructor is a major pest in world beekeeping. It was first detected in Madagascar in 2010 on the endemic honeybee Apis mellifera unicolor. To evaluate V. destructor spread dynamics in Madagascar a global survey was conducted in 2011–2012. A total of 695 colonies from 30 districts were inspected for the presence of mites. 2 years after its introduction, nine districts were found infested. Varroa destructor spread was relatively slow compared to other countries with a maximum progression of 40 km per year, the five newly infested districts being located next to the first infested ones. The incidence of mite infestation was also investigated by monitoring 73 colonies from five apiaries during 1 year (2011–2012). Sixty percent of local colony mortality was recorded after 1 year of survey. Varroa destructor strain determination was done by partial sequencing of the cytochrome oxidase I gene of 13 phoretic mites sampled in five districts. A single V. destructor mitochondrial haplotype was detected, the Korean type, also present in the closest African countries. A global pathogen survey was also conducted on the colonies inspected for mite presence. The greater wax moth, Galleria mellonella has been found in all colonies all over the country. Two other pathogens and morphological abnormalities in workers, such as deformed wings, were found associated with only V. destructor presence. A prevention management plan must be implemented to delimit mite spread across the island.     

L925I Mutation in the Para-Type Sodium Channel Is Associated with Pyrethroid Resistance in Triatoma infestans from the Gran Chaco Region

Background

Chagas'' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil.The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species.

Methods and Findings

In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field.

Conclusions and Significance

The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas'' disease transmission.     

First data on resistance mechanisms of Varroa jacobsoni (OUD.) against tau-fluvalinate

In 1991, the first losses of efficacy of tau-fluvalinate against the honeybee ectoparasite Varroa jacobsoni Oud. were recorded in Sicily. Since then, diminished efficacy with available pyrethroid treatments has been encountered in many regions of Italy. The aim of this study was to investigate the type of resistance in V. jacobsoni to the pyrethroid tau-fluvalinate by focusing on metabolic resistance mechanisms (detoxication). After developing a suitable application method, two synergists were used: piperonyl butoxide (PBO), as an inhibitor of the microsomal monooxygenases of the cytochrome P450 complex and S,S,S-tributylphosphorotrithioate (DEF), which blocks esterases. A significant decrease in the LC₅₀ values of the susceptible and of the resistant mite strains after the application of PBO was observed. A slight decrease of the LC₅₀ values was also observed after the application of DEF. However, this decrease was not significant. These results indicate that the resistance of Varroa mites to tau-fluvalinate can partly be explained by an increased detoxication due to the monooxygenases in the P450 system, which is blocked by PBO. Esterases seems to play a negligible role. Whether glutathione-S-transferases are involved, is still unknown, but other mechanisms, such as the modification of the binding sites and/or reduced uptake might be involved as well.     

Culturing chelifers (Pseudoscorpions) that consume Varroa mites

Chelifers (Pseudoscorpions) are generalist predators of small prey such as mites. Their occasional presence in honeybee hives suggests potential to exploit them as part of a management programme against Varroa mites (Varroa destructor), a significant pest of honeybees. Two species of native New Zealand chelifers Nesochernes gracilis and Heterochernes novaezealandiae, shown to consume Varroa mites, were collected from commercial nucleus hives or in litter surrounding the hives. Methods for mass‐rearing the chelifers were developed to provide specimens for research and introduction into beehives for biological control of Varroa. Cultures were fed aphids and fruit fly larvae in vented containers containing sand and bark. N. gracilis was maintained at 14°C, 18°C, and 22°C. At 18°C, 1423 nymphs were reared from 140 N. gracilis adults, with 84.8% of all nymphs produced at this temperature. H. novaezealandiae was maintained at 18°C and 22°C, with 5 nymphs raised from 12 adults at 18°C and none at the higher temperature.     

The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae)

Varroa destructor, a key biotic threat to the Western honey bee, has played a major role in colony losses over the past few years worldwide. Overuse of traditional acaricides, such as tau-fluvalinate and flumethrin, on V. destructor has only increased its tolerance to them. Therefore, the application of essential oils in place of traditional pesticides is an attractive alternative, as demonstrated by its high efficiency, lack of residue and tolerance resistance. To study the acaricidal activity of essential oils, we used clove oil (Syzygium aromaticum L.), a typical essential oil with a wide range of field applications, and examined its effects on the enzyme activities of Ca²⁺-Mg²⁺-ATPase, glutathione-S-transferase (GST) and superoxide dismutase (SOD) and its effects on the water-soluble protein content of V. destructor body extracts after exposure to 0.1 μl and 1.0 μl of clove oil for 30 min. Our results showed that the water-soluble protein content significantly decreased after the treatments, indicating that the metabolism of the mites was adversely affected. The bioactivity of GSTs increased significantly after a low dosage (0.1 μl) exposure but decreased at a higher dosage (1.0 μl), while the activities of SOD and Ca²⁺-Mg²⁺-ATPase were significantly elevated after treatments. These results suggest that the protective enzyme SOD and detoxifying enzymes Ca²⁺-Mg²⁺-ATPase and GST contributed to the stress reaction of V. destructor to the essential oils and that the detoxification ability of V. destructor via GST was inhibited at higher dosages. Our findings are conducive to understanding the physiological reactions of V. destructor to treatment with essential oils and the underlying mechanisms behind the acaricidal activities of these natural products.     

Honey bee colony collapse disorder is possibly caused by a dietary pyrethrum deficiency

Industrialized farming relies on bee keepers transporting hives to the vicinity of large areas of mono-crops for crop pollination. Hives are typically moved multiple times per growing season to satisfy the pollination need. A phenomenon wherein colonies of honey bees collapse in large numbers has been threatening crops in North America. Honey bees are hosts to at least two pathogenic mites; Varroa destructor and Acarapis woodi (a tracheal mite). Pyrethrums are a group of flowering plants which include Chrysanthemum coccineum, Chrysanthemum cinerariifolium, Chrysanthemum marschallii, and related species. These plants produce potent insecticides, also named pyrethrums, which are powerful mite toxins. We believe that a honey bee dietary deficiency of pyrethrums and other micro-nutrients from pyrethrum producing plants allows parasitic mites to either kill the honey bees directly or reduce honey bee resistance to other pathogens. Intermittent feeding of honey bees on pyrethrum producing plants might reverse or prevent colony collapse disorder.     

烟粉虱对拟除虫菊酯杀虫剂的抗性机理

通过增效剂生物测定、生化分析以及钠离子通道基因ⅡS4-6 cDNA片段的RT-PCR扩增,探讨了烟粉虱Bemisia tabaci（Gennadius）对拟除虫菊酯杀虫剂的抗性机理。结果表明：对于采自田间的6个烟粉虱抗性品系,磷酸三苯酯（TPP）和胡椒基丁醚（PBO）对氯氰菊酯、溴氰菊酯、氯氟氰菊酯和甲氰菊酯均有显著的增效作用,而DEM对4种拟除虫菊酯杀虫剂均无明显的增效作用。烟粉虱抗性品系的α-NA羧酸酯酶和β-NA羧酸酯酶活性分别是敏感品系的2.16～2.65倍和1.22～1.41倍,抗性品系的谷胱甘肽S转移酶活性与敏感品系没有差异,表明羧酸酯酶和多功能氧化酶在烟粉虱对拟除虫菊酯类杀虫剂的抗性中具有重要的作用,而谷胱甘肽S转移酶与抗性无关。通过RT-PCR克隆了6个烟粉虱田间抗性品系的钠离子通道结构域ⅡS4-6 cDNA片段的序列（420 bp）,发现与敏感品系相比,有2个位点发生突变,分别为L925I突变和I917V突变,L925I突变在所有6个烟粉虱田间抗性种群中均有发生,该位点突变已被证实与拟除虫菊酯类杀虫剂密切相关,表明神经不敏感性可能是烟粉虱对拟除虫菊酯产生抗性的另一个重要因子。     

Live Varroa jacobsoni (Mesostigmata: Varroidae) fallen from honey bee (Hymenoptera: Apidae) colonies

The proportion of Varroa jacobsoni Oudemans that were alive and mobile when they fell from honey bees, Apis mellifera L., in hives was measured during a 20-wk period to determine the potential use of systems that prevent these mites from returning to the bees. Traps designed to discriminate between the live, fallen mites and those that are dead or immobile were used on hive bottom boards. A large fraction of the fallen mites was alive when acaricide was not in use and also when fluvalinate or coumaphos treatments were in the hives. The live proportion of mitefall increased during very hot weather. The proportion of mitefall that was alive was higher at the rear and sides of the hive compared with that falling from center frames near the hive entrance. More sclerotized than callow mites were alive when they fell. A screen-covered trap that covers the entire hive bottom board requires a sticky barrier to retain all live mites. This trap or another method that prevents fallen, viable mites from returning to the hive is recommended as a part of an integrated control program. It also may slow the development of acaricide resistance in V. jacobsoni and allow the substitution of less hazardous chemicals for the acaricides currently in use.     

Multiple origins of pyrethroid resistance in sympatric biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)

The two most damaging biotypes of Bemisia tabaci, B and Q, are sympatric in the Mediterranean basin and show high resistance to pyrethroids synergized by organophosphates. Previous work showed that in the B biotype, this resistance is associated with the L925I mutation in the para-type voltage gated sodium channel. Here we identified two mutations in the para-type voltage gated sodium channel associated with resistance to pyrethroids synergized by organophosphates in the Q biotype: the L925I mutation that occurs in the B biotype, and substitution of threonine to valine, at position 929 (T929V). To determine if the L925I and T929V mutations have single or multiple origins, we sequenced the DNA regions flanking the mutations from 13 B and Q strains collected worldwide. The survey identified five resistant alleles and five susceptible alleles. In the resistant alleles, the nucleotide diversity was low within biotypes (0.001), but high between biotypes (0.033). Nucleotide diversity in susceptible alleles was high between the two biotypes (0.028). These observations are consistent with multiple independent origins of resistance. Although the B and Q biotypes coexist in several regions of the Mediterranean basin, divergence in their DNA sequences at the para-type voltage gated sodium channel locus suggests gene flow between these biotypes is low or nil.     

Prospective Large-Scale Field Study Generates Predictive Model Identifying Major Contributors to Colony Losses

Over the last decade, unusually high losses of colonies have been reported by beekeepers across the USA. Multiple factors such as Varroa destructor, bee viruses, Nosema ceranae, weather, beekeeping practices, nutrition, and pesticides have been shown to contribute to colony losses. Here we describe a large-scale controlled trial, in which different bee pathogens, bee population, and weather conditions across winter were monitored at three locations across the USA. In order to minimize influence of various known contributing factors and their interaction, the hives in the study were not treated with antibiotics or miticides. Additionally, the hives were kept at one location and were not exposed to potential stress factors associated with migration. Our results show that a linear association between load of viruses (DWV or IAPV) in Varroa and bees is present at high Varroa infestation levels (>3 mites per 100 bees). The collection of comprehensive data allowed us to draw a predictive model of colony losses and to show that Varroa destructor, along with bee viruses, mainly DWV replication, contributes to approximately 70% of colony losses. This correlation further supports the claim that insufficient control of the virus-vectoring Varroa mite would result in increased hive loss. The predictive model also indicates that a single factor may not be sufficient to trigger colony losses, whereas a combination of stressors appears to impact hive health.     

First detection of <Emphasis Type="Italic">Varroa destructor</Emphasis> resistance to coumaphos in Argentina

In Argentina, studies on Varroa destructor resistance to coumaphos are still unknown. At present, high infestation levels of V. destructor are being detected in colonies of Apis mellifera after treatment with this acaricide. The aim of the present study was to determine the LC₅₀ of coumaphos in V. destructor from four apiaries with high mite density after treatment with coumaphos. The LC₅₀’s were 112, 319, 127 and 133 μg/Petri dish for mites from the four apiaries. Significant LC₅₀ differences were detected between resistant and susceptible mites. LC₅₀ increased 197–559-fold when compared to the corresponding baseline, suggesting the development of resistance. These results are the first report of resistance to coumaphos in V. destructor in Argentina.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Evaluations of the Removal of <Emphasis Type="Italic">Varroa destructor</Emphasis> in Russian Honey Bee Colonies that Display Different Levels of <Emphasis Type="Italic">Varroa</Emphasis> Sensitive Hygienic Activities

The removal of Varroa destructor was assessed in Russian honey bee (RHB) colonies with known levels of Varroa Sensitive Hygienic (VSH) and brood removal activities. The expression of grooming behaviour using individual bees was also measured using three groups of RHB displaying different VSH levels: low hygiene (RHB-LH, < 35% VSH), medium hygiene (RHB-MH, 35–70%) and high hygiene (RHB-HH, > 70%). Italian colonies (5.43–71.62% VSH) served as control. Our results demonstrated, for the first time, significant relationships between two hygienic responses (VSH activity measured as percent change in infestation and the actual brood removal of Varroa-infested donor comb) and two measurements of mite fall (trapped old mites/trapped mites or O/T and trapped young mites/trapped mites or Y/T). However, these relationships were only observed in RHB colonies. In addition, the RHB colonies that displayed the highest levels of hygiene (RHB-HH) also groomed longer in response to the presence of a V. destructor mite based on individual bee assays. The positive regressions between the two hygienic measurements and O/T and their negative regressions with Y/T suggest that the removal of infested brood prevented successful mite reproduction, ultimately suppressing V. destructor infestations in the RHB colonies. In addition, it is demonstrated that RHB resistance to V. destructor rests on both an increased hygienic response and the removal of phoretic mites, released by hygienic behaviour, through grooming. Both resistance traits are reflected in the O/T and Y/T ratios found in trapped mites from RHB colonies. None of the measurements involving mite injuries were associated with any measurements of hygiene and colony infestations.     

Efficacy of strips coated with Metarhizium anisopliae for control of Varroa destructor (Acari: Varroidae) in honey bee colonies in Texas and Florida

Strips coated with conidia of Metarhizium anisopliae (Metschinkoff; Deuteromycetes: Hyphomycetes) to control the parasitic mite, Varroa destructor (Anderson and Trueman) in colonies of honey bees, Apis mellifera (Hymenoptera: Apidae) were compared against the miticide, tau-fluvalinate (Apistan) in field trials in Texas and Florida (USA). Apistan and the fungal treatments resulted in successful control of mite populations in both locations. At the end of the 42-day period of the experiment in Texas, the number of mites per bee was reduced by 69-fold in bee hives treated with Apistan and 25-fold in hives treated with the fungus; however mite infestations increased by 1.3-fold in the control bee hives. Similarly, the number of mites in sealed brood was 13-fold and 3.6-fold higher in the control bee hives than in those treated with Apistan and with the fungus, respectively. Like the miticide Apistan, the fungal treatments provided a significant reduction of mite populations at the end of the experimental period. The data from the broodless colonies treated with the fungus indicated that optimum mite control could be achieved when no brood is being produced, or when brood production is low, such as in the early spring or late fall. In established colonies in Florida, honey bee colony development did not increase under either Apistan or fungal treatments at the end of the experimental period, suggesting that other factors (queen health, food source, food availability) play some major role in the growth of bee colonies. Overall, microbial control of Varroa mites with fungal pathogens could be a useful component of an integrated pest management program for the honey bee industry.     

Pseudomonas contamination of a fungus-based biopesticide: Implications for honey bee (Hymenoptera: Apidae) health and Varroa mite (Acari: Varroidae) control

The ectoparasitic mite Varroa destructor is a major honey bee pest, and its control using pathogen-based biopesticides would resolve many of the problems, such as contamination and pesticide resistance, experienced with chemical control. A biopesticide, formulated with commercially-prepared conidia of a strain of Beauveria bassiana isolated from V. destructor was tested against the mites in bee colonies in southern France. The impact of treatment on hive survivorship, weight and mite infestation levels were very different from those of previous experiments using laboratory-prepared conidia: bee hives treated with the biopesticide died at a higher rate, lost more weight, and had higher mite densities at the end of the study than control hives. The biopesticide was subsequently found to be contaminated with bacteria. Two strains of bacteria were identified, by biotyping and sequencing data of the 16S rRNA and rpoB regions, and while the strains were distinct both were Pseudomonas sp. belonging to the P. fluorescens group. In dual cultures B. bassiana growth was slowed or suppressed when bacterial cfu density was about equal or greater than that of B. bassiana. Experiments using caged adult bees showed that bees ingesting diet and sugar solution treated with B. bassiana and kept at 30 °C had significantly lower survival times than those treated with one of the bacterial strains, but the opposite was true at 33 °C. Because one arthropod (honey bees) was treated for infestation by another (V. destructor), the impact of bacterial contamination was likely more noticeable than in most uses of biopesticides, such as treating plants against phytophagous insects. To reduce such risk in biopesticide development, a systematic screening for bacterial contamination prior to field application is recommended.