Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera

Ecotoxicology and Environmental Safety 142 (2017) 69–78

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Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera

MARK

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Eva Vianey Alquisira-Ramíreza, Guadalupe Peña-Chorab, , Víctor Manuel Hernández-Velázquezc, Andrés Alvear-Garcíaa, Iván Arenas-Sosad, Ramón Suarez-Rodríguezc a

Facultad de Ciencias Agropecuarias, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Colonia Chamilpa, Cuernavaca, Morelos C.P. 62209, Mexico Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, Cuernavaca, Morelos C.P. 62209, Mexico c Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, Cuernavaca, Morelos C.P. 62209, Mexico d Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Campus Morelos, Av. Universidad 2001, Cuernavaca, Morelos CP: 62210, Mexico b

A R T I C L E I N F O

A B S T R A C T

Keywords: Bacillus thuringiensis Varroa destructor Apis mellifera Sublethal effect Cypermethrin

The sublethal effects of two strains of Bacillus thuringiensis, which were virulent in vitro to Varroa destructor, were measured on Apis mellifera. The effects of five concentrations of total protein (1, 5, 25, 50 and 100 μg/mL) from the EA3 and EA26.1 strains on larval and adult honey bees were evaluated for two and seven days under laboratory conditions. Based on the concentrations evaluated, total protein from the two strains did not affect the development of larvae, the syrup consumption, locomotor activity or proboscis extension response of adults. These same parameters were also tested for the effects of three concentrations (1, 10 and 15 μg/kg) of cypermethrin as a positive control. Although no significant differences were observed after two days of treatment with cypermethrin, a dose-response relationship in syrup consumption and locomotor activity was observed. A significant reduction in the proboscis extension response of the bees treated with cypermethrin was also observed. Therefore, in contrast to cypermethrin, our results indicate that the EA3 and EA26.1 strains of B. thuringiensis can be used in beehives to control V. destructor and reduce the negative effects of this mite on colonies without adverse effects on the larvae and adults of A. mellifera. Additionally, the overuse of synthetic miticides, which produce both lethal and sublethal effects on bees, can be reduced.

1. Introduction The honey bees Apis mellifera Linnaeus (Hymenoptera: Apidae) are very important both socially and economically, but it is most important ecologically because the honey bees are the primary pollinating insect and makes significant contributions to the maintenance and development of biodiversity. Honey bees are essential in the production of plant-based foods and are widely used in modern agriculture for crop pollination (Pham-Delègue et al., 2002; VanEngelsdorp et al., 2008). Unfortunately, millions of colonies of A. mellifera are disappearing worldwide annually, primarily in Asia, Europe and North America (Neumann and Carreck, 2010; Seitz et al., 2016). For example, in 2008, the loss of hives in the United States reached 36%, which is well above an acceptable level (17%) (VanEngelsdorp et al., 2010). This loss is due to many factors, including poisoning by synthetic pesticides and the

incidence of parasites such as Varroa destructor (Koch and Weisser, 1997; Neumann and Carreck, 2010; Rortais et al., 2005; Topolska et al., 2008), which is the most destructive pest facing beekeeping worldwide (Anderson and Trueman, J W, 2000; Sammataro et al., 2000). The results of several studies suggest that the interaction of V. destructor with various diseases caused by fungi, bacteria, viruses and protozoa plays a very important role in the loss of the colonies (Boecking and Genersch, 2008; De Rycke et al., 2002; Glinski and Jarosz, 1992; Le Conte et al., 2010; Martin et al., 2010; Zhang et al., 2007). To control V. destructor, beekeepers have relied on chemical miticides, such as pyrethroids (fluvalinate, flumetrina, acrinathrin), amitraz, cimiazol, and coumaphos; however, the mites have developed resistance to most of these chemicals (Colin et al., 1997; Elzen et al., 2000; Spreafico et al., 2001; Thompson et al., 2002). Additionally, the miticides kill bees, affect their behavior and leave residues in honey and

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Corresponding author. E-mail addresses: [email protected] (E.V. Alquisira-Ramírez), [email protected] (G. Peña-Chora), [email protected] (V.M. Hernández-Velázquez), [email protected] (A. Alvear-García), [email protected] (I. Arenas-Sosa), [email protected] (R. Suarez-Rodríguez). http://dx.doi.org/10.1016/j.ecoenv.2017.03.050 Received 12 September 2016; Received in revised form 28 January 2017; Accepted 29 March 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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other bee products (Kochansky et al., 2001; Martel et al., 2007; Mullin et al., 2010; Pettis et al., 2004; Wallner, 1999). Organic products such as oxalic acid, formic acid and essential oils are an alternative for the control of V. destructor. However, the use of organic products has not been fully accepted by many beekeepers because they are difficult to apply, their effectiveness depends on the ambient temperature, and the sudden release of certain products may affect the bee brood and even cause the death of adults (Bogdanov et al., 2002; Desneux et al., 2007; Gregorc et al., 2004; Milani, 1999). In recent decades, the use of biological control agents such as bacteria, viruses and fungi as a new form of pest control has gained worldwide interest (Bale et al., 2008; Lacey et al., 2001). Among the biological control agents, the bacterium Bacillus thuringiensis is the most successful (Lacey et al., 2015), one of the reasons is that B. thuringiensis is a thermoresistive bacteria and can be found in many habitats (Petras and Casida, 1985). The bacterium has primarily been used in genetically modified (GM) plants against a variety of pest insects (RamirezRomero et al., 2005). Additionally, B. thuringiensis has effects against nematodes and mites (De Maagd et al., 2003; Schnepf et al., 1998). In a previous report using in vitro bioassays, we found the total protein of native B. thuringiensis strains was highly pathogenic to V. destructor, with mortalities above 80% 48 h after treatment (Alquisira-Ramírez et al., 2014). A notable feature of biological control agents such as B. thuringiensis is high specificity against the target pests and no negative effects on beneficial organisms (Bale et al., 2008; Chandler et al., 2001; Lacey et al., 2001; Milani, 1999). For example, as reported by AlquisiraRamírez et al. (2014), the strains EA3 and EA26.1 were virulent in vitro to V. destructor but did not produce mortality in A. mellifera larvae or adults despite concentrations that were 14- and 67-fold higher than LC50 for the mite. Both these strains were isolated from dead V. destructor. With a concentration of 20 μg/mL the EA3 strain produced a 93% of mite mortality 48 h after treatment, the LC50 was 7.1 μg/mL. With a concentration of 10 μg/mL the EA26.1 strain produced a 97% mite mortality 36 h after treatment, the LC50 was 1.50 μg/mL. The different concentrations used for the calculation of LC50 can be observed in previous investigations reported by Alquisira-Ramírez et al. (2014). The evaluation of the sublethal effects caused by B. thuringiensis is also very important because they can negatively affect the physiological condition or the behavior of bees. With the use of different tests, it is possible to measure the sublethal effects on bees caused by various chemical pesticides and some of the toxins from B. thuringiensis. For example, sublethal effects can be measured by changes in locomotor and foraging activity and food consumption (Cox and Wilson, 1984; Decourtye et al., 2004a). Additionally, a test as Proboscis Extension Response (PER) has been extensively used in behavioral physiology experiments to explore primarily the associative learning process of odors (Decourtye et al., 2004b, 2005; Ramírez-Romero et al., 2008; Taylor et al., 1987). To develop an alternative method to control V. destructor that is based on B. thuringiensis and is harmless to A. mellifera, it is important to determine whether there are deleterious effects on honey bees. In this work, we evaluated the in vitro effects of different concentrations of total protein of the EA3 and EA26.1 strains of B. thuringiensis on the weight of larvae and the syrup consumption, locomotor activity and Proboscis Extension Response (PER) of adults. Additionally, the effects of these strains were compared with a synthetic insecticide, cypermethrin.

the University of the State of Morelos, Mexico. As described by Alquisira-Ramírez et al. (2014), the strains were isolated from dead adult females of V. destructor and subsequently evaluated in vitro against the mites. The EA3 strain produced 93% mite mortality 48 h after treatment, with an LC50 of 7.1 μg/mL, and the EA26.1 strain produced 97% mite mortality 36 h after treatment, with an LC50 of 1.50 μg/mL (Alquisira-Ramírez et al., 2014). Bacillus thuringiensis strains were grown in Petri dishes with solid HCT medium for 72 h at 30 °C. The culture was recovered in 1 mL of sterile distilled water and 1 mM PMSF (phenyl methanesulfonyl fluoride) was added; total protein (spore-crystal complex) was quantified by the Bradford technique (Bradford, 1976). With a micropipette, total protein of B. thuringiensis strains were dissolved in a solution of 50% sucrose and 0.1% surfactant (adult bee bioassays) or larval diet (larval bioassays) to obtain the desired concentrations. Five concentrations (1, 5, 25, 50 and 100 μg/mL) were used to determine whether there was a dose–response relationship of the sublethal effects caused by B. thuringiensis on bees. These concentrations were previously evaluated and do not cause significant mortality in larvae or adult bees despite being up to 14- and 67-fold higher than LC50 for V. destructor (Alquisira-Ramírez et al., 2014). We used the cypermethrin (99%) as the positive control, which was provided by the National Center for Research in Veterinary Parasitology INIFAP, Morelos, Mexico. We evaluated previously different concentrations of cypermethrin (0.1, 0.3, 1, 10, 15, 30, 50 and 100 μg/kg) and we selected those that did not produce more than 30% of mortality (1, 10 and 15 μg/kg = part per billion [ppb]) in a period of two days (48 h). These concentrations were dissolved in a solution of 50% sucrose.

2. Materials and methods

2.3. Laboratory bioassay to exposure the adult A. mellifera to B. thuringiensis strains and cypermethrin

2.2. Laboratory bioassay to exposure the larvae of A. mellifera to B. thuringiensis strains The larval diet was prepared according to Kaftanoglu et al. (2011), with a composition of: 53% royal jelly, 4% glucose, 8% fructose, 1% yeast extract and 34% distilled water. The diet was divided into 2 mL centrifuge tubes and stored at −18 °C in a freezer until they were used. The diet was thawed and brought to 34 °C in a water bath immediately before feeding. With a micropipette, total protein of B. thuringiensis was dissolved in larval diet to obtain the desired concentrations. A total of 450 2.5- to 3-day-old larvae were used; the study had five treatment groups (90 larvae per treatment), with three replicates per treatment and 30 larvae in each replicate. One control group was fed a diet without the bacterial protein. The larvae were exposed to the protein on the first two days of feeding; each larva was fed with 10 μL of diet, for this, six aliquots of 50 μL of larval food with different concentrations of protein were placed in a polyethylene Petri dish (9 cm diameter) and five larvae were grafted onto each aliquot (30 larvae per Petri dish) (Alquisira-Ramírez et al., 2014). After the third day, the larvae were fed gradually as described by Kaftanoglu et al. (2011). In our case, the days: three, four, five and six, the five larvae were fed on aliquots of 100, 150, 200 and 250 μL of larval diet respectively. Every day, the larvae were placed in new Petri dishes and the larvae were gently placed on top of the fresh food (Alquisira-Ramirez, 2014; Kaftanoglu et al., 2011). The Petri dishes were transferred into a humidity chamber and maintained at 34 °C and 90% RH. On the seventh day, when stopped feeding and pupal phase began (9.5 or 10 days of age) the prepupae were transferred to filter paper and weighed (Aupinel et al., 2005; Kaftanoglu et al., 2011).

2.1. Sources of B. thuringiensis strains and preparation of inoculum To obtain bees with the same age for the bioassays, combs containing mature worker bee pupae were removed from two mite-free colonies and maintained at 34 °C and 70% humidity (Evans et al.,

The EA3 and EA26.1 strains of B. thuringiensis were provided by the Vegetal Parasitology Laboratory at the Center of Biological Research at 70

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4 °C for four min or until they showed the first signs of immobility. Each bee was mounted into a small plastic tube, with strips of adhesive tape attached between the head and thorax and over the abdomen. Thus, a harnessed bee could only move its antennae and mouthparts, including the proboscis. The bioassay for the PER was performed with each antenna touched with a droplet of water or sucrose solution. Assays began 30 min after the last bee of each trial was prepared (Pankiw and Page, 1999). We used the PER to measure responsiveness to the following sucrose concentrations: 0.1%, 0.3%, 1%, 10%, 30% and 50%, which were offered in ascending order to reduce the potential sensitization that can occur with higher concentrations of sucrose (Scheiner and Arnold, 2010). When a bee showed proboscis extension, the PER was recorded, and the total number of proboscis responses to the six sucrose stimulations was counted for each individual. The total number of responses was the gustatory response score of a bee. Solutions were applied to antennae with a 2-min, inter-trial interval. Small movements of the proboscis that did not result in full extension were not counted as a response.

2009; Pankiw et al., 2004; Scheiner and Arnold, 2010). Groups of 10 newly emerged adult bees (> 24 h old) were transferred to plastic containers (clear, plastic 14 oz tumblers); four square centimeters were removed and covered with mosquito mesh for ventilation (AlquisiraRamírez et al., 2014; Evans et al., 2009). The experimental unit was three plastic containers with 10 bees each and three replicates of 30 bees were used for each concentration, for a total of 90 bees per treatment (Shaw et al., 2002). Two hours after being transferred to the containers, the bees were fed with different concentrations of protein of B. thuringiensis or cypermethrin mixed in a solution of 50% sucrose and 0.1% surfactant (25% alkyl phenol polyoxyethylene ether, 1% antifoam agent, 74% diluent). The solution was deposited into a feeding device on the top of the plastic tumbler and delivered ad libitum. One control group was fed with 50% sucrose and 0.1% surfactant without the bacterial protein or cypermethrin. When the spore-crystal complex of B. thuringiensis is ingested by the susceptible insect the toxic effect is triggered, due to the action mechanism, the insect dies in a few hours (Bravo et al., 2004; Whalon and Wingerd, 2003). For this reason and in accordance with the criteria from the United States Environmental Protection Agency (US EPA, 1996) the bioassay of acute toxicity was developed during two days. Also, in order to evaluate the effect of chronic toxicity we performed a second test with duration of seven days (AlquisiraRamírez et al., 2014). The bioassays were maintained in an incubator at 34 °C and 40% RH. The average in the syrup consumption was estimated by measuring the difference in volume between the first and last days of treatment then it was divided among the surviving bees (Evans et al., 2009; Higes et al., 2007; Papadopoulou-Karabela et al., 1992; Ramirez-Romero et al., 2008; US EPA, 1996).

2.6. Statistical analyses Due to the distribution of values obtained in each pathogenicity bioassay, the data of larval development, the syrup consumption and the locomotor activity were analyzed by ANOVA tests. The data of flights and Proboscis Extension Response (PER) were analyzed by Kruskal-Wallis tests (B. thuringiensis bioassays) and ANOVA tests (cypermethrin bioassays). Also, multiple comparisons of means were performed using Tukey’s test, with a significance level of 0.05. In these cases, we used the SAS statistical program version 9.0. Angular transformation of data was performed previously.

2.4. Locomotion assay 3. Results After exposure to B. thuringiensis or cypermethrin, when bees were three and eight days old, the locomotor activity of A. mellifera was evaluated. For each concentration, three replicates of 10 bees were randomly taken from the different plastic containers, for a total of 150 adult bees from the five B. thuringiensis treatments and 90 adult bees from the cypermethrin treatments. One control group (30 adult bees) fed without bacterial protein or cypermethrin was evaluated. The bees were placed individually in the center of a clean polystyrene Petri dish (14 cm diameter), which was placed on a paper grid (1.5 cm2 pattern). The activity was evaluated for two minutes; the number of grid marks crossed by the bee was recorded with a manual counter. Only grids that were fully crossed by the center of the bee’s thorax were recorded. Grid marks crossed twice because of a reversal in direction were not recounted. Additionally, the bees were introduced individually into a square wooden cage (30 cm on each side) covered with mosquito mesh, and the number of flights performed by each bee was recorded for two minutes. All measurements were obtained in a room with ambient lighting (Aliouane et al., 2009; Humphries et al., 2005).

After the bioassay of acute toxicity in adult bees, the B. thuringiensis strains evaluated in the present study do not cause mortality (AlquisiraRamirez, 2014). Also in the bioassay of chronic toxicity, the mortalities recorded by the EA3 strain were considerably lower (between 2.2% and 5.6%) and the EA26.1 strain caused mortality between 2.2% and 8.9%, no statistical differences were found in relation to the control group (Alquisira-Ramírez et al., 2014). During in vitro rearing of A. mellifera, mortality between 18% and 32% was observed, however, it was not attributed to B. thuringiensis, because the control group larvae also had a mortality rate of 18%, which was not statistical different from the treatments (AlquisiraRamírez et al., 2014). In cypermethrin bioassays, treatment with 1 μg/kg presented 2% mortality, while the treatments with 10 and 15 μg/kg recorded 23% and 29% mortality respectively and were statistically different in relation to the control group (Alquisira-Ramirez, 2014). 3.1. Effect on development of larval Apis mellifera To observe the effects of B. thuringiensis on larval development, when the larvae stopped feeding, they were removed from the feeding dishes and weighed. The results are shown in Table 1. The development of larvae treated with strain EA3 was not negatively affected (F =0.36, 5 df; P=0.86), and the larvae weighed 83 mg on average. Similarly, the development of larvae treated with strain EA26.1 was not affected, and larvae weighed 80 mg (F =0.73, 5 df; P=0.61). For the evaluated concentrations of B. thuringiensis, the results indicated that the development of A. mellifera larvae was not affected.

2.5. Sucrose sensitivity assay After exposure to B. thuringiensis or cypermethrin, the sucrose sensitivity of A. mellifera was evaluated, bees were three and eight days old (see following discussion). For each concentration, three replicates of 10 bees were randomly taken from the different plastic containers, for a total of 150 adult bees from the five B. thuringiensis treatments and 90 adult bees from the cypermethrin treatments. One control group (30 adult bees) fed without bacterial protein or cypermethrin was evaluated. Proboscis Extension Response (PER) is reflexive of bees all ages in response to antennal stimulation with solutions of sucrose. To facilitate their manipulation, the bees were stored in a refrigerator maintained at

3.2. Syrup consumption After exposure to B. thuringiensis or cypermethrin, the syrup 71

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were observed for the bees that were treated for seven days with different concentrations of B. thuringiensis protein.

Table 1 Mean larval weights (mg) after feeding for seven days on different total protein concentrations of B. thuringiensis. B. thuringiensis strains

3.3. Locomotor activity EA3 Concentrations (µg/mL)A

Weights ± SEM

Control 1 5 25 50 100

89 ± 17 86 ± 20 82 ± 30 70 ± 11 67 ± 4 106 ± 30

EA26.1

The movements of adults treated for two days with concentrations of total protein of strain EA3 were not significantly different from those of the control group (F =0.53; 5 df; P=0.748), which were between 109 and 139 grid marks crossed (Fig. 3a). Similarly, the bees treated with strain EA26.1 crossed between 116 and 144 marks, and no differences were observed compared with the control group crossings (F =0.45; 5 df; P=0.805). Additionally, in Fig. 3a, the movements of bees treated with cypermethrin are shown. With exposure to the miticide, the most crossings, 131, were observed in the control group. When the chemical concentration increased, the activity was reduced, and the bees treated with the highest concentration of cypermethrin recorded an average of only 90 crossings, although no significant differences were recorded (F =2.45; 3 df; P=0.14). To further evaluate locomotor activity, the number of times that the bees flew in two minutes was recorded (Fig. 3b). The number of flights for bees treated with protein concentrations of the EA3 strain ranged between three and five, with no significant differences (X2 =3.94; df =5; P=0.557). The number of flights for bees treated with concentrations of the EA26.1 strain ranged between four and eight, with no significant differences (X2 =11.01; df =5; P=0.088). The mean number of flights of bees treated with cypermethrin is shown in Fig. 3b. The control group recorded the most flights with six, and when the concentration was increased to 15 μg/kg, the number of flights was reduced to two; however, no significant differences were detected (F =0.84, df =3; P=0.50). After seven days of continuous feeding with the concentrations of the EA3 strain proteins, each bee crossed between 146 and 164 marks (Fig. 4a). The number of marks crossed by bees treated with EA26.1 strain proteins ranged between 155 and 179. No significant differences were found between the treatment and control groups (F =1.12; 5 df; P=0.400 and F =0.42; 5 df; P=0.826, respectively). Similarly, the number of flights of bees treated with concentrations of EA3 strain proteins ranged between five and nine (Fig. 4b), without statistically significant differences (X2 =4.21; 5 df; P=0.518). Additionally, no effects were recorded in the number of flights of bees treated with the EA26.1 strain 285 (X2 =4.47; 5 df; P=0.48). All tests of bee locomotor activity found no dose–response relationship for either strain. Therefore, unlike cypermethrin, honey bees that were treated for seven days with different concentrations of B. thuringiensis proteins

82 ± 12 76 ± 10 74 ± 3 92 ± 13 68 ± 10 89 ± 11

Tukey test showed not significant difference between treatments and control groups (P > 0.05).

consumption of adult bees was quantified, and the results are shown in Fig. 1. Bees treated for two days with concentrations of total protein of strain EA3 consumed between 44 and 54 μL of syrup daily, and the bees treated with concentrations of strain EA26.1 consumed between 57 and 62 μL of syrup (Fig. 1). For both strains, no significant differences in syrup consumption were observed between the five treatments and the control (F =0.17; 6 df; P=0.980 and F =0.16; 6 df; P=0.984, respectively). As shown in Fig. 2, syrup consumption of bees treated with cypermethrin was also recorded, and unlike the treatments with B. thuringiensis, a dose-response relationship was observed for the effects of cypermethrin on syrup consumption. Bees in the control group consumed an average of 67 μL of syrup, which was progressively reduced in treatments with cypermethrin until the concentration of 15 μg/kg (56 μL of syrup per bee); however, the differences between treatments and the control were not statistically significant (F =1.34; 4 df; P=0.32). After seven days of continuous feeding on the concentrations of protein of the EA3 strain, the daily consumption of each bee was between 26 and 29 μL of syrup, whereas the bees treated with the proteins of the EA26.1 strain consumed between 25 and 31 μL (Fig. 2). No significant differences were found between the treatments and the control (F =0.12; 6 df; P=0.99 and F =0.79; 6 df; P=0.59, respectively). The syrup consumption of bees treated with cypermethrin was not measured because unlike B. thuringiensis protein at seven days after treatment, more than 30% of the bees had died. Similarly, the effects on locomotor activity and sucrose sensitivity were not measured. In contrast to cypermethrin, no adverse effects on syrup consumption

Fig. 1. Mean syrup consumption ( ± SEM) two days after feeding bees with different concentrations of B. thuringiensis proteins or cypermethrin. NS: not significantly different from the control (P > 0.05).

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Fig. 2. Mean syrup consumption ( ± SEM) seven days after feeding bees with different total protein concentrations of two strains of B. thuringiensis. NS: not significantly different from the control (P > 0.05).

Fig. 3. Mean number of crossings (a) and flights (b) ( ± SEM) two days after treating bees with different concentrations of B. thuringiensis proteins or cypermethrin. NS: not significantly different from the control (P > 0.05).

ranged between 40% (treatment with 50 μg/mL) and 77% (treatment with 100 μg/mL). Following statistical analysis of the responses, no significant differences were found between the treatments and the control (X2 =4.78; 5 df; P=0.442). Similarly, for the PER of bees treated with EA26.1 (Fig. 5b), the treatment with 100 μg/mL had the highest response rate (50%); however, no significant differences were found between the treatments and the control (X2 =1.39; 5 df; P=0.925). Although PER began with a sucrose concentration of 1% in bees treated with cypermethrin (Fig. 5c), the response rate was

showed no adverse effects on locomotor activity. 3.4. Sucrose sensitivity PER was quantified forty-eight hours after exposure to B. thuringiensis or cypermethrin, and the results are shown in Fig. 5. For bees treated at different concentrations of total protein of the EA3 strain (Fig. 5a), in most treatments, a concentration of 10% sucrose initiated the PER. When bees were stimulated with 50% sucrose, the responses 73

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Fig. 4. Mean number of crossings (a) and flights (b) ( ± SEM) seven days after treating bees with different total protein concentrations of two strains of B. thuringiensis. NS: not significantly different from the control (P > 0.05).

no significant differences were found between treatments and the control (X2 =3.31; 5 df; P=0.651). In the analysis of the responses of bees treated with proteins of strain EA26.1, no significant differences were recorded in the responsiveness to sucrose (X2 =7.82; 5 df; P=0.165). Unlike cypermethrin, no adverse effects on sucrose sensitivity were observed for bees that were treated for seven days with different concentrations of B. thuringiensis proteins.

significantly affected by the increase in concentration (F =5.55; 3 df; P=0.02). The PER of bees in the control group was 60%, whereas bees treated with the concentration of 15 μg/kg cypermethrin had a PER of only 23%. Subsequently, the responses of bees treated for seven days with the EA3 strain were analyzed. The highest percentage of PER was 60%, which was observed in the treatment with 1 μg/mL (Fig. 6a and b), but

Fig. 5. PER after two days of exposure to B. thuringiensis proteins or cypermethrin. The different letters associated with treatments indicate differences among treatments as determined by Tukey’s test (P < 0.05).

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Fig. 6. PER seven days after treating bees with different concentrations of total protein of two strains of B. thuringiensis. NS: not significantly different from the control (P > 0.05).

4. Discussion

needed to develop this theory. In comparison with other reports we observed slightly lower weights in larval rearing in vitro (between 67 and 106 mg), however, are within the observed by other authors. Hendriksma et al. (2011) found an average weight of 141 mg, while Aupinel et al. (2005) reported weights ranging from 97 to 124 mg. On the other hand, Kaftanoglu et al. (2011) reported weights between 85 and 313 mg of larvae fed with different diets. The differences obtained in the larvae weight may be due to types of diet that are administered to them, because each author is making modifications to existing protocols in the different investigations (Silva et al., 2009). The causes of the observed variance in weight could also be due to practical experimenter skills, different degrees of brood stress, genetic variation and larval age heterogeneity at grafting (Hendriksma et al., 2011). Due to the action mechanism of B. thuringiensis, when the sporecrystal complex is ingested the insect dies in a few hours (Bravo et al., 2004; Whalon and Wingerd, 2003). For this reason and in accordance with criteria from the United States Environmental Protection Agency (US EPA, 1996) the bioassay of acute toxicity was developed during two days, also, in order to evaluate the effect of chronic toxicity we performed a second test with duration of seven days (AlquisiraRamírez et al., 2014). Gut paralysis is one symptom of some insects after exposure to the Cry toxins of B. thuringiensis; therefore, these insects stop feeding and ultimately die (Oppert et al., 2012). However, according to the results of this study, although bees were treated with high concentrations of total protein of B. thuringiensis, the consumption of syrup was not affected. On the seventh day of treatment, the average syrup consumption was 28 μL per bee per day. These results were similar to those obtained by Babendreier et al. (2005), who observed that the mean consumption per day ranged from 33 to 38 μL at days after feeding bees a sucrose solution that contained more Cry1Ab protein (360 μg/25 mL). Additionally, Malone et al. (1999) and Malone et al. (2004) found that bees fed Cry1Ba toxin consumed an average of 32 μL of syrup daily. In all of these cases, Cry toxins did not negatively affect the food consumption of bees. Statistical analysis did not show differences between cypermethrin treatments, but we believe it is important to make it clear that unlike the treatments with B. thuringiensis a dose-response relationship was observed for the effects of cypermethrin on syrup consumption (the bees consumed less syrup with the increase in concentration). After two days, more than 30% of the bees died; therefore, we could not assess the effects of cypermethrin at seven days (Alquisira-Ramirez, 2014). In order to find sublethal concentrations of cypermethrin on bees during a period exposure of seven-days, lower concentrations were used for

The decline of A. mellifera colonies is causing concern about the future of biodiversity and agricultural production, and most researchers agree that the interactions of V. destructor with diseases caused by microorganisms and pesticide residues, among others, play a decisive role in the annual loss of millions of colonies of A. mellifera worldwide (Boecking and Genersch, 2008; De Rycke et al., 2002; Glinski and Jarosz, 1992; Martin et al., 2010; Zhang et al., 2007). Therefore, new methods of pest control are required that do not pollute and are more environmentally friendly. Alquisira-Ramírez et al. (2014) showed that the use of entomopathogenic microorganisms such as B. thuringiensis offers an alternative to control V. destructor because the bacterium is virulent to the mite but does not cause mortality in bees. Therefore, the focus of this study was to evaluate potential sublethal effects on bees caused by two strains of B. thuringiensis that were described by Alquisira-Ramírez et al. (2014). The artificial rearing of bees using diets contaminated with toxic substances is an in vitro method to evaluate the weight and other variations in developing larvae (Hendriksma et al., 2011). In this study, the effect of different concentrations of the total protein of B. thuringiensis (strains EA3 and EA26.1) on the weight of A. mellifera larvae was determined. Based on the results, the two strains evaluated had no negative effects on larval development. In a previous report, the Cry1Ab toxin of B. thuringiensis in the pollen of transgenic plants also had no negative effects on the size of four- to five-day-old larvae (Hanley et al., 2003). To date, no toxic effects of the proteins of B. thuringiensis have been demonstrated for the larvae and adults of A. mellifera (Chilcutt and Tabashnik, 1999; Duan et al., 2008; Jia et al., 2016; Walker et al., 2007). The absence of toxicity could be because the pH of the bee intestine is usually acidic, and B. thuringiensis toxins, in addition to those of other bacillus pathogens, are activated at alkaline pH values (Mohr and Tebbe, 2006; Rausell et al., 2004). The survival of bees could also be due to different defense barriers, including the immune system, the antimicrobial properties of the food (Brødsgaard et al., 1998; Crailsheim and Riessberger-Galle, 2001; Feldlaufer et al., 1993; Spivak and Gilliam, 1998; Wedenig et al., 2003) and some anatomical structures (proventriculus) (Han et al., 2012; Peng and Marston, 1986; Smith, 2012). Although no significant differences between the weights of larvae treated with B. thuringiensis and the control group were detected, larval development increased in larvae fed 100 μg/mL of total protein. The food that worker bees use to feed larvae contains a high percentage of protein from the pollen of flowers; thus, B. thuringiensis is a protein-rich source that larvae could use for development, but more studies are 75

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example 0.1, 0.3 and 1 μg/kg, however we believe that the comparison with the entomopathogen would not be proportional because the concentrations of B. thuringiensis used were much higher. Other pyrethroids, such as deltamethrin, also produce high mortality and a significant reduction in the consumption of contaminated syrup a few days after starting treatment (Decourtye et al., 2004b; Ramirez-Romero et al., 2005). Such a reduction in food consumption can lead to serious damage to honey bees because food consumption is directly correlated with the hive productivity. The ultimate result of reduced consumption is the poor collection of nectar and pollen. Our results indicated that the strains of B. thuringiensis evaluated in this study, unlike some chemicals such as cypermethrin, were safe to A. mellifera, even when the bees were fed for several days with high concentrations of total protein. We also observed a remarkable decrease in food consumption of seven-day-old bees (average consumption of 28 μL) compared with that of two-day-old bees (between 49 and 59 μL). Malone et al. (2004) assessed the food consumption of bees using seven different diets for ten days, which included some toxins of transgenic plants and found that the consumption of seven-day-old bees was approximately 40% lower than that of two-day-old bees. Although the authors did not discuss this decline, this phenomenon could be due to changes in the eating habits of bees based on polyethism and nutritional needs; however, further investigations are required to test this hypothesis. Deterioration in locomotor activity is one of the symptoms observed in bees poisoned with chemical or biochemical substances; therefore, we conducted a study in which the effect on locomotion was evaluated in bees that fed on the total protein of two strains of B. thuringiensis. Some insects show a general paralysis a few hours after exposure to the Cry1Ab of B. thuringiensis, which can last for several hours or even days (Oppert et al., 2012). In this study, we observed that the locomotor activity (grid mark crossings and flights) of bees treated with different total protein concentrations of B. thuringiensis was not adversely affected. Although no significant differences were observed for the bees treated for two days with cypermethrin, the bees consumed less syrup at high concentrations, and compared with the control group, locomotor activity was reduced more than 30% in bees fed with 15 μg/ kg of cypermethrin. According to some reports, cypermethrin at low doses causes problems in carbohydrate metabolism (Bendahou et al., 1999); thus, the bees are deprived of a primary source of energy, which may limit foraging activity, generate problems in dance communication and ultimately affect hive development and cause its death (El Hassani et al., 2008; Lambin et al., 2001). Therefore, unlike cypermethrin, B. thuringiensis did not endanger the performance of bees in the different tasks within the hive. Additionally, at seven days, the bees had more movements 137–171 crosses on the grid and 5–10 flights) than those of bees at two days of age 126–127 crosses on the grid and 3–8 flights). This difference was likely because the locomotion reflex is low in newborn bees and they do not move in response to light; for example, newly emerged bees (40–90 min) only perform between 25 and 50 crosses on the grid in the two minutes (Humphries et al., 2005). Pankiw and Page (1999) noted that bees mature during the first five days of adulthood, after which they are more sensitive to light and are more active. The technique of Proboscis Extension Response (PER) is used in experiments to assess sublethal effects of some chemical pesticides and transgenic products on the olfactory learning ability of bees (Decourtye et al., 2004b; El Hassani et al., 2008; Han et al., 2010; Dai et al., 2012; Ramirez-Romero et al., 2008). The PER is a test carried out mainly in bees over 14–15 days old, before worker bees become foragers (Decourtye et al., 2005; Scheiner and Arnold, 2010), however, the proboscis extension response is part of the eating behavior of bees of all ages. The purpose of the present study was to demonstrate that EA3 and EA26.1 strains of B. thuringiensis that were virulent in vitro to V. destructor do not affect bees of any age, with this, in the future a formulation could be developed and applied inside

the hive without side effects on bees. On this occasion, we use the PER in bees of three and eight days old because this technique has shown that the three main worker castes including nurses associatively learn the smell of food that has been collected by only a few fodder, therefore, olfactory information is shared and acquired by bees of all ages, which influences behavior inside and outside the hive (Grüter et al., 2006; Pankiw et al., 2004). Ramirez-Romero et al. (2008), mention that certain pesticides can produce repellency of the contaminated syrup towards the bees. Currently, PER capacity also has been used for testing olfactory repellency of certain chemicals to insects such as butyric acid and DEET (N,N-diethyl-3 methylbenzamide) mixture (Abramson et al., 2010) and a natural product as citronella (Malerbo-Souza and Nogueira-Couto, 2004). In the present study, treatment with B. thuringiensis strains EA3 and EA26.1 did not affect the PER. This result is consistent with a previous study in which the Cry1Ah toxin of B. thuringiensis had no adverse effect on the PER of A. mellifera ligustica (Dai et al., 2012). Additionally, the Cry1Ab toxin at a concentration of 3 μg/kg does not modify the response; however, honey bees exposed to Cry1Ab at 5000 μg/kg show a higher percentage of PER compared with that of the control group (Ramirez-Romero et al., 2008). Rapid responses were also found for bees that had previously been exposed to conventional insecticides (Weick and Thorn, 2002) and some transgenic products (Picard-Nizou et al., 1997; Pham-Delègue et al., 2000). In this study, two days after feeding bees with sucrose and cypermethrin at concentrations of 10 and 15 μg/kg, a reduction was observed in the PER of 22% and 61%, respectively. Taylor et al. (1987) evaluated six different pyrethroids, and based on their results, the treated bees respond only until the concentration of the stimuli reaches 60%. Some chemical pesticides, such as imidacloprid, affect the first stage of bee memory (short-term memory) when information is stored and then interfere with memory consolidation (long-term memory), most likely due to the physiological effects that occur in the brain neuropiles, which are an essential element in the role of olfactory memory (Decourtye et al., 2004a). The tests performed with the EA3 and EA26.1strains showed that B. thuringiensis do not cause effects of repellency or alteration in the PER; thus, it was unlikely that the sensory process was disturbed in the development of memory (Ramires-Romero et al., 2008). Unlike some synthetic insecticides such as cypermethrin and some concentrations of B. thuringiensis toxins found in GM plants, the total protein concentrations of the B. thuringiensis strains evaluated in this investigation did not affect the proboscis extension response. The results of this study will be a valuable contribution to beekeeping worldwide because with the strains tested, we can develop new formulations based on B. thuringiensis to control V. destructor, the primary vector of microbial diseases of A. mellifera. With the new formulations, the overuse of synthetic miticides can be reduced, which produce both lethal and sublethal effects on bees. Thus, in the future, our scientific contributions may help reduce losses of honey bee colonies. Acknowledgments E. V.A.R. acknowledges the Consejo Nacional de Ciencia y Tecnología for a PhD fellowship and the anonymous reviewers for critical comments to improve the manuscript. References Abramson, C.I., Giray, T., Mixson, T.A., Nolf, S.L., Wells, H., Kence, A., Kence, M., 2010. Proboscis conditioning experiments with honeybees, Apis mellifera caucasica, with butyric acid and DEET mixture as conditioned and unconditioned stimuli. J. Insect Sci. 10 (122), 1–17. Aliouane, Y., El Hassani, A.K., Gary, V., Armengaud, C., Lambin, M., Gauthier, M., 2009. Subchronic exposure of honeybees to sublethal doses of pesticides: effects on behavior. Environ. Toxicol. Chem. 28 (1), 113–122.

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