Avoidance Behavior of Honey bee, Apis mellifera from Commonly used Fungicides, Acaricides and Insecticides in Apple Orchards

INTRODUCTION

Honey bees are vegetarians, usually consuming only nectar and pollen from plant blossoms or sweets like sugar syrup and honey dew (Atkin and Anderson. 1976 (1970)). These insects are highly social and a colony may contain as many as 10,000 to 100,000 individuals, depending on the time of year, prevailing weather conditions and availability of nectar and pollen sources (Robinson, 1979; Proctor et al., 1996). Honey bees provide not only the income sources for beekeepers but also pollination services for crop production (Degrandi-Hoffman, 1978; NRCS, 2005; Jung, 2008; Jung, 2014). Amongst beneficial insects, honey bees are considered as the most efficient and reliable pollinators of various agricultural crops (McGregor, 1976). Recently increased concerns on honey bee population decline have wide spread not only within Korea but over the world (Jung, 2014). There are several factors contributing to the decline of honey bees as well as the pollination services (Goulson et al., 2015), but considered as the combined stresses from parasites, pesticides and lack of foraging sources. Habitat destruction, including nesting and mating sites, and alternative forage, is the main issue in the decline of pollinators (Batra, 1995). Bee poisonings from diverse chemicals especially of insecticides are another important source of honeybee population decline together with parasitic pests and diseases (Crane and Walker, 1983; Choi and Lee, 1986). Pesticides constitute an important component of modern agriculture without which the increase in agricultural production could never be achieved (Atkins et al., 1970). However, the large scale and indiscr-iminate use of pesticides has resulted in great conflict of interests because of simultaneously occurrences of harmful effects to biotic and abiotic agricultural environments (Kevan and Plowright, 1995). Many agrochemicals sprayed in the fields for crop protection against insect and disease pests are injurious to honeybees (Johansen, 1977). Impacts of these chemicals can occur directly as the mortality on contact or ingestion or indirectly elicit behavioral disorder or abnormal development of immatures. In many cases of acute toxicity, poisoned bees often become irritable (likely to sting), paralyzed or stupefied, appear to be ‘chilled’ or exhibit other abnormal behavior (Dharmawardena, 1994).

Avoidance behavior is one of the behavioral survival mechanisms of organism together repair or tolerance of molecular or physiological damage from toxic chemicals (Bowler et al., 1992; Li-Byarlay et al., 2016), Avoidance behavior includes irritancy which is occurring after physical contact and repellency occurring without physical contact but often with olfactory means (Chareonviriyaphap et al., 1997). Here we assessed the avoidance tendencies of relatively low toxic pesticides such as acaricides and fungicides commonly used in apple production in Korea. Choice test given only 50% sucrose solution and pesticidemixed sucrose solution as food estimated the avoidance in laboratory and field assessments were measured after spraying each selected chemical.

MATERIALS AND METHODS

Pesticide avoidance test in the laboratory

Pesticides: Sixty three pesticides (Table 1, 2, 3; 16 fungicides, 12 acaricides and 35 insecticides) were tested for the study. From the registry of pesticides on apple, selection was based on the chemicals that could be used in blooming season as the first priority and then mostly common use as the second priority.

Honeybee: Honey bees used in the experiments were from the experimental apiary of Andong National University. About 30 colonies of Apis mellifera, hybrids of A. m. ligustica were maintained with conventional methods but limited pesticide uses. For the avoidance assay, mixed ages of worker bees were collected from randomly chosen hives.

Cylindrical cages made of stainless wire ( 13.5 x H10.5cm) were used for this test. Five honey bees were kept in one cage considered as single replication and total 5 replications per pesticide were conducted. No food or water was provided for 2 hours before the experiments. Then, 50% sucrose solution and pesticide-mixed 50% sucrose solutions retaining recommended dose of pesticides were provided into two separate pots maintaining 9cm distance for detecting pesticide avoidance under 25±3°C, 50~80% RH and dark condition. Choice of each food source was checked for 5 minutes at the intervals of 1, 2 and 3 hours. When bee is on the food item and showed feeding behavior of touching food and retracting proboscis, it was considered as choice. The avoidance index was calculated by the formula:

$\[ \text{AI=}\frac{{\text{N}}_{\text{p}}}{{\text{N}}_{\text{c}}\text{+}{\text{N}}_{\text{p}}} \]$

Table 1.

List of fungicides with chemical class, common name, amount of active ingredient (AI, %), formulation type and recommended concentration (g or ml of formulated product/20L) used in the laboratory test and in the field (**)

Table 2.

List of acaricides with chemical class, common name, amount of active ingredient (AI, %), formulation type and recommended concentration (g or ml of formulated product/20L) used in the laboratory test and in the field (**)

Table 3.

List of insecticides with chemical class, common name, amount of active ingredient (AI, %), formulation type and recommended concentration (g or ml of formulated product/20L) used in the laboratory test and in the field (**)

where AI = avoidance index, N_p = number of bees chosen pesticide-mixed sucrose solution, and N_c = number of bees chosen sucrose solution. The lower the avoidance index (AI) value, the higher the avoidance tendency. If the value is significantly lower than 0.5, it was considered as avoiding.

RESULTS AND DISCUSSION

Pesticide avoidance test in the laboratory

In this study, honeybee showed significant avoidance to all tested fungicides (Fig. 1, P<0.05) in the laboratory test. This means that honeybee discriminate fungicide-mixed sucrose solution. Only to iminocatadine triacetate, the AI values was marginally significant (P=0.07). This chemical is used for apple bitter rot or valsa canker management. AI index of most fungicides used in apple orchards showed ranges from 0.12 to 0.37 with carbendazim lowest. Carbendazim is relatively non-toxic to honeybee in contact and feeding test (Kang, 2009).

Fig. 1.

Avoidance indices (Mean±SE) of honeybees to fungicides. Value significantly lower than 0.5 means high avoidance.

Most of acaricides tested in the experiment showed lower than 0.2 AI values indicating significant avoidance tendency (Fig. 2). AI value of phosphamidon was not significant which means honeybee does not discriminate the chemical (P=0.24). Phosphamidon is an organophosphate insecticide developed in 1960s. It acts as a choline esterase inhibitor, and showed high toxicity to honeybee in the laboratory experiments (Kang, 2009), while the lowest avoidance index was observed from acequinocyl (0.07), which showed very low toxicity to honeybee in the contact and feeding test (Kang, 2009).

Fig. 2.

Avoidance indices (Mean±SE) of honeybees to acaricides. Value significantly lower than 0.5 means high avoidance.

Honeybees showed significant avoidance to most of the insecticide tested (Fig. 3, P<0.05), except chlorpyrifos (P=0.06), tebufenpyrad (P=0.07) and diflubenzuron (P=0.17). The avoidance index was found lowest (0.09) in cyhexatin and highest in diflubenzuron (0.37).

Fig. 3.

Avoidance indices (Mean±SE) of honeybees to insecticides. Neonicotinoid insecticides were marked with *. Value significantly lower than 0.5 means high avoidance.

Interestingly, avoidance behavior to neonicotinoid insecticides showed bifurcated patterns; highly avoided (AI<1.2) to thiacloprid, acetamiprid while less avoided (AI>2.5) to thiacloprid, imidacloprid, thiamethoxam and dinotefuran. Imidacloprid and thiaclopriod are much more toxic to honeybee than acetamiprid or thiacloprid in the laboratory contact or feeding trials or field, leading EU ban of those chemicals (Cressey, 2013; Lee et al., 2016). Less discrimination of these chemicals by honeybee could result in synergized toxicity in the field to honeybee (Woodcock et al., 2016; Alburaki et al., 2017) or bumblebee (Arce et al., 2017). If bees are exposed to guttation of the plants where neonicotinoids were applied into the soil such as seed coating, the negative impact could exacerbated by the increased concentration of the systemic pesticide (Girolami et al., 2009).

Pesticides avoidance test in the field

From the field trials evaluating avoidance tendency of honeybee from pesticide-treated flower blossoms, flower visiting rates were variable on treatments. Fenarimol, diflubenzuron and carbary showed no difference on the flower visiting rates of honey bees measured 2-4 DAT and 8-10 DAT (Fig. 4). This implies that those chemicals would not elucidate the behavioral changes of honeybee foraging possibly via contact or olfactory cures. Flower visiting rates were relatively lower in acequinocyl, amitraz, fenthion, acephate, diclorovos and abamectin. All treatments showed avoidance. AI values were ranged from 0.23 to 0.42 (Fig. 4). The responses were categorized into three groups; highly, moderately and marginally avoided. Abamectin, fenthion, amitraz and acequinocyl showed highly avoided which showed high avoidance in the laboratory test, too. While fungicide of fenarimol, acaricides of acrinathrin and phosphamidon, IGR insecticide of diflubenzuron and neonicotinoid insecticide, imidacloprid showed less avoidance in the field test. Interestingly, fewer numbers of honeybees were foraging abamectin and fenthion sprayed flowers, but high numbers of honeybees were found forging on flowers where difulbenzuron and carbaryl were sprayed (Fig. 4). Recently more apple growers spay carbaryl for flower and fruit thining, higher toxic effects on foraging honeybee as well as broods are expected. This result could partly explain the massive bee poisoning during apple flowering season. Kim et al. (2014) reported that carbaryl is the most responsible chemical imposing greater risk to honeybee during apple flowering season.

Fig. 4.

The comparison of visiting rate of honeybees to the apple flowers at 2 to 4 and 8 to 10 days after spray. Sixteen pesticides were sprayed and flower visiting rates (No. bees/blossoms/10 min) were estimated. On the same chemical, different letters on the bar indicate significant difference of visiting rates in two-time frames.

Fig. 5.

Avoidance tendency of honeybees from the flower blossoms where 16 pesticides were sprayed in apple orchards.

Avoidance is an adaptive behavior through which an organism could escape the hazard for better survival. Through the series of laboratory and field experiments, we found that honeybee showed significant avoidance tendencies from the toxic pesticides. Interestingly, avoidance tendencies were much strong to fungicides and acaricides than to insecticides. Lower physiological susceptibility to fungicides or acaricides together with strong avoidance would benefit to survival of honeybees. But weaker avoidance to some insecticides including neonicotinoids of imidacloprid or thiamethoxam could lead to danger of bee poisoning.

Acknowledgments

This work was partly supported from RDA agenda project (PJ01303701) on evaluation of honey pollination services for crop production.

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