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Journal of Apiculture - Vol. 38 , No. 4
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[ Original research article ]
Journal of Apiculture - Vol. 37, No. 2, pp. 135-141
Abbreviation: J. Apic.
ISSN: 1225-0252 (Print)
Print publication date 30 Jun 2022
Received 25 Nov 2021 Revised 20 Apr 2022 Accepted 21 Apr 2022
DOI: https://doi.org/10.17519/apiculture.2022.06.37.2.135

Longevity-enhancing Effects of Rosmarinic Acid Feeding on Honey bees (Apis mellifera L.) after Exposure to Some Pesticides Used in Strawberry Greenhouse
Delgermaa Ulziibayar1 ; Tekalign Begna1 ; Daniel Bisrat2, 3 ; Chuleui Jung1, 3, *
1Department of Plant Medicals, Andong National University, Andong 36729, Republic of Korea
2Department of Pharmaceutical Chemistry and Pharmacognosy, School of Pharmacy, College of Health Sciences, Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia
3Agriculture Science and Technology Research Institute, Andong National University, Andong 36729, Republic of Korea

Correspondence to : * E-mail: cjung@andong.ac.kr

Abstract

Bee pollination plays important roles in increasing the yield, weight, and quality of strawberries grown in greenhouses. Use of pesticides is often included in the pest management strategy to control insects, fungi and weeds. However, these pesticides can negatively affect bees, compromising greenhouse pollination programs. In this study, we investigate the effect of rosmarinic acid (RA) feeding on the longevity of honey bees after oral and contact exposure of bees to some selected 14 different pesticides and G-3KM (commercial product) was used as a positive control. Among the 14 pesticides tested, six were found to be highly toxic to honey bee workers as indicated by their 48h-LD50 (μg/bee) values obtained from oral and spray bioassays; thiamethoxam (0.0002/0.0011), dinotefuran (0.005/0.018), emamectin benzoate (0.002/0.0002), spinetoram (0.018/0.001), sulfoxaflor (0.04/0.01), and cyantraniliprole (0.1/0.03), respectively. It was interesting to note that RA and G-3KM-supplemented feeding reduced honey bees mortality by 20-30% after they were exposed to high concentrations of the 6 highly toxic pesticides. The results indicate that RA could be used effectively in reducing honey bee mortality caused by pesticides.

Keywords: Rosmarinic acid, G-3KM, Apis mellifera, Pesticides, Longevity
INTRODUCTION

Insect pollination for fruit set and seed development accounts 75% of agricultural crops of the world (Klein et al., 2007). Managed honey bees are used in greenhouse for pollinating vegetable crops due to their greater availability and low cost (Kalev et al., 2002) and the case of strawberry cultivation in greenhouse is not an exception. Increase in foraging activity and interval of visitation of honey bees (Apis mellifera) in strawberry greenhouse play a role for successful pollination which in turn enhances the quality of fruit (Begna et al., 2020). In this regards, managed species of honey bees like A. mellifera and A. cerena were recognized as essential pollinators for strawberry flower (Abrol et al., 2019). Pollination by these bee species enhances the quantity of strawberry fruit in Europe and Southern Asia (Sommeijer and Ruijter, 2000). Klatt et al. (2014) indicated that bee pollination plays an important role not only in increasing the fruit set, and weight, but also the quality of greenhouse-grown strawberries.

Strawberry are highly susceptible to pests. Among these, Western tarnished plant bug, Lygus hesperus Knight aphids (green peach aphid, Myzus persicae Sulzer, strawberry aphid, Chaetosiphon fragaefolii (Cockerell), greenhouse whitefly, Trialeurodes vaporariorum (Westwood) and Western flower thrips, Frankliniella occidentalis (Pergande) are economically important pests to strawberry (Zalom et al., 2014). These pests are causing a huge damage to strawberry, and they are responsible for strawberry production declines (Zalom et al., 2014; Dara, 2015). Pest management is important for producing high yields and aesthetic standard desired by consumers. The management of strawberry is mainly depended on chemical pesticides; generally limited to the rotation of pesticides of different modes of action (He et al., 2015; Dara, 2016). However, these pesticides can negatively affect bees, compromising greenhouse pollination programs (Gradish et al., 2010). For instance, abamectin, thiamethoxam, spinetoram and novaluron used to control mite, aphids, thrips, and caterpillars during strawberry crop production in Brazil affect stingless bees (Piovesan et al., 2020). Another study by Costa et al. (2014) showed that some commonly used pesticides (abamectin, acetamiprid, cartap chloride, chlorfenapyr, cyromazin, deltamethrin, thiamethoxam, flufenoxuron, and pyriproxyfen) in conventional melon production systems in Brazil had negative impacts on honey bees.

Metabolic detoxification is a major mechanism accounting for insect resistance to xenobiotics, including insecticides (Aupinel et al., 2007). Cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and carboxylesterases (COEs) are three major enzyme that play a role in detoxification in mammals (Li et al., 2007). RA has been reported to possesses several biological activities, including health enhancing activities (Chun et al., 2014; Teruel et al., 2015), immune responses, anti-inflammatory, and antioxidant activities (Kim et al., 2013; Alagawany and Abd El-Hack, 2015; Alagawany et al., 2017). In the present study, we first investigate the oral and spray toxicities of different pesticides on honey bee worker, and followed by the effect of RA-supplemented feeding in reducing honey bee mortality after honey bees were intoxicated with highly toxic pesticides.

MATERIALS AND METHODS
1. Worker bees

Adult worker bees A. mellifera were collected from the healthy colonies from the Experimental Apiary of Andong National University, Korea by using a small amount of smoke, brushing them from the combs and transferring them into plastic cups.

2. Chemicals

Commercial formulations of pesticides used in this study are listed below in Table 1. Additionally, rosmarinic acid (purity>96%, CAS-No. 20283-92-5; Sigma-Aldrich, Korea), acetone (purity 99.5%, CAS No. 67-64-1; Daejung, South Korea) and G-3KM detoxicant (Dogo Medical Company Seoul, South Korea) were purchased and kept in the refrigerator prior to use.

Table 1. 
Pesticide name, manufacturer, percentage of active ingredient, commercial formulation type, field concentration and mode of action
No Pesticides IRAC group MoA1 Sub-groups a.i.2
(%)		 RC3
(a.i.ppm)
1 Imicyafos 1b (AChE) inhibitors Organophosphates 30 75
2 Thiacloprid 4a (nAChR) competitive modulators Neonicotinoids 10 50
3 Acetamiprid 4a Neonicotinoids 8 40
4 Dinotefuran 4a Neonicotinoids 20 100
5 Thiamethoxam 4a Neonicotinoids 10 50
6 Sulfoxaflor 4c Sulfoximines 7 35
7 Spinetoram 5 (nAChR) allosteric modulators Site I Spinosyns 5 25
8 Emamectin benzoate 6 (GluCl) allosteric modulators Avermectin 2.15 10.75
9 Lufenuron 15 Inhibitors of chitin biosynthesis affecting CHS1 Benzoylureas 5 25
10 Acequinocyl 20b Mitochondrial complex III electron transport inhibitors Acequinocyl 15 150
11 Metaflumizone 22b Voltage-dependent sodium channel blockers Semicarbazones 20 100
12 Cyflumetofen 25a Mitochondrial complex II electron transport inhibitors Beta-ketonitrile derivatives 20 100
13 Cyantraniliprole 28 Ryanodine receptor modulators Diamides 5 50
14 Fluxametamide 30 GABA-gated chloride channel allosteric modulators Isoxazolines 9 45
1Mode of action, 2Active ingredient, 3Recommended concentration

3. Rosmarinic acid treatment

Prior to the treatment, we prepared 10 bees in each cage of treatment groups by using CO2 gas as an anesthetic and kept in the experimental room at 25℃ prior to the test. All pesticides were diluted in sugar syrup for oral test or pure water for spray test to prepare the test concentrations (from the producer’s recommended concentration to 10-6 times dilution). Each pesticides had six concentrations and each concentration replicated five times. Ten honey bees were used per replicate (cage). Mortality was observed and recorded at 48 hours after the treatment.

For LD50 estimation, we measured feeders with the treated sugar solution before and after pesticides exposure for oral test and bees (n=10) weighed before and immediately after being sprayed with pesticides in spray bioassay.

1) Oral test

Caged bees (10 individuals per cage) were starved for 2 hrs at room temperature (25℃), RH 50-70% prior to the test. Bees were fed sugar solution (50%) with different concentrations of pesticides for 1 hour (Laurino et al., 2011) using feeder and weighed (FX-200i, A&D, Korea) to estimate the amount of pesticides taken up by bees. Control bees were fed only sugar solution (50%). Followed starvation, the worker bees were fed with syrup adulterated with different pesticides using a feeder at the bottom of the cup for 1 hr. Subsequently, the worker bees were fed with sugar syrup supplemented with rosmarinic acid (100 μg/mL) for 48 hrs, and this procedure was repeated five times for each treatment. The feeder unit containing syrup alone was considered as control. The numbers of dead bees were recorded 48 hrs after feeding with syrup supplemented with rosmarinic acid. Worker bees were considered dead if they did not move after being touched with the fine-tipped brush.

2) Spray test

After transferring 10 individual honey bees onto the Petri dish (15 cm in diameter), we sprayed (from 15 cm distance, 10 times) the pesticide solution with 600 mL hand sprayers (KOMAX G600, Sansoo Co., LTD, Korea). Control bees were sprayed only pure water. Then those bees were transferred into testing plastic cages. One group were fed with 50% sugar solution (control group) and the other group (treatment group) were fed with 50% sugar solution with RA or G-3KM. The number of bee mortality were counted 48 hr after the treatment.

3) Statistical analysis

The LD50 values of 48 hr post exposure to 14 different pesticides against honey bee workers as well as those of 48 hr post exposure to sugar, RA or G-3KM with 6 more toxic pesticides were calculated using the Probit analysis in SPSS version 26 (IBM Corp., 2011) to determine the dose-mortality response curves. LC50 values of contact (spray) exposure were also calculated by Probit analysis considering 4.52 μL amount of pesticides deposited on each honey bees body during spray bioassay, the spray LD50 was presumed from LC50. We used the LD50, when calculating Hazard Quotients (HQ). HQ=field application rate/oral or contact (LD50) relative to the field application adopted for field concentration determination (Halm et al., 2006; Stoner and Eitzer, 2013; Abdu-Allah and Pittendrigh, 2018). If the HQ<50: harmless; 50<HQ<2500: slight to moderately toxic; HQ>2500: dangerous for bees (Villa et al., 2000).

A two-way analysis of variance (ANOVA) was conducted, followed by Tukey multiple-range tests at p<0.05 for mean separation, using Embedded on SPSS Statistics 26 (IBM Corp., 2011) to compare the statistical significance of differences in mortality between control, sugar, RA and G-3KM groups.

RESULTS
1. Oral and spray toxicity (48 h-LD50/LC50)

Honey bees (A. mellifera L.) exhibited different levels of susceptibility to 14 tested pesticides as shown in Table 2. The results of toxicity of 14 pesticides, belonging to different classes, to honey bees (A. mellifera L.) are summarized in Table 2 for the oral and spray bioassays. As indicated in Table 2 for the oral application, thiamethoxam was found to be the most toxic to honey bees (48h-LD50=0.0002 μg/bee), followed by emamectin benzoate (48h-LD50=0.002 μg/bee). Five of the tested pesticides (acequinocyl, cyflumetofen, fluxametamide, lufenuron, metaflumizone) showed the lowest toxicity to honey bees (LD50>100 μg/bee) on oral toxicity assay. When honey bees were subjected to 14 pesticides on spray toxicity assay, we observed similar toxicity profile as of the oral toxicity tests as indicated in Table 2.

Table 2. 
Lethal concentration (LC50), lethal dose (LD50) and hazard quotient (HQ) of used pesticides to honey bees, for the oral and spray toxicity, at 48 hr
No Pesticides 48 hr-LC50 (μg/mL) 48 hr-LD50 (μg/bee) HQ
Oral Spray Oral Spray Oral Spray
1 Imicyafos 157.7 55.53 2.7 0.3 5.0 45.0
2 Thiacloprid >10,000 696.7 - 3.177 - 0.46
3 Acetamiprid 337.8 5741.8 6.756 26.183 0.11 0.03
4 Dinotefuran 0.35 3.93 0.005 0.018 30.0 16.7
5 Thiamethoxam 0.01 0.248 0.0002 0.0011 5000 909.09
6 Sulfoxaflor 1.45 1.55 0.04 0.01 17.5 70.0
7 Spinetoram 0.93 0.28 0.018 0.001 32.5 650.0
8 Emamectin benzoate 0.1 0.04 0.002 0.0002 344.0 3440.0
9 Lufenuron >10,000 >10,000 >100 >100 <1 <1
10 Acequinocyl >10,000 >10,000 >100 >100 <1 <1
11 Metaflumizone >10,000 >10,000 >100 >100 <1 <1
12 Cyflumetofen >10,000 >10,000 >100 >100 <1 <1
13 Cyantraniliprole 5.43 7.4 0.1 0.03 10 33.3
14 Fluxametamide >10,000 >10,000 >100 >100 <1 <1

Among the 14 tested pesticides, six (thiamethoxam, dinotefuran, emamectin benzoate, spinetoram, sulfoxaflor, and cyantraniliprole) were highly toxic to honey bees. Especially, thiamethoxam, emamectin benzoate and spinetoram showed high risk (HQ>2500) against A. mellifera (Table 2). The imicyafos and acetamiprid were moderately toxic in oral bioassay, and the other pesticides were non-toxic (>100 μg/bee) for both tests to A. mellifera adults (Table 2). In general, our data showed that oral application of the pesticides was more toxic than that of spray except for the cases of imicyfos, spinetoram, and emamectin benzoate. The toxicity of pesticides after 48 h of oral treatment followed the order of: thiamethoxam>emamectin benzoate>dinotefuran>spinetoram>sulfoxaflor>cyantraniliprole>imicyafos> acetamiprid.

2. RA in reducing honey bee mortality

Six highly toxic pesticides of different mode of action groups were selected for further investigation of RA-supplemented feeding in reducing honey bee mortality after oral and contact pesticide intoxication. The results of RA-supplemented feeding in reducing honey bee mortality are summarized in Table 3. It was interesting to note that RA-supplemented feeding responses to worker bees intoxicated with imicyafos and cyantraniliprole were found to be higher than G-3KM, and sugar (control) treatments (p=0.03 and p=0.09, respectively) in oral bioassays (Table 3). Both RA and G-3KM were found to be significantly effective with sulfoxaflor and emamectin benzoate in spray test (p<0.005).

Table 3. 
48h-LD50 and Hazard Quotient (HQ) estimated for each group (sugar=control, RA and G-3KM) in the honey bees with pesticides
No Pesticides Groups 48 hr-LD50 (μg/bee) HQ
Oral* Spray* Oral Spray
1 Imicyafos Sugar 2.7a 0.3a 5.0 45.0
RA 33.75b 0.9a 0.004 15.0
G-3KM 24.4b 13.7b 0.6 1.0
2 Dinotefuran Sugar 0.01a 0.018a 30.0 16.7
RA 0.01a 0.025a 30.0 12.0
G-3KM 0.02a 0.015a 15.0 20.0
3 Sulfoxaflor Sugar 0.04a 0.01a 17.5 70.0
RA 0.07b 0.02a 10.0 35.0
G-3KM 0.07b 0.12b 10.0 5.8
4 Spinetoram Sugar 0.02a 0.001a 32.5 650.0
RA 0.02a 0.002b 32.5 325.0
G-3KM 0.04b 0.003b 16.3 216.7
5 Emamectin benzoate Sugar 0.002a 0.0002a 344.0 3440.0
RA 0.003a 0.0005b 229.3 1376.0
G-3KM 0.01b 0.0007b 68.8 982.9
6 Cyantraniliprole Sugar 0.1a 0.03a 9.5 31.7
RA 3.1b 0.05b 0.3 19.0
G-3 0.5a 0.07b 1.9 13.6
Note:*Values in the same column for the same pesticide followed by different lowercase letters are significantly different (p<0.05).

It was also noted that RA and G-3KM-supplemented feeding reduced honey bees mortality by 20-30% after they were exposed to high concentrations of the 6 highly toxic pesticides. The results indicated that RA was effective in reducing the mortality of honey bees caused by pesticides.

DISCUSSION

A total fourteen pesticides of different groups tested on A. mellifera adults by the oral and spray bioassays. The order of pesticides toxicity was thiamethoxam>emamectin benzoate>dinotefuran>spinetoram>sulfoxaflor>cyantraniliprole>imicyafos>acetamiprid. Thiamethoxam and dinotefuran (nitro-neonicotinoid), emamectin benzoate (Avermectin), spinetoram (Spinosyns), sulfoxaflor (Sulfoximine), and cyantraniliprole (Diamide) pesticides were found to be highly toxic (<2 μg/bee), for both of oral and spray bioassays. Imicyafos (organophosphate) and acetamiprid (cyano-neonicotinoid) were moderately toxic (2<11 μg/bee) in oral bioassay test, and the other pesticides were non-toxic (>11 μg/bee) for both bioassay tests to A. mellifera adults (Table 2) which is in agreement to the previous studies (Hardstone and Scott, 2010). Thiamethoxam, emamectin benzoate and spinetoram were dangerous (HQ>2500) for A. mellifera adults.

The nitro-neonicotinoids (thiamethoxam and dinotefuran) are highly toxic to bees, with acute LD50 from 0.004 to 0.075 μg/bee (Iwasa et al., 2004; Cresswell, 2011). When thiamethoxam orally administrated, the 48h-LD50 value was 5 ng, and 24h-LD50 for contact administration was reported to be 29 ng (Decourtye et al., 2005). In our findings, the cyano-neonicotinoids (thiacloprid and acetamiprid) are much less toxic than the other pesticides, which is consistent with a similar report by Iwasa et al. (2004). Abdu-Allah and Pittendrigh (2018) reported that macrocyclic lactone-class pesticides, such as emamectin benzoate and spinetoram were more toxic topically (LD50=0.0006 and 0.0023 μg/bee, respectively) and orally (LD50=0.66 and 4.99 μg/bee, respectively) for honey bees. As shown in Table 3, emamectin benzoate was the least safest to honey bees as indicated by the highest Hazard Quotient value, which was also supported by several other similar investigations with bees and other insects (Lumaret et al., 2012; Abdu-Allah and Pittendrigh, 2018). However, lufenuron, acequinocyl, metaflumizone, cyflumetofen and fluxametamide were non-toxic (>100 μg/bee) for both bioassay tests to A. mellifera adults.

RA-supplemented feeding reduced honey bees mortality by 20-30% after they were exposed to toxic pesticides. This reduction in mortality might be due to the induction of detoxification or antioxidant mechanism in honey bees. Honey bees activate detoxification and antioxidant mechanisms when they are exposed to toxic pesticides (Johnson et al., 2010). Enhancing the production level of acetylcholinesterase (AChE) is one of the main mechanisms when individuals are exposed to pesticides such as organophosphates (Walker et al., 2005) and neonicotinoids (Boily et al., 2013). The mortality of A. mellifera workers supplemented with RA and G-3KM was reduced. Bisrat et al. (2020) also reported that G-3KM treatment was effective in reducing the mortality of honey bees when they were first intoxicated with pesticides.

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