Method Article

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

DOI:

10.3791/55296

⸱

April 1st, 2017

 ,  , 
1Department of Biotechnology and Animal Science, National Ilan University

In This Article

Summary

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Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

Abstract

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The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Introduction

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The presence of pesticides in the environment is one of the most serious problems that impacts the life of a honey bee1,2,3. Several studies have demonstrated the common presence of pesticide residues in honey bee colonies and bee products. In Taiwan, the average application of pesticides was 11-12 kg/ha every year (from 2005 to 2013). The amount of pesticides used in Taiwan is higher than that of the EU countries, and the Latin American countries4,5. In other words, the apicultural environment is suffering serious pesticide stress, especially in Taiwan and possibly in other countries.

The honey bee Apis mellifera is one of the major pollinators in agricultural systems6 and it also produces valuable products such as honey. However, honey bees are exposed to various pesticides and these pesticides can be brought back to beehives after foraging on flowers that have been sprayed with pesticides when collecting nectar and pollen7,8. They also can be exposed to pesticides by the beekeepers themselves aiming to control pest problems inside the hives9,10,11. Because honey bee larvae are fed by nurse bees for their development, larvae, drones and even the queen may be exposed to these pesticide-contaminated nectars and pollen12. The toxicity of various pesticides to honey bees needs to be addressed13.

Many efforts have been made to evaluate the issues of environmental pesticide residues. Yang et al. tested the influence of the neurotoxic insecticide imidacloprid on the development of honey bee larvae in the beehive and reported that a sub-lethal dose of imidacloprid resulted in olfactory associative behavior of the adult bees14. Also, Urlacher et al. examined the sub-lethal effects of an organophosphate pesticide, chlorpyrifos, on a honey bee worker's learning performance under laboratory conditions15. In our previous study, we evaluated the impact of an insect growth regulator (IGR), pyriproxyfen (PPN), on larval honey bees16.

In this paper, we present methods for evaluating the chemical impacts on the development of honey bees. Honey bee feeding methods were described and applied to either individual honey bees or to a colony. At first, we tested different concentrations of pesticide-contaminated basic larval diet (BLD) on larvae in the colonies to evaluate the impact of the pesticide on individual honey bees in vivo. We then proceeded to simulate the natural conditions of the pesticide by using pesticide-contaminated syrup within beehives. In this method, PPN, which is widely used against pest insects17 and is harmful to the development of honey bee larvae and pupae16,18,19, will be an indicator to represent the negative effect of the pesticide in the field.

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Protocol

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1. Preparations

  1. Make 1 L of 50% sugar syrup. Dissolve 1 kg sucrose in 1 L ddH2O.
  2. Prepare pyriproxyfen (PPN) solution in BLD. Make 1.1 L of 10,000 ppm PPN stock solution and dilute 100 mL PPN solution in 1 L sterilized ddH2O. Store at 4 °C.
  3. Dilute the PPN stock solution to final concentrations of 0.1, 1, 10 and 100 mg/kg (ppm) in the BLD for the following experiment.
  4. Make PPN-syrup (for the colony level). Dilute the PPN stock to final concentrations of 10 and 100 ppm in 50% sugar syrup for the following experiment.
  5. Honey bee rearing.
    Note: Here, the experimental location is the National Ilan University (NIU) apiary, Yi-Lan City, Taiwan; (GPS coordinates: N24.747278, E121.746200).
    1. Check honey bee (Apis mellifera L.) colonies weekly for food quantity and feed with 1 liter 50% sugar syrup if necessary (the honey storage area is empty). Define a healthy colony as 9 frames of honey bee combs in each colony with a queen laying eggs normally.
  6. Prepare 100 mL of basic larval diet (BLD). Dissolve 6% D-glucose, 6% fructose, and 1% yeast extract in sterilized distilled deionized water (ddH2O) and supplement with 50% royal jelly. Store at 4 °C but not for more than 3 days.
    Note: Pre-warm to 35 °C before experiment and use within 3 days.

2. In Vivo Feeding Method

Note: In vivo feeding method has been modified from Hanley et al.20

  1. Honey bee larvae selection and labeling.
    1. Insert a queen excluder to divide 9 frames of a healthy colony into 4 frames and 5 frames while limiting the queen to the 4 frame section. Leave at least one empty frame in the 4 frame section for laying eggs.
    2. After the queen laying eggs for 1 day, check the frames for the appearance of eggs and keep the eggs inside the beehives for 72 h (the 4th day) until 1 day-old larvae hatch. Take one of the frames containing 1-day-old worker larvae (hatched within 24 h) out from the test hive by hand and remove the honey bee workers from the frame with a bee brush.
    3. Cover the frame with a transparent slide (Size = Length x Width x thick = 29.7 mm x 21 mm x 0.1 mm) and nail the transparent slide on the edge of frames with thumbtacks (Figure 1A).
    4. For each treatment, randomly select 50 one day-old larvae (the 4th day) and mark each brood cell by using permanent marker pens on the transparent slide (Figures 1A and 1B).
      Note: Write the information of each treatment on the frame and transparent slide as well by using permanent marker pens to avoid the confusion between different treatments. Remove the marked slides and keep for the in vivo feeding and observation.
  2. In vivo feeding.
    1. Add different concentrations of PPN-BLD (0.1, 1, 10 and 100 ppm) to each labeled brood cell by pipetting at day 1, 2 and 3 with 10 μL, 10 μL and 20 μL, respectively, according to the intake quantity of the larvae age. Add the same amount of BLD (no PPN) to a control group. Thereby, the total dose of PPN-BLD in each labeled brood cell accumulates to 4, 40, 400, and 4,000 pg.
      NOTE: Recognize the labeled brood cells by the marked transparent slides and remove the marked transparent slides after feeding. Use fresh BLD for feeding to prevent the cleaning of the brood cells by the worker bees.
    2. Return the PPN-treated frames to the original colonies for further observations.
      NOTE: Each treatment has four biological repeats in four colonies.
  3. Observation of treated larvae at day 7.
    1. To observe the PPN-treated larvae, return the labeled transparent slides to the frames to record the mortality and capping rates in the labeled brood cells.
  4. Observation of treated pupae and eclosion rates at day 13.
    1. Remove the bee wax of capped brood cells.
    2. Put the soft tipped tweezers into brood cell and clamp the pupae very slightly then take pupae out gently.
    3. Transfer the pupae into 24-well tissue culture plates with a double-layer of laboratory tissues beneath each well. Record the damage and mortality during pupal transfer.
    4. Keep the 24-well tissue culture plates in an incubator at 34 °C and 70% relative humidity until emergence (ca. 8 days).
    5. Observe and record the pupae and emerged honey bees.
  5. Statistics
    1. Calculate the recorded data and present as a mean ± S.D.
    2. Analyze the data using analysis of variance (ANOVA) by SAS and use the least significant difference (LSD) test to analyze the differences between two means of different treatments. Define statistically significant as P-value <0.05. Different letters in the same column of the table showed a significant effect by the statistical analysis.

3. Toxicity of PPN at Colony Level in Beehive

  1. Set up the honey bee groups.
    1. Insert a queen excluder vertically to divide 9 frames of a healthy colony into 4 frames (Part A) and 5 frames (Part B) while limiting the queen to the 4 frame section . Leave at least one empty frame in the 4-frame section for laying eggs.
      Note: Put another queen excluder the on top of the queen part to prevent the queen from moving between the parts.
    2. After the queen laying eggs for 1 day, check the frames for the appearance of eggs. Take the appropriate frames containing eggs out from part A by hand and remove the honey bee workers from the frame with a bee brush.
    3. Cover the frame with transparent slide and nail the transparent slide to the edge of the frame with thumbtacks.
    4. Randomly select 100 brood cells containing eggs and mark each brood cell by using permanent marker on the transparent slide. Assign these 100 brood cells as Group 1. Write the information of each treatment on the frame and transparent slide using permanent marker to avoid the confusion between different treatments.
    5. Return the labeled frame to part A for 3 days and then transfer the queen to part B to lay eggs.
    6. After 1 day, check the frames of part B, choose one appropriate frame and label 100 brood cells containing eggs as described in step 3.1.4. Assign these 100 brood cells as Group 2.
    7. Return the labeled frame to part B for 3 days and then transfer the queen to part A to lay eggs.
    8. Repeat the queen exchange between Part A and Part B 6 more times and assign groups numerically, respectively (Figure 2). There should be total of 9 groups.
  2. Treat honey bee colony with PPN sugar syrup on day 13 (Figure 2).
    1. Add 1 L 50% PPN sugar syrup containing 10 or 100 ppm in a plastic bee feeder box (W x L x H = 20 cm x 30 cm x 3.5 cm) and put the box on top of the frames in the experimental colonies.
      Note: Group 1 does not receive the PPN at 13 days as the brood cells have been sealed.
    2. Feed the control group with 1 L 50% sugar syrup (no PPN) as described in step 2.2.
  3. Count 1 day-old larvae and record as the egg hatching rate of 100 labeled brood cells for each group at day 5 (Figure 2). Honey bee eggs usually take 3 days to hatch into 0 day larvae; therefore, check and label 100 eggs-containing brood cells at day 1 and count the number of 1 day-old larvae for each group at day 5 to obtain the percentage of hatching rate.
  4. Count the capped brood cells and record as the larval capping rate of 100 labeled brood cells for each group at day 11 (Figure 2). 6 to 7 day-old larval brood cells will be capped with bee wax by honey bee workers for larvae pupating.
  5. Observe pupae maturation and record the eclosion rate of 100 labeled brood cells for each group at day 17 (Figure 2).
    1. Remove the bee wax of capped brood cells and take pupae out with soft tipped tweezers gently. Transfer the pupae into a 24-well tissue culture plates with a double-layer of laboratory tissues beneath each well.
    2. Keep the 24-well tissue culture plates in an incubator at 34 °C and 70% RH until emergence.
    3. Observe and record the pupae and emerged honey bees for each group until Group 9 (49 experimental days).
      Note: Each treatment has four biological repeats.
  6. Statistics
    1. Calculate the recorded data and present as the mean ± SD.
    2. Analyze the significant differences between pairs of treatments (e.g. 0 ppm/10 ppm, 10 ppm/100 ppm and 0 ppm/100 ppm) in each group by using the Student's two-tailed t-test. Define as statistically significant if P-value < 0.05.

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Results

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For the honey bee field test, a queen was limited to the 4-frame section for laying eggs. This step could increase the brood density in one frame and facilitate subsequent observations. Each treatment was labelled, and the honey bees' development was clearly observed through a transparent slide. In vivo feeding of PPN-BLD to honey bee larvae in the beehive was performed to precisely evaluate the influence of PPN on the development of honey bees in the colony. Using the in viv...

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Discussion

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The queen-limited egg-laying method and queen-exchange method are critical steps for setting up honey bee groups for field testing within this protocol. The queen-limited egg-laying method permits synchronization of the life cycle of honey bees. Consequently, researchers can select 1 day-old larvae of the same age for treatment with different doses of pesticide. For the queen-exchange method, the queen was exchanged between part A (4 frames) and B (5 frames) to obtain different developmental stages of honey bee for field...

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Disclosures

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The authors declare that they have no competing financial interests.

Acknowledgements

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This research was supported by Grant 105AS-13.2.3-BQ-B1 from the Bureau of Animal and Plant Health Inspection and Quarantine, the Council of Agriculture, Executive Yuan and Grant 103-2313-B-197-002-MY3 from the Ministry of Science and Technology (MOST).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Honey bee boxSAN-YI Honey FactoryW1266Honeybees rearing
Queen excluder (between frames)SAN-YI Honey FactoryI1575Queen limitation 
Queen excluder (on top)SAN-YI Honey FactoryI1566Queen limitation on top 
Bee brushSAN-YI Honey Factory, TaiwanW1414clean the bees on frame gently
Bee feederSAN-YI Honey Factory, TaiwanP0219feed sugar syrup to colony
Transparent slideWan-Shih-Chei, Taiwan (http://www.mbsc.com.tw/a01goods.asp?s_id=40)1139Mark the larval area on the frames (Material: Polyethylene Terephthalate, PET) (Size = Length*Width*thick = 29.7 mm * 21 mm * 0.1 mm)
24 well tissu culture plateGuangzhou Jet Bio-Filtration Co., LtdTCP011024Rearing pupae from extraction
AutoclaveTomin medical equipmenco., LTD.TM-321Make sterilized distilled deionized water (ddH2O)
P20 pipetmanGilsonF123600Add PPN into bee larval food pool
Incubator Yihder Co., Ltd.LE-550RDRearing pupae from extraction
KimwipesCOW LUNG INSTRUMENT CO., LTDKCS34155Rearing pupae from extraction
Royal jellyNational Ilan University (NIU)NIUMake basic larval diet (BLD)
D-(+)-GlucoseSigmaG8270Make basic larval diet (BLD)
D-(-)-FructoseSigmaF0127Make basic larval diet (BLD)
Yeast extractCONDA, pronadisa1702Make basic larval diet (BLD)
SucroseTaiwan sugar coporationE01071010Make sugar syrup for bee food
Pyriproxyfen (11%)LIH-NUNG CHEMICAL CO.. LTD.Registration No. 1937Insect growth regulator (IGR) used in the experiment

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Tags

Pesticide ExposurePyriproxyfen TreatmentColony Feeding MethodLarval Stage AnalysisPupae Handling TechniqueEclosion Rate MeasurementBrood Cell TreatmentEnvironmental Chemical TestingHoneybee Colony Assessment

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