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.
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.
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.
1. Preparations
2. In Vivo Feeding Method
Note: In vivo feeding method has been modified from Hanley et al.20
3. Toxicity of PPN at Colony Level in Beehive
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 vivo feeding method facilitated the observation of the impacts of the chemical treatments on the beehive.
For each dose of PPN-BLD, a total of fifty brood cells were marked and treated. After adding PPN-BLD, the treated frames were returned to the original colonies for observation under natural conditions. The negative effects of PPN-BLD on larval-stage honey bees were easily observed. A dosage-dependent effect was observed. Table 1 present the number of honey bees that died at the larval stage at the two higher doses of 10 and 100 ppm. As shown in Table 1, at the two doses (0.1 and 1 ppm), the capping rates, days to emergence and eclosion rates were not significantly different from 0 (BLD food added) or the unfed control, with only a significantly higher percentage of bees with deformed wings at 1 ppm. Melanization of pupae was observed at low concentrations of PPN. Furthermore, the proportion of adult honey bees appeared with deformed wings increased with higher doses of PPN (Table 1).
To simulate honey bee colonies suffering from environmental PPN residue, feeding of PPN syrup to the entire honey bee colony was performed. Before treatment, each colony was divided into 9 different temporal groups at 3 day intervals by two queen excluders (Figure 2). Therefore, the effects of PPN on the rates of hatching, capping and eclosion were observed simultaneously under conditions similar to those in the natural environment.
The colonies were fed syrup with 10 or 100 ppm PPN at day 13 (indicated by the red dotted in Figure 2). Theoretically, group 1 was a PPN-free control because the PPN-syrup was fed at day 13 and the honey bees in group 1 were in the pupal stage and capped; the honeybees in the larval stage began to be affected in groups 2 and 3, and the eggs will be affected in groups 4-9 due to the longer exposure time in the PPN-contaminated beehives (Figure 2).
In this trial, we fed honey bee colonies with PPN syrup and observed development when the adult honeybees consumed the syrup and presumably fed the queen and larvae. This assumption was confirmed by the hatching, capping and eclosion rates, and deformed winged bees were observed after the PPN treatments, as shown in Figures 3A–3D. All parameters differed significantly at the higher dose (100 ppm) starting with group 3, except the hatching rate (Figure 3A–3D). Moreover, after the PPN-syrup treatments, many pupae died with black cuticles or by failing to emerge. In the 100 ppm PPN treatment, the capped cells were destroyed, and the injured pupae were removed from the colony (Figure 3E).
Figure 1: Schematics of brood-cells labeling. (A) 1 day-old worker larvae covered by transparent slide papers and were labeled by permanent marker. (B) Labeled 1 day-old worker larval brood cells; the white arrow indicates a 1 day-old worker larva inside. Please click here to view a larger version of this figure.
Figure 2: Timeline for when PPN is started relative to the 9 experimental groups. Different concentrations of PPN were fed at day 13 (red dotted line). The treated colony was divided into Part A and Part B. The queen exchanges from part A to part B and vice versa for laying eggs are shown. This has been reproduced with permission from Elsevier16. Please click here to view a larger version of this figure.
Figure 3: Development of honey bee larvae before and after feeding 1 kg PPN syrup into tested bee colonies. A total of nine groups were surveyed in this experiment: (A) hatching rate; (B) capping rate; (C) eclosion rate; and (D) malformed wing rate. Mean ± SD are presented; Red arrows indicate the time during which PPN may start acting on bees. Black asterisks show statistical significance compared to the control (0 ppm). Red asterisks show statistical significance between 10 and 100 ppm; (E) 100 ppm PPN syrup treated bee colony showed uncapped cells, presumably deformed pupae and black and deformed pupae. This has been reproduced with permission from Elsevier16. Please click here to view a larger version of this figure.
PPN-BLD (ppm) | Larvae No. | Larval development | ||
Capped rate (%) | Eclosion rate (%) | Malformed wings (%) | ||
100 | 140 | 0b | 0b | – |
10 | 139 | 22.2 ± 33.2b | 0b | – |
1 | 140 | 72.3 ± 17.9a | 67.7 ± 17.6a | 7.7 ± 5.7a |
0.1 | 136 | 78.3 ± 17.5a | 75.4 ± 22.8a | 1.4 ± 2.8b |
0 | 138 | 78.9 ± 5.4a | 78.9 ± 5.4a | 0b |
Non-feeding | 122 | 87.8 ± 9.1a | 86.1 ± 7.2a | 0.8 ± 1.5b |
Table 1: Effects of continued 3 days feeding of PPN on 1 day-old larvae. To each larval cell, 10, 10 and 20 μL of BLD were added from days 1 to 3, respectively. Each assay contained 25-38 larvae in a colony and 4 colonies were tested. Mean ± SD are presented. Different letters in the same column are significantly different by the least square difference test (P < 0.05) after ANOVA showed a significant effect. This has been reproduced with permission from Elsevier16.
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 testing to evaluate the impacts of pesticide and pesticide residues. Moreover, a large number of selected brood cells were recorded in the field test using transparent slides for labelling. However, egg over-laying occasionally results in insufficient brood cells for the queen laying eggs. Therefore, the preparation of empty frames is required for the queen-exchange method. Alternatively, the queen-limited egg-laying method could be also used to prepare different honey bee groups for testing in the beehive. The separation of the frames into 2 frames in part A and 7 frames in part B and the placement of the queen in part A (2 frames) may limit the eggs laid by the queen within the 2 frames.
For the in vivo feeding method, PPN-BLD was added to each brood cell. The honey bees exhibited higher acceptance of BLD than syrup16,20. Honey bee larvae can survive and grow on an artificial diet composed of royal jelly, sugars, yeast extract, and distilled water22,23. The glucose and fructose composition of the BLD did not affect the survival rates of the larvae in vitro21, and therefore, BLD would be more stable for honey bee growth in the beehive. Notably, the use of fresh BLD for feeding could also prevent the worker bees' larval exclusion during the larval feeding experiment. Moreover, during the feeding process, gentle feeding is initially required to avoid the death of 1 day-old larvae.
During field tests, chemically contaminated BLD in the beehive occasionally caused larval exclusion due to the high olfactory sensitivity of nursing bees. The sugar composition significantly affects the average larval survival, pre-pupal larval weights, adult weights, and ovariole numbers21. When syrup was used to deliver transgenic pollen or a positive control pesticide diazinon to honey bee larvae, the workers removed few larvae from the brood cells containing added or contaminated food21. Thus, the sugar composition should be noted. Increasing the number of 1 day-old larvae or dispersing the tested individual brood cell sites may also improve the problems. Indeed, based on the observation of the PPN-BLD treatment processes and the dramatic dose-dependent impacts of PPN-BLD on the development of honey bees, we assumed that the nurses did not remove the artificially added BLD to the brood cells. Low concentrations of PPN cause melanization of pupae, possibly due to increased phenoloxidase activity, which regulates melanization and pupation24,25,26. Based on the use of an artificial feeding method for each brood cell, the dramatic impacts of PPN-BLD on the development of honey bees might be due to direct contact with PPN from the first day of hatching.
The chemical dosages used in the feeding experiments could be designed based on survey data for pesticide residues in fresh pollen samples collected from honey bee hives. There are a variety of routes of pesticide contamination of colonies in the natural environment. Chemical contaminants could be brought back to the honey bee colonies and ingested by larvae due to the feeding motion of worker honey bees, eventually influencing the development of larvae. To evaluate the impact of PPN at the colony level in the beehive, syrup was used instead of pollen for feeding in this study as the fastest and most direct way to ensure that the honey bees consumed the chemical. Furthermore, the condition of the syrup can be defined, whereas the content of pollen is difficult to control (e.g., pathogens or pesticide contamination).
Under field conditions, different developmental stages (eggs, larvae, pupae and adult bees) of honey bees in a honey bee colony suffer the same environment factor. In our experimental setup, natural conditions were simulated to evaluate the impacts of the chemical on the development stages of honey bees in a dynamic environment. Therefore, after PPN treatment (13 days), we could observe the uninfluenced capping rate in group 1, the influenced capping rate in the other eight groups, and the influenced larval stages in groups 2 and 3, etc. Under these conditions, the true effects within the colony and the route of dispersal of the chemical to the whole colony are clear.
The honey bee colonies treated with PPN syrup exhibited distortion of capped cells and removal of pupae from the colony. Furthermore, extensive melanization of pupae was observed in the 100 ppm PPN treatment. Thus, the PPN-syrup feeding method allowed the dynamic impact of chemicals on the life cycle of honey bees in the beehive to be observed. In this trial, the syrup taken up by the worker and nursing bees was presumably fed to the queen and larvae. For confirmation, our future studies will use chemical markers (such as an edible dye) in the chemical-containing syrup to further facilitate the study of the dynamics of chemically contaminated beehives.
The food source of nurse bees should come from foragers or in beehive and larvae are fed mandibular and hypopharyngeal gland secretions produced by nurse bees21,25,26,27,28. When larvae are fed by nurse bees, secretions produced by the hypopharyngeal gland in the mandibles may dilute the PPN-syrup. This biological dilution effect more closely resembles the natural conditions but explains the differing results to the in vivo feeding method. Several materials including other pesticides, heavy metal and honey bee pathogens could be applied to this feeding method for evaluating and addressing the roles in honey bee populations.
The authors have nothing to disclose.
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).
Honey bee box | SAN-YI Honey Factory | W1266 | Honeybees rearing |
Queen excluder (between frames) | SAN-YI Honey Factory | I1575 | Queen limitation |
Queen excluder (on top ) | SAN-YI Honey Factory | I1566 | Queen limitation on top |
Bee brush | SAN-YI Honey Factory, Taiwan | W1414 | clean the bees on frame gently |
Bee feeder | SAN-YI Honey Factory, Taiwan | P0219 | feed sugar syrup to colony |
Transparent slide | Wan-Shih-Chei, Taiwan (http://www.mbsc.com.tw/a01goods.asp?s_id=40) | 1139 | Mark the larval area on the frames (Material: Polyethylene Terephthalate, PET) (Size= Length*Width*thick= 29.7mm*21mm*0.1mm) |
24 well tissu culture plate | Guangzhou Jet Bio-Filtration Co., Ltd | TCP011024 | Rearing pupae from extraction |
Autoclave | Tomin medical equipmenco., LTD. | TM-321 | Make sterilized distilled deionized water (ddH2O) |
P20 pipetman | Gilson | F123600 | Add PPN into bee larval food pool |
Incubator | Yihder Co., Ltd. | LE-550RD | Rearing pupae from extraction |
Kimwipes | COW LUNG INSTRUMENT CO., LTD | KCS34155 | Rearing pupae from extraction |
Royal jelly | National Ilan University (NIU) | NIU | Make basic larval diet (BLD) |
D-(+)-Glucose | Sigma | G8270 | Make basic larval diet (BLD) |
D-(-)-Fructose | Sigma | F0127 | Make basic larval diet (BLD) |
Yeast extract | CONDA, pronadisa | 1702 | Make basic larval diet (BLD) |
Sucrose | Taiwan sugar coporation | E01071010 | Make sugar syrup for bee food |
Pyriproxyfen (11%) | LIH-NUNG CHEMICAL CO.. LTD. | Registration No. 1937 | Insect growth regulator (IGR) used in the experiment |