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).

1. Preparations
<ol>
	<li>Make 1 L of 50% sugar syrup. Dissolve 1 kg sucrose in 1 L ddH2O.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Make PPN-syrup (for the colony level). Dilute the PPN stock to final concentrations of 1...

<ol>
	<li>Ruffi nengo, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+pest+management+to+control+Varroa+destructor+and+its+implications+to+Apis+mellifera+colonies.">Integrated pest management to control Varroa destructor and its implications to Apis mellifera colonies.</a> Zootecnia Trop. 32 (2), 149-168 (2014).</li><li>Mullin, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+miticides+and+agrochemicals+in+North+American+apiaries,+implications+for+honey+bee+health.">High levels of miticides and agrochemicals in North American apiaries, implications for honey bee health.</a> PLoS One. 5, e9754 (2010).</li><li>Lu, C. A., Chang, C. H., Tao, L., Chen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributions+of+neonicotinoid+insecticides+in+the+Commonwealth+of+Massachusetts,+a+temporal+and+spatial+variation+analysis+for+pollen+and+honey+samples.">Distributions of neonicotinoid insecticides in the Commonwealth of Massachusetts, a temporal and spatial variation analysis for pollen and honey samples.</a> Environ. Chem. 13, 4-11 (2016).</li><li>Tsai, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+coupling+the+pesticide+use+reduction+with+environmental+policy+for+agricultural+sustainability+in+Taiwan.">Analysis of coupling the pesticide use reduction with environmental policy for agricultural sustainability in Taiwan.</a> Environ. &amp; Pollut. 2, 59-65 (2013).</li><li>Weng, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+market+status+and+development+trend+(in+Chinese).">Pesticide market status and development trend (in Chinese).</a> PRIDE. , (2016).</li><li>Kevan, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollinators+as+bioindicators+of+the+state+of+the+environment,+species,+activity+and+diversity.">Pollinators as bioindicators of the state of the environment, species, activity and diversity.</a> Agric. Ecosys. Environ. 74 (1-3), 373-393 (1999).</li><li>Kevan, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forest+application+of+the+insecticide+fenitrothion+and+its+effect+on+wild+bee+pollinators+(Hymenoptera:+Apoidea)+of+lowbush+blueberries+(Vaccinium+SPP.)+in+Southern+New+Brunswick,+Canada.">Forest application of the insecticide fenitrothion and its effect on wild bee pollinators (Hymenoptera: Apoidea) of lowbush blueberries (Vaccinium SPP.) in Southern New Brunswick, Canada.</a> Biol. Conserv. 7, 301-309 (1975).</li><li>Crane, E., Walker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The impact of pest management on bees and pollination. , (1983).</li><li>Haouar, M., Decormis, L., Rey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluvalinate+applied+to+flowering+apple+trees-contamination+of+honey-gathering+bees+and+hive+products.">Fluvalinate applied to flowering apple trees-contamination of honey-gathering bees and hive products.</a> Agronomie. 10 (2), 133-137 (1990).</li><li>Chauzat, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+pesticide+residues+in+pollen+loads+collected+by+honey+bees+in+France.">A survey of pesticide residues in pollen loads collected by honey bees in France.</a> J. Econ. Entomol. 99 (2), 253-262 (2006).</li><li>Bonzini, S., Tremolada, P., Bernardinelli, I., Colombo, M., Vighi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+pesticide+fate+in+the+hive+(part+1),+experimentally+determined+&#964;-fluvalinate+residues+in+bees,+honey+and+wax.">Predicting pesticide fate in the hive (part 1), experimentally determined &#964;-fluvalinate residues in bees, honey and wax.</a> Apidologie. 42 (3), 378 (2011).</li><li>Sanchez-Bayo, F., Goka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+residues+and+bees-a+risk+assessment.">Pesticide residues and bees-a risk assessment.</a> PLoS One. 9 (4), e94482 (2014).</li><li>Johnson, R. M., Ellis, M. D., Mullin, C. A., Frazier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticides+and+honey+bee+toxicity-USA.">Pesticides and honey bee toxicity-USA.</a> Apidologie. 41, 312-331 (2010).</li><li>Yang, E. C., Chang, H. C., Wu, W. Y., Chen, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+olfactory+associative+behavior+of+honeybee+workers+due+to+contamination+of+imidacloprid+in+the+larval+stage.">Impaired olfactory associative behavior of honeybee workers due to contamination of imidacloprid in the larval stage.</a> PLoS One. 7, e49472 (2012).</li><li>Urlacher, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+chlorpyrifos+levels+in+forager+bees+and+comparison+with+levels+that+disrupt+honey+bee+odor-mediated+learning+under+laboratory+conditions.">Measurements of chlorpyrifos levels in forager bees and comparison with levels that disrupt honey bee odor-mediated learning under laboratory conditions.</a> J. Chem. Ecol. 42 (2), 127-138 (2016).</li><li>Chen, Y. W., Wu, P. S., Yang, E. C., Nai, Y. S., Huang, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+pyriproxyfen+on+the+development+of+honey+bee+(Apis+mellifera+L.)+colony+in+field.">The impact of pyriproxyfen on the development of honey bee (Apis mellifera L.) colony in field.</a> J. Asia Pac. Entomol. 19 (3), 589-594 (2016).</li><li>Dennehy, T. J., DrGain, B. A., Harpold, V. S., Brink, S. A., Nichols, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Whitefly Resistance to Insecticides in Arizona: 2002 and 2003 Results. , 1926-1938 (2004).</li><li>Yang, E. C., Wu, P. S., Chang, H. C., Chen, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sub-lethal+dosages+of+insecticides+on+honey+bee+behavior+and+physiology.">Effect of sub-lethal dosages of insecticides on honey bee behavior and physiology.</a> , (2010).</li><li>Fourrier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+exposure+to+the+juvenile+hormone+analog+pyriproxyfen+disrupts+acceptance+of+and+social+behavior+performance+in+adult+honey+bees.">Larval exposure to the juvenile hormone analog pyriproxyfen disrupts acceptance of and social behavior performance in adult honey bees.</a> PLoS One. 10, e0132985 (2015).</li><li>Hanley, A. V., Huang, Z. Y., Pett, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dietary+transgenic+Bt+corn+pollen+on+larvae+of+Apis+mellifera+and+Galleria+mellonella.">Effects of dietary transgenic Bt corn pollen on larvae of Apis mellifera and Galleria mellonella.</a> J. Apicult.Res. 42 (4), 77-81 (2003).</li><li>Kaftanoglu, O., Linksvayer, T. A., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+honey+bees,+apis+mellifera,+in+vitro+1,+effects+of+sugar+concentrations+on+survival+and+development.">Rearing honey bees, apis mellifera, in vitro 1, effects of sugar concentrations on survival and development.</a> J. Insect Sci. 11 (96), 1-10 (2011).</li><li>Vandenberg, J. D., Shimanuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+rearing+worker+honey+bees+in+the+laboratory.">Technique for rearing worker honey bees in the laboratory.</a> J. Apicult. Res. 26 (2), 90-97 (1987).</li><li>Peng, Y. S. C., Mussen, E., Fong, A., Montague, M. A., Tyler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chlortetracycline+on+honey+bee+worker+larvae+reared+in+vitro.">Effects of chlortetracycline on honey bee worker larvae reared in vitro.</a> J. Invertebr.Pathol. 60 (2), 127-133 (1992).</li><li>Bitondi, M. M., Mora, I. M., Simoes, Z. L., Figueiredo, V. 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Dev. 29 (2), 111-119 (2000).</li><li>Santos, A. E., Bitondi, M. M., Simoes, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone-dependent+protein+patterns+in+integument+and+cuticular+pigmentation+in+Apis+mellifera+during+pharate+adult+development.">Hormone-dependent protein patterns in integument and cuticular pigmentation in Apis mellifera during pharate adult development.</a> J. Insect Physiol. 47 (11), 1275-1282 (2001).</li><li>Brouwers, E. V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose/Fructose+ratio+in+the+food+of+honeybee+larvae+during+caste+differentiation.">Glucose/Fructose ratio in the food of honeybee larvae during caste differentiation.</a> J. Apicult.Res. 23 (2), 94-101 (1984).</li><li>Howe, S. R., Dimick, P. S., Benton, A. 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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...

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&#39;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.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:193px;">Honey bee box</td>
			<td style="width:243px;">SAN-YI Honey Factory</td>
			<td style="width:155px;">W1266</td>
			<td style="width:297px;">Honeybees rearing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:193px;">Queen excluder (between frames)</td>
			<td style="width:243px;">SAN-YI Honey Factory</td>
			<td style="width:155px;">I1575</td>
			<td style="width:297px;">Queen limitation&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:193px;">Queen excluder (on top )</td>
			<td style="width:243px;">SAN-YI Honey Factory</td>
			<td style="width:155px;">I1566</td>
			<td style="width:297px;">Queen limitation on top&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:193px;">Bee brush</td>
			<td style="width:243px;">SAN-YI Honey Factory, Taiwan</td>
			<td>W1414</td>
			<td style="width:297px;">clean the bees on frame gently</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:193px;">Bee feeder</td>
			<td style="width:243px;">SAN-YI Honey Factory, Taiwan</td>
			<td>P0219</td>
			<td style="width:297px;">feed sugar syrup to colony</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Transparent slide</td>
			<td style="width:243px;">Wan-Shih-Chei, Taiwan (http://www.mbsc.com.tw/a01goods.asp?s_id=40)</td>
			<td>1139</td>
			<td style="width:297px;">Mark the larval area on the frames (Material: Polyethylene Terephthalate, PET) (Size= Length*Width*thick= 29.7mm*21mm*0.1mm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:193px;">24 well tissu culture plate</td>
			<td style="width:243px;">Guangzhou Jet Bio-Filtration Co., Ltd</td>
			<td style="width:155px;">TCP011024</td>
			<td style="width:297px;">Rearing pupae from extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:193px;">Autoclave</td>
			<td style="width:243px;">Tomin medical equipmenco., LTD.</td>
			<td style="width:155px;">TM-321</td>
			<td style="width:297px;">Make sterilized distilled deionized water (ddH2O)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">P20 pipetman</td>
			<td style="width:243px;">Gilson</td>
			<td style="width:155px;">F123600</td>
			<td style="width:297px;">Add PPN into bee larval food pool</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:193px;">Incubator&#160;</td>
			<td style="width:243px;">Yihder Co., Ltd.</td>
			<td style="width:155px;">LE-550RD</td>
			<td style="width:297px;">Rearing pupae from extraction</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:193px;">Kimwipes</td>
			<td style="width:243px;">COW LUNG INSTRUMENT CO., LTD</td>
			<td style="width:155px;">KCS34155</td>
			<td style="width:297px;">Rearing pupae from extraction</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:193px;">Royal jelly</td>
			<td style="width:243px;">National Ilan University (NIU)</td>
			<td style="width:155px;">NIU</td>
			<td style="width:297px;">Make basic larval diet (BLD)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">D-(+)-Glucose</td>
			<td style="width:243px;">Sigma</td>
			<td style="width:155px;">G8270</td>
			<td style="width:297px;">Make basic larval diet (BLD)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">D-(-)-Fructose</td>
			<td style="width:243px;">Sigma</td>
			<td style="width:155px;">F0127</td>
			<td style="width:297px;">Make basic larval diet (BLD)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:193px;">Yeast extract</td>
			<td style="width:243px;">CONDA, pronadisa</td>
			<td style="width:155px;">1702</td>
			<td style="width:297px;">Make basic larval diet (BLD)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">Sucrose</td>
			<td style="width:243px;">Taiwan sugar coporation</td>
			<td style="width:155px;">E01071010</td>
			<td style="width:297px;">Make sugar syrup for bee food</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:193px;">Pyriproxyfen (11%)</td>
			<td style="width:243px;">LIH-NUNG CHEMICAL CO.. LTD.</td>
			<td style="width:155px;">Registration No. 1937</td>
			<td style="width:297px;">Insect growth regulator (IGR) used in the experiment</td>
		</tr>
	</tbody>
</table>

evaluating the effect of environmental chemicals on honey bee development from the individual to colony level

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.

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&#39; 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...

Watch this Scientific Journal Video about Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level at JoVE.com

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

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 ...

The overall goal of this experiment is to evaluate how pesticide residues in the environment affect individual honeybees in the natural condition of beehive colonies. The main advantage of this method is that it surveys the effects of pesticides or other contaminants of interest on both individual honeybees and the whole colonies. Visual demonstration of this method is critical as the manipulation of pupae is difficult because they are fragile.Demonstrating the procedure will be Ko Chong-Yu a PHD student from my laboratory. Prior to the experiment, feed each colony a liter of 50%sugar syrup. Replenish the food reservoir weekly, a healthy colony will have nine frames of bee combs and one queen laying eggs.To begin, locate the queen in the colony and replace at least one frame on her side of the colony with an empty frame. Then insert the queen excluders to isolate the queen to a four frame section containing at least one empty frame. The next day, check the frames for freshly laid eggs and put the eggs apart from the queen for 72 hours.Then remove these frames, brush off the worker bees, and prepare to treat the larvae in the brood cells. For each treatment cover 50 randomly selected one day old larvae in the frame with transparent slides. Secure the slides with thumbtacks, then make identifying information on the slides with permanent marker.Now using BLD warmed to 35 degrees celsius, make different concentrations of PPN treatment ranging from 0.1 to 100 parts per million in 100 mL volumes. For the next three days, treat the brood cell groups. To treat the larvae, uncover them and add 10 microliters of the PPN BLD solution to each brood cell.Repeat the treatment the next day using 10 microliters of treatment solution, and repeat the treatment on the third day using 20 microliters of treatment solution. After the third treatment, let them develop in the colony In the experimental design, replicate each treatment group four times using different colonies. On day seven, inspect the PPN treated larvae, record the mortality and capping rates in the treatment groups.On day 13, record the eclosion rate of each treatment group. First, remove the bee wax from the capped brood cells. Then remove the pupae using tipped tweezers to grip them very lightly, and gently tug them out.When removing the pupae, use grip between head and thorax and take pupae out very gently. It will be necessary to practice many times to obtain a survival rate of at least 90%prior to running experiment. Transfer the pupae into 24 welled tissue culture plates lined with two layers of laboratory tissue.Be sure to note any damage in mortality that occurs during the transfer. Keep the culture plates at 34 degrees celsius with 70%relative humidity until emergence. Over the next eight days, record the eclosion rate.Set up as before with a healthy nine frame colony where the queen is limited to a four frame section containing at least one empty frame. Call the four frame section part A and the five frame section part B.Be sure the queen excluders divide the colony completely so the queen cannot move between sections. This division is vital to collecting groups of one day old larvae.The next day, label a group of 100 brood cells with freshly laid eggs as before. This is group one, return it to part A for three days. Two days later, transfer the queen to part B to lay eggs.The next day, label 100 freshly laid eggs in part B as group two and return the section to part B in the colony. After two more days, move the queen back to part A.Now repeat the process to make another group and continue exchanging the queen between part A to part B every three days until nine groups of one day old eggs have been assigned on three day intervals. During the first five days of development, score the hatching rate of each labeled group of brood cells.After 11 days of development, start recording the number of capped brood cells in each group. On day 13 of the experiment, treat the colony with 50%sugar syrup containing PPN at 10 or 100 parts per million. Load a feed box with one liter of treatment and connect it to the colony.Group one is an untreated control as those cells should now be capped. Starting on day 17 record the eclosion rate of each group. To do this, transfer the larvae as previously demonstrated to a 24 well plate, then incubate the plate and later record the eclosion rate.After 49 days, the eclosion data from all nine groups should be known. Individual brood cells were treated as described with PPN BLD. The negative effect of the pesticide on the larval stage honeybees were easily observed and were dosage dependent.These effects included melanization and wing deformation. To simulate honey bee colonies suffering from environmental PPN residue, entire colonies were fed PPN syrup. Group one is a control as these bees developed to adults prior to treatment.Group two and three were initially effected in the larva stage, and groups four through nine were initially effected as eggs. Changes in hatching, capping, eclosion, and deformities occurred with PPN treatments. 100 parts per million of PPN was more effective than 10 parts per million, except on the hatching rate.Moreover, after the treatments, many pupae died with black cuticles or by failing to emerge. In the 100 ppm PPN treatment, capped cells were destroyed and the injured pupae were removed from the colony. Once mastered, the colony treatment procedure can be done in about one month, and the daily manipulation of pupa can be finished in about two hours.While attempting this procedure, it's important to remember to regularly check the condition of your colony and become well-practiced at handling pupae before you perform an experiment. Don't forget that working with honeybees can be hazardous due to bee venom allergies. Precautions such as wearing protecting clothing should always be taken while performing this procedure.

evaluating-effect-environmental-chemicals-on-honey-bee-development

Research

JoVE Journal

Environment

9.0K vistas.  National Ilan University. The overall goal of this experiment is to evaluate how pesticide residues in the environment affect individual honeybees in the natural condition of beehive colonies. The main advantage of this method is that it surveys the effects of pesticides or other contaminants of interest on both individual honeybees and the whole colonies. Visual demonstration of this method is critical as the manipulation of pupae is difficult because they are fragile.Demonstrating the procedure will be Ko Cho...