Name: evaluating the effect of environmental chemicals on honey bee development from the individual to colony level
Uploaded: 2017-04-01T14:00:00
Description: 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

Cette recherche a été soutenue par Grant 105AS-13.2.3-BQ-B1 du Bureau de santé animale et végétale inspection et de quarantaine, le Conseil de l&#39;agriculture, Yuan exécutif et Grant 103-2313-B-197-002-MY3 du ministère de la science et de la technologie (MOST).

1. Préparation <ol><li> Faire 1 L de 50% de sirop de sucre. Dissoudre 1 kg de saccharose dans 1 L ddH 2 O. </li><li> Préparer la solution pyriproxyfène (PPN) dans BLD. Faire 1,1 L de 10 000 ppm solution PPN de stock et une solution diluée à 100 ml PPN dans 1 litre stérilisé ddH 2 O. Conserver à 4 ° C. </li><li> Diluer la solution mère PPN à des concentrations finales de 0,1, 1, 10 et 100 mg / kg (ppm) dans le BLD pour l&#39;expérience suivante. </li><li> Faire PPN-sirop (pour le nivea...

<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. 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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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La méthode de ponte reine limitée et la méthode queen-échange sont des étapes essentielles pour la mise en place des groupes d&#39;abeilles de miel pour les essais sur le terrain dans ce protocole. La méthode de ponte reine limitée permet la synchronisation du cycle de vie des abeilles. Par conséquent, les chercheurs peuvent choisir 1 jour larves âgées du même âge pour le traitement avec différentes doses de pesticides. Pour la méthode queen-échange, la reine a été échangée entre la partie A (4 cadres...

La présence de pesticides dans l&#39;environnement est l&#39; un des problèmes les plus graves qui influe sur la vie d&#39;une abeille 1, 2, 3. Plusieurs études ont démontré la présence commune des résidus de pesticides dans les colonies d&#39;abeilles et les produits apicoles. A Taiwan, l&#39;application moyenne des pesticides était 11-12 kg / ha chaque année (de 2005 à 2013). La quantité de pesticides utilisés à Taiwan est supérieure à celle des pays de l&#39; UE, et les pays d&#39; Amérique latine 4, 5. En d&#39;autres termes, l&#39;environnement apicole souffre de stress grave des pesticides, en particulier à Taiwan et peut-être dans d&#39;autres pays. L&#39;abeille Apis mellifera est l&#39; un des principaux insectes pollinisateurs dans les systèmes agricoles 6 et elle produit également des produits de valeur tels que le miel. Cependant, les abeilles sont Exposed à divers pesticides et ces pesticides peut être ramené à la recherche de nourriture après ruchers sur les fleurs qui ont été traitées avec des pesticides lors de la collecte nectar et de pollen 7, 8. Ils peuvent également être exposés aux pesticides par les apiculteurs eux - mêmes visant à contrôler les problèmes de ravageurs à l&#39; intérieur des ruches 9, 10, 11. Parce que les larves d&#39;abeilles sont alimentés par les abeilles nourrices pour leur développement, les larves, les drones et même la reine peut être exposé à ces nectars contaminées par les pesticides et le pollen 12. La toxicité des différents pesticides pour les abeilles doit être adressée 13. De nombreux efforts ont été faits pour évaluer les problèmes de résidus de pesticides sur l&#39;environnement. Yang et al. testé l&#39;influence de l&#39;insecticide imidaclopride neurotoxiques sur le développement des larves d&#39;abeilles dans leruche et a rapporté qu&#39;une dose sublétale de imidaclopride a donné lieu à un comportement associatif olfactif des abeilles adultes 14. En outre, Urlacher et al. a examiné les effets sublétaux d&#39;un pesticide organophosphoré, chlorpyrifos, sur la performance de l&#39; apprentissage d&#39;un travailleur d&#39;abeille de miel dans des conditions de laboratoire 15. Dans notre étude précédente, nous avons évalué l&#39;impact d&#39;un régulateur de croissance des insectes (IGR), pyriproxyfène (PPN), sur les abeilles larves 16. Dans cet article, nous présentons les méthodes d&#39;évaluation des impacts chimiques sur le développement des abeilles. méthodes d&#39;alimentation des abeilles au miel ont été décrits et appliqués soit à abeilles domestiques individuels ou à une colonie. Dans un premier temps , nous avons testé différentes concentrations de l&#39; alimentation des larves de base contaminées par des pesticides (BLD) sur les larves dans les colonies pour évaluer l&#39;impact des pesticides sur les abeilles individuelles in vivo. Nous avons ensuite simuler le Condit natureldes ions du pesticide à l&#39;aide de sirop contaminées par des pesticides à l&#39;intérieur des ruches. Dans cette méthode, PPN, qui est largement utilisé contre les insectes nuisibles 17 et est nocif pour le développement des larves d&#39;abeilles et pupes 16, 18, 19, sera un indicateur pour représenter l&#39;effet négatif du pesticide sur le terrain.

<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.

Pour l&#39;essai sur le terrain de l&#39;abeille, une reine était limitée à la section 4 cadre pour la ponte des œufs. Cette étape pourrait augmenter la densité de géniteurs dans un cadre et faciliter les observations suivantes. Chaque traitement a été marqué, et le développement des abeilles a été clairement observée à travers une lame transparente. Dans l&#39; alimentation in vivo de PPN-BLD aux larves d&#39;abeilles dans la ruche a été réalisée pou...

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

Nous présentons ici un procédé pour alimenter des aliments contaminés par les pesticides à la fois une abeille individuelle et une colonie ruche. La procédure évalue l&#39;effet des pesticides sur les abeilles individuelles par l&#39; alimentation in vivo de l&#39; alimentation des larves de base et sur la condition naturelle de la colonie de ruche.

Évaluation de l&#39;incidence des produits chimiques sur l&#39;environnement Miel de développement Abeille de l&#39;individu à la colonie Niveau

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.

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