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

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. 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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. 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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

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study evaporation under various surface boundary conditions to improve our current understanding and characterization of this multiphase phenomenon as well as to validate numerical heat and mass transfer theories that couple Navier-Stokes flow in the atmosphere and Darcian flow in the porous media. Experimental data were collected using a unique soil tank apparatus interfaced with a small climate controlled wind tunnel. The experimental apparatus was instrumented with a suite of state of the art sensor technologies for the continuous and autonomous collection of soil moisture, soil thermal properties, soil and air temperature, relative humidity, and wind speed. This experimental apparatus can be used to generate data under well controlled boundary conditions, allowing for better control and gathering of accurate data at scales of interest not feasible in the field. Induced airflow at several distinct wind speeds over the soil surface resulted in unique behavior of heat and mass transfer during the different evaporative stages.

A protocol for the design and construction of a soil tank interfaced to a small climate controlled wind tunnel to study the effects of atmospheric forcings on evaporation is presented. Both the soil tank and wind tunnel are instrumented with sensor technologies for the continuous in situ measurement of environmental conditions.

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...