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 10 and 100 ppm in 50% sugar syrup for the following experiment.</li>
	<li>Honey bee rearing. 
	Note: Here, the experimental location is the National Ilan University (NIU) apiary, Yi-Lan City, Taiwan; (GPS coordinates: N24.747278, E121.746200).
	<ol>
		<li>Check honey bee (Apis mellifera L.) colonies weekly for food quantity and feed with 1 liter 50% sugar syrup if necessary (the honey storage area is empty). Define a healthy colony as 9 frames of honey bee combs in each colony with a queen laying eggs normally.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of basic larval diet (BLD). Dissolve 6% D-glucose, 6% fructose, and 1% yeast extract in sterilized distilled deionized water (ddH2O) and supplement with 50% royal jelly. Store at 4 &#176;C but not for more than 3 days. 
	Note: Pre-warm to 35 &#176;C before experiment and use within 3 days.</li>
</ol>
2. In Vivo Feeding Method
Note: In vivo feeding method has been modified from Hanley et al.20
<ol>
	<li>Honey bee larvae selection and labeling.
	<ol>
		<li>Insert a queen excluder to divide 9 frames of a healthy colony into 4 frames and 5 frames while limiting the queen to the 4 frame section. Leave at least one empty frame in the 4 frame section for laying eggs.</li>
		<li>After the queen laying eggs for 1 day, check the frames for the appearance of eggs and keep the eggs inside the beehives for 72 h (the 4th day) until 1 day-old larvae hatch. Take one of the frames containing 1-day-old worker larvae (hatched within 24 h) out from the test hive by hand and remove the honey bee workers from the frame with a bee brush.</li>
		<li>Cover the frame with a transparent slide (Size = Length x Width x thick = 29.7 mm x 21 mm x 0.1 mm) and nail the transparent slide on the edge of frames with thumbtacks (Figure 1A).</li>
		<li>For each treatment, randomly select 50 one day-old larvae (the 4th day) and mark each brood cell by using permanent marker pens on the transparent slide (Figures 1A and 1B). 
		Note: Write the information of each treatment on the frame and transparent slide as well by using permanent marker pens to avoid the confusion between different treatments. Remove the marked slides and keep for the in vivo feeding and observation.</li>
	</ol>
	</li>
	<li>In vivo feeding.
	<ol>
		<li>Add different concentrations of PPN-BLD (0.1, 1, 10 and 100 ppm) to each labeled brood cell by pipetting at day 1, 2 and 3 with 10 &#956;L, 10 &#956;L and 20 &#956;L, respectively, according to the intake quantity of the larvae age. Add the same amount of BLD (no PPN) to a control group. Thereby, the total dose of PPN-BLD in each labeled brood cell accumulates to 4, 40, 400, and 4,000 pg. 
		NOTE: Recognize the labeled brood cells by the marked transparent slides and remove the marked transparent slides after feeding. Use fresh BLD for feeding to prevent the cleaning of the brood cells by the worker bees.</li>
		<li>Return the PPN-treated frames to the original colonies for further observations. 
		NOTE: Each treatment has four biological repeats in four colonies.</li>
	</ol>
	</li>
	<li>Observation of treated larvae at day 7.
	<ol>
		<li>To observe the PPN-treated larvae, return the labeled transparent slides to the frames to record the mortality and capping rates in the labeled brood cells.</li>
	</ol>
	</li>
	<li>Observation of treated pupae and eclosion rates at day 13.
	<ol>
		<li>Remove the bee wax of capped brood cells.</li>
		<li>Put the soft tipped tweezers into brood cell and clamp the pupae very slightly then take pupae out gently.</li>
		<li>Transfer the pupae into 24-well tissue culture plates with a double-layer of laboratory tissues beneath each well. Record the damage and mortality during pupal transfer.</li>
		<li>Keep the 24-well tissue culture plates in an incubator at 34 &#176;C and 70% relative humidity until emergence (ca. 8 days).</li>
		<li>Observe and record the pupae and emerged honey bees.</li>
	</ol>
	</li>
	<li>Statistics
	<ol>
		<li>Calculate the recorded data and present as a mean &#177; S.D.</li>
		<li>Analyze the data using analysis of variance (ANOVA) by SAS and use the least significant difference (LSD) test to analyze the differences between two means of different treatments. Define statistically significant as P-value &#60;0.05. Different letters in the same column of the table showed a significant effect by the statistical analysis.</li>
	</ol>
	</li>
</ol>
3. Toxicity of PPN at Colony Level in Beehive
<ol>
	<li>Set up the honey bee groups.
	<ol>
		<li>Insert a queen excluder vertically to divide 9 frames of a healthy colony into 4 frames (Part A) and 5 frames (Part B) while limiting the queen to the 4 frame section . Leave at least one empty frame in the 4-frame section for laying eggs. 
		Note: Put another queen excluder the on top of the queen part to prevent the queen from moving between the parts.</li>
		<li>After the queen laying eggs for 1 day, check the frames for the appearance of eggs. Take the appropriate frames containing eggs out from part A by hand and remove the honey bee workers from the frame with a bee brush.</li>
		<li>Cover the frame with transparent slide and nail the transparent slide to the edge of the frame with thumbtacks.</li>
		<li>Randomly select 100 brood cells containing eggs and mark each brood cell by using permanent marker on the transparent slide. Assign these 100 brood cells as Group 1. Write the information of each treatment on the frame and transparent slide using permanent marker to avoid the confusion between different treatments.</li>
		<li>Return the labeled frame to part A for 3 days and then transfer the queen to part B to lay eggs.</li>
		<li>After 1 day, check the frames of part B, choose one appropriate frame and label 100 brood cells containing eggs as described in step 3.1.4. Assign these 100 brood cells as Group 2.</li>
		<li>Return the labeled frame to part B for 3 days and then transfer the queen to part A to lay eggs.</li>
		<li>Repeat the queen exchange between Part A and Part B 6 more times and assign groups numerically, respectively (Figure 2). There should be total of 9 groups.</li>
	</ol>
	</li>
	<li>Treat honey bee colony with PPN sugar syrup on day 13 (Figure 2).
	<ol>
		<li>Add 1 L 50% PPN sugar syrup containing 10 or 100 ppm in a plastic bee feeder box (W x L x H = 20 cm x 30 cm x 3.5 cm) and put the box on top of the frames in the experimental colonies. 
		Note: Group 1 does not receive the PPN at 13 days as the brood cells have been sealed.</li>
		<li>Feed the control group with 1 L 50% sugar syrup (no PPN) as described in step 2.2.</li>
	</ol>
	</li>
	<li>Count 1 day-old larvae and record as the egg hatching rate of 100 labeled brood cells for each group at day 5 (Figure 2). Honey bee eggs usually take 3 days to hatch into 0 day larvae; therefore, check and label 100 eggs-containing brood cells at day 1 and count the number of 1 day-old larvae for each group at day 5 to obtain the percentage of hatching rate.</li>
	<li>Count the capped brood cells and record as the larval capping rate of 100 labeled brood cells for each group at day 11 (Figure 2). 6 to 7 day-old larval brood cells will be capped with bee wax by honey bee workers for larvae pupating.</li>
	<li>Observe pupae maturation and record the eclosion rate of 100 labeled brood cells for each group at day 17 (Figure 2).
	<ol>
		<li>Remove the bee wax of capped brood cells and take pupae out with soft tipped tweezers gently. Transfer the pupae into a 24-well tissue culture plates with a double-layer of laboratory tissues beneath each well.</li>
		<li>Keep the 24-well tissue culture plates in an incubator at 34 &#176;C and 70% RH until emergence.</li>
		<li>Observe and record the pupae and emerged honey bees for each group until Group 9 (49 experimental days). 
		Note: Each treatment has four biological repeats.</li>
	</ol>
	</li>
	<li>Statistics
	<ol>
		<li>Calculate the recorded data and present as the mean &#177; SD.</li>
		<li>Analyze the significant differences between pairs of treatments (e.g. 0 ppm/10 ppm, 10 ppm/100 ppm and 0 ppm/100 ppm) in each group by using the Student&#39;s two-tailed t-test. Define as statistically significant if P-value &#60; 0.05.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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><tbody><tr><td>Honey bee box</td><td>SAN-YI Honey Factory</td><td>W1266</td><td>Honeybees rearing</td></tr><tr><td>Queen excluder (between frames)</td><td>SAN-YI Honey Factory</td><td>I1575</td><td>Queen limitation&amp;nbsp;</td></tr><tr><td>Queen excluder (on top)</td><td>SAN-YI Honey Factory</td><td>I1566</td><td>Queen limitation on top&amp;nbsp;</td></tr><tr><td>Bee brush</td><td>SAN-YI Honey Factory, Taiwan</td><td>W1414</td><td>clean the bees on frame gently</td></tr><tr><td>Bee feeder</td><td>SAN-YI Honey Factory, Taiwan</td><td>P0219</td><td>feed sugar syrup to colony</td></tr><tr><td>Transparent slide</td><td>Wan-Shih-Chei, Taiwan (http://www.mbsc.com.tw/a01goods.asp?s_id=40)</td><td>1139</td><td>Mark the larval area on the frames (Material: Polyethylene Terephthalate, PET) (Size = Length*Width*thick = 29.7 mm * 21 mm * 0.1 mm)</td></tr><tr><td>24 well tissu culture plate</td><td>Guangzhou Jet Bio-Filtration Co., Ltd</td><td>TCP011024</td><td>Rearing pupae from extraction</td></tr><tr><td>Autoclave</td><td>Tomin medical equipmenco., LTD.</td><td>TM-321</td><td>Make sterilized distilled deionized water (ddH&lt;sub&gt;2&lt;/sub&gt;O)</td></tr><tr><td>P20 pipetman</td><td>Gilson</td><td>F123600</td><td>Add PPN into bee larval food pool</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Yihder Co., Ltd.</td><td>LE-550RD</td><td>Rearing pupae from extraction</td></tr><tr><td>Kimwipes</td><td>COW LUNG INSTRUMENT CO., LTD</td><td>KCS34155</td><td>Rearing pupae from extraction</td></tr><tr><td>Royal jelly</td><td>National Ilan University (NIU)</td><td>NIU</td><td>Make basic larval diet (BLD)</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G8270</td><td>Make basic larval diet (BLD)</td></tr><tr><td>D-(-)-Fructose</td><td>Sigma</td><td>F0127</td><td>Make basic larval diet (BLD)</td></tr><tr><td>Yeast extract</td><td>CONDA, pronadisa</td><td>1702</td><td>Make basic larval diet (BLD)</td></tr><tr><td>Sucrose</td><td>Taiwan sugar coporation</td><td>E01071010</td><td>Make sugar syrup for bee food</td></tr><tr><td>Pyriproxyfen (11%)</td><td>LIH-NUNG CHEMICAL CO.. LTD.</td><td>Registration No. 1937</td><td>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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

9.0K Views.  National Ilan University. 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.

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

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 ...

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 ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Neuroscience

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Biochemistry

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Biology

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

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

Summary

Explore More Videos

Chapters in this video

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

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

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

Assessing Agrochemical Risk to Mated Honey Bee Queens

Histology Basics and Cell Death Detection in Honeybee Tissue