The authors warmly thank K. Anderson, J.J. Adamczyk, E. Beren, I Carstensen, M. Giansiracusa B. Mott, N. Holst and A.R. Stilwell for discussions on design and execution and for help in the field.

The protocols described below follow the animal care guidelines of USDA Agricultural Research Service.
1. Full Evaluation
NOTE: A full evaluation is necessary to determine 1) the weights of all hive parts, including the lid, cover, boxes, bottom board, frames, and 2) the amounts of brood and food resources. The weights of all hive parts will be summed and subtracted from the total hive weight when all bees were present to yield the weight of all the adult bees. Data on the weights of hive parts that are largely constant, such as the lid, bottom board and box(es), can be retained and used in future evaluations, thus expediting the evaluations. Conduct a full evaluation at or near the beginning of the study. Wooden parts may change in terms of moisture content24 so repeat full evaluations are recommended after long periods. Weigh a part of the hive, such as the lid, at each evaluation to estimate changes in other hive parts. Place a screened vestibule around the workers and hive during evaluation if other hives are close by and robbing pressure is a concern.
<ol>
	<li>Weigh all hive parts. Use a scale with a precision of 1 g or less.
	<ol>
		<li>Weigh the lid, inner cover, queen excluder, syrup feeder, and any other parts on the top of the hive.</li>
		<li>Using a spatula or hive tool, remove, and weigh any food or treatment material, such as protein supplement or grease patty, resting on the tops of the frames. Replace afterward if necessary.</li>
		<li>Place an empty hive box (hereafter the &#34;temporary box&#34;) next to the hive to serve as the transitional location for the frames. Place the temporary box on top of a flat surface, such as a lid or a bottom board, to prevent bees, and particularly the queen, from falling out of the box onto the ground.</li>
		<li>Move the frames rapidly and gently, preferably in groups of two or three to retain parts of the bee cluster, to the temporary box so the frames are in the same order as they were in the original box. Once all the frames have been moved, shake any remaining bees onto the frames in the temporary box and weigh the hive box.</li>
		<li>If the box was the lowest box, weigh the bottom board, entrance reducer, and any other hive parts after shaking the bees into the temporary box. Number frames with an indelible marker before moving them so they can be replaced in their original orientation.</li>
		<li>Reinstall the bottom board, entrance reducer, brood box, and other equipment. Move the frames from the temporary box back to the original brood box in their original order and orientation, again taking care to avoid unnecessary disturbance of the bees.</li>
	</ol>
	</li>
	<li>Remove a frame from one end of the box and visually inspect both sides of the frame for the presence of the queen. If the queen is found, place her gently in a part of the box that has already been evaluated, to avoid further disturbance. Shake worker bees gently but firmly into the box between frames, rather than on top, if possible.</li>
	<li>Weigh the frame and photograph both sides of the entire frame with a digital camera, getting as close as possible to the frame so capped brood and honey cells are easily distinguished, but at a sufficient distance so the entire frame is included. Photograph the frame while another worker holds it.
	<ol>
		<li>Use a &#39;frame holder&#39; (any jig that holds the frame upright while on the scale and does not mar the comb) on the scale. Weigh the within-hive syrup feeders. 
		Note: The size of the frame in the photograph will be used to convert surface areas from relative to absolute measures.</li>
	</ol>
	</li>
	<li>Replace the frame in its original position and orientation in the hive box. Remove the next frame prior to reinstalling the previous frame to provide more space to safely shake bees within the box. Check for the presence of the queen, shake to remove bees, weigh, photograph, and replace each subsequent frame until all frames have been processed.</li>
	<li>If the hive has two or more boxes, weigh and photograph frames from the lowest box first.
	<ol>
		<li>Remove the upper box or boxes sequentially, stacking them next to the hive in reverse to their original order. Once the bottom box has been evaluated, place the next box to be evaluated on top of the bottom box, place the temporary box on top of the remaining unevaluated boxes, and move frames from the second box to the temporary box, as above. Move the frames back to their original box, in their original orientation. 
		NOTE: By inspecting the lower boxes first, shaken bees will fall into a hive box that will no longer be disturbed.</li>
	</ol>
	</li>
	<li>Obtain the total hive weight during a recent period of inactivity, such as the night or the early morning before the bees are flying on the day of the evaluation. This weight represents the entire colony including the foragers.</li>
	<li>Calculate the adult bee mass by summing the weights of all the hive parts (woodenware, frames, bee feed, etc.) and then subtracting that sum from the &#34;total hive weight&#34; value obtained in the previous step. The difference between the two is an estimate of the adult bee mass. 
	NOTE: Once the full evaluation is conducted, subsequent evaluations can be conducted on a partial basis, assuming the non-frame hive parts, such as the boxes and the bottom board, have not changed appreciably in weight. Partial evaluations take less time.</li>
	<li>To conduct a partial evaluation for a hive with a single box, remove, photograph, weigh, and replace the frames without moving frames to a temporary box. In a hive with multiple boxes, the follow the procedure as described above (step 1.5), but without weighing the hive boxes.</li>
</ol>
2. Installation of Hives on Scales
NOTE: The hive scales used in this study are outdoor electronic bench scales with a maximum capacity of at least 100 kg, a load cell precision of about one part in 5000 with temperature compensation, and a pan size of 460 mm x 610 mm. These scales are connected to a 24 VDC indicator and a 12-bit datalogger. Indicator output may be digital or analog; scale systems used here had output in mA and required calibration curves to convert data to kg.
<ol>
	<li>Install scales on a firm, level surface. 
	NOTE: Elevating the scales from the ground using concrete or ceramic blocks, or pieces of kiln-dried wood, will keep the scale and electrical connections from coming into contact with soil or water.</li>
	<li>Cover the scale pan on top with plastic, cloth, cardboard, or other material to prevent excessive light reflection and heating of the hive entrance.</li>
	<li>Ensure that entrances to hives that are close together face different directions to reduce drift among colonies.</li>
	<li>Calibrate hive scales upon installation and periodically thereafter, particularly if the scales are moved.
	<ol>
		<li>Place scale calibration weights on the weighing pan and record the scale output. Ensure that the total weights exceed 50% of the maximum capacity of the scale. 
		NOTE: If the scale output is voltage or current units, rather than mass units, then use the slope and intercept of the calibration curve (relationship between total known weight on the pan and scale output) to convert scale output to mass units.</li>
	</ol>
	</li>
</ol>
3. Installation of Temperature Sensors
NOTE: Temperature sensors used in this study were of two types: 1) thermocouples attached to cables directly connected to a battery-powered datalogger (which may be placed inside or outside the hive, if the cable is long enough); and 2) small, battery-powered devices with integrated sensors and dataloggers and which required removal from the hive for data to be downloaded.
<ol>
	<li>Obtain a temperature sensor that can fit between frames, is resistant to the high humidity conditions in the interior of a hive, and has sufficient battery power and memory (or is accessible, via cable or wireless connection).</li>
	<li>Place sensors that might be affected by wax or propolis in a protective container, such as a tissue embedding cassette (small, disposable plastic box with a snap-shut lid and with ventilation slots). The protective container reduces wax and propolis build-up that can clog sensor access ports of sensors or otherwise affect data download.</li>
	<li>Attach the sensor just beneath the top bar in the center of the box to provide informative data on brood rearing (see below). 
	NOTE: A short piece of wire is attached to the container with the sensor and the wire stapled to the top of the frame, so the container hangs down one side. Sensors placed directly on the brood mass may interfere with brood and comb maintenance, and sensors placed on frames near the exterior, or placed on inner covers or lids, may be affected a great deal by external conditions, particularly during cool weather.</li>
</ol>
4. Preparation of Syrup Treatment
NOTE: Make treatment solutions fresh the day of each feeding using analytical-grade imidacloprid. Imidacloprid is highly water soluble and therefore easily incorporated in syrup; some pesticides have low water solubility and should be applied using other means. Formulations of 1 kg of treatment solution were applied using a 1 L plastic bottle as follows:
<ol>
	<li>Mix control (no pesticide) sucrose solution 1:1 w:w (e.g. 500 g sucrose:500 mL distilled water). Dissolve sucrose in distilled water using a large mixing flask with mixing bar on a hot plate heated to no more than 60 &#176;C.</li>
	<li>For solutions that will include imidacloprid, mix sucrose solution as above, but withhold 100 mL (thus &#34;short&#34;) to allow for the added volume of respective imidacloprid &#34;spike&#34; solutions.
	<ol>
		<li>Dissolve 500 g of sugar in 400 mL of distilled water to allow for the addition of a 100 mL spike to achieve 1 kg of treatment solution. For convenience in the field, transfer 900 g of &#34;short&#34; sugar solution to another bottle, then add the spike to each individual bottle.</li>
	</ol>
	</li>
	<li>Prepare a 10 ppm imidacloprid stock solution by measuring 1.0 mg of pesticide on an analytical balance and dissolving it in 100 mL of distilled water using a mixing bar without heat. 
	NOTE: To avoid problems with static electricity, weigh the imidacloprid into small, nonreactive plastic receptacles and place those receptacles directly in the solution. Stir the solution and remove the receptacles after the imidacloprid has dissolved.</li>
	<li>For a 5 ppb solution, mix 0.5 mL of the stock solution into 99.5 mL of distilled water to achieve 100 mL of spike solution. Add this to 900 g of the short sucrose solution to achieve 1 kg of 5 ppb syrup. For the 20 ppb solution, mix 2.0 mL of stock solution into 98.0 mL of distilled water to produce the 100 mL spike solution. For a 100 ppb solution mix, 10.0 mL of stock solution into 90.0 mL of distilled water for the spike solution.</li>
</ol>
5. Preparation of Bee Hives and Application of Treatment
<ol>
	<li>Evaluate colonies to determine the adult bee and brood populations prior to and after application of treatment. Sample material(s) of interest, such as honey, bee bread, bees, or wax, to determine baseline pesticide levels prior to treatment.
	<ol>
		<li>To sample wax, open a 50 mL centrifuge tube, select a section of empty comb, and scrape the open mouth of the tube along the comb until the desired amount of wax has been collected. Avoid touching the wax in the tube. Replace the cap on the tube and label the tube. Replace the frame in the hive.</li>
		<li>To sample honey or nectar, open a centrifuge tube and press the mouth of the tube against the section of comb containing the honey or nectar. Allow the material to flow into the tube, rather than scraping the tube, to reduce the amount of wax in the sample. Replace the cap on the tube and label the tube. Replace the frame in the hive.</li>
		<li>To sample bee bread, select a frame containing bee bread and use a clean metal or plastic spatula to remove the contents of several cells of bee bread and place the material in a centrifuge tube. Replace the frame in the hive. 
		NOTE: Collecting sufficient material will usually take several minutes per frame. A typical sample for pesticide analysis has at least 3 g of material; sample the honey, wax, and bee bread at least 2 or 3 different points in each hive and combine those subsamples for each hive.</li>
	</ol>
	</li>
	<li>Shortly after the evaluation, such as the next day, remove any broodless frames containing honey and nectar and replace those frames with either foundation or empty drawn comb to facilitate syrup storage. 
	NOTE: The objective is to increase both empty storage capacity and demand for nectar. If treatment is to be applied mixed in a protein supplement, removal of food frames may not be necessary.</li>
	<li>Ensure the hive has a feeder. As soon as possible after the pre-treatment evaluation, apply the treatment by pouring the syrup (step 4.4) into the feeder as rapidly as possible. Take care not to spill any syrup outside the hive where bees from other colonies might find it. 
	NOTE: Internal feeders may work best for reducing robbing; some pesticides affect adult bee activity and treated hives may be less vigilant in protecting feeders from robbing bees9.</li>
</ol>
6. Analysis of Hive Frame Photographs
NOTE: Frame photographs are made during each hive evaluation. Information can be extracted from the photographs using the following protocol.
<ol>
	<li>Activate an image analysis software, such as ImageJ, that allows for selecting a shape on a photograph with a tracing tool and measuring the area of the selected shape.</li>
	<li>Retrieve a digital photograph of a frame. Select &#39;File&#39; and click &#39;Open&#39;. Navigate to the folder where the photographs are kept to select a photo for analysis. The image will now be opened within the ImageJ program, and the ImageJ toolbar will be active.</li>
	<li>Select the &#39;Polygon&#39; tool from the toolbar. Use the mouse to click on each corner to define the area of the frame covered with comb. The shape will snap together when the starting corner is reached.</li>
	<li>Once the comb area is defined using the polygon tool, click on &#39;Analyze&#39; and select &#39;Measure&#39;; a new dialog box will pop up with the measurement. Use the column &#39;Area&#39; to measure the pixels within the defined area that was drawn. 
	NOTE: The first time the program is used, define the desired results to be reported.
	<ol>
		<li>Click on &#39;Results&#39; in the Results dialog box, and select &#39;Set Measurements&#39;. Check area, and uncheck all others.</li>
	</ol>
	</li>
	<li>To define the area covered by capped brood, select the &#39;Freehand&#39; tool. Depress the button on the mouse at a starting point and trace around the brood. When the starting point is reached, release the mouse button; the shape will snap into place.
	<ol>
		<li>Click &#39;Analyze&#39; and select &#39;Measure&#39;. If the brood pattern is not solid, due to factors such as disease, use the &#39;Multi-point&#39; selection tool to count the cells instead. Convert cell counts directly to cm2 for ease of comparison; there are typically about 4.01 cells/cm2 20.</li>
	</ol>
	</li>
	<li>Divide the value of the traced brood shape by the value of the interior of the frame to obtain a percent frame coverage of capped brood.</li>
	<li>Measure the interior of representative frames to obtain a standard so the brood surface area can be converted to cm2. 
	NOTE: A typical Langstroth deep frame has a surface area of about 880 cm2 20. For example, if 20% of the interior of a Langstroth deep frame is found to be covered by brood, then the surface area would be 0.20 x 880 cm2 = 176 cm2.</li>
	<li>Sum the brood surface area for the hive. To convert this measure to mass, multiply the surface area by 0.77 g/cm2 20.</li>
	<li>Calculate the surface area of honey, uncapped nectar, and stored pollen, for example, using the method described in steps 6.3 to 6.6. 
	NOTE: If nectar and pollen are considered together as &#34;food stores&#34; this reduces the components of a frame to two (food stores and brood). A typical Langstroth frame with plastic foundation and empty drawn comb weighs about 556 g20, so to calculate the weight of food stores, sum the weight of the frame with empty drawn comb and the weight of the brood mass, and subtract that weight from the observed weight of the frame in question.</li>
</ol>

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G., Holst, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+continuous+monitoring+of+honey+bee+colonies.">Application of continuous monitoring of honey bee colonies.</a> Apidologie. 46, 10-22 (2015).</li><li>De Smet, L., 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=Stress+indicator+gene+expression+profiles,+colony+dynamics+and+tissue+development+of+honey+bees+exposed+to+sub-lethal+doses+of+imidacloprid+in+laboratory+and+field+experiments.">Stress indicator gene expression profiles, colony dynamics and tissue development of honey bees exposed to sub-lethal doses of imidacloprid in laboratory and field experiments.</a> PLoS ONE. 12 (2), e0171529 (2017).</li><li>Danka, R. G., Rinderer, T. E., Hellmich, R. A., Collins, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The thermology of wintering honey bee colonies. , (1971).</li><li>Szabo, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermology+of+wintering+honey-bee+colonies+in+4-colony+packs.">Thermology of wintering honey-bee colonies in 4-colony packs.</a> Am. Bee J. 189, 554-555 (1989).</li><li>Human, H., Nicolson, S. W., Dietemann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+honeybees,+Apis+mellifera+scutellata,+regulate+humidity+in+their+nest?.">Do honeybees, Apis mellifera scutellata, regulate humidity in their nest?.</a> Naturwissenschaften. 93, 397-401 (2006).</li></ol>

The authors have no competing financial interests.

Just as a crop scientist requires accurate and precise data to evaluate the effects of different treatments on plant growth and yield, honey bee researchers require accurate and precise data to evaluate bee colony growth and activity. These kinds of data are particularly important when the treatment effects may be subtle and long term, such as might be expected when bees are exposed to low concentrations of pesticides. 
Continuous hive weight data contain much information on the growth and activity of the colonies being studied, as well as the response of those colonies to exogenous disturbances, such as from robbing bees and from the addition of sugar syrup to a feeder9. However, proper interpretation of continuous weight and temperature data requires some knowledge of the size of the colony on a periodical basis. While the size of the adult bee population can be estimated from the daily amplitudes of detrended hourly data during a nectar flow, due to the increased forager traffic, outside of a nectar flow the relationship does not hold well20. Likewise, colony weight changes due to foraging success, food consumption, and bee population increases and decreases (e.g. bee kills) are partly a function of colony size. Smaller colonies may be healthy but show lower growth rates and less forager mass, for example, simply due a smaller forager population. 
Identifying a consistent and informative fixed position for a temperature sensor within the hive is crucial for producing reproducible results, particularly with overwintering. Previous research has shown that temperature monitoring at the top rail of a central frame in the brood (bottom) box were less correlated with ambient (external) temperatures than several other positions in the hive and were more influenced by the colony itself20. During winter, temperature differences between the colony bee cluster and ambient would expected to be high, and so differences among positions within the hive would likewise be expected to be greater than during warmer months. In addition, the bee cluster will move to different locations within the hive to exploit food resources25,26. The winter study described here showed that temperatures at horizontal positions, from the top middle to top outer frames, were significantly different while vertical differences on the middle frame were not. Horizontal air movement across frames is relatively restricted while vertical movement along a frame is not, so vertical mixing would be expected to be greater. These results confirm that a sensor position on the top rail of the middle frame is likely to be informative with respect to temperature influences from the colony.
Hive evaluations as they are described here resolve some of these issues. By subtracting the sum of the hive parts from the total hive weight measured during inactivity, such as at night, the estimate of the adult bee mass is independent of the number of foraging bees and thus insensitive to the time of evaluation. Recording data from a scale allows little room for subjectivity, and frame photographs provide a permanent record, so analyses of brood or food surface areas can be revisited if necessary, thus reducing the dependence on the training of the person doing the analyses. 
The methods described here are not without sources of error. Hive parts need to be as free of bees as possible, and any significant pieces of comb or other material that is removed should be weighed before discarded. The moisture content of wooden components can change over time and empty hives can exhibit measurable daily oscillations in weight due to daily changes in ambient relative humidity24. A hive containing a colony will probably not change as much because honey bees tend to maintain a relatively constant humidity inside the hive27, which would attenuate the ambient effects. These methods also depend on working rapidly. Once a hive is taken apart, the temperature and gas conditions change drastically and cool temperatures can impact brood health14. Taking apart a hive without a screen to keep out bees from neighboring colonies can promote robbing, particularly in times of nectar dearth. 
Modifications of these procedures are occasionally necessary. While it is in the interest of the researchers to gather as much data as possible, on some occasions, such as when many hives must be inspected, when robbing pressure is high, or when weather conditions are not optimal, workers must move quickly and forgo some data. If data on food reserves are not crucial, frames containing only honey and/or pollen, or frames with only wax or foundation, should be weighed but not photographed. Levels of uncapped larvae and eggs are also of interest to some researchers and they are often detected in frame photographs; however, in order to avoid sampling bias a researcher must be confident in being able to detect them in all photographs. Often only capped brood can be reliably and consistently identified on photographs taken under field conditions. Regarding the application of treatment, some hives, particularly those fed higher doses of imidacloprid, can become lethargic, causing a reduction in both their consumption of syrup and their ability to defend their hive. If a colony fails to consume its syrup after 5-7 d, the syrup should be removed, weighed, and discarded (to avoid contamination due to fermentation) and the colony offered fresh syrup. By weighing the discarded syrup, researchers will have record of how much each colony consumed, and thus can calculate total dosage. Finally, the application of treatments in sugar syrup is most effective when there are few or no alternative nectar sources. Alternative nectar sources would dilute the treatment to an unknown degree. Researchers should bear in mind, as was pointed out in the protocol section, that pesticides such as imidacloprid are associated with reduced activity levels, including hive protection, depending on the concentration. Reduced colony defense may also lead to increased robbing and possible confounding of treatments.
The results presented here show that continuous monitoring of hive weight and internal temperature are sensitive to colony-level behaviors, even with respect to behavioral changes induced by very low (5 ppb) concentrations of a neurotoxic pesticide. Further application of these methods to other neurotoxins and to pesticides with other modes of action, such as insect growth regulators, would increase our understanding of the effects of field-realistic doses on honey bee colonies.

Concerns have been raised recently about the exposure of honey bees to low concentrations of agrochemicals, particularly pesticides. Honey bees in the field may be exposed via direct application on foragers in the field or even the colonies themselves, by contact with treated surfaces, by dust from seed treatments, and by consumption of plant products such as pollen, nectar, and exudate1,2,3,4,5. Acute toxicity is clearly detrimental to honey bees, but the concentrations that causes 50% mortality in test insects (LC50) of many pesticides, including neonicotinoids such as imidacloprid, are far higher than that observed in bee bread and wax6 and higher than what is considered a &#34;field realistic&#34; range7. However, recent work has shown significant effects of sublethal exposure to pesticides on the colony level8, even for concentrations as low as 5 parts per billion (ppb)9.
Honey bee colonies have been referred to as &#34;superorganisms&#34; because many functions associated with an individual animal, including food procurement, reproduction (which honey bee colonies conduct via colony fission), and temperature control, are conducted by groups of adult worker bees10,11,12,13,14. Honey bee colonies, considered as organisms, offer a unique opportunity for study because the structures that support the colonies can be disassembled, examined, and reassembled with, if done carefully, little, or no adverse effects. Beekeepers and researchers take advantage of this by disassembling hives to inspect them for, among other things, the presence and status of the queen, pests, and pathogens, sufficiency of food stores, quantity of brood, and other factors15. Bee colonies have another important characteristic in that, unlike most homeotherms, they are typically stationary and obtain resources by deploying forager bees that can collect food at distances up to 10 km16. Bee hives can thus be maintained indefinitely on electronic scales, and the resulting continuous weight data has been shown to provide information on honey storage, foraging activity, foraging success, swarming, syrup consumption, lethal and sublethal pesticide effects, pest infestation and robbing9,17,18,19,20. Because all parts of the colony, including brood, can be accessed, sensors for temperature, humidity, CO2, sound, and other factors can be installed within the hive and used to obtain continuous data which inform on the health and status of the colony21.
Estimates of the size of the adult bee and brood populations are useful if not critical for interpreting many kinds of continuous data. Visual assessments are fast and widely applied in field studies22 but have shortcomings. Visual inspections are usually conducted during the day and thus do not account for foraging bees, which can compose a significant part of the population23. The precision and accuracy of estimates from visual inspections also depend on the training and experience of the inspector(s). Objectivity is another consideration, particularly if the hive inspectors are aware of which hives are in which treatment groups; assumptions about the distribution of the error of such measures may be difficult to justify.
Rate of hive weight gain, size of the foraging population, and within-hive temperatures, to provide a few examples, would be expected to be quantitatively different between large and small colonies, so measuring and controlling for colony size often greatly facilitates the detection of treatment effects in randomized experiments. For research purposes, a precise and objective measure of the adult bees was developed based in the principle that the difference between the total hive weight during a period of inactivity, such as at night, and the sum of the weight of all the hive parts (including woodenware, wax, honey, and brood) is the weight of the total adult bee mass. By weighing frames and measuring brood mass using frame photographs, food resources can likewise be estimated by subtracting the brood mass from the frame weight, after controlling for the weights of the empty frame and the wax. These methods are intended to improve on the precision expected from visual assessments in the field.

<table border="0" cellpadding="0" cellspacing="0" width="814">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:427px;">Langstroth hive equipment (box)</td>
			<td style="width:301px;">Mann Lake Ltd, Hackensack, MN</td>
			<td style="width:87px;">KD-700</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (frame)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td>WW-900</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (lid and cover)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td style="width:87px;">WW-302 
			&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (base)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td>WW-316</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (internal feeder)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td>FD-505</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cordovan Italian queens and bee packages</td>
			<td>C.F. Koehnen &#38; Sons, Glenn, CA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scale, bench (100 kg max. capacity): model B-2418&#160;</td>
			<td>TEKFA, Copenhagen, Denmark</td>
			<td>discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scale, bench (100 kg max. capacity): Diamond Series</td>
			<td>Avery Weigh-Tronix , Fairmont, MN</td>
			<td>1824-200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidacloprid, analytical-grade CAS # 138261-41-3</td>
			<td>Sigma-Aldrich, St. Louis, MO 63103</td>
			<td align="right">37894</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electronic scale, precision (precision = 0.1 mg): Adventurer Pro 260</td>
			<td>Ohaus,&#160; Parsippany, NJ</td>
			<td>AV264C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electronic scale, portable (15 kg max. cap.): Ranger Count 3000</td>
			<td>Ohaus, Parsippany, NJ</td>
			<td>RC31P15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermocouple probe: TMC6-HD</td>
			<td>Onset Computer Corp., Bourne, MA</td>
			<td>TMC6-HD</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Datalogger, 12-bit: Hobo U-12</td>
			<td>Onset Computer Corp., Bourne, MA</td>
			<td>U12-012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature and r.h. datalogger: iButton Hygrochron</td>
			<td>Baulkham Hills, NSW 2153, Australia</td>
			<td>DS1923</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature datalogger: iButton Thermochron</td>
			<td>Baulkham Hills, NSW 2153, Australia</td>
			<td>DS1922L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nalgene plastic bottle</td>
			<td>Thermo Scientific, Rochester, NY</td>
			<td>2104-0032</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue embedding cassette</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>B1000731WH</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital camera: Pentax K-01</td>
			<td>Ricoh Imaging Co., Ltd.</td>
			<td align="right">15241</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ version 1.47 software</td>
			<td>W. Rasband, National Institutes of Health, USA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge tubes, 50 ml</td>
			<td>Fisher Scientific, Asheville, NC</td>
			<td>14-959-49A</td>
		</tr>
	</tbody>
</table>

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.

Honey bee colony growth and phenology, measured using regular hive inspections with continuous weight monitoring, revealed significantly lower brood production among colonies exposed to imidacloprid at 100 ppb. 
Hive inspections showed that adult bee masses were not significantly affected by exposure to both sublethal concentrations of 5 and 100 ppb, but analysis of photographs of brood frames revealed that brood production in the 100 ppb treatment was significantly lower (Figure 1). Continuous hive weight data showed different colony growth rates among groups of hives exposed to different concentrations of imidacloprid. The continuous hive weight data were divided into two parts: the 25 h running average data, which is related to colony growth and foraging success, and difference between the hourly raw data and the 25 h running average, the hourly detrended data. The daily amplitudes of the detrended data are related to foraging activity19,20 (Figure 2). While weighing hives occasionally may have detected average weight differences at some point, continuous weighing provided detrended data for information on colony behavior.
The top rail of the middle frame in a typical commercial hive is an effective location for temperature sensors. 
Adult worker bees in colonies generate and maintain high temperatures (33-36&#176;C) especially in the presence of brood14 and the cluster itself is mobile to take advantage of food stores in different parts of the hive25,26. To reduce heat loss, clusters avoid having a large contact surface with exterior parts of the hive, such as the sides or bottom, so the distance from the top center of the box to the cluster is seldom large. The top center of the brood box has been shown to have reduced temperature variability compared to other locations in the hive, such as the top center of an outer frame and the top of a second box (&#34;super&#34;) in summer and fall conditions20. In winter conditions, in which the difference between the ambient temperature and the cluster would be expected to be the greatest, the same location was found to have the highest temperature and lowest variability compared to ambient conditions, indicating the greatest contribution from the cluster itself (Figures 3 and 4; Table 1). Differences in temperature regimes could not be attributed to differences in the adult bee mass (Table 2).
Amplitudes of sine curves fit to continuous temperature data were significantly higher in the 100 ppb treatment than in the 5 ppb treatment while neither treatment group was significantly different from the control group9. 
Amplitudes are directly proportional to temperature variability, so the higher amplitudes indicate more variable temperatures at that point in the hive. Lower amplitudes reflect the higher brood production in the 5 ppb and control treatment groups compared to the 100 ppb treatment group (Figure 5). These results, combined with those from the hive inspections and continuous hive weight data, confirm that colony-level behavior was significantly affected by exposure to 100 ppb imidacloprid.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56355/56355fig1.jpg" /> 
Figure 1: Adult bee and brood measures. Hive inspection data from May, 2014, to March, 2015, for colonies exposed to syrup containing 0 (control), 5 and 100 ppb imidacloprid near Tucson, AZ. (A) Average (+ SEM) total adult bee mass; (B) Average (+ SEM) sealed brood surface area. Gray zone indicates treatment period (4-6 kg of treated sucrose syrup per week per hive for 6 weeks). Four colonies per treatment group; one colony in the 100 ppb died during winter. Adult bee masses were not recorded in August. Data here was previously published and is used here with permission9. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56355/56355fig2.jpg" /> 
Figure 2: Honey bee hive weight data. Continuous weight data from June to December, 2014, for honey bee colonies exposed to syrup containing 0, 5 and 100 ppb imidacloprid near Tucson, AZ. (A) Total hive weight (&#177; SEM); (B) Amplitudes of sine curves fit to detrended weight data from the same period. Gray zone indicates treatment period (4-6 kg of treated sucrose syrup per week per hive for 6 weeks). Four colonies per treatment group; one colony in the 100 ppb died during winter. Data here was previously published and is used here with permission9. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56355/56355fig3.jpg" /> 
Figure 3: Within-hive temperatures at different locations. Daily average (&#177; SEM) temperatures (&#176;C) for 3 sensor locations inside honey bee hives. (A) 4 single-box hives kept at a high altitude site (MLEM: 2412 m); and (B) 3 hives kept at a lower altitude site (SRER: 719 m) near Tucson, AZ from December 2013 to February 2014. Hives were wooden Langstroth deep boxes (43.65 l capacity) fitted with wooden inner covers and metal-lined telescoping lids. Thermocouple temperature probes were attached to the top bars of the frames, and an integrated datalogger/temperature sensor was placed on the bottom rail of the middle frame. These data show that the sensor position at the top of the middle frame was consistently higher than ambient compared to the other sensor positions and thus informative about temperature management by the bee colonies. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56355/56355fig4.jpg" /> 
Figure 4: Within-hive temperature variability at different locations. Daily average (&#177; SEM) amplitudes for sine curves fit to hourly detrended temperature data for 3 sensor locations inside honey bee hives. (A) 4 hives kept at at high altitude site (MLEM: 2412 m); and (B) 3 hives kept at a lower altitude site (SRER: 719 m) near Tucson, AZ from December 2013 to February 2014. Hives were wooden Langstroth deep boxes (43.65 l capacity) fitted with wooden inner covers and metal-lined telescoping lids. Thermocouple temperature probes were attached to the top bars of the frames, and an integrated datalogger/temperature sensor was placed on the bottom rail of the middle frame. These data show that the sensor position at the top of the middle frame was consistently lower in variability (here measured as the amplitude of sine curves fit to hourly detrended data) than ambient compared to the other sensor positions and thus informative about temperature management by the bee colonies. Scales for the ambient temperature (solid black line) are on the right of each graph. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56355/56355fig5.jpg" /> 
Figure 5: Within-hive temperatures for treated colonies. Continuous temperature data from June to December, 2014, for honey bee colonies exposed to syrup containing 0, 5 and 100 ppb imidacloprid near Tucson, AZ. (A) Average hive temperature (&#177; SEM); (B) Amplitudes of sine curves fit to detrended temperature data from the same period. Gray zone indicates treatment period (4-6 kg of treated sucrose syrup per week per hive for 6 weeks). Four colonies per treatment group; one colony in the 100 ppb died during winter. Data here was previously published and is used here with permission (Meikle et al. 2016a). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Comparison</td>
			<td>t test</td>
			<td>adj. P</td>
			<td>Site</td>
			<td>Avg. diff. (&#176;C)</td>
		</tr>
		<tr>
			<td>Pos. 2 - Pos. 1</td>
			<td>2.05</td>
			<td>0.0485</td>
			<td>MLEM</td>
			<td>1.00 &#177;0.25</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>SRER</td>
			<td>4.38 &#177;0.12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pos. 2 - Pos. 3</td>
			<td>0.84</td>
			<td>1</td>
			<td>MLEM</td>
			<td>2.05 &#177;0.21</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>SRER</td>
			<td>0.12 &#177;0.11</td>
		</tr>
	</tbody>
</table>
Table 1: Average temperature differences and post hoc comparisons among different sensor positions within honey bee hives. Position 1: top outer frame; Position 2: top middle frame; Position 3: bottom middle frame. 4 single-box hives were kept at a high altitude site (MLEM: 2412 m) and 3 hives were kept at a lower altitude site (SRER: 719 m) near Tucson, AZ from December 2013 to February 2014. Hives were wooden Langstroth deep boxes (43.65 l capacity) fitted with wooden inner covers and metal-lined telescoping lids. Thermocouple temperature probes were attached to positions 1 and 2, and an integrated datalogger/temperature sensor was attached to position 3.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group</td>
			<td>Date</td>
			<td>Average (&#177; SEM) adult bee mass (g)</td>
			<td>Average (&#177; SEM) brood surface area (cm2)</td>
		</tr>
		<tr>
			<td>MLEM</td>
			<td>18 Nov. 2013</td>
			<td>2119 &#177;412</td>
			<td>1372 &#177;396</td>
		</tr>
		<tr>
			<td>SRER</td>
			<td>15 Nov. 2013</td>
			<td>2270 &#177;312</td>
			<td>53 &#177;30</td>
		</tr>
		<tr>
			<td>MLEM</td>
			<td>13 Feb. 2014</td>
			<td>2171 &#177;105</td>
			<td>0</td>
		</tr>
		<tr>
			<td>SRER</td>
			<td>11 Feb. 2014</td>
			<td>2027 &#177;487</td>
			<td>867 &#177; 79</td>
		</tr>
	</tbody>
</table>
Table 2: Average adult bee masses and brood masses, estimated using the protocols described here, for four hives kept at a high altitude site (MLEM: 2412 m); and three hives kept at a lower altitude site (SRER: 719 m) near Tucson, AZ from December 2013 to February 2014. Hives were wooden Langstroth deep boxes (43.65 l capacity) fitted with wooden inner covers and metal-lined telescoping lids. Thermocouple temperature probes were attached to positions 1 and 2, and an integrated datalogger/temperature sensor was attached to position 3.

Watch this Scientific Journal Video about Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees at JoVE.com

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

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 ...

monitoring-colony-level-effects-sublethal-pesticide-exposure-on-honey

Nanyang Technological University

Research

JoVE Journal

Biology

9.0K Views.  Carl Hayden Bee Research Center. 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.

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

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...