The authors thank Kimball Clark and Tim Krogh for providing Osmia nesting reeds, Meredith Nesbitt and Molly Bidwell for assistance in the lab, Drs. Cameron Currie, Christelle Gu&#233;dot, Terry Griswold, Michael Branstetter and three anonymous reviewers for their useful comments that improved the manuscript. This work was supported by USDA-Agricultural Research Service appropriated funds (Current Research Information System #3655-21220-001), Wisconsin Department of Agriculture, Trade, and Consumer Protection (#197199), National Science Foundation (under Grant No. DEB-1442148), the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494).

1. Prepare Propiconazole Solutions for Fungicide Exposure Experiments
<ol>
	<li>Prepare 0.1x fungicide solution by dissolving appropriate volumes of commercially purchased propiconazole 14.3% in sterile water the day of the experiment. Ensure that only freshly prepared fungicide solution is used for all treatments.</li>
	<li>Add 23 &#181;L of 0.1x fungicide solution per gram of pollen provision to obtain the maximum concentration of propiconazole previously reported from bee-collected pollen24 (0.361 PPM or &#181;g of active ingredient g-1 of pollen).</li>
</ol>
2. Harvest Eggs and Host Pollen Provisions from Osmia Reeds
<ol>
	<li>Using a sterilized scalpel, dissect freshly plugged nesting reeds of Osmia, splitting it into two parts along the length of the reed to expose the individual chambers. 
	NOTE: Each nest may contain between 8 to 14 chambers and a single egg within a chamber.</li>
	<li>Inspect the reeds visually to identify the chambers containing male eggs based on previously published guidelines77. Use a sterilized bent needle to remove each pollen provision along with the associated egg from the nesting reed, and place in a clean weigh boat.</li>
	<li>Gently separate the egg from the provision using a clean fine paint brush and record the fresh weight of the pollen provision and egg using a standard laboratory balance. Calculate the average weight of the male pollen provisions.</li>
	<li>Perform the subsequent steps with minimum delay to reduce the chances of damage to the egg from exposure to excess temperature and dehydration.</li>
</ol>
3. Prepare Host Plant Pollen Provisions
<ol>
	<li>Visually inspect the maternally collected host-plant pollen excavated from the nesting chambers to ensure that no parasites are present78. In order to reduce any potential maternal bias, combine the pollen provisions into a single mass in a sterile petri dish and mix well using a sterilized needle.</li>
	<li>Divide the combined mass into new pollen provisions, ensuring that the weight of each reconstituted provision is approximately equal to the average weight of a naturally allocated male provision (Mean &#177; SE, 0.35 &#177; 0.01 g, N = 42). 
	NOTE: Because Osmia sp. allocates smaller pollen provisions to the male offspring, this results in lower body weights of the male larvae compared to that of females77. To avoid any such bias resulting from sex-specific differences, only use male eggs in the experiments.</li>
</ol>
4. Prepare Non-Host Plant Pollen Provision
<ol>
	<li>Pulverize commercially purchased honey bee-collected pollen to a fine powder using a standard laboratory ball-mill.</li>
	<li>Based on the moisture content of maternally-collected host pollen provisions (~20%), hydrate the pollen powder using appropriate volumes of 40% sterilized sugar solution79 and mix thoroughly to form a dough-like consistency.</li>
	<li>Divide into individual pollen masses, each weighing approximately the same as the average weight of a naturally allocated male provision. 
	NOTE: Moisture content of maternally collected host pollen provisions can be standardized in prior by comparing the fresh and dry weight of pollen provisions from 30 randomly selected male chambers80. To obtain the dry weight, pollen provisions should be freeze-dried in a lyophilizer (1.5 Pa for 72 h).</li>
</ol>
5. Prepare Multiwell Cell Culture Plates
<ol>
	<li>Line individual wells of sterile 48-well culture plate with autoclaved tin cups (5 &#215; 9 cm). Using sterile forceps, gently flair out the top rim of the capsule so that it may accommodate the pollen provision.</li>
	<li>Place a single mass of host or non-host pollen provision inside the tin cup using sterile tools based on the treatment group. 
	NOTE: To avoid cross-contamination, use separate plates for treatment and control groups.</li>
</ol>
6. Add Fungicides
<ol>
	<li>Make a centrally placed depression within the pollen mass using a sterile wooden stick. Use a new stick for each pollen provision.</li>
	<li>Add appropriate volumes of fungicide solution (for treatment), or sterile water (for controls) into the depression. Pinch the opening of the depression using sterile forceps to minimize surface contact between the fungicide/ sterile water and the egg.</li>
	<li>Ensure that the factorial set-up of the experimental design aligns with that depicted in the schematic representation (Figure 4).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57876/57876fig4.jpg" /> 
Figure 4. Schematic representation of the experimental setup. The experiment represents a fully-crossed 2 &#215; 2 factorial setups. Factor 1 represents Fungicide exposure and consists of 2 levels: (i) No fungicide (N = 10), and (ii) Fungicide (N = 10). Factor 2 represents Pollen source and consists of 2 levels: (i) Host pollen (N = 8), and (ii) Non-host pollen (N = 8). <a href="/files/ftp_upload/57876/57876fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Rear and Observe Larvae
<ol>
	<li>Place a randomly selected male egg on the top surface of the pollen provision using a clean fine paint brush. Once eggs have been placed on all the provisions, replace the lid of the cell culture plate, securing it with labeling tape on the corners.</li>
	<li>Place the well plates on a clean tray and cover it with a dark cloth to obstruct contact with direct light. Place a 6 well plate containing 30 mL of sterile water within the tray to prevent desiccation. Leave incubation trays undisturbed inside an incubator&#160;at room temperature.</li>
	<li>Observe well plates daily under a dissecting microscope without removing the lid of the well plates. Ensure that the larvae are alive by checking for movement. If no movement is detected, discard the tin cup containing the dead larvae and the remaining pollen provision. Allow all surviving larvae to develop undisturbed within the well plates till they reach the prepupal stage.</li>
	<li>Remove the larva from the tin cup once it reaches the prepupal stage41. Use a brush to clean any defecate from the silk cocoon. Carefully cut through the silk cocoon using a dissecting microscope and extract the prepupa with rubber forceps.</li>
	<li>Handle the prepupa gently to ensure that the tools do not pierce the soft body. Record the fresh weight of the prepupa (prepupal biomass) and the developmental time from egg to the prepupal stage (larval developmental time). 
	NOTE: Any dead larva should be discarded immediately to prevent undesired microbial growth on the cadaver and leftover pollen provision. This reduces the risk of infection to the remaining healthy larvae.</li>
</ol>

The authors have nothing to disclose.

Rearing bees outside their natural nesting reeds, under laboratory conditions, allows the testing of multiple hypotheses pertaining to larval fitness. To the extent that unidentified factors continue to cause bee mortality, risk assessment studies using in vitro experiments can help identify potential threats and inform management practices for this species-rich group of wild pollinators 12,38,49,<sup class="...

Given their role as the dominant group of insect pollinators1, the global loss in bee populations poses a threat to food security and ecosystem stability2,3,4,5,6,7. The declining trends in both managed and wild bee populations have been attributed to several shared risk factors including habitat fragmentation, emerging parasites and pathogens, loss of genetic diversity, and the introduction of invasive species3,4,7,8,9,10,11,12. In particular, the dramatic increase in the use of pesticides, (e.g., neonicotinoids) has been directly linked to detrimental effects among bees13,14,15. Several studies have shown that the synergism between neonicotinoids and ergosterol-biosynthesis-inhibiting (EBI) fungicides can lead to high mortality across multiple bee species16,17,18,19,20,21,22. Nevertheless, fungicides, long considered to be &#39;bee-safe&#39;, continue to be sprayed on in-bloom crops without much scrutiny23. Foraging bees have been documented to routinely bring back pollen loads contaminated with fungicide residues24,25,26. The consumption of such fungicide-ladenpollen can cause high mortality among larval bees27,28,29,30, and a suite of sub-lethal effects among adult bees16,31,32,33,34. A recent study suggests that fungicides may cause bee losses by altering the microbial community within hive-stored pollen, thereby disrupting the critical symbioses between bees and pollen-borne microbes35.
Although solitary bees are vital for the pollination of several wild and agricultural plants36,37,38, this diverse group of pollinators has received much less attention in pesticide monitoring studies. The nest of an adult solitary female contains 5-10 sealed brood chambers, each stocked with a finite mass of maternally-collected pollen and nectar, and a single egg39. After hatching, the larvae rely on the allocated pollen provision, and the associated pollen-borne microbiota to obtain adequate nutrition40,41. Because they lack the benefits of a social lifestyle, solitary bees may be more vulnerable to pesticide exposure42. For instance, while deficits in social bees following a spray may be compensated to some extend by workers and newly emerging brood, the death of a single adult solitary female ends all reproductive activity43. Such differences in susceptibility highlight the need to incorporate diverse bee taxa in ecotoxicological studies to ensure adequate protection for managed and wild bees alike. However, aside from a handful of studies, investigations into the effects of fungicide exposure has primarily focused on social bees18,23,32,44,45,46,47,48,49.
Solitary bees belonging to genus Osmia (Figure 1) have been used worldwide as efficient pollinators of several important fruit and nut crops39,50,51,53,53. As with other managed pollinator groups24,54,55,56,57,58, adult Osmia bees are routinely exposed to fungicides sprayed on in-bloom crops44. Adult females foraging on recently sprayed crops may collect and stock their brood chambers with fungicide-laden pollen, which later forms the sole diet for the developing larvae. Consuming the contaminated pollen provisions can subsequently expose the larvae to fungicide residues42. The risk of exposure may be higher among oligolectic species that forage only on a few closely related host plants59,60,61. Certain megachilid bees, for example, appear to preferentially forage for low-quality Asteraceae pollen, as a means of reducing parasitism62. However, the extent to which fungicides impact larval fitness among oligolectic solitary bees has not been empirically quantified. The goal of this study is to develop a protocol to test the main and interactive effects of fungicide exposure and pollen source on the fitness of in vitro reared solitary bees. To investigate, eggs of O. ribifloris sensu lato (s.l.) can be obtained commercially (Table of Materials). This population is ideal because of its importance as a native pollinator, and its strong predilection for the nectar-rich Mahonia aquifolium (Oregon grape) found within the region53,63,64 (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57876/57876fig1.jpg" /> 
Figure 1. A high-resolution photo of an adult Osmia ribifloris. Photo credit Dr. Jim Cane, Research Entomologist, USDA-ARS <a href="/files/ftp_upload/57876/Figure_1.pdf" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57876/57876fig2.jpg" /> 
Figure 2. Phragmite nesting reeds of Osmia ribifloris (s.l.) with a nesting female in the foreground. Chamber partitions and terminal plugs for the reeds are constructed from masticated leaves. Photo credit Mr. Kimball Clark, NativeBees.com <a href="/files/ftp_upload/57876/57876fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The first objective of this study is to evaluate the effect of consuming fungicide-treated pollen on larval fitness (measured in terms of development time and prepupal biomass). While exposure to the commonly applied fungicide propiconazole has been linked to increased mortality among adult bees across several species 23,24,32,44,45,54,55,56,57,58,65,66,67, its impact on larval bees is less known. The second objective of this study is to evaluate the effects of consuming non-host pollen on larval fitness. Previous studies indicate that larvae of oligolectic bees fail to develop when forced to consume non-host pollen68. Such results may be attributed to variations in bee physiology69, pollen biochemistry70, and the beneficial microbiome associated with natural pollen provisions71. The third objective of this study is to evaluate the interactive effects of fungicide treatment and dietary pollen on larval fitness.
Numerous biological traits including maternal body size, provisioning rate, foraging strategy, and pollen quantity72,73,74,75 are known to affect larval fitness among solitary bees. These factors can introduce significant variability between reeds, which poses a challenge in developing defensible experimental designs when assessing larval health. Moreover, given that larval development occurs inside sealed nesting reeds, the effects of such variability on the progeny are difficult to visualize and quantified without using non-lethal techniques (Figure 3). To overcome this challenge, all hypotheses within this study are tested using larvae reared outside of their nesting reeds. The experimental design represents a fully crossed 2 &#215; 2 factorial set-ups, with each factor consisting of 2 levels; Factor 1: Fungicide exposure (Fungicide; No fungicide); Factor 2: Pollen source (Host pollen, Non-host pollen). Bees are raised from the egg to the prepupal stage within sterile multiwell cell culture plates under controlled laboratory conditions. Each well is individually stocked with a standardized amount of pollen provision and a single egg. After hatching, the larva feeds on the allocated pollen within the well, completes larval development, and initiates pupation. Past studies have shown that unexplained mortality is lower among bees raised within this artificial rearing environment than that encountered in the wild49,76. The use of in vitro-reared bees has several advantages over field-based studies: 1) it minimizes the confounding effects of natural variability and uncontrolled factors typically associated with field-based studies; 2) it allows multiple levels of manipulation for each factor(s) of interest to be tested simultaneously across treatment groups; 3) the number of replicates can be predetermined, and experimental factors for each replicate can be individually manipulated; 4) larval response variables can be easily visualized and recorded independently without disturbing adjacent larvae; 5) the protocol can be modified to accommodate more complex experimental designs involving multiple factors and response variables.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57876/57876fig3.jpg" /> 
Figure 3. Contents within a natural nesting reed of Osmia ribifloris (s.l.). Close up of (A) a dissected reed showing individual chambers, pollen provisions, and partitions, and (B) freshly harvested pollen provisions, and the associated eggs (indicated with a black circle). <a href="/files/ftp_upload/57876/57876fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>eggs of O. ribifloris sensu lato (s.l.)</td>
			<td></td>
			<td></td>
			<td>Kaysville, Davis County, Utah, USA</td>
		</tr>
		<tr>
			<td>Osmia reeds</td>
			<td>Nativebees.com</td>
			<td>NA</td>
			<td>Freshly plugged reeds</td>
		</tr>
		<tr>
			<td>Dissection set</td>
			<td>VWR</td>
			<td>89259-964</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>Long Nose Pliers</td>
			<td>Husky</td>
			<td>1006</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well culture plates</td>
			<td>VWR</td>
			<td>10062-892</td>
			<td>Sterile sealed</td>
		</tr>
		<tr>
			<td>48 well culture plates</td>
			<td>VWR</td>
			<td>10062-898</td>
			<td>Sterile sealed</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>25373100</td>
			<td>Sterile sealed</td>
		</tr>
		<tr>
			<td>Square Weighing Boats</td>
			<td>VWR</td>
			<td>10770-448</td>
			<td></td>
		</tr>
		<tr>
			<td>Camel Hair Brush</td>
			<td>Bioquip</td>
			<td>1153A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tin capsules</td>
			<td>EA Consumables</td>
			<td>D1021</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>470302-808</td>
			<td></td>
		</tr>
		<tr>
			<td>Propiconazole 14.3</td>
			<td>Quali-Ppro</td>
			<td>60207-90-1</td>
			<td>Propiconazole 14.3%</td>
		</tr>
		<tr>
			<td>Honey bee pollen</td>
			<td>Bee energised</td>
			<td>897098001244</td>
			<td>Untreated, natural, raw pollen</td>
		</tr>
		<tr>
			<td>Microbalance</td>
			<td>VWR</td>
			<td>10204-990</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulverisette</td>
			<td>LAB SYNERGY INC.</td>
			<td>30334913</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden sticks</td>
			<td>VWR</td>
			<td>470146908</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>VWR</td>
			<td>89097-912</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>VWR</td>
			<td>89403-384</td>
			<td></td>
		</tr>
		<tr>
			<td>Planting tray</td>
			<td>VWR</td>
			<td>470150-632</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>BDH1158-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>VWR</td>
			<td>21008936</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe</td>
			<td>Cole-Palmer</td>
			<td>UX-07940-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber tweezer</td>
			<td>Amazon</td>
			<td>B0135HWPN4</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needles</td>
			<td>VWR</td>
			<td>89219-334</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze drier</td>
			<td>Labcono</td>
			<td>LFZ-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical software</td>
			<td>SPSS</td>
			<td>Version 21.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Larval fitness was quantified using three metrics (i) larval developmental time, (ii) prepupal biomass, and (iii) percent survivorship. A two-way ANOVA was conducted using Fungicide exposure (two levels: No fungicide, Fungicide) and Pollen source (two levels: Host pollen, Non-host pollen) as the independent variables, and larval developmental time as the dependent variable. The main effect for Fungicide exposure (F1,28 = 1.24, P = 0.28) was non-significant bet...

Watch this Scientific Journal Video about In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors at JoVE.com

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.

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 ...

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8.1K Views.  University of Wisconsin-Madison. 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.

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

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...