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 (...

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 ...

Solitary bee species represent the most diverse group of bees on the planet but it can be quite difficult to successfully rear the larvae under highly controlled conditions. We have created a method for in vitro culturing of solitary bee species, allowing us to isolate and measure risk factors associated with their microbial symbionts. Among certain stem-nesting solitary bee species it is critical that the culturing techniques cater to the young larvae as well as their pollen-born microbial symbionts.Here we have illuminated the main and interactive effects of multiple stressors, allowing us to discern whether the stressors are largely acting alone or in concert. Demonstrating the procedure will be Molly Bidwell and Prarthana Dharampal, both from my laboratory. Using a sterilized scalpel, dissect freshly plugged Osmia nesting reeds.Split the reeds into two parts lengthwise to expose the individual chambers. Nests typically contain eight to 14 chambers with one egg per chamber. Identify the male eggs using the standard method.Only male eggs are used in this experiment because of sex bias in pollen provision size. Male provisions are typically smaller than female provisions. Then with a sterilized instrument, remove each pollen provision along with the associated egg and place them in a clean weigh boat.In the weigh boat, gently separate the egg from the provision. Then weigh the pollen provision and egg separately. Work quickly with the Osmia eggs to minimize their time off the pollen.For host pollen provision preparations, first visually inspect the maternally collected host plant pollen excavated from the nesting chambers for parasites. Then to reduce any potential maternal bias, combine the pollen provisions into a single mass and mix them using a sterilized needle. Next, divide the host pollen into new provisions of standard mass.To prepare non-host plant pollen provisions pulverize commercially purchased honey bee-collected pollen to a fine powder using a standard laboratory bone mill. Then, based on the moisture content of host pollen provisions, hydrate the pollen powder using sterilized 40%sugar solution. Mix this to a dough-like consistency.Then make non-host pollen provisions that are the same weight as the maternally collected provisions. For the treatment plate, line individual wells of a 48-well culture plate with autoclaved tin cups. Prepare separate plates for the treatment and control groups.Then using sterile forceps gently flare out the top rim of the cups so that they can accommodate the pollen provision. Next, load single pollen provisions into the tin cups. Now within each pollen mass make a centrally placed depression using a sterile wooden stick.Use a new stick for each pollen provision. Into the depression, add the appropriate volumes of treatment solution such as fungicide and sterile water for controls. After each addition, pinch the depression closed using clean sterile forceps.To begin, load each treatment plate well with a randomly selected male egg. Place it on top of the pollen provision using a clean fine paintbrush. Once the eggs have been placed, secure the lid onto the plate using tape on the corners.Then, place each prepared plate into an incubator maintained at room temperature. Include one six-well plate loaded with sterile water to prevent desiccation. Leave the plates undisturbed inside the incubator.Check the plate daily under a dissecting microscope without removing the lid. If the larvae are alive, they will be moving. If no movement is detected, discard the tin cup containing the dead larvae and the remaining pollen provision.When a larva reaches the prepupal stage, remove it from the tin cup and use a brush to clean frass off the silk cocoon. Then using a dissecting microscope carefully cut through the silk cocoon and extract the prepupa using featherweight forceps. Handle the prepupa gently to prevent any damage to its body.Now record the mass of the prepupa and the developmental time from the egg to the prepupal stage. Larval fitness was quantified using larval developmental time and prepupal biomass. There was a significant negative correlation between larval development time and prepupal biomass among fungicide treated and untreated groups.Although there was a significant negative correlation for larvae raised on non-host pollen, no such relationship was observed for larvae raised on host pollen. Pollen source had a significant main effect on bee fitness. When raised on non-host pollen larval developmental time increased significantly, whereas prepupal biomass decreased significantly among both fungicide treated and untreated groups compared to larvae raised on host pollen.Fungicide exposure did not have significant main effect on fitness. There was no statistically significant difference in developmental time and prepupal biomass for treatments raised on host and non-host pollen across fungicide treated and untreated groups. There was no significant interactive effect of fungicide exposure and pollen source on larval developmental time and prepupal biomass.After watching this video you should have a good understanding of how to successfully rear solitary bee larvae under in vitro conditions. Once mastered, this technique can be used to raise bees under in vitro conditions from the egg to the pupal stage while causing minimal mortality from handling stress. While using this procedure, it is important to remember to use eggs or very young larvae.This ensures that the larvae are raised almost entirely on the intended manipulation of diet treatments. This protocol can be extended to several other species of solitary bees and wasps and can be easily modified to test the main and interactive effects of multiple risk factors impacting pollinator health. By providing a means for in vitro culture for solitary bee larvae, this technique can help identify previously unknown risk factors among vulnerable bee populations.For instance, some of their experiments based on this protocol can help ascertain the role of the pollen microbiome in conferring varying degrees of resistance to xenobiotic stressors. As an accessible and inexpensive technique, this protocol represents a versatile tool for hypothesis testing which can be easily modified by investigators to address their own research objectives.

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8.0K 조회수.  University of Wisconsin-Madison. Solitary bee species represent the most diverse group of bees on the planet but it can be quite difficult to successfully rear the larvae under highly controlled conditions. We have created a method for in vitro culturing of solitary bee species, allowing us to isolate and measure risk factors associated with their microbial symbionts. Among certain stem-nesting solitary bee species it is critical that the culturing techniques cater to the young larvae as well as their pollen-born microbial s...