This study was supported by the National Key R&#38;D Program of China (2017YFD0200400), Major Scientific and Technological Innovation Project (2017CXGC0214), Bee Industry Innovation Team of Shandong Province, Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2019G01), and Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2021B13).

1. Preparation of the feeding tube
<ol>
	<li>Punch a hole (~0.3 mm diameter) into the lid of a 2 mL centrifuge tube using an electric winding iron (see Table of Materials). Use such a centrifuge tube to maintain an O. excavata larva and its provision mass.</li>
</ol>
2. Preparation of pesticide
<ol>
	<li>Dissolve the technical-grade pesticide (see Table of Materials) in acetone to acquire stock solutions of 1 x 104 &#181;g a.i. mL-1. Then, perform gradient dilutions of the solution to more than five concentrations. 
	NOTE: Chlorpyrifos at 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 &#181;g a.i. mL-1 were used in this study.</li>
</ol>
3. Preparation of the provisions
<ol>
	<li>Acquire plastic bee tubes containing provisions (see Table of Materials) and newly hatched larvae of O. excavata from a mass-rearing program. 
	NOTE: No pesticides were used from 20 days before flowering to the entire flowering period; chemical analysis results showed that commonly used pesticide contents in randomly selected fifty provisions were both below the minimum test levels.</li>
	<li>Separate provisions and larvae gently using a soft brush. Select female larvae based on provision size and cell position within the nest9. Then, place uniform-sized provisions and selected female larvae in Petri dishes (60 mm diameter) and set them aside for use. 
	NOTE: Fifty provisions were randomly selected to analyze the contents of commonly used pesticides: chlorpyrifos, imidacloprid, fendifenuron, phoxim, avermectin. The soft brush parameters are (a) diameter of the brush: 0.3 mm, (b) length of the brush: 2 cm, (c) length of the pen: 18 cm.</li>
</ol>
4. Provision treatment with pesticide
<ol>
	<li>Soak the selected evenly sized provisions (from step 3.2) in diluted pesticide (from step 2.1; chlorpyrifos at 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 &#181;g a.i. mL-1) for 10 s using a cage. Soak the control check (CK) in 0.2% solvent (acetone in this study). 
	NOTE: There are three replicates per concentration treatment, and each replicate consisted of 60 provisions. The difference of dosage of each provision can be reduced by selecting evenly sized provisions.</li>
	<li>Measure the volume of pesticide solution before and after treating the provisions with the pesticide. Then, calculate the immersed volume of insecticide in each treatment, including 60 mass provisions (Supplementary Table 1). Place the provisions in separate centrifugal tubes with holes (from step 1.1) after air-drying on a sterile worktable. 
	NOTE: Before the experiment, put the cages containing the provisions into the pesticide solution, and then measure the volume of pesticide solution before and after soaking to eliminate the error.</li>
	<li>Transfer female larvae individually to the surface of naturally dried provisions using a soft brush. 
	NOTE: One larva in one tube.</li>
</ol>
5. Growth conditions
<ol>
	<li>Rear the larvae of O. excavata in a growth chamber in the dark, 65%-75% relative humidity, and 25 &#177; 2 &#176;C16.</li>
</ol>
6. Examination of the results
<ol>
	<li>The acute lethal toxicity test
	<ol>
		<li>Measure the mortality of the larvae after placing them onto the treated and the control (CK) provisions for 48 h. 
		NOTE: The death criteria: when the larvae did not respond to mild touch using a soft brush under black-light lamps22. Black-light lamps were used to simulate the dark growth conditions of larvae and avoid the influence of light on the larvae when checking growth indicators. For eliminating human error, the mortalities with and without removal of larvae from the provisions after 48 h in control groups were also measured.</li>
		<li>Weigh 60 provisions before and after 48 h of insect rearing trials to determine the amount of provision consumed by each larva.</li>
		<li>Calculate the dose of pesticide at each concentration consumed by each larva according to the percentage of provision eaten and the pesticide content in each provision. 
		NOTE: The equation for dose calculation is23: 
		<img alt="Equation 1" src="/files/ftp_upload/62946/62946eq01.jpg" /> 
		where, D is the consumed dose of pesticide by each larva; W1 is the weight of 60 provisions before infusion of pesticide; W2 is the remaining weight of 60 provisions after 48 h; V1 is the volume of pesticide before immersion for 60 provisions; V2 is the volume of pesticide after immersion for 60 provisions; C is the concentration of the pesticide.</li>
	</ol>
	</li>
	<li>The sublethal toxicity test
	<ol>
		<li>Weigh the larvae before rearing trials and after 14 days of treatments to determine the larval weight gain.</li>
		<li>Observe O. excavata daily during cocooning under black-light lamps to measure the larval development duration.</li>
		<li>Weigh the remaining portions of provisions after 14 days of feeding on treated and CK provisions to calculate the consumption and the efficiency of conversion of ingested food (ECI)24.</li>
		<li>Examine the number of eclosions by sniping the cocoons using a small scissor when the control bees emerge into adults.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to declare.

For adult pollinators, there are two main methods for measuring the ecotoxicity of pesticides. One is the contact method, in which the pesticide is applied to the prothorax of the adult insects; the other is the gastric toxicity method, in which the adult pollinators are fed with honey water containing pesticide25,26. In recent years, it has been found that the pollination effect and eclosion rate of O. excavata are relatively low27</su...

Pollinators play a critical role in the ecosystem services of modern global agriculture. While honey bees (Apis mellifera; Hymenoptera: Apidae) have traditionally been considered as the essential economic pollinators of crops, recent research suggests that Osmia (Hymenoptera: Megachilidae) is also very important in improving pollination for certain crops, increasing fruit size and number of seeds, and reducing the proportion of asymmetric fruit in commercial orchards in different parts of the world1. Osmia excavata has been considered an ideal species for apple pollination, mainly in Asia, like in north and northwest China and Japan2,3,4. It can provide pollination services for certain crops with similar or sometimes with greater efficiency. In this respect, they have been shown to replace or work in synergy with the honey bees4,5,6.
The biological characteristics of O. excavata are unique compared with social bees. Its univoltine, solitary, and nesting activity occurs mainly in spring and early summer. The nests of O. excavata are usually found in preexisting holes, typically in deadwood, hollow plants, straw tubes, and bamboo stem in the natural condition3. The adult O. excavata emerges from its cocoon to mate, gather pollen, and build a nest to lay eggs, which begin to hatch a week later. The fertilized eggs develop into females, while the unfertilized eggs develop into males3. Females are distributed in the bottom of the bee tube, and the corresponding provisions are more significant. In contrast, males were in the proximity of tube exit with minor provisions7, so the males come out first, and the females come out later. The female mixes pollen with a small amount of nectar into a moist blob, the only food source for each larva in the cell8.
Several studies have reported a decrease in the population of pollinating insects9,10. The extensive use of pesticides has been identified as one of the main factors for reducing pollinator abundance and diversity and may also endanger pollination services11,12. To reduce and mitigate the adverse effects of pesticides, it is necessary to conduct a pesticide risk assessment for pollinators. Some countries have established regulatory frameworks to ensure safety to bees from the pesticides used13,14. Recent studies have shown that Osmia was more susceptible to pesticides than honey bees1,15.
Interestingly, most risk assessments were focused on adult honey bees11,12; little research has been conducted on O. excavata, especially the larvae. Furthermore, the mortality of Osmia directly caused by pesticides is most commonly considered16. Still, the chronic toxicities such as larval weight gain, developmental duration, feeding patterns, eclosion ability, subsequent adult behavior, and fecundity may have the same harm as the acute lethal toxicities and are often ignored because of a lack of an effective experimental method for the solitary bees17.
Up to now, two methods are used to evaluate the effects of pesticides on the larvae of solitary bees: (1) an appropriate amount of pesticide was applied in the localized spot of provisions without removing the egg of solitary bees1,18,19,20; (2) replacing provisions with artificial pollen-nectar mixtures containing a specific amount of pesticide21. However, there are some limitations to the above two methods. The former can only measure acute toxicity, but not chronic toxicity because the larvae ingested the entire dose in a short period of time; the latter would lead to a high mortality rate because of human manipulation1. Here, the immersion method was described to study the ecotoxicity of pesticides to O. excavata under highly controlled research conditionsby simulating the behavior of larval feeding on residual pesticide in the provisions in the real environment. The method of this study solves the disadvantages of the above two methods and is suitable for measuring the effects of a hazardous substance on acute and chronic toxicity.

<table><tbody><tr><td>Abamectin</td><td>Jinan Lvba Pesticide Co. Ltd</td><td></td><td></td></tr><tr><td>Black-light lamps</td><td>Kanghua Medical Device Co., Ltd</td><td></td><td></td></tr><tr><td>Centrifugal tube box with 100 Wells</td><td>Shanghai Rebus Network Technology Co., Ltd</td><td></td><td></td></tr><tr><td>Centrifuge tube</td><td>Shanghai Rebus Network Technology Co., Ltd</td><td></td><td>2 mL; &amp;nbsp;Serve as bee tube</td></tr><tr><td>Electric soldering iron</td><td>Kunshan Kaipai Hardware Electromechanical Co., Ltd</td><td></td><td></td></tr><tr><td>Electronic scale</td><td>Sartorius Scientific Instruments (Beijing) Co., Ltd</td><td>3137510295</td><td></td></tr><tr><td>Graduated cylinder</td><td>Anhui Weiss Experimental Equipment Co. Ltd</td><td></td><td></td></tr><tr><td>Petri dishes (60 mm diameter)</td><td>Qingdao jindian biochemical equipment co., LTD</td><td></td><td></td></tr><tr><td>Pollen provision</td><td>Yantai Bifeng Agricultural Science and Technology Co. Ltd</td><td></td><td></td></tr><tr><td>Soft brush</td><td>Wengang Wenhai painting material factory</td><td></td><td></td></tr><tr><td>Solitary bees</td><td>Yantai Bifeng Agricultural Science and Technology Co. Ltd</td><td></td><td></td></tr></tbody></table>

evaluating the effect of pesticides on the larvae of the solitary bees

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

The contents of commonly used pesticides, chlorpyrifos, imidacloprid, fendifenuron, phoxim, avermectin in provisions were less than the limit of quantification (0.01-0.02 mg kg-1) in the control group; these results excluded the influence of pesticide residues on each treatment. The mortality with and without removing larvae from provisions after 48 h in control groups was evaluated; the results showed no significant differences (Table 1), indicating a minor human error.
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Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

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

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

evaluating-the-effect-of-pesticides-on-the-larvae-of-the-solitary-bees

Research

JoVE Journal

Biochemistry

2.1K Views.  Shandong Academy of Agricultural Sciences. Dit protocol kan worden gebruikt om de nadelige effecten van pesticiden op solitaire bijen te voorspellen en te beperken. Het belangrijkste voordeel van deze techniek is het simuleren van larvale voeding op restgif in proviand. Visuele demonstratie van deze methode is van cruciaal belang omdat de biologische kenmerken van Osmia-opgravingen uniek zijn in vergelijking met sociale bijen en er weinig onderzoek is gedaan naar O.exvata, met name de larve.Om de voedingssonde voor te bereiden,...

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

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

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

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

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

Environment

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

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

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

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

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

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

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

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

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

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

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

Biology

Assessing Agrochemical Risk to Mated Honey Bee Queens

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

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

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

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

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