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 10<sup...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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; &#160;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, ...

This protocol can be used to predict and mitigate the adverse effects of pesticides on solitary bees. The main advantage of this technique is simulating larval feeding on residual pesticide in provisions. Visual demonstration of this method is critical because the biological characteristics of Osmia excavata are unique compared with social bees, and little research has been conducted on O.excavata, especially the larva.To prepare the feeding tube, punch a 0.3 millimeter diameter hole into the lid of a two milliliter centrifuge tube using an electric winding iron. To prepare the pesticide, dissolve the technical grade pesticide in acetone to acquire stock solutions of 10 milligrams per milliliter, then perform gradient dilutions of the solution to more than five concentrations. Next, acquire plastic bee tubes containing provisions and newly hatched Osmia excavata larva from a mass rearing program.Using a soft brush, gently separate the larva from the provisions, select female larvae based on provision size and cell position within the nest, then place the uniform size provisions with the selected female larva in 60 millimeter diameter Petri dishes and set them aside for use. Before proceeding to the pesticide treatment, weigh the provisions. To treat the provisions with pesticides, first, measure the volume of pesticide solution.Then soak the selected evenly sized provisions in the diluted pesticide solutions for 10 seconds. After treating the provisions with the pesticide, measure the volume of the pesticide solutions again. After air drying the provisions on a sterile work table, add them to the prepared centrifuge tubes with holes.Next, weigh 60 female larvae and using a soft brush, transfer the larvae to the surface of the naturally dried provisions, adding one larva per tube. Rear the larvae in a growth chamber in the dark at 25 plus or minus two degrees Celsius with 65 to 75%relative humidity. For the acute lethal toxicity test, measure the mortality of the larva after being placed onto the treated and the control provisions for 48 hours.Next, weigh the provisions remaining after 48 hours of insect rearing to determine the amount of provision consumed by each larva, then using this formula, 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. For the sublethal toxicity test, observe the O.excavata daily during cocooning under black light lamps to measure the larval development duration. Then weigh the larvae after 14 days of treatments to determine the larval weight gain.Next, weigh the provisions remaining after 14 days of feeding to calculate the consumption and the efficiency of conversion of ingested food. And examine the number of eclosion by sniping the cocoons using a small scissor when the control bees emerge into adults. The acute lethal toxicity test showed that the LD50 value of the chlorpyrifos for the O.excavata larvae was 0.001 micrograms per bee.In the sublethal toxicity test, as the dose increased, the index values of larval weight gain, consumption, and efficiency of conversion of ingested food decreased for treatments, with the lowest values relative to the control observed in the 0.013 micrograms chlorpyrifos per bee. Conversely, the most extended larval developmental duration was observed in 0.016 micrograms chlorpyrifos per bee, compared to the control treatment. Evaluation of the impact of chlorpyrifos on eclosion rate revealed a significant negative linear relationship between ingested dosages of chlorpyrifos and the eclosion rate of O.excavata.The eclosion rate was considerably lower when the ingested dosages exceeded 0.02 micrograms per bee than those in the control treatment. In this protocol, screening for provisions of uniform size is critical to minimize the test error. This technique is helpful in exploring the effects of pesticides on the adult flight and fecundity of solitary bees, thus solving problems of low recovery rate and population decline.

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

Research

JoVE Journal

Biochemistry

2.0K visualizações.  Shandong Academy of Agricultural Sciences. Este protocolo pode ser usado para prever e mitigar os efeitos adversos dos pesticidas sobre abelhas solitárias. A principal vantagem dessa técnica é simular a alimentação larval em pesticidas residuais em provisões. A demonstração visual desse método é fundamental porque as características biológicas da Escavada de Osmia são únicas em comparação com as abelhas sociais, e pouca pesquisa tem sido realizada na O.excavata, especialmente na larva.Para preparar o tubo de ali...