Name: evaluating the effect of pesticides on the larvae of the solitary bees
Uploaded: 2021-10-15T14:20:00
Description: Watch this Scientific Journal Video about Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees at JoVE.com

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>
	<li>Sgolastra, F., Tosi, S., Medrzycki, P., Porrini, C., Burgio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+spirotetramat+on+solitary+bee+larvae,+Osmia+cornuta+(hymenoptera:+megachilidae),+in+laboratory+conditions.">Toxicity of spirotetramat on solitary bee larvae, Osmia cornuta (hymenoptera: megachilidae), in laboratory conditions.</a> Journal of Apicultural Science. 59 (2), 73-83 (2015).</li><li>Wei, S. G., Wang, R., Smirle, M. J., Xu, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+Osmia+excavata+and+Osmia+jacoti+(Hymenoptera:+Megachilidae)+for+apple+pollination.">Release of Osmia excavata and Osmia jacoti (Hymenoptera: Megachilidae) for apple pollination.</a> TheCanadian Entomologist. 134 (3), 369-380 (2002).</li><li>Men, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+characteristics+and+pollination+service+of+Mason+bee.">Biological characteristics and pollination service of Mason bee.</a> Chinese Journal of Applied Entomology. 55 (6), 973-983 (2018).</li><li>Bosch, J., Kemp, W. P., Trostle, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bee+population+returns+and+cherry+yields+in+an+orchard+pollinated+with+Osmia+lignaria+(Hymenoptera:+Megachilidae).">Bee population returns and cherry yields in an orchard pollinated with Osmia lignaria (Hymenoptera: Megachilidae).</a> Journal of Economic Entomology. 99 (2), 408-413 (2006).</li><li>Winfree, R., Williams, N. M., Dushoff, J., Kremen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+bees+provide+insurance+against+ongoing+honey+bee+losses.">Native bees provide insurance against ongoing honey bee losses.</a> Ecology Letters. 10 (11), 1105-1113 (2007).</li><li>Garibaldi, L. A., Steffan-Dewenter, I., Winfree, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild+pollinators+enhance+fruit+set+of+crops+regardless+of+honey+bee+abundance.">Wild pollinators enhance fruit set of crops regardless of honey bee abundance.</a> Science. 339 (6127), 1608-1611 (2013).</li><li>Bosch, J., Sgolastra, F., Kemp, W. P., James, R. R., Pitts-Singer, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+ecophysiology+of+Osmia.+mason+bees+used+as+crop+pollinators.">Life cycle ecophysiology of Osmia. mason bees used as crop pollinators.</a> Bee Pollination in Agricultural Ecosystems. , 83-104 (2008).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+investigation+and+restriction+factors+analyses+of+Osmia+excavata+Alfken+in+Jiaodong.">Population investigation and restriction factors analyses of Osmia excavata Alfken in Jiaodong.</a> Apiculture of China. 69 (9), 68-71 (2018).</li><li>Biesmeijer, J. C., Roberts, S. P. M., Reemer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+declines+in+pollinators+and+insect-pollinated+plants+in+Britain+and+the+Netherlands.">Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands.</a> Science. 313 (5785), 351-354 (2006).</li><li>Potts, S. G., Biesmeijer, J. C., Kremen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+pollinator+declines:+trends,+impacts+and+drivers.">Global pollinator declines: trends, impacts and drivers.</a> Trends in Ecology &amp; Evolution. 25 (6), 345-353 (2010).</li><li>Chen, L., Yan, Q., Zhang, J., Yuan, S., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joint+toxicity+of+acetamiprid+and+co-applied+pesticide+adjuvants+on+honeybees+under+semi-field+and+laboratory+conditions.">Joint toxicity of acetamiprid and co-applied pesticide adjuvants on honeybees under semi-field and laboratory conditions.</a> Environmental Toxicology and Chemistry. 38 (9), 1940-1946 (2019).</li><li>Sgolastra, F., Medrzycki, P., Bortolotti, L., Renzi, M. T., Bosch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+mortality+between+a+neonicotinoid+insecticide+and+an+ergosterol-biosynthesis-inhibiting+fungicide+in+three+bee+species.">Synergistic mortality between a neonicotinoid insecticide and an ergosterol-biosynthesis-inhibiting fungicide in three bee species.</a> Pest Management Science. 73 (6), 1236-1243 (2017).</li><li>Bireley, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preface:+Workshop+on+pesticide+exposure+assessment+paradigm+for+non-Apis+bees.">Preface: Workshop on pesticide exposure assessment paradigm for non-Apis bees.</a> Environmental Entomology. 48 (1), 1-3 (2019).</li><li>European Food Safety Authority. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EFSA+Guidance+Document+on+the+risk+assessment+of+plant+protection+products+on+bees+(Apis+mellifera,+Bombus+spp.+and+solitary+bees).">EFSA Guidance Document on the risk assessment of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees).</a> EFSA Journal. 11 (7), 3295 (2013).</li><li>Rundlof, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+coating+with+a+neonicotinoid+insecticide+negatively+affects+wild+bees.">Seed coating with a neonicotinoid insecticide negatively affects wild bees.</a> Nature. 521 (7550), 77-80 (2015).</li><li>Yuan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+and+hazard+assessment+of+six+neonicotinoid+insecticides+on+Osmia+excavata+(hymenoptera:megachilidae).">Toxicity and hazard assessment of six neonicotinoid insecticides on Osmia excavata (hymenoptera:megachilidae).</a> Acta Entomologica Sinica. 61 (8), 950-956 (2018).</li><li>Lin, Z., Meng, F., Zheng, H., Zhou, T., Hu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neonicotinoid+insecticides+on+honeybee+health.">Effects of neonicotinoid insecticides on honeybee health.</a> Acta Entomologica Sinica. 57 (5), 607-615 (2014).</li><li>Gradish, A. E., Scott-Dupree, C. D., Cutler, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Megachile+rotundata+to+insecticides+used+in+wild+blueberry+production+in+Atlantic+Canada.">Susceptibility of Megachile rotundata to insecticides used in wild blueberry production in Atlantic Canada.</a> Journal of Pest Science. 85, 133-140 (2012).</li><li>Hodgson, E. W., Pitts-Singer, T. L., Barbour, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+insect+growth+regulator,+novaluron+on+immature+alfalfa+leafcutting+bees,+Megachile+rotundata.">Effects of the insect growth regulator, novaluron on immature alfalfa leafcutting bees, Megachile rotundata.</a> Journal of Insect Science. 11, 43 (2011).</li><li>Konrad, R., Ferry, N., Gatehouse, A. M. R., Babendreier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+effects+of+oilseed+rape+expressing+oryzacystatin-1+(OC-1)+and+of+purified+insecticidal+proteins+on+larvae+of+the+solitary+bee+Osmia+bicornis.">Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis.</a> PLoS ONE. 3 (7), 2664 (2008).</li><li>Abbott, V. A., Nadeau, J. L., Higo, H. A., Winston, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+and+sublethal+effects+of+imidacloprid+on+Osmia+lignaria+and+clothianidin+on+Megachile+rotundata+(Hymenoptera:+megachilidae).">Lethal and sublethal effects of imidacloprid on Osmia lignaria and clothianidin on Megachile rotundata (Hymenoptera: megachilidae).</a> Journal of Economic Entomology. 101, 784-796 (2008).</li><li>Yan, Z., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublethal+effect+of+abamectin+on+3rd+instar+larvae+of+Prodenia+litura.">Sublethal effect of abamectin on 3rd instar larvae of Prodenia litura.</a> Chinese Journal of Tropical Crops. 32 (10), 1945-1950 (2011).</li><li>Song, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+ecotoxicity+of+insecticides+with+different+modes+of+action+to+Osmia+excavata+(Hymenoptera:+Megachilidae).">Comparative ecotoxicity of insecticides with different modes of action to Osmia excavata (Hymenoptera: Megachilidae).</a> Ecotoxicology and Environmental Safety. 212 (5), 112015 (2021).</li><li>Chen, F. J., Wu, G., Ge, F., Parajulee, M. N., Shrestha, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+elevated+CO2+and+transgenic+Bt+cotton+on+plant+chemistry,+performance,+and+feeding+of+an+insect+herbivore,+the+cotton+bollworm.">Effects of elevated CO2 and transgenic Bt cotton on plant chemistry, performance, and feeding of an insect herbivore, the cotton bollworm.</a> Entomologia Experimentalis Et Applicata. 115 (2), 341-350 (2005).</li><li>Cang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+and+safety+evaluation+of+pesticides+commonly+used+in+strawberry+production+to+bees.">Toxicity and safety evaluation of pesticides commonly used in strawberry production to bees.</a> Zhejiang Agricultural Sciences. (4), 785-787 (2009).</li><li>Cang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+toxicity+and+safety+assessment+of+chiral+fipronil+against+Apis+mellifera+and+Trichogramma+ostriniae.">Acute toxicity and safety assessment of chiral fipronil against Apis mellifera and Trichogramma ostriniae.</a> Ecotoxicology. 7 (3), 326-330 (2012).</li><li>Liu, X., Pan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+to+ensure+pollination+effect+and+cocoon+recovery+rate+of+Osmia+excavata+in+apple+orchard.">Measures to ensure pollination effect and cocoon recovery rate of Osmia excavata in apple orchard.</a> Northwest Horticulture. (3), 20-21 (2017).</li><li>Meikle, W. G., Adamczyk, J. J., Weiss, M., Ross, J., Beren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublethal+concentrations+of+clothianidin+affect+honey+bee+colony+growth+and+hive+CO2+concentration.">Sublethal concentrations of clothianidin affect honey bee colony growth and hive CO2 concentration.</a> Scientific Reports. 11 (1), 4364 (2021).</li><li>Meikle, W. G., Adamczyk, J. J., Weiss, M., Ross, J., Beren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublethal+concentrations+of+clothianidin+affect+honey+bee+colony+behavior+and+interact+with+landscapes+to+affect+colony+growth.">Sublethal concentrations of clothianidin affect honey bee colony behavior and interact with landscapes to affect colony growth.</a> BioRxiv. , (2020).</li><li>Wang, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+effects+of+three+neonicotinoid+pesticides+on+physiology+and+survival+of+honey+bees+(Apis+mellifera+L).">Combination effects of three neonicotinoid pesticides on physiology and survival of honey bees (Apis mellifera L).</a> Journal of Environmental Entomology. 41 (3), 612-618 (2019).</li><li>Kopit, A. M., Pitts-Singer, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routes+of+pesticide+exposure+in+solitary,+cavity-nesting+bees.">Routes of pesticide exposure in solitary, cavity-nesting bees.</a> Environmental Entomology. 47 (3), 499-510 (2018).</li><li>Cheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+oral+toxicity+of+chlorpyrifos+and+imidacloprid+to+adult+honey+bees+(Apis+mellifera+L).">Chronic oral toxicity of chlorpyrifos and imidacloprid to adult honey bees (Apis mellifera L).</a> Asian Journal of Ecotoxicology. 11 (2), 715-719 (2016).</li><li>Li, M., Ma, C., Xiao, L., Li, Z., Su, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chlorpyrifos+on+behavior+response+of+Apis+mellifera+and+Apis+cerana.">Effects of chlorpyrifos on behavior response of Apis mellifera and Apis cerana.</a> Apicultural Science Association of China. , (2016).</li><li>Cresswell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+meta-analysis+of+experiments+testing+the+effects+of+a+neonicotinoid+insecticide+(imidacloprid)+on+honey+bees.">A meta-analysis of experiments testing the effects of a neonicotinoid insecticide (imidacloprid) on honey bees.</a> Ecotoxicology. 20 (1), 149-157 (2011).</li><li>Nauen, R., Ebbinghaus-Kintscher, U., Schmuck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+and+nicotinic+acetylcholine+receptor+interaction+of+imidacloprid+and+its+metabolites+in+Apis+mellifera+(Hymenoptera;+Apidae).">Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera; Apidae).</a> Pest Management Science. 57 (7), 577-586 (2001).</li><li>Colin, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+quantify+and+analyze+the+foraging+activity+of+honey+bees:+relevance+to+the+sublethal+effects+induced+by+systemic+insecticides.">A method to quantify and analyze the foraging activity of honey bees: relevance to the sublethal effects induced by systemic insecticides.</a> Archives of Environmental Contamination and Toxicology. 47 (3), 387-395 (2004).</li><li>Decourtye, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+sublethal+toxicity+of+nine+pesticides+on+olfactory+learning+performances+of+the+honeybee+Apis+mellifera.">Comparative sublethal toxicity of nine pesticides on olfactory learning performances of the honeybee Apis mellifera.</a> Archives of Environmental Contamination &amp; Toxicology. 48 (2), 242-250 (2005).</li><li>Williamson, S. M., Wright, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+multiple+cholinergic+pesticides+impairs+olfactory+learning+and+memory+in+honeybees.">Exposure to multiple cholinergic pesticides impairs olfactory learning and memory in honeybees.</a> Journal of Experimental Biology. 216 (10), 1799-1807 (2013).</li><li>Henry, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+pesticide+decreases+foraging+success+and+survival+in+honey+bees.">A common pesticide decreases foraging success and survival in honey bees.</a> Science. 336 (6079), 348-350 (2012).</li><li>Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+in+homing+flights+in+the+honey+bee+Apis+mellifera+after+a+sublethal+dose+of+neonicotinoid+insecticides.">Reduction in homing flights in the honey bee Apis mellifera after a sublethal dose of neonicotinoid insecticides.</a> Bulletin of Insectology. 66 (1), 1-9 (2013).</li></ol>

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.
<p class="jove_con...

Watch this Scientific Journal Video about Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees at JoVE.com

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

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. ...

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 ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

Defining Hsp33&#039;s Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &amp;#181;s-ms Timescale

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &amp;#945;-Synuclein Monomer at a Time

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...