JoVE Journal > Environment
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Ambiente

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Published: October 09, 2017
doi:
10.3791/54631
, , , , ,
1Vegetable Crop Research Unit,USDA-ARS, 2Department of Entomology,University of Wisconsin-Madison, 3Department of Horticulture,University of Wisconsin-Madison, 4Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, 5Department of Bacteriology,University of Wisconsin-Madison, 6Laboratory of Genetics,Genome Center of Wisconsin, 7DOE Great Lakes Bioenergy Research Center,Wisconsin Energy Institute, 8J.F. Crow Institute for the Study of Evolution,University of Wisconsin-Madison

Summary

黄蜂蜂巢内的微生物联合体丰富和保存蜂幼虫的花粉。使用下一代测序, 连同实验室和实地实验, 这份手稿描述的协议用于测试的假说, 杀菌剂残留改变花粉微生物群, 和群体人口统计, 最终导致殖民地损失.

Abstract

种植者经常在开花时使用杀菌剂来保护农作物免受疾病的危害, 从而使蜜蜂暴露于杀菌剂残留物中。虽然被认为是 “蜜蜂安全”, 但有越来越多的证据表明花粉中的杀菌剂残留物与蜜蜂的下降有关 (蜂蜜和蜜蜂的种类)。虽然这些机制仍然不为人知, 但研究人员推测, 蜜蜂微生物共生的参与。微生物在保存和/或处理花粉方面起着举足轻重的作用, 它们作为幼虫的营养。通过改变微生物群落, 杀菌剂可能破坏这些微生物介导的服务, 从而危害蜜蜂的健康。这份手稿描述了用于研究杀菌剂可能导致蜂群衰退的间接机制的协议。笼子试验揭露蜜蜂对杀菌剂处理过的花朵已经提供了第一个证据, 即杀菌剂造成了本地大黄蜂 (熊凤仙花) 的严重蜂群损失。利用田间相关剂量的杀菌剂, 进行了一系列的实验, 以提供更精细的描述微生物群落动态的杀菌剂暴露花粉。通过下一代测序和基因分析, 研究了花粉微生物群中真菌和细菌组合结构组成的变化。本文所开发的实验, 旨在提供一个机械的了解如何杀菌剂影响花粉的微生物-规定。最终, 这些发现应该揭示出杀菌剂可能导致蜂群衰退的间接途径。

Introduction

管理和野生蜜蜂物种正经历着广泛的下降, 对自然和农业系统的主要影响1。尽管共同努力, 以了解这一问题的原因, 驱动蜂蜜蜂下降的因素仍然没有很好地理解2,3,4。对于某些种类的野生, 本地蜜蜂, 情况已经变得可怕5,6。如果蜜蜂种群在与工业农业相交时不能持续, 其种群数量将继续下降, 而需要传粉者的作物 (全球总产量的 35%7) 将会减少收成。

虽然许多潜在的因素, 如农药暴露, 疾病和栖息地损失1,4,8,9,10都牵连到蜜蜂的衰落,在农业系统内部或附近, 这些压力源对本地蜜蜂健康的交互作用的了解相对较少。许多目前的研究工作继续侧重于杀虫剂, (例如, 烟碱11,12), 尽管过去的研究表明, 杀菌剂也可能通过损害记忆形成而在蜜蜂减少中发挥作用,嗅觉接收13, 巢识别14, 酶活性和代谢功能15,16,17。在全球范围内, 杀菌剂继续应用于开花的农作物。最近的研究表明, 蜜蜂通常会将杀菌剂残留物带回蜂巢18, 事实上, 研究显示, 有很大比例的被测试的蜂箱含有杀菌剂残留物19,20。进一步的工作表明, 杀菌剂残留与蜜蜂幼虫死亡率的高比率21,22,23和在殖民地内存在 “被埋葬的花粉”, 虽然无毒,缺乏微生物活性, 营养受损24。尽管杀菌剂长期以来被认为是 “蜜蜂安全” 的事实, 但现在有证据表明, 单独接触杀菌剂可能会在本地大黄蜂物种中造成严重的蜂群损失,熊凤仙花25。

为了建立杀菌剂暴露与群体死亡率之间的因果关系, 需要确定这些化学品的作案手法。如土壤26, 沉积物27和水生环境28, 通过瞄准真菌, 杀菌剂最有可能改变真菌丰度和花粉中的多样性-规定, 从而调用一个主要的社区转移,可能强烈偏爱细菌。没有真菌竞争者或拮抗剂, 某些致病细菌可以增殖相对不受控制, 促进花粉变质的规定。过去的研究表明, 微生物, 特别是酵母菌和丝状真菌, 作为营养共生的蜜蜂29,30,31, 防止寄生虫和病原体32 ,33, 并提供花粉存储的长期保存。因此, 杀菌剂可能会通过破坏微生物群落来间接地伤害幼蜂, 因为它们需要提供这些服务和/或增加对机会性病原体和寄生虫的易感性12。随着对粮食生产的需求日益增加, 世界各地的农作物每年都在开花时喷洒杀菌剂, 这突出了需要了解这种杀菌剂引起的影响的严重性。

到目前为止, 与本地蜜蜂微生物生态学有关的主要知识鸿沟可以由以下问题来表示: 在何种程度上杀菌剂改变蜂花粉中的微生物群落-规定？什么是消耗花粉与一个深刻改变的微生物群落的下游影响？与这些生态学上相关的问题, 实验开发了以显露的主要目标 1) 仅杀菌剂残余可能导致严重殖民地衰落在本地蜂种;2) 花粉中微生物群落的程度–规定被杀菌剂改变, 3) 蜜蜂的健康受到严重改变的微生物群落的影响。实验目标的定义是通过结合实验室和实地实验来解决上述问题。本研究利用先进的基因和分子技术和传统的野外观察方法, 将杀菌剂对蜜蜂健康的潜在影响结合起来。

这项研究的第一个目的是证明单独的杀菌剂暴露会在本地蜜蜂物种中造成巨大的群体损失。一项涉及大型田间网箱的研究被用来调查杀菌剂暴露对熊凤仙花群体生长的影响, 这是一个无处不在的、丰富的美国本土蜜蜂 (图 1、图 2、图 3)。据推测, 与医护蜂房相比, 杀菌剂治疗的蜂箱会呈现较低的适应性和不典型的人口学。从这个实验中获得的数据证实了这一假说, 证明花粉中的杀菌剂残留物可能是造成本地大黄蜂物种严重群体损失的唯一原因25。本研究的第二个目的是探讨花粉微生物对杀菌剂暴露的反应。据推测, 花粉中微生物的群落组成–暴露在杀菌剂中的成分会与未经处理的花粉不同。虽然真菌的丰度和多样性预计会显著下降, 但细菌和/或单一的优势真菌物种在没有其他竞争真菌的情况下可能会无节制地生长。通过一系列的体内试验, 将利用基因分析微生物群落组成的这些变化。

Protocol

1. 使用田间网箱试验检查杀菌剂照射对蜂群成功的影响 在燕麦种植场中设置了十网笼。在每个笼子周围挖一条沟, 把网笼的四条边挖到地上, 以确保蜜蜂不能逃脱。将笼子与盆栽, 开花的植物, 是已知的吸引蜜蜂 ( 如 荞麦, 琉璃, alyssum, 宇宙和向日葵) ( 图 2 )。 用一个单托盘 (36 厘米 x 42 厘米) 的盛开三叶草补充笼子。集群花卉资源在笼子的一个角落里, ?…

Representative Results

野外网箱研究: 从笼型实验中获得的数据表明, 大黄蜂的蜂群对杀菌剂暴露有明显的反应。杀菌剂处理的蜂箱比控制单元 (43.2 ± 11.2, F19= 6.8, p = 0.03) (图 4) 产生的工人数量显著减少 (12.2 ± 3.8, 平均± SE)。此外, 杀菌剂处理的蜂箱的蜂生物量 (0.91 克± 0.15) 明显低于对照蜂房 (2.36 克± …

Discussion

对杀菌剂对蜜蜂健康的影响的调查仍然是害虫管理战略的一个理解方面。我们的研究目的是通过一套互补的技术来弥合这一知识鸿沟, 这一方法明确地隔离了推动蜜蜂衰落的潜在因素。下面详细介绍这些实验的规划、基本原理和渲染。

重要的是要确保不允许蜜蜂逃脱笼实验的网格, 因为这将损害人口学分析。同样重要的是, 人工巢有足够的绝缘, 以防止雨水和阳光直射。应注意…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者 (s) 感谢威斯康星大学生物技术中心 DNA 测序设施, 提供放大和测序设施和服务, 凯特琳卡尔森, 珍妮弗诀窍, 杰克奥托, 和最大 Haase 提供技术援助分子分析。这项工作得到了农业部-农业研究处拨款资助 (目前的研究信息系统 #3655-21220-001)。国家科学基金会提供进一步支助 (赠款 No。DEB-1442148), 美国能源部大湖生物能源研究中心 (能源部 DE-FC02-07ER64494), 和美国农业部国家食品和农业研究所 (孵化项目 1003258)。C.T.H. 是一个皮尤学者在生物医学科学和阿尔弗雷德克劳斯·特普费尔教员研究员, 支持的皮尤慈善信托基金和亚历山大·冯洪堡基金会分别。

Materials

Natupol Beehive Koppert USRESM1 16 hives
Propiconazole 14.3 Quali-Ppro 60207-90-1 Propiconazole 14.3%
Abound Syngenta 4033540 Azoxystrobin 22.9%
Chlorothalonil Syngenta 3452 Fungicide used for trials
Pollen granules Bee rescued B004D5650C 3X 16oz bottles, pollen for trials
Bacterial strains for inoculation Currie Lab
Yeast strains for inoculation Hittinger lab
Primer pairs UW Biotech Center
DNA Isolation Kit Mo Bio 12830-50 Commercial DNA isolation kit
Qubit dsDNA HS Assay Kit Thermo Fisher Q32851 DNA quantification tool
Select Master Mix for CFX Thermo Fisher 4472952 Used to perform real-time PCR using SYBR GreenER dye.
Real-Time PCR Detection System Bio Rad 1855196 Instrument used for PCR amplification
PCR Clean-Up Kit, Axygen 10159-696 Used for efficient removal of unincorporated dNTPs, salts and enzymes
DNA 1000 Kit Agilent 5067-1504 Used for sizing and analysis of DNA fragments
MiSeq Sequencer Illumina Used for next-generation sequencing
Assorted glassware (beaker, flasks, pipettes, test tubes, repietters) VWR
DOWNLOAD MATERIALS LIST

Referências

  1. Potts, S. G., Biesmeijer, J. C., Kremen, C., Neumann, P., Schweiger, O., Kunin, W. E. Global pollinator declines: Trends, impacts and drivers. Trends Ecol Evolut. 25 (6), 345-353 (2010).
  2. Vanengelsdorp, D., Meixner, M. D. A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them. J Invertebr Pathol. 103, S80-S95 (2010).
  3. Ellis, J. D., Evans, J. D., Pettis, J. Colony losses, managed colony population decline, and Colony Collapse Disorder in the United States. J. Apic. Res. 49 (1), 134-136 (2010).
  4. Vanbergen, A. J. Threats to an ecosystem service: pressures on pollinators. Front Ecol Environ. 11 (5), 251-259 (2013).
  5. Cameron, S. A., et al. Patterns of widespread decline in North American bumble bees. Proc. Natl. Acad. Sci. U.S.A. 108 (2), 662-667 (2011).
  6. Szabo, N. D., Colla, S. R., Wagner, D. L., Gall, L. F., Kerr, J. T. Do pathogen spillover, pesticide use, or habitat loss explain recent North American bumblebee declines?. Conser Lett. 5 (3), 232-239 (2012).
  7. Klein, A. -. M., et al. Importance of pollinators in changing landscapes for world crops. Proc R Soc Lond [Biol]. 274 (1608), 303-313 (2007).
  8. Sánchez-Bayo, F., Goulson, D., Pennacchio, F., Nazzi, F., Goka, K., Desneux, N. Are bee diseases linked to pesticides? – A brief review. Environ Int. 89, 7-11 (2016).
  9. Kwong, W. K., Moran, N. A. Gut microbial communities of social bees. Nature Rev. Microbiol. 14 (6), 374-384 (2016).
  10. Engel, P., et al. The Bee Microbiome: Impact on Bee Health and Model for Evolution and Ecology of Host-Microbe Interactions. mBio. 7 (2), e02164-e02115 (2016).
  11. Henry, M., et al. A common pesticide decreases foraging success and survival in honey bees. Science. 336 (6079), 348-350 (2012).
  12. Pettis, J. S., vanEngelsdorp, D., Johnson, J., Dively, G. Pesticide exposure in honey bees results in increased levels of the gut pathogen Nosema. Die Naturwissenschaften. 99 (2), 153-158 (2012).
  13. Williamson, S. M., Wright, G. A. Exposure to multiple cholinergic pesticides impairs olfactory learning and memory in honeybees. J. Exp. Biol. 216 (10), 1799-1807 (2013).
  14. Artz, D. R., Pitts-Singer, T. L. Effects of fungicide and adjuvant sprays on nesting behavior in two managed solitary bees, Osmia lignaria and Megachile rotundata. PLoS ONE. 10 (8), (2015).
  15. Johnson, R. M., Wen, Z., Schuler, M. A., Berenbaum, M. R. Mediation of Pyrethroid Insecticide Toxicity to Honey Bees (Hymenoptera: Apidae) by Cytochrome P450 Monooxygenases. J. Econ. Entomol. 99 (994), 1046-1050 (2006).
  16. Pilling, E. D., Bromleychallenor, K. A. C., Walker, C. H., Jepson, P. C. Mechanism of synergism between the pyrethroid insecticide lambda-cyhalothrin and the imidazole fungicide prochloraz, in the honeybee (Apis mellifera L). Pest Biochem Physiol. 51 (1), 1-11 (1995).
  17. Iwasa, T., Motoyama, N., Ambrose, J. T., Roe, R. M. Mechanism for the Differential Toxicity of Neonicotinoid Insecticides in the Honey Bee Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera. Crop Protection. , (2016).
  18. Mullin, C. A., et al. High levels of miticides and agrochemicals in North American apiaries: implications for honey bee health. PLoS One. 5 (3), e9754 (2010).
  19. Pettis, J. S., Lichtenberg, E. M., Andree, M., Stitzinger, J., Rose, R., Vanengelsdorp, D. Crop pollination exposes honey bees to pesticides which alters their susceptibility to the gut pathogen Nosema ceranae. PLoS One. 8 (7), e70182 (2013).
  20. David, A., et al. Widespread contamination of wildflower and bee-collected pollen with complex mixtures of neonicotinoids and fungicides commonly applied to crops. Environ Int. 88, 169-178 (2016).
  21. Zhu, W., Schmehl, D. R., Mullin, C. A., Frazier, J. L. Four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae. PLoS One. 9 (1), e77547 (2014).
  22. Simon-Delso, N., Martin, G. S., Bruneau, E., Minsart, L. A., Mouret, C., Hautier, L. Honeybee colony disorder in crop areas: The role of pesticides and viruses. PLoS ONE. 9 (7), (2014).
  23. Park, M. G., Blitzer, E. J., Gibbs, J., Losey, J. E., Danforth, B. N. Negative effects of pesticides on wild bee communities can be buffered by landscape context. Proc R Soc Lond [Biol]. 282 (1809), (2015).
  24. van Engelsdorp, D., et al. “Entombed Pollen”: A new condition in honey bee colonies associated with increased risk of colony mortality. J Invertebr Pathol. 101 (2), 147-149 (2009).
  25. Bernauer, O. M., Gaines-Day, H. R., Steffan, S. A. Colonies of bumble bees (Bombus impatiens) produce fewer workers, less bee biomass, and have smaller mother queens following fungicide exposure. Insects. 6 (2), 478-488 (2015).
  26. Tu, C. M. Effect of fungicides, captafol and chlorothalonil, on microbial and enzymatic activities in mineral soil. J Environ Sci Health B. 28 (B28), 67-80 (1993).
  27. Huang, C. -. Y., Ho, C. -. H., Lin, C. -. J., Lo, C. -. C. Exposure effect of fungicide kasugamycin on bacterial community in natural river sediment. J Environ Sci Health B. 45 (5), 485-491 (2010).
  28. Artigas, J., et al. Effects of the fungicide tebuconazole on microbial capacities for litter breakdown in streams. Aquat. Toxicol. 122, 197-205 (2012).
  29. Goerzen, D. W. Microflora associated with the alfalfa leafcutting bee, Megachile rotundata (Fab) (Hymenoptera: Megachilidae) in Saskatchewan, Canada. Apidologie. 22 (5), 553-561 (1991).
  30. Anderson, K. E., Sheehan, T. H., Eckholm, B. J., Mott, B. M., DeGrandi-Hoffman, G. An emerging paradigm of colony health: Microbial balance of the honey bee and hive (Apis mellifera). Insectes Sociaux. 58 (4), 431-444 (2011).
  31. Crotti, E., et al. Microbial symbionts of honeybees: a promising tool to improve honeybee health. N. Biotechnol. 30 (6), 716-722 (2013).
  32. Koch, H., Schmid-Hempel, P. Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. Proc. Natl. Acad. Sci. U.S.A. 108 (48), 19288-19292 (2011).
  33. Anderson, K. E., et al. Microbial ecology of the hive and pollination landscape: bacterial associates from floral nectar, the alimentary tract and stored food of honey bees (Apis mellifera). PloS One. 8 (12), e83125 (2013).
  34. Evans, E. C., Spivak, M. Effects of Honey Bee (Hymenoptera: Apidae) and Bumble Bee (Hymenoptera: Apidae) Presence on Cranberry (Ericales: Ericaceae) Pollination. J Econ Entomol. 99 (3), 614-620 (2006).
  35. Goulson, D., et al. Can alloethism in workers of the bumblebee, Bombus terrestris, be explained in terms of foraging efficiency?. Anim. Behav. 64 (1), 123-130 (2002).
  36. User Guide: Qubit dsDNA HS Assay Kits. Available from: https://tools.thermofisher.com/content/sfs/manuals/Qubit_dsDNA_HS_Assay_UG.pdf (2010)
  37. Khadempour, L., LeMay, V., Jack, D., Bohlmann, J., Breuil, C. The Relative Abundance of Mountain Pine Beetle Fungal Associates Through the Beetle Life Cycle in Pine Trees. Microbial Ecol. 64 (4), 909-917 (2012).
  38. Dorn-In, S., Hölzel, C. S., Janke, T., Schwaiger, K., Balsliemke, J., Bauer, J. PCR-SSCP-based reconstruction of the original fungal flora of heat-processed meat products. Int J Food Microbiol. 162 (1), 71-81 (2013).
  39. Klindworth, A., et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41 (1), (2013).
  40. White, T., Bruns, T., Lee, S., Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. 18 (1), 315-322 (1990).
  41. Kõljalg, U., et al. Towards a unified paradigm for sequence-based identification of fungi. Mol Ecol. 22 (21), 5271-5277 (2013).
  42. Wang, Q., Garrity, G. M., Tiedje, J. M., Cole, J. R. Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy. Appl. Environ. Microbiol. 73 (16), 5261-5267 (2007).
  43. Team, R. C. . R: A language and environment for statistical computing [Computer software]. , (2015).
  44. Kaltenpoth, M., Engl, T. Defensive microbial symbionts in Hymenoptera. Funct Ecol. 28 (2), 315-327 (2014).
  45. Gerth, M., Saeed, A., White, J. A., Bleidorn, C. Extensive screen for bacterial endosymbionts reveals taxon-specific distribution patterns among bees (Hymenoptera, Anthophila). FEMS Microbiol Ecol. 91 (6), (2015).
  46. Smith, C. J., Osborn, A. M. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 67 (1), 6-20 (2009).
  47. Kim, M., Morrison, M., Yu, Z. Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes. J Microbiol Methods. 84 (1), 81-87 (2011).
  48. DeSantis, T. Z., et al. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl. Environ. Microbiol. 72 (7), 5069-5072 (2006).
  49. Langille, M. G. I., et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nature Biotechnol. 31 (9), 814-821 (2013).
  50. Malik, S., Beer, M., Megharaj, M., Naidu, R. The use of molecular techniques to characterize the microbial communities in contaminated soil and water. Environ Int. 34 (2), 265-276 (2008).
  51. Ladurner, E., Bosch, J., Kemp, W. P., Maini, S. Assessing delayed and acute toxicity of five formulated fungicides to Osmia lignaria and Apis mellifera. Apidologie. 36 (3), 449-460 (2005).
  52. Huntzinger, C. I., James, R. R., Bosch, J., Kemp, W. P. Fungicide Tests on Adult Alfalfa Leafcutting Bees (Hymenoptera: Megachilidae). J Econ Entomol. 101 (4), 1088-1094 (2008).
  53. Gradish, A. E., Scott-Dupree, C. D., Shipp, L., Harris, C. R., Ferguson, G. Effect of reduced risk pesticides for use in greenhouse vegetable production on Bombus impatiens (Hymenoptera: Apidae). Pest Manag. Sci. 66 (2), 142-146 (2010).
  54. Calderone, N. W. Insect pollinated crops, insect pollinators and US agriculture: trend analysis of aggregate data for the period 1992-2009. PloS One. 7 (5), e37235 (2012).
  55. Ollerton, J., Winfree, R., Tarrant, S. How many flowering plants are pollinated by animals?. Oikos. 120 (3), 321-326 (2011).

Tags

Keywords: BumblebeesFungicidesMicrobiomeColony HealthPollinatorField ExperimentMetagenomicsEmpirical Techniques
check_url/pt/54631?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Steffan, S. A., Dharampal, P. S., Diaz-Garcia, L., Currie, C. R., Zalapa, J., Hittinger, C. T. Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health. J. Vis. Exp. (128), e54631, doi:10.3791/54631 (2017).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image