这项研究是由格兰特105AS-13.2.3-BQ-B1从动物局动植物卫生检验检疫，农业委员会，行政院和格兰特103-2313-B-197-002-MY3从支持部科学技术部（MOST）。

1.准备<ol><li>使1升50％的糖浆。溶解1公斤蔗糖在1L的DDH 2 O. </li><li>准备在BLD吡丙醚（PPN）的解决方案。使1.1升的10,000ppm的PPN原液，并稀释于无菌的DDH 2 O储存于4℃1 L 100毫升PPN溶液。 </li><li>稀释在BLD的PPN原液至0.1，1，10和100mg / kg的（PPM）最终浓度为下述实验。 </li><li>让PPN糖浆（为殖民地的水平）。稀释PPN股票的50％糖浆10和100ppm的最终浓度为以下实验。 </li><li>蜜蜂饲养。 注:在这里，实验地点是国立宜兰大学（NIU）养蜂场，艺兰市台; （GPS坐标:N24.747278，E121.746200）。 <ol><li>检查蜜蜂（ 意大利蜜蜂 L.）菌落每周食物量并在必要时（蜂蜜存储区是空的）用1升50％的糖浆进料。定义一个健康集落在每个集落，用大号产卵通常蜜蜂梳子的9个帧。 </li></ol></li><li>制备100毫升基本幼虫饮食（BLD）的。溶解6％D-葡萄糖，6％果糖，和1％的酵母提取物在无菌蒸馏水去离子水（DDH 2 O）和补充有50％蜂王浆。储存在4°C，但不超过3天。 注意:预温热至35℃实验前并在3天内使用。 </li></ol> 2. 体内送入方法注意: 体内喂养方法已经从汉利等改性。 20 <ol><li> 蜜蜂幼虫的选择和标签。 <ol><li>插入一个大号排阻到健康群体的9个帧分割成4个帧和5帧，同时限制王后到4帧区间。留在4帧部分的至少一个空帧产卵。 </li><li>蜂王产卵1天后，检查蛋的外观的帧并保持蜂箱72个小时（ 第 4天）内的鸡蛋直到1天老幼虫孵化。采用包含1日龄工人幼虫（24小时内画影线）出用手从测试配置单元中的帧中的一个和从与蜂刷框架拆除蜜蜂工人。 </li><li>覆盖有透明的滑动件（尺寸=长×宽×厚=29.7毫米×21毫米×0.1毫米）所述框架和钉对帧与图钉（ 图1A）的边缘上的透明幻灯片。 </li><li>对于每个处理，随机选择50一天的老幼虫（ 第 4天），并通过使用在透明滑动永久记号笔标记每个育雏细胞（ 图1A和1B）。 注意:通过使用永久记号笔，以避免不同的处理之间的混乱写帧和透明幻灯片上每个处理的信息，以及。除去标记的幻灯片，并保持用于体内&lt;/ em&gt;的进料和观察。 </li></ol></li><li> 体内喂养。 <ol><li>不同浓度的PPN-BLD（0.1，1，10和100ppm）的每个标记的育雏细胞，通过移液在1天，2和3加以分别为10μL，10μL和20μL，根据的吸气量幼虫龄。 BLD（无PPN）相同量添加到对照组。由此，PPN-BLD的每个标记的细胞育雏总剂量累积到4，40，400，和4000微克。 注意:通过标记透明载玻片识别标记的育雏细胞和喂食后除去标记透明幻灯片。使用新鲜BLD供给由工蜂防止育雏细胞的清洗。 </li><li>返回PPN处理帧到初始菌落作进一步的观察。 注:每个处理有四个殖民地4个生物重复。 </li></ol></li><li> 7天处理的幼虫观察。 <ol><li>观察日ËPPN处理幼虫，标记的透明滑动返回到帧以记录死亡率和封盖率标记的育雏细胞。 </li></ol></li><li> 13天治疗蛹羽化和利率的观察。 <ol><li>删除封顶育雏细胞的蜂蜡。 </li><li>把软尖镊子进入育雏细胞和夹紧蛹非常轻微地再取出来蛹轻轻。 </li><li>与实验室组织的双层下方各孔转移到蛹24孔组织培养板中。记录蛹转移过程中的损伤和死亡。 </li><li>保持在培养箱中在24孔组织培养板在34℃和70％相对湿度，直至出现（ 约 8天）。 </li><li>观察并记录蛹和出现的蜜蜂。 </li></ol></li><li> 统计 <ol><li>计算所记录的数据并呈现为平均值±SD </li><li>分析由SA使用方差分析（ANOVA）分析数据S和用最少的差异显著（LSD）检验，以分析不同处理两种手段的区别。定义为P值&lt;0.05具有统计学显著。不同的字母在表的同一列显示，通过统计分析显著的效果。 </li></ol></li></ol> 3. PPN的毒性在殖民地水平蜂箱<ol><li>成立蜜蜂群体。 <ol><li>插入一个大号排除器垂直于健康的群体的9个帧分割成4个帧（A部分）和5帧（B部分），同时限制王后到4帧区间。在用于产卵的4帧部分保留至少一个空帧。 注:换个女王逃生的女皇部分的顶部，以防止女王从零部件之间移动。 </li><li>女王铺设1天的鸡蛋后，检查帧鸡蛋的外观。用手取含有鸡蛋从部分A进行适当的帧并取出蜜蜂工人从与蜂刷帧。 </li><li>盖上透明滑动框架和钉透明幻灯片与图钉框架的边缘。 </li><li>随机地选择含有鸡蛋100个育雏细胞，并通过使用在透明幻灯片永久性记号笔标记每个育雏细胞。分配这些100个育雏细胞作为组1.收件使用永久标记，以避免不同的处理之间的混淆的帧和透明幻灯片上每个处理的信息。 </li><li>返回标记的帧部分用于3天，然后王后转移到B部分产卵。 </li><li> 1天后，检查部分B的帧中，选择一个合适的帧和标签如步骤3.1.4所述的含有鸡蛋100个育雏细胞。分配这些100个育雏细胞作为第2组。 </li><li>返回标记的帧到B部分3天，然后转移王后A部分产卵。 </li><li>反复A部分和B部分之间的女王交换6次以上和数值分配团，分别（ 图2）。应该有共9组。 </li></ol></li><li> ŤREAT蜂蜜蜂群与在第13天PPN糖浆（ 图2）。 <ol><li>加入1升含有在塑料蜂料箱10或100ppm（宽×长×高= 20厘米×30厘米×3.5厘米）的50％PPN糖浆和把盒子上在实验菌落的帧的顶部。 注:第1组没有13天内收到的PPN为育雏细胞已被查封。 </li><li>饲料对照组如步骤2.2中所述的1L 50％糖浆（无PPN）。 </li></ol></li><li>共1日龄幼体和记录作为每一组在第5天（ 图2）100个标记的育雏细胞的蛋孵化率。蜜蜂卵一般需要3天才能孵化成0天幼虫;因此，检查和标签100个鸡蛋含有育雏细胞在第1天，并在第5天计数1天老幼虫的数目对于每个组，以获得孵化率百分比。 </li><li> C指望。2-15封端的育雏细胞和记录作为每一组第11天（ 图2）100个标记的育雏细胞的幼虫封盖率。 6〜7日龄幼虫窝细胞会通过蜜蜂工人蜜蜂蜡幼虫化蛹为上限。 </li><li>观察蛹成熟和记录标记100个育雏细胞的羽化率对于每个组17天（ 图2）。 <ol><li>删除封顶育雏细胞的蜂蜡，并采取了蛹轻轻用软尖镊子。转移蛹于24孔组织培养板用实验室组织的双层之下每个孔中。 </li><li>保持在培养箱中在24孔组织培养板在34℃和70％RH，直到出现。 </li><li>观察并记录蛹和出现蜜蜂对于每个组，直到第9组（49实验天）。 注:每个处理有四个生物重复。 </li></ol></li><li>统计<ol><li>计算所记录的数据并呈现为平均±标准差。 </li><li>分析对治疗之间的显著差异（ 例如为0ppm / 10 ppm时，为10 ppm / 100ppm的和为0ppm / 100ppm的）每个组中通过使用学生双尾t检验。定义如果P值&lt;0.05为有统计学显著。 </li></ol></li></ol>

<ol>
	<li>Ruffi nengo, S. 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=Integrated+pest+management+to+control+Varroa+destructor+and+its+implications+to+Apis+mellifera+colonies.">Integrated pest management to control Varroa destructor and its implications to Apis mellifera colonies.</a> Zootecnia Trop. 32 (2), 149-168 (2014).</li><li>Mullin, C. 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=High+levels+of+miticides+and+agrochemicals+in+North+American+apiaries,+implications+for+honey+bee+health.">High levels of miticides and agrochemicals in North American apiaries, implications for honey bee health.</a> PLoS One. 5, e9754 (2010).</li><li>Lu, C. A., Chang, C. H., Tao, L., Chen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributions+of+neonicotinoid+insecticides+in+the+Commonwealth+of+Massachusetts,+a+temporal+and+spatial+variation+analysis+for+pollen+and+honey+samples.">Distributions of neonicotinoid insecticides in the Commonwealth of Massachusetts, a temporal and spatial variation analysis for pollen and honey samples.</a> Environ. Chem. 13, 4-11 (2016).</li><li>Tsai, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+coupling+the+pesticide+use+reduction+with+environmental+policy+for+agricultural+sustainability+in+Taiwan.">Analysis of coupling the pesticide use reduction with environmental policy for agricultural sustainability in Taiwan.</a> Environ. &amp; Pollut. 2, 59-65 (2013).</li><li>Weng, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+market+status+and+development+trend+(in+Chinese).">Pesticide market status and development trend (in Chinese).</a> PRIDE. , (2016).</li><li>Kevan, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollinators+as+bioindicators+of+the+state+of+the+environment,+species,+activity+and+diversity.">Pollinators as bioindicators of the state of the environment, species, activity and diversity.</a> Agric. Ecosys. Environ. 74 (1-3), 373-393 (1999).</li><li>Kevan, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forest+application+of+the+insecticide+fenitrothion+and+its+effect+on+wild+bee+pollinators+(Hymenoptera:+Apoidea)+of+lowbush+blueberries+(Vaccinium+SPP.)+in+Southern+New+Brunswick,+Canada.">Forest application of the insecticide fenitrothion and its effect on wild bee pollinators (Hymenoptera: Apoidea) of lowbush blueberries (Vaccinium SPP.) in Southern New Brunswick, Canada.</a> Biol. Conserv. 7, 301-309 (1975).</li><li>Crane, E., Walker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The impact of pest management on bees and pollination. , (1983).</li><li>Haouar, M., Decormis, L., Rey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluvalinate+applied+to+flowering+apple+trees-contamination+of+honey-gathering+bees+and+hive+products.">Fluvalinate applied to flowering apple trees-contamination of honey-gathering bees and hive products.</a> Agronomie. 10 (2), 133-137 (1990).</li><li>Chauzat, M. P., 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+survey+of+pesticide+residues+in+pollen+loads+collected+by+honey+bees+in+France.">A survey of pesticide residues in pollen loads collected by honey bees in France.</a> J. Econ. Entomol. 99 (2), 253-262 (2006).</li><li>Bonzini, S., Tremolada, P., Bernardinelli, I., Colombo, M., Vighi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+pesticide+fate+in+the+hive+(part+1),+experimentally+determined+&#964;-fluvalinate+residues+in+bees,+honey+and+wax.">Predicting pesticide fate in the hive (part 1), experimentally determined &#964;-fluvalinate residues in bees, honey and wax.</a> Apidologie. 42 (3), 378 (2011).</li><li>Sanchez-Bayo, F., Goka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+residues+and+bees-a+risk+assessment.">Pesticide residues and bees-a risk assessment.</a> PLoS One. 9 (4), e94482 (2014).</li><li>Johnson, R. M., Ellis, M. D., Mullin, C. A., Frazier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticides+and+honey+bee+toxicity-USA.">Pesticides and honey bee toxicity-USA.</a> Apidologie. 41, 312-331 (2010).</li><li>Yang, E. C., Chang, H. C., Wu, W. Y., Chen, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+olfactory+associative+behavior+of+honeybee+workers+due+to+contamination+of+imidacloprid+in+the+larval+stage.">Impaired olfactory associative behavior of honeybee workers due to contamination of imidacloprid in the larval stage.</a> PLoS One. 7, e49472 (2012).</li><li>Urlacher, 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=Measurements+of+chlorpyrifos+levels+in+forager+bees+and+comparison+with+levels+that+disrupt+honey+bee+odor-mediated+learning+under+laboratory+conditions.">Measurements of chlorpyrifos levels in forager bees and comparison with levels that disrupt honey bee odor-mediated learning under laboratory conditions.</a> J. Chem. Ecol. 42 (2), 127-138 (2016).</li><li>Chen, Y. W., Wu, P. S., Yang, E. C., Nai, Y. S., Huang, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+pyriproxyfen+on+the+development+of+honey+bee+(Apis+mellifera+L.)+colony+in+field.">The impact of pyriproxyfen on the development of honey bee (Apis mellifera L.) colony in field.</a> J. Asia Pac. Entomol. 19 (3), 589-594 (2016).</li><li>Dennehy, T. J., DrGain, B. A., Harpold, V. S., Brink, S. A., Nichols, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Whitefly Resistance to Insecticides in Arizona: 2002 and 2003 Results. , 1926-1938 (2004).</li><li>Yang, E. C., Wu, P. S., Chang, H. C., Chen, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sub-lethal+dosages+of+insecticides+on+honey+bee+behavior+and+physiology.">Effect of sub-lethal dosages of insecticides on honey bee behavior and physiology.</a> , (2010).</li><li>Fourrier, J., 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=Larval+exposure+to+the+juvenile+hormone+analog+pyriproxyfen+disrupts+acceptance+of+and+social+behavior+performance+in+adult+honey+bees.">Larval exposure to the juvenile hormone analog pyriproxyfen disrupts acceptance of and social behavior performance in adult honey bees.</a> PLoS One. 10, e0132985 (2015).</li><li>Hanley, A. V., Huang, Z. Y., Pett, W. L. <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+dietary+transgenic+Bt+corn+pollen+on+larvae+of+Apis+mellifera+and+Galleria+mellonella.">Effects of dietary transgenic Bt corn pollen on larvae of Apis mellifera and Galleria mellonella.</a> J. Apicult.Res. 42 (4), 77-81 (2003).</li><li>Kaftanoglu, O., Linksvayer, T. A., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+honey+bees,+apis+mellifera,+in+vitro+1,+effects+of+sugar+concentrations+on+survival+and+development.">Rearing honey bees, apis mellifera, in vitro 1, effects of sugar concentrations on survival and development.</a> J. Insect Sci. 11 (96), 1-10 (2011).</li><li>Vandenberg, J. D., Shimanuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+rearing+worker+honey+bees+in+the+laboratory.">Technique for rearing worker honey bees in the laboratory.</a> J. Apicult. Res. 26 (2), 90-97 (1987).</li><li>Peng, Y. S. C., Mussen, E., Fong, A., Montague, M. A., Tyler, T. <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+chlortetracycline+on+honey+bee+worker+larvae+reared+in+vitro.">Effects of chlortetracycline on honey bee worker larvae reared in vitro.</a> J. Invertebr.Pathol. 60 (2), 127-133 (1992).</li><li>Bitondi, M. M., Mora, I. M., Simoes, Z. L., Figueiredo, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Apis+mellifera+pupal+melanization+program+is+affected+by+treatment+with+a+juvenile+hormone+analogue.">The Apis mellifera pupal melanization program is affected by treatment with a juvenile hormone analogue.</a> J. Insect Physiol. 44 (5-6), 499-507 (1998).</li><li>Zufelato, M. S., Bitondi, M. M., Simoes, Z. L., Hartfelder, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+juvenile+hormone+analog+pyriproxyfen+affects+ecdysteroid-dependent+cuticle+melanization+and+shifts+the+pupal+ecdysteroid+peak+in+the+honey+bee+(Apis+mellifera).">The juvenile hormone analog pyriproxyfen affects ecdysteroid-dependent cuticle melanization and shifts the pupal ecdysteroid peak in the honey bee (Apis mellifera).</a> Arthropod Struct. Dev. 29 (2), 111-119 (2000).</li><li>Santos, A. E., Bitondi, M. M., Simoes, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone-dependent+protein+patterns+in+integument+and+cuticular+pigmentation+in+Apis+mellifera+during+pharate+adult+development.">Hormone-dependent protein patterns in integument and cuticular pigmentation in Apis mellifera during pharate adult development.</a> J. Insect Physiol. 47 (11), 1275-1282 (2001).</li><li>Brouwers, E. V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose/Fructose+ratio+in+the+food+of+honeybee+larvae+during+caste+differentiation.">Glucose/Fructose ratio in the food of honeybee larvae during caste differentiation.</a> J. Apicult.Res. 23 (2), 94-101 (1984).</li><li>Howe, S. R., Dimick, P. S., Benton, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+freshly+harvested+and+commercial+royal+jelly.">Composition of freshly harvested and commercial royal jelly.</a> J. Apicult. Res. 24 (1), 52-61 (1985).</li></ol>

作者宣称，他们没有竞争的经济利益。

王后限制产蛋方法和王后交换法是用于获得本协议内的现场测试设置蜜蜂基团的关键步骤。王后限制产蛋方法允许蜜蜂的生命周期的同步。因此，研究人员可以选择相同的年龄与不同剂量农药的治疗1日龄幼虫。对于女王交换法，王后被部分A（4帧）和B（5帧）以获得蜜蜂的不同发育阶段的现场测试，以评估农药和农药残留的影响之间进行交换。此外，大量的选择育雏细胞被记录在使用透明幻灯片?...

农药在环境中的存在是最严重的问题之一是影响一个蜜蜂1，2，3的使用寿命。一些研究表明农药残留量的蜂蜜蜂群和蜜蜂产品的共同存在。在台湾，农药的平均应用为11-12公斤/公顷，每年（2005至13年）。农药在台湾使用量是比欧盟国家的高，以及拉丁美洲国家的4，5。换句话说，在养蜂环境正在遭受严重的农药压力，特别是在台湾，可能在其他国家。 在西方蜜蜂是农业系统6的主要传粉昆虫之一，它也产生有价值的产品，如蜂蜜。然而，蜜蜂是博览会ED各种杀虫剂和这些农药能觅食上采集花蜜和花粉7,8时已喷洒杀虫剂花后带回蜂箱。他们还可以通过自己的目的来控制荨麻疹9，10，11内的虫害问题养蜂人暴露于杀虫剂。由于蜜蜂幼虫被护士蜜蜂为他们的发展，幼虫，无人机喂，甚至女王可能会接触到这些农药污染的花蜜和花粉12。需要解决的13多种农药对蜜蜂的毒性。 许多已作出努力，以评估环境农药残留的问题。杨等人。测试的蜜蜂幼虫在发展中的神经毒性杀虫剂吡虫啉的影响蜂箱和报道，吡虫啉亚致死剂量导致成年蜂14的嗅觉关联行为。此外，Urlacher 等。检查的有机磷农药，毒死蜱，对蜜蜂工人的学习表现在实验室条件下15的亚致死效应。在我们以前的研究中，我们评估的昆虫生长调节剂（IGR）的影响，吡丙醚（PPN），幼虫蜜蜂16。 在本文中，我们提出，以评估对蜜蜂的发展化学影响的方法。进行了描述并应用于任何个别蜜蜂或菌落蜜蜂馈送方法。起初，我们在殖民地幼虫上测试了不同浓度的农药污染的基本幼虫饲料（BLD），以评估在体内个别蜜蜂农药的影响。我们接着来模拟自然康迪特通过使用蜂箱内农药污染的糖浆中的农药的离子。在该方法中，PPN，它被广泛用于对抗有害昆虫17和有害蜜蜂幼虫和蛹16，18，19的发展，将表示该领域的农药的负面影响的指标。

<table><tbody><tr><td>Honey bee box</td><td>SAN-YI Honey Factory</td><td>W1266</td><td>Honeybees rearing</td></tr><tr><td>Queen excluder (between frames)</td><td>SAN-YI Honey Factory</td><td>I1575</td><td>Queen limitation&amp;nbsp;</td></tr><tr><td>Queen excluder (on top)</td><td>SAN-YI Honey Factory</td><td>I1566</td><td>Queen limitation on top&amp;nbsp;</td></tr><tr><td>Bee brush</td><td>SAN-YI Honey Factory, Taiwan</td><td>W1414</td><td>clean the bees on frame gently</td></tr><tr><td>Bee feeder</td><td>SAN-YI Honey Factory, Taiwan</td><td>P0219</td><td>feed sugar syrup to colony</td></tr><tr><td>Transparent slide</td><td>Wan-Shih-Chei, Taiwan (http://www.mbsc.com.tw/a01goods.asp?s_id=40)</td><td>1139</td><td>Mark the larval area on the frames (Material: Polyethylene Terephthalate, PET) (Size = Length*Width*thick = 29.7 mm * 21 mm * 0.1 mm)</td></tr><tr><td>24 well tissu culture plate</td><td>Guangzhou Jet Bio-Filtration Co., Ltd</td><td>TCP011024</td><td>Rearing pupae from extraction</td></tr><tr><td>Autoclave</td><td>Tomin medical equipmenco., LTD.</td><td>TM-321</td><td>Make sterilized distilled deionized water (ddH&lt;sub&gt;2&lt;/sub&gt;O)</td></tr><tr><td>P20 pipetman</td><td>Gilson</td><td>F123600</td><td>Add PPN into bee larval food pool</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Yihder Co., Ltd.</td><td>LE-550RD</td><td>Rearing pupae from extraction</td></tr><tr><td>Kimwipes</td><td>COW LUNG INSTRUMENT CO., LTD</td><td>KCS34155</td><td>Rearing pupae from extraction</td></tr><tr><td>Royal jelly</td><td>National Ilan University (NIU)</td><td>NIU</td><td>Make basic larval diet (BLD)</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G8270</td><td>Make basic larval diet (BLD)</td></tr><tr><td>D-(-)-Fructose</td><td>Sigma</td><td>F0127</td><td>Make basic larval diet (BLD)</td></tr><tr><td>Yeast extract</td><td>CONDA, pronadisa</td><td>1702</td><td>Make basic larval diet (BLD)</td></tr><tr><td>Sucrose</td><td>Taiwan sugar coporation</td><td>E01071010</td><td>Make sugar syrup for bee food</td></tr><tr><td>Pyriproxyfen (11%)</td><td>LIH-NUNG CHEMICAL CO.. LTD.</td><td>Registration No. 1937</td><td>Insect growth regulator (IGR) used in the experiment</td></tr></tbody></table>

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.

为蜜蜂现场测试中，大号仅限于4帧部分用于产卵。此步骤可能会增加育雏密度在一帧和促进随后的观测。每个处理标记，和蜜蜂的发展显然通过透明幻灯片观察。 在 PPN-BLD的体内料到在蜂箱蜜蜂幼虫进行精确评估PPN对蜜蜂的殖民地发展的影响。使用体内供给方法促进了在蜂箱的化学处理的影响的观察。 对?...

Watch this Scientific Journal Video about Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level at JoVE.com

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

在这里我们提出 的方法，以农药污染的食物输送到两个单独的蜜蜂和蜂箱菌落。过程评估由基本幼虫饮食的体内喂养和也对蜂窝菌落的天然条件对个体蜜蜂的农药效果。

从个体到群体度评价环境化学物质对蜜蜂发展的影响

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 ...

evaluating-effect-environmental-chemicals-on-honey-bee-development

Research

JoVE Journal

Environment

9.0K 次观看.  National Ilan University. 在这里我们提出 的方法，以农药污染的食物输送到两个单独的蜜蜂和蜂箱菌落。过程评估由基本幼虫饮食的体内喂养和也对蜂窝菌落的天然条件对个体蜜蜂的农药效果。 

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

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

科研

环境科学

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.

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

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

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.

体外孤蜂饲养: 一种评估幼虫危险因素的工具

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

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

神经科学

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 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.

评估农药对孤独蜜蜂幼虫的影响

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

生物化学

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

单队列殖民地和工蜂的激素治疗的准备来分析生理学与关联作用和/或内分泌系统

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

SwarmSight: 利用共同的准备和常规硬件实时跟踪昆虫的天线运动和喙的延伸反射

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

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.

蜜蜂致死农药暴露对蜂群水平的影响

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

生物学

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.

评估交配蜜蜂女王的农用化学品风险

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

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

蜜蜂组织中的组织学基础和细胞死亡检测

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...