The authors thank Mona Chambers, Geoffrey Hidalgo and Maurissa Two Two for their technical assistance. We also thank Dr. Earl White for amino acid analysis, Dr. Roger Simonds for analysis of pollen for pesticides, and Drs. Stanley S. Schneider, Vanessa Corby-Harris and Kirk Anderson for their reviews and discussion of earlier versions of the manuscript and many helpful suggestions.

1. AHB和EHB殖民地获得Beebread <ol><li>将花粉过滤器对蜜蜂的殖民地，并收集花粉。使用研磨咖啡研磨机花粉成细粉（类似于花粉花药棚）。 </li><li>建立每个AHB和EHB 5殖民地在一个封闭的飞行区（EFA），这样蜜蜂觅食，只能在规定的花粉。为了防止工人EHB和AHB殖民地之间漂移，分全民教育成独立的部分，使蜜蜂无法将它们之间的交叉。将个人EHB或AHB菌落3,500-4,000工蜂，蜡梳花蜜，蜂蜜，未成熟的育雏和空梳在全民教育的各个部分。 注意:当建立的殖民地不具备存储花粉。花粉中存储的速率可以增加不包括在所述的菌落一个铺设女王。 </li><li>通过放置一个托盘，在全民教育的各个部分花粉饲料地面花粉殖民地。每个托盘使foragin散布约60g花粉克蜜蜂可以收集它作为corbicular负载和花粉保存在他们的殖民地为beebread。继续每天提供新鲜花粉每个托盘上3周。 </li><li>参阅来自欧洲的菌落作为欧洲beebread（EBB），并从非洲化菌落作为非洲化beebread（ABB）至beebread。 </li></ol>在笼子里饲养2蜜蜂<ol><li>从单独的笼子里出现AHB和EHB殖民地密封工人育雏的环境室内放置帧定在32-34℃，相对湿度40％.. </li><li>当工人出现，大约24小时的时候，建立12有机玻璃笼生物测定（尺寸= 11.5 X 7.5 X16.5厘米3），并添加或者100新兴EHB或100新兴AHB工蜂每个笼子里。放置梳的截面与任意EBB或ABB的细胞在每笼一已知数量（每笼24-30个细胞）以产生下列治疗组合:AHB馈ABB，EHB馈ABB，AHB馈EBB和EHB馈EBB。 （4个处理; 6每次治疗笼;共24箱）。 </li><li>加水的小瓶和50％的蜂蜜和水的溶液体积为每个笼子制定。每天笔芯蜂蜜和水的小瓶11天的学习期。 </li></ol> 3.采样工人蜜蜂和Beebread和消费估计<ol><li>样品10新兴EHB和AHB工人将它们放置在鸡笼前。参阅这些作为第0天的蜜蜂，并让它们作为血淋巴蛋白浓度的基准。 </li><li>从每个网箱中取出10蜜蜂后，他们喂养EBB或ABB 4，7，和11天。 </li><li>放置在单个微量离心管活蜜蜂和冰袋设置。选择四个蜜蜂血淋巴蛋白浓度的分析子样本。 </li><li>在第一天，11取样蜜蜂后，计数梳细胞仍然含有beebread的数目。这是beebread消费的相对度量。 </li><li>从细胞中每个网箱中取出剩余beebread和存储组合通道根据笼吃离心管。保持beebread样品在-80℃下直到用于pH，可溶性蛋白浓度和氨基酸含量进行分析。 </li></ol> 4. pH值估计花粉和Beebread的<ol><li>取供给到在EFA蜜蜂花粉六个随机0.3克样品和溶解于300微升蒸馏水中。采用防水双结pH值矛与0.01的精度测量pH值。 </li><li>就拿beebread 0.3 g样品是留在每个网箱的11天喂养一段时间后。溶解在300微升蒸馏水和测量pH值的beebread所描述的花粉（4.1）。 </li></ol> 5.蛋白质分析<ol><li>采取花粉六个样品和EBB和ABB从每笼的样本。商店的样品在-20℃，直到用于可溶性蛋白浓度进行分析。 </li><li>混合20毫克或者花粉或beebread与1000微升的0.1M磷酸缓冲液（PBS）中。 </li><li> VORTEX持续10秒和离心机将混合物在571.2×g离心1分钟。 </li><li>除去上清液并将其放置在96孔平底的EIA / RIA聚苯乙烯板的孔中的10微升样品。复制每个样品的3口井中。 </li><li>从通过将20微升毛细管（已加热并拉至针尖点）插入邻近附着的翅膀的点胸廓的右侧部分从每笼收集蜜蜂绘制血淋巴。收集其他血淋巴，如果需要的话，通过将相同的管进入腹腔背甲之间的膜。 </li><li>加入1μl血淋巴至9微升0.1M的PBS中。储存在-20℃的血淋巴溶液直至分析可溶性蛋白。 </li><li>确定使用市售Bradford蛋白测定试剂盒总可溶性蛋白的浓度在花粉，beebread，和血淋巴样品。按照制造商的说明。 </li><li>建立的标准曲线来估计可溶性蛋白Concentration通过测定蛋白质吸光度与已知蛋白质浓度的牛血清白蛋白（BSA）的样品中。使用分光光度计测量蛋白质吸光度在595nm处。 </li></ol> 6.氨基酸分析<ol><li>从每一集落的梳细胞池个体样本来创建EBB和ABB的用于分析有代表性的样品。 </li><li>取50毫克，花粉或beebread样品称入自动进样器小瓶中，并加入1毫升蒸馏水的小瓶中，沿着用100μl的50毫微克/微升内标由-d 4中丙氨酸的溶液中，D 23 -lauric酸， 13 C 6葡萄糖和D 39 -arachidiac酸。 </li><li>盖上样品并超声5分钟。 </li><li>通过加入1毫升甲醇调节的HLB盒中，平衡加入1毫升蒸馏水，然后加入1毫升的beebread或花粉的样品。用1毫升5.0％甲醇/ H 2 O和埃卢洗仓德用1ml的80％的MeOH / H 2 O. </li><li>蒸发在氮气流中的样品至干燥。重构用50μl吡啶和100微升的N，O-双（三甲基硅基）三氟乙酰胺+三甲基氯硅烷（BSTFA + TMCS）的样品。 </li><li>帽和孵育所述样品在70℃下进行30分钟。 </li><li>使样品冷却，并转移到一个干净的自动进样器小瓶中。 </li><li>帽并将样品放入一个质量选择检测器接口到气相色谱仪来分析对挥发性化合物和有机酸的样品。分离糖和有机酸以下的TMS衍生使用一个柱（30米×0.25毫米内径）用1.0微米的膜厚度BSTFA + TMCS。 </li><li>设置列烘箱中于50℃2分钟，然后线性地增加温度至290℃以5℃/分钟。并保持7分钟。将GC注射器和GC / MS接口，以250℃，290℃，分别。 <ol><li>使用氦气作为载体在FLO为1.0毫升/分钟瓦特率。设置该MS源温度至230℃。 </li></ol></li><li>调整和每天用全氟三丁胺（PFTBA）校准质谱仪。使用在全扫描（35-700 AMU）正离子模式下的1微升注射PFTBA，以获得对所述在场数据和氨基酸浓度。 </li></ol>

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The authors have nothing to disclose.

使用上述的方法，我们发现，通过AHB所作的beebread被消耗在更大量由两个AHB和EHB。虽然EHB和AHB类似消费量的每种类型beebread的，AHB有较高的血淋巴中的蛋白质滴度。根据我们的方法的调查结果是相似的过去的报告，其中血淋巴蛋白水平在AHB比在EHB虽然都饲喂相同日粮26更高。通过测量EBB和ABB其中被消耗以不同的速率由两个EHB和AHB的消耗，它被确定在每个亚种血淋巴蛋白浓度无法通过增加食物消耗上升。似乎有一个高原在护士蜂年龄工人血淋巴蛋白浓度以及为高原设定点是AHB比EHB高。 有用于建立菌落beebread生产，这将优化花粉存储的速率几个重要的条件。首先，将共lonies需要开放育雏帧。没有开放的育雏饲料，工人不会收集花粉多。其次，殖民地必须queenless因此不需要额外的育雏产生。育雏饲养需要大量的花粉，只有多余的花粉被存储。在建立了全民教育的小菌落，会有一点花粉被存储为beebread如果育雏领域不断扩大使殖民地必须queenless。最后，对于要作出beebread，花粉必须收集作为corbicular负荷和存储在梳细胞。如果花粉被收集在花粉陷阱，它必须被研磨成细粉它呈现给蜜蜂，这样他们可以收集它作为corbicular载荷之前。 的方法来测量beebread的消耗产生定性而不是绝对的估计。这是数只消费时，细胞完全排空的蜂粮。总蜂粮消费的一个更准确的估计可能会被删除蜂BR获得EAD从细胞并使其成能之前和研究期间之后被测量肉饼。但是，我们要保持蜂粮的细胞，使蜜蜂可以在上面饲料，因为他们会在一个殖民地，也许继续在研究期间处理它。前和研究后的重量梳子部的差异不是作为消费的估计中使用，因为体重可能增加，因为蜜蜂把稀释蜂蜜在某些细胞饲喂给他们。 工人们可能还增加了一些稀释的蜂蜜蜜蜂面包。由于这些原因，含有前后馈送周期的蜂面包大致相等量的细胞进行计数，并产生一个质量测量。尽管如此，仍有在空的ABB细胞后第11天用EBB相比计数的数量的两种类型beebread之间的显着差异。 当确定存储花粉变得beebread可迪fficult因为蜜蜂不断增加花粉细胞。用于生产beebread的殖民地，建立开放式育雏的框架，以便将蜜蜂采集花粉。然而，菌落queenless所以有成立菌落后幼虫喂养只有约9天。在3周时间内的剩余时菌落呈全民教育，所收集的蜜蜂被存储并转换为beebread花粉。保持在梳子细胞所存储的花粉为它在笼子里饲养时对蜜蜂额外11天还可继续花粉的处理，beebread。花粉的蜜蜂面包的转换需要大约7天8。送入EHB和AHB的beebread有较低的pH值和降低蛋白质浓度的花粉美联储相比。转换beebread后改变花粉类似的调查结果已报告了其他7,10,27。我们的研究结果与以往不同的报告然而，在有浓度差异Øbeebread和花粉F之间的某些氨基酸。在蛋白质和氨基酸的浓度的变化可能是由于蛋白水解酶的活性，其中的源可能是蜜蜂本身或设立于beebread 7,8,28,29的微生物群落。 用于测量蛋白质浓度的方法是类似于先前描述以确定对非洲化饮食蛋白质和欧洲蜜蜂26的影响。作为该方法的延伸，我们能够估计可溶性蛋白在花粉和beebread。这些方法产生类似的结果之前的报道7,10,27。我们的研究结果提供了额外的证据表明，AHB更有效地吸收食物的蛋白质比EHB，并认为这可能是AHB的大部分地区的生态优势，其中已移居，并成为建立30-32的关键因素。

营养在蜂蜜蜂群的健康和活力，并在其设立的人口基础性作用。从食物中营养物质提供能量和必要的育雏饲养，体温调节的生化成分，觅食和免疫反应。对于蜜蜂的殖民地，营养生长所需的菌落群和维护他们的健康来自花蜜和花粉。花蜜提供碳水化合物和花粉耗材的剩余饮食要求，如蛋白质，脂质，维生素和矿物质1。 蜜蜂亚种可以根据营养菌落级参数，如工人长寿，育雏饲养和社会免疫力2-6的机制不同。这些差异可能与食物如何，特别是花粉由菌落处理并消化在个人。花粉被存储在梳细胞和通过微生物介导的乳酸发酵是化学变化<sup&gt; 7-10。发酵花粉被称为beebread。有可能是亚种间在他们从他们收集的花粉和存储获得营养物质的能力基因型差异，这可能会影响菌落生长和生存。食品加工和营养收购基因型效应已被记录在其他生物造成了一些个人同时消耗同样的食物11-14获得比别人更多的营养和热量。 在这里，我们描述了用于比较由蜜蜂亚种不同的制作成分和beebread的食用方法。方法测量也被描述所得血淋巴蛋白滴度工蜂。在beebread的营养成分先前的研究已经做完了欧洲蜜蜂（EHB）10,15,16。然而，可能会有在beebread由不同亚种蜂制成甚至当它们以相同的花粉的差异。 EHB和AHB进行比较，因为这些潜艇pecies有可能与食品加工和营养采集17独特行为和生理差异。其中最显着的区别在于AHB收集和消耗比EHB更多的花粉，似乎更容易转化为育雏18。 AHB殖民地有更高的蜂拥率比EHB和潜逃当食物资源变得有限19-23。潜逃是罕见的EHB。 AHB也有较高的代谢率比EHB 24。为EHB和AHB之间的菌落水平的差异的营养基础可能与花粉采集的速率，并以它的营养成分（ 例如 ，氨基酸和蛋白质）之后它被转换为beebread。 Beebread消耗和由此产生的蛋白质收购也可能在EHB和AHB之间的群体水平的差异的作用。使用开发的方法，EHB和AHB从相同花粉源所做beebread。该beebread然后反馈到EAC的蜜蜂ħ亚种和可能确定是否蜜蜂采集蛋白从beebread在独特的，以它们的亚种或往beebread源的方式进行。

<table><tbody><tr><td>waterproof double junction pH spear&amp;nbsp;</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Coffee Grinder</td><td>Mr. Coffee&amp;nbsp;</td><td>model 1DS77</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffer solution</td><td>Emd-millipore</td><td>BSS-1005-B</td><td></td></tr><tr><td>EIA/RIA polystyrene plate</td><td>Sigma-Aldrich-Corning</td><td>CLS3590-100EA</td><td></td></tr><tr><td>microcapillary pipets</td><td>Kimble Glass Inc.</td><td></td><td></td></tr><tr><td>Quick Start Bradford Protein Assay Kit 2&amp;nbsp;</td><td>Bio-Rad</td><td>#500-0202</td><td></td></tr><tr><td>Spectrophotometer</td><td>Biotek Synergy HT&amp;nbsp;</td><td></td><td></td></tr><tr><td>Mass Selective Detector&amp;nbsp;</td><td>Agilent</td><td>5973N</td><td></td></tr><tr><td>HLB cartridge</td><td></td><td></td><td></td></tr><tr><td>gas chromatograph&amp;nbsp;</td><td>Agilent</td><td>6930</td><td></td></tr><tr><td>&amp;nbsp;gas chromatography column&amp;nbsp;</td><td>A J&amp;amp;W Scientific&amp;nbsp;</td><td>DB-1701</td><td></td></tr><tr><td>d&lt;sub&gt;4&lt;/sub&gt;-alanine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>488917</td><td></td></tr><tr><td>d&lt;sub&gt;23&lt;/sub&gt;-lauric acid</td><td>Sigma-Aldrich</td><td>451401</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;-glucose</td><td>Sigma-Aldrich</td><td>389374</td><td></td></tr><tr><td>Pyridine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>270970</td><td></td></tr><tr><td>N,O-Bis (trimethylsilyl)trifluoroacetamide +&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Trimethylchlorosilane (BSTFA + TMCS)</td><td>Sigma-Aldrich</td><td>33148</td><td></td></tr><tr><td>Perfluorotributylamine (PFTBA)</td><td>Sigma-Aldrich</td><td>442747-U</td><td></td></tr><tr><td>&lt;sup&gt;d39&lt;/sup&gt;-arachidiac acid</td><td>Cambridge Isotope&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

methods for comparing nutrients in beebread made by africanized and european honey bees and the effects on hemolymph protein titers

蜜蜂获得蜂巢的beebread花粉，他们收集和储存的营养物质。我们开发的方法来控制花粉源蜜蜂采集并转换通过将菌落在一个专门建造的封闭飞行区域beebread。方法被开发来分析花粉和beebread的蛋白质和氨基酸组成。我们还描述了如何在beebread的消耗量，测定和方法中使用的出苗后喂食beebread为4,7和11天后，以确定成年工蜂血淋巴蛋白滴度。方法被应用，以确定是否基因型影响花粉转化为beebread和蜜蜂消耗，并从中获得的蛋白质的速度。两个亚种（欧洲和非洲化蜜蜂;分别EHB和AHB）被提供有相同的花粉源。基于所开​​发的方法，beebread由两个亚种制成具有较低的蛋白质浓度和pH值下比花粉。在一般情况下，氨基酸CONcentrations在beebread由两种或EHB AHB取得相近，且发生在较高水平beebread比花粉。无论AHB和EHB消耗显著更由AHB比EHB的beebread的。虽然EHB和AHB类似消费量的每种类型beebread的，血淋巴蛋白浓度AHB均高于EHB高。在AHB和EHB之间的蛋白质收购的差异可能反映了相关的地理区域，其中每个亚种进化的环境适应性。这些差异可能有助于成功建立，因为对育雏饲养和菌落生长的影响AHB人口的新世界。

<html> Beebread贮存在-80℃下不到一个月的pH和蛋白质浓度被分析之前，而对于前氨基酸分析约4个月。 Beebread从在pH和蛋白质浓度（ 图1）的花粉不同。 beebread的pH比花粉下因为是蛋白质浓度。既EHB和AHB消耗更多的ABB比EBB（ 图2）。 可溶性蛋白在AHB的血淋巴水平显著高于EHB无论beebread它们所消耗的类型（ 图3）的。发生这些差异血淋巴中的蛋白质水平，即使EHB和AHB类似消费量的每种类型beebread的。蜜蜂在采样时的年龄显著影响可溶性蛋白的浓度在血淋巴。有一天-7或11，没有差异相比，蛋白质浓度显著低于一天4蜜蜂。 _content"&gt;的10个氨基酸，这对蜜蜂必不可少，但所有的组氨酸中的花粉进行检测。在大多数情况下，在beebread测定氨基酸的浓度比在花粉更高（ 图4）。例如，浓度亮氨酸和苏氨酸是在beebread高出约60％的花粉进行比较，缬氨酸浓度分别高出约25％。丙氨酸，天冬氨酸，谷氨酰胺和甲硫氨酸水平也均在beebread高于花粉。氨基酸浓度没有之间有很大的不同无论使用哪种EBB或花粉相比ABB和EBB除苯丙氨酸和半胱氨酸。苯丙氨酸水平高一倍左右的ABB。半胱氨酸浓度较低的EBB与ABB或花粉相比，色氨酸是存在于高浓度的唯一氨基酸花粉比EBB或脯氨酸的花粉和ABB的ABB。浓度比在EBB高。 图1:pH值（A）和可溶性蛋白质含量（B）中的花粉和欧洲（EHB）或非洲化（AHB）蜜蜂所做的beebread的比较。花粉的pH比beebread如通过方差分析确定显著更高（F 2,12 = 3725，P &lt;0.0001），接着是Tukeys W-多重比较检验。在花粉蛋白质浓度较beebread由EHB（EBB）或AHB（ABB）制成显著更高（F 2,27 = 16.49; P &lt;0.0001）。手段其次是在同一封信都没有显著的不同在0.05的水平。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig2highres.jpg" width="600" /> 图2:CEL的平均百分比LS含有beebread即完全由笼蜜蜂消耗在11天的间隔。该beebread是由使 ​​用相同的花粉源无论是欧洲（EHB）或非洲化（AHB）蜜蜂。装置从每个治疗5笼估计;那些具有相同的字母不显著的不同在0.05的水平由单向方差分析（F 3,16 = 7.3，P = 0.003）和杜克的读写测试来确定。该图已被修改为25。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig3highres.jpg" width="600" /> 图3:蛋白血淋巴欧洲（EHB）或非洲化的平均浓度（AHB）蜜蜂喂beebread 4，7，11天由欧洲（EBB）或非洲化（ABB）蜜蜂的重复测量分析。差异表明中4 TREA显著差异tment组（F = 3,20 19.7，P &lt;0.001）。在AHB美联储ABB可溶性蛋白的水平显著高于EHB美联储ABB（P = 0.008）或EBB（P = 0.018）。蜜蜂在采样时的年龄显著影响可溶性蛋白的浓度在血淋巴。有一天-7（P &lt;0.0001）或11（P = 0.001）相比，水平显著低于一天4蜜蜂。 7日和day11蜜蜂没有差异（P = 0.149）。这个数字已经被修改25。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig4highres.jpg" width="600" /> 图4:氨基酸浓度（每克花粉微克或beebread）的花粉，或从它的EBB 所作的beebread是beebread由欧洲蜜蜂和ABB是由非洲化蜜蜂。色氨酸，半胱氨酸，苯丙氨酸和脯氨酸分别绘制在预先清楚起见senting的金额。这个数字已经被修改25。

Watch this Scientific Journal Video about Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers at JoVE.com

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

对于在比较营养Beebread由非洲化和欧洲蜜蜂的方法和淋巴蛋白质滴度影响

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.6K 次观看.  USDA-ARS. Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies. 

视频: Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

管理和检测蛋白标记的节肢动物的扩散研究

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

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 ...

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

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

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

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 ...

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 ...

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

蜂蜜蜂 (api 蜜蜂) 中下咽腺性腺泡大小的测定

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（<em&gt;欧洲熊蜂</em&gt;）

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

神经科学

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 ...

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

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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 ...