この研究は、動物の局と植物衛生検査検疫、農業委員会、行政院とグラント103から2313-B-197から002-MY3から省からの助成金105AS-13.2.3-BQ-B1によってサポートされていました科学技術（MOST）の。

1.準備<ol><li> 50％の砂糖シロップの1リットルを作ります。 1リットルのddH 2 Oに1kgのスクロースを溶解</li><li> BLDにおけるピリプロキシフェン（PPN）溶液を調製します。 10,000ppmのPPNストック溶液1.1 Lを作成し、4℃でのddH 2 Oストア滅菌1L中100mLのPPN溶液を希釈します。 </li><li>以下の実験のためにBLD 0.1、1、10及び100mg / kgの（PPM）の最終濃度にPPNストック溶液を希釈します。 </li><li> （コロニーレベル用）PPN-シロップを作ります。以下の実験のために50％の砂糖シロップに10と100 ppmの最終濃度にPPNストックを希釈します。 </li><li>ミツバチ飼育。 注：ここでは、実験場所は国立宜蘭大学（NIU）養蜂場、李-LAN市、台湾です。 （GPS座標：N24.747278、E121.746200）。 <ol><li>ミツバチ（ ミツバチ L.）は食物量毎週コロニーをチェックして（蜂蜜記憶領域が空である）、必要に応じて1リットルの50％砂糖シロップとフィード。健康の定義通常産卵クイーン各コロニーにおけるミツバチコームの9つのフレームとしてコロニー。 </li></ol></li><li>基本的な幼虫の食（BLD）の100ミリリットルを準備します。滅菌蒸留脱イオン水（のddH 2 O）中の6％D-グルコース、6％フルクトース、および1％酵母抽出物を溶解し、50％ローヤルゼリーで補います。 4°Cで保管してくださいではなく、3日以上のため。 実験の前に35°Cまでの事前暖かく、3日以内に使用します。注意してください。 </li></ol> インビボ供給方法2. 注意： インビボでの供給方法は、ハンリーらから変更されています。 20 <ol><li> ミツバチの幼虫の選択とラベリング。 <ol><li> 4フレーム区間に女王を制限しながら4つのフレームと5つのフレームに健康なコロニーの9つのフレームを分割するクイーン圧排を挿入します。産卵するための4フレーム区間内の少なくとも1つの空のフレームを残します。 </li><li>女王は1日の卵を産卵した後、、卵の外観のためのフレームをチェックし、1日齢幼虫が孵化するまで72時間（4 日目 ）のためのハチの巣の内部に卵を保ちます。手で試験ハイブから（24時間以内に斜線）1日齢ワーカー幼虫を含むフレームのうちの1つを取り、蜂ブラシ付きフレームからミツバチの労働者を削除します。 </li><li>透明スライド（サイズ=縦×横×厚さ= 29.7ミリメートル×21ミリメートル×0.1ミリメートル）でフレームをカバーし、画鋲（ 図1A）とフレームのエッジに透明スライドを爪。 </li><li>各治療のために、ランダムに50一日齢の幼虫（4 日目 ）を選択し、透明スライド上に油性マジックペンを使用して、各ひな細胞をマークする（ 図1Aおよび1B）。 注：異なる処理の間での混乱を避けるために、恒久的なマーカーペンを使用することにより、同様のフレームと透明スライド上の各処理の情報を書き込みます。マークされたスライドを削除し、in vivoでのために保ちます&lt;/ em&gt;の給餌と観察。 </li></ol></li><li> 生体内給餌で 。 <ol><li>の摂取量に応じて、それぞれ10μL、10μL、20μL、一日1,2および3にピペッティングすることによって、各標識ひなセルにPPN-BLD（0.1、1、10及び100 PPM）の異なる濃度を追加幼虫時代。対照群にはBLD（無PPN）の同量を追加します。これにより、各標識ひなセルにおけるPPN-BLDの総用量は、4、40、400、4000 PGに蓄積します。 注：マーク透明スライドによって標識ひな細胞を認識し、給餌後にマーク透明スライドを削除します。働きバチによってひな細胞の洗浄を防ぐために供給するための新鮮なBLDを使用してください。 </li><li>さらに観測の元植民地にPPN処理されたフレームを返します。 注：各処理は、4個のコロニーの4つの生物学的反復配列を持っています。 </li></ol></li><li> 7日目の治療の幼虫の観察。 <ol><li>目を観察するために、電子PPN処理した幼虫は、標識されたひな細胞に死亡し、キャッピング率を記録するためのフレームにラベルされた透明のスライドを返します。 </li></ol></li><li> 13日目での治療蛹および羽化率の観察。 <ol><li>頂いたひな細胞のミツロウを削除します。 </li><li>ひなセルに柔らかい先端がピンセットを入れて、クランプ蛹を非常にわずかにそっと蛹を取ります。 </li><li>各ウェルの下に実験室組織の二層を有する24ウェル組織培養プレートに蛹を転送します。蛹転送時の損傷や死亡率を記録します。 </li><li> （ 約 8日間）出現するまで、34℃、相対湿度70％のインキュベーターで24ウェル組織培養プレートを保ちます。 </li><li>観察し、蛹と登場ミツバチを記録。 </li></ol></li><li> 統計 <ol><li>記録したデータを計算し、平均値±SDとして存在</li><li> SAによって分散分析（ANOVA）を使用してデータを分析Sとは異なる治療法の二つの手段の違いを分析するために最小有意差（LSD）テストを使用します。 P -値&lt;0.05として統計的に有意で定義します。テーブルの同じ列における異なる文字は、統計分析によって有意な効果を示しました。 </li></ol></li></ol>ビーハイブでコロニーレベルでのPPNの3毒性<ol><li>ミツバチのグループを設定します。 <ol><li> 4フレーム区間に女王を制限しながら4つのフレーム（パートA）及び5つのフレーム（パートB）に健全なコロニーの9つのフレームを分割する垂直クイーン圧排を挿入します。産卵するための4フレーム部に少なくとも1つの空のフレームを残します。 注意：別の女王は、部品間を移動するから女王を防ぐために、女王の部分の上にエクスクルー置きます。 </li><li>女王は1日の卵を産卵した後、卵の出現のためのフレームをご確認ください。手でパートAから卵を含む適切なフレームを取り、ミツバチの労働者を削除します蜂ブラシでフレームから。 </li><li>透明スライドとフレームをカバーし、画鋲のフレームのエッジに透明スライドを爪。 </li><li>ランダムに卵を含む100個のひな細胞を選択し、透明スライド上の永久的なマーカーを使用して、各ひなセルをマーク。グループ1は、異なる処理間の混乱を避けるために、恒久的なマーカーを使用して、フレームと透明スライド上の各処理の情報を書くように、これら100個のひな細胞を割り当てます。 </li><li> 3日間パートAにラベルされたフレームを返し、その後、卵を産むためにパートBに女王を転送します。 </li><li> 1日後、パートBのフレームをチェックする1つの適切なフレームを選択し、ステップ3.1.4で説明したように卵を含む100個のひな細胞を標識します。グループ2としてこれらの100個のひな細胞を割り当てます。 </li><li> 3日間パートBへの標識フレームを返し、その後、卵を産むためにパートAに女王を転送します。 </li><li> 6回パートAとパートBとの間のクイーン交換を繰り返し、数値グループを割り当て、それぞれ（ 図2）。 9基の合計があるはずです。 </li></ol></li><li> 13日目にPPN糖シロップとT reatミツバチコロニー（ 図2）。 <ol><li> 1 Lの50％プラスチック蜂フィーダボックス10または100 ppm含有PPN糖シロップ（幅×長さ×H = 20センチメートルX 30センチメートル×3.5 cm）を追加し、実験コロニーにおけるフレームの上にボックスを置きます。 注意：ひな細胞が封入されているとして、グループ1は13日でPPNを受信しません。 </li><li>ステップ2.2で説明したように1 Lの50％砂糖シロップ（NO PPN）と対照群を供給する。 </li></ol></li><li> 5日目に、各グループの100個のラベルされたひな細胞の卵孵化率（ 図2）として1日齢幼虫やレコードをカウントします。ミツバチの卵は、通常、0日の幼虫に孵化するために3日かかります。そのため、チェックして、100個の卵を含む1日目にひなの細胞を標識し、孵化率の割合を得るために、5日目に、各グループのために1日齢幼虫の数を数えます。 </li><li> C11日目に各群の100個の標識ひな細胞の割合をキャッピング幼虫（ 図2）のようにキャップされたひな細胞およびレコードをount。 6〜7日齢の幼生ひな細胞は幼虫pupatingのためのミツバチの労働者によってミツロウでキャップされます。 </li><li>蛹の成熟を観察した（ 図2）、17日目に各群の100個の標識ひな細胞の羽化率を記録します。 <ol><li>頂いたひな細胞のミツロウを外し、優しく柔らかな先端ピンセットで蛹を取り出します。各ウェルの下に実験室組織の二層を有する24ウェル組織培養プレートに蛹を転送します。 </li><li>出現するまで34℃、70％RHのインキュベーター中で24ウェル組織培養プレートを保ちます。 </li><li>観察し、記録蛹をし、グループ9（49実験日）まで、各グループのミツバチが登場。 注意：各処理は4つの生物学的反復配列を持っています。 </li></ol></li><li>統計<ol><li>記録したデータを計算し、平均値として存在します±SD。 </li><li>スチューデントの両側tの検定を用いて、各グループの治療のペア間の有意差（ 例えば 、0 PPM / 10ppmで、10 PPM / 100ppmの0 PPM / 100 ppm）を分析します。 P -値&lt;0.05かのように、統計的に有意で定義します。 </li></ol></li></ol>

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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>

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

クイーン制限産卵法、クイーン交換方法は、このプロトコル内のフィールドテストのためのミツバチのグループを設定するための重要なステップです。クイーン制限産卵方法はミツバチのライフサイクルの同期が可能になります。その結果、研究者は、農薬の異なる用量での治療のための同じ年齢の1日齢幼虫を選択することができます。クイーン交換法のために、女王は、農薬及び農薬残留...

環境中の農薬の存在が影響ミツバチ1、2、3の人生で最も深刻な問題の一つです。いくつかの研究は、ミツバチのコロニーとミツバチ製品中の残留農薬の一般的な存在を証明しています。台湾では、農薬の平均的なアプリケーションは、（2005年から2013年まで）キログラム/ヘクタール、毎年11〜12でした。台湾で使用される農薬の量は、EU諸国のそれよりも高く、そしてラテンアメリカ諸国4、5です。言い換えれば、養蜂環境は、特に台湾では、おそらく他の国では、深刻な農薬ストレスを受けています。 ミツバチミツバチは農業システム6の主要な花粉媒介の一つであり、それはまた、蜂蜜などの貴重な製品を生産しています。しかし、ミツバチは、博覧会です様々な農薬やこれらの農薬へのedは蜜や花粉7、8を収集する際に農薬を噴霧してきた花で採餌した後、ハチの巣に戻すことができます。彼らはまた、自身がじんましん9、10、11内部の害虫問題を制御することを目指し養蜂家によって農薬に暴露することができます。ミツバチの幼虫は、その開発のための看護師ミツバチによって供給されるので、幼虫、ドローンとも女王は、これらの農薬汚染ネクターと花粉12に露出されてもよいです。ミツバチへの各種殺虫剤の毒性は13を対処する必要があります。 多くの努力は、環境残留農薬の問題を評価するために行われています。 Yang ら。でミツバチの幼虫の開発に神経毒性殺虫剤イミダクロプリドの影響をテストしました蜂の巣はとイミダクロプリドのサブ致死量は成人の蜂14の嗅覚連想行動をもたらしたことを報告しました。また、アーラッカーら。実験室条件の下で15ミツバチの労働者の学習パフォーマンス上の有機リン系農薬、クロルピリホス、の亜致死影響を調べました。私たちの以前の研究では、幼虫のミツバチ16上（PPN）ピリプロキシフェン昆虫成長調節（IGR）の影響を、評価しました。 本稿では、ミツバチの開発に化学的影響を評価するための方法を提示します。ミツバチ供給方法が記載され、いずれかの個々のミツバチまたはコロニーに適用しました。最初に、我々は、in vivoでの個々のミツバチへの農薬の影響を評価するためにコロニーに幼虫に殺虫剤に汚染された基本的な幼虫の食（BLD）の異なる濃度をテストしました。私たちは、その後、自然コンディットをシミュレートするために進めハチの巣内農薬汚染シロップを使用することによって殺虫剤のイオン。この方法では、広く害虫17に対して使用され、ミツバチの幼虫および蛹16、18、19の発展に有害であるPPNは、フィールド内の農薬の負の効果を表す指標であろう。

<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.

ミツバチのフィールドテストのために、女王が産卵するための4フレーム部に限られていました。このステップは、1つのフレーム内ひな密度を増加させ、後続の観察を容易にすることができます。各処理は標識し、そしてミツバチの開発は明らかに透明スライドで観察しました。蜂の巣でミツバチの幼虫にPPN-BLDの生体送りで正確にコロニーにおけるミツバチの?...

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

ここで我々が提示 個々ミツバチと蜂の巣コロニーの両方に農薬汚染された食物を供給するための方法。手順は、基本的な幼虫の食事のとも蜂の巣コロニーの自然条件にin vivoで供給することにより、個々のミツバチへの農薬の影響を評価します。

個々のコロニーレベルにミツバチ開発に関する環境化学物質の影響を評価します

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

9.0K ビュー.  National Ilan University. ここで我々が提示 個々ミツバチと蜂の巣コロニーの両方に農薬汚染された食物を供給するための方法。手順は、基本的な幼虫の食事のとも蜂の巣コロニーの自然条件にin vivoで供給することにより、個々のミツバチへの農薬の影響を評価します。 

ビデオ: 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.

環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択

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

環境学

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.

実証、メタゲノムと計算手法を照らす殺菌剤妥協ミツバチ健康メカニズム

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.

体外孤独な蜂の飼育: 幼虫の危険因子を評価するためのツール

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.

ミツバチにおけるRNAiによる二重遺伝子ノックダウンと味覚知覚測定（<em&gt;ミツバチ</em&gt;）

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

神経科学

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.

孤独なミツバチの幼虫に対する農薬の効果の評価

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

生化学

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.

シングルコホートコロニーと労働者ミツバチのホルモン治療の準備は役割および/または内分泌系との生理学関連を分析します

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: 昆虫アンテナの動きと共通の準備と従来のハードウェアを使用して吻伸展反射のリアルタイム追跡

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.

致死の農薬暴露の蜂蜜の蜂のコロニー レベルの効果を監視

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.

交配ミツバチクイーンズへの農薬リスクの評価

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.

ミツバチ組織における組織学の基礎と細胞死検出

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

個々のコロニーレベルにミツバチ開発に関する環境化学物質の影響を評価します

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環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択

実証、メタゲノムと計算手法を照らす殺菌剤妥協ミツバチ健康メカニズム

体外孤独な蜂の飼育: 幼虫の危険因子を評価するためのツール

ミツバチにおけるRNAiによる二重遺伝子ノックダウンと味覚知覚測定（

孤独なミツバチの幼虫に対する農薬の効果の評価

シングルコホートコロニーと労働者ミツバチのホルモン治療の準備は役割および/または内分泌系との生理学関連を分析します

SwarmSight: 昆虫アンテナの動きと共通の準備と従来のハードウェアを使用して吻伸展反射のリアルタイム追跡

致死の農薬暴露の蜂蜜の蜂のコロニーレベルの効果を監視

交配ミツバチクイーンズへの農薬リスクの評価

ミツバチ組織における組織学の基礎と細胞死検出

個々のコロニーレベルにミツバチ開発に関する環境化学物質の影響を評価します

要約

さらに動画を探す

この動画の章

環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択

実証、メタゲノムと計算手法を照らす殺菌剤妥協ミツバチ健康メカニズム

体外孤独な蜂の飼育: 幼虫の危険因子を評価するためのツール

ミツバチにおけるRNAiによる二重遺伝子ノックダウンと味覚知覚測定（

孤独なミツバチの幼虫に対する農薬の効果の評価

シングルコホートコロニーと労働者ミツバチのホルモン治療の準備は役割および/または内分泌系との生理学関連を分析します

SwarmSight: 昆虫アンテナの動きと共通の準備と従来のハードウェアを使用して吻伸展反射のリアルタイム追跡

致死の農薬暴露の蜂蜜の蜂のコロニー レベルの効果を監視

交配ミツバチクイーンズへの農薬リスクの評価

ミツバチ組織における組織学の基礎と細胞死検出

致死の農薬暴露の蜂蜜の蜂のコロニーレベルの効果を監視