Name: evaluating the effect of environmental chemicals on honey bee development from the individual to colony level
Uploaded: 2017-04-01T14:00:00.000+00:00
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Description: 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

Bu araştırma Grant 105AS-13.2.3-BQ-B1 Bakanlığı&#39;ndan Hayvan Bürosu ve Bitki Sağlığı Teftiş ve Karantina, Tarım Konseyi, Yürütme Yuan ve Grant 103-2313-B-197-002-My3 dan destek verdi Bilim ve Teknoloji (EN) ait.

1. hazırlıklar <ol><li> % 50 şeker şurubu 1 L olun. 1 L&#39;lik GKD 2 O 1 kg sukroz çözülür </li><li> BLD piriproksifen (PPN) çözeltisi hazırlayın. 10,000 ppm PPN stok çözeltisi 1.1 L yapmak ve 4 ° C &#39;de GKD 2 O Mağazada steril 1 L için 100 mL PPN seyreltilir. </li><li> Aşağıdaki deney için BLD içinde 0.1, 1, 10 ve 100 mg / kg (ppm) nihai konsantrasyonlara PPN stok çözeltisi ile seyreltilir. </li><li> (Koloni düzeyi için) PPN-şurup yapın. Aşağıdaki deney için,% 50 şeker şurubu, 10 ve 100 ppm nihai konsantrasyonlara PPN stokunu. </li><li> Bal arısı yetiştirme. Not: Burada, deneysel konum Ulusal İlan Üniversitesi (NIU) apiary Yi-Lan Şehir, Tayvan olduğu; (GPS koordinatları: N24.747278, E121.746200). <ol><li> Bal arısı (Apis mellifera L.) yiyecek miktarına haftalık kolonilerinin kontrol ve (bal depolama alanı boş) gerektiğinde 1 litre% 50 şeker şurubu ile beslenir. Sağlıklı bir tanımlaNormalde yumurtlamaya kraliçe her kolonideki bal arısı tarakların 9 kare gibi koloni. </li></ol></li><li> Temel larva diyet (BLD) 100 mL hazırlayın. (GKD 2 O) steril distile deiyonize su içinde% 6, D-glikoz,% 6 fruktoz ve% 1 maya ekstratı çözülür ve% 50 arı sütü ile tamamlar. 4 ° C&#39;de saklayın ama en fazla 3 gün. deneyden önce 35 ° C&#39;ye kadar Öncesi sıcak ve 3 gün içinde kullanın: Not. </li></ol> İn Vivo Besleme Yöntem 2. Not: İn vivo besleme yöntemi Hanley ve arkadaşları modifiye edilmiştir. 20 <ol><li> Bal arısı larvası seçimi ve etiketleme. <ol><li> 4 çerçeve bölümüne kraliçe sınırlandırırken 4 çerçeveleri ve 5 kare, sağlıklı bir koloni 9 kare bölmek için bir kraliçe dışlayıcı yerleştirin. yumurtalar için 4 çerçeve bölümünde en az bir adet boş çerçeve bırakın. </li><li> Queen 1 gün için yumurtlamaya sonra, Yumurta görünüm için çerçeveler kontrol etmek ve 1 günlük bir larva kadar 72 saat (4. gün) için kovan içinde yumurta tutun. elle Test kovandan dışarı (24 saat içinde yumurtadan çıkartılmıştır) 1-günlük larvaları işçi içeren çerçevelerin birini alın ve bir arı fırça ile çerçeveden bal arısı işçi çıkarın. </li><li> Şeffaf bir sürgünün (Boyut = Uzunluk x Genişlik x kalınlığında = 29.7 mm x 21 mm x 0,1 mm) ile çerçeve kapağı ve raptiyeler (Şekil 1A) ile çerçeve kenarında şeffaf slayt tırnak. </li><li> Her bir muamele için, rasgele 50 adet günlük larva (4. gün) seçmek ve şeffaf bir slayt üzerinde kalıcı marker kullanılarak her kuluçka hücresi işareti (Şekil 1A ve 1B). Not: Farklı tedaviler arasında karışıklığı önlemek için kalıcı işaretleyici kalem kullanarak hem çerçeve ve şeffaf slaytta her tedavinin bilgisini yazın. İşaretli slaytları çıkarın ve in vivo tutmak &lt;/ Em&gt; besleme ve gözlem. </li></ol></li><li> In vivo besleme olarak. <ol><li> emme miktarına göre, sırasıyla, 10 uL, 10 uL, 20 uL, günün 1, 2 ve 3 de pipetleme her etiketli kuluçka hücresine PPN-BLD (0.1, 1, 10 ve 100 ppm), farklı konsantrasyonlarda ekleme larvalar yaşı. Bir kontrol grubuna BLD (hayır PPN) aynı miktarda ekleyin. Bu şekilde, her bir etiketli kuluçka hücresine PPN-BLD toplam dozu 4, 40, 400 ve 4000 pg birikir. NOT: işaretli şeffaf slaytlar tarafından etiketli kuluçka hücrelerini tanır ve beslendikten sonra işaretli şeffaf slaytları çıkarın. işçi arılar tarafından kuluçka hücrelerinin temizlenmesi önlemek için beslenme için taze BLD kullanın. </li><li> ayrıca gözlemler için orijinal kolonilerine PPN&#39;ler ile tedavi kareleri döndürür. NOT: Her bir işlem dört kolonilerde dört biyolojik tekrarları vardır. </li></ol></li><li> 7. günde tedavi larva Gözlem. <ol><li> th gözlemlemekE etiketli kuluçka hücrelerinde ölüm ve kapatma oranları kaydedilmiştir çerçevelere etiketli şeffaf slaytlar dönün larvaları PPN ile tedavi. </li></ol></li><li> Gün 13 tedavi pupa ve eclosion oranlarının gözlenmesi. <ol><li> şapkalı kuluçka hücrelerinin arı mumu kaldırın. </li><li> kuluçka hücre içine yumuşak uçlu cımbız koyun ve kelepçe pupa çok hafif sonra yavaşça pupa çıkar. </li><li> her bir altındaki laboratuar dokuların çift tabaka ile 24 oyuklu doku kültürü plakalarına pupa aktarın. pupa transferi sırasında hasar ve mortalite kaydedin. </li><li> (Yaklaşık 8 gün) ortaya çıkmasına kadar 34 ° C&#39;de ve% 70 nispi nemde bir kuluçka makinesi içinde 24 oyuklu doku kültürü plakaları tutun. </li><li> Gözlemleyin ve pupa ve ortaya çıkan bal arıları kaydedin. </li></ol></li><li> istatistik <ol><li> ± SD ortalaması olarak kaydedilmiş verileri ve mevcut hesaplama </li><li> SA varyans analizi (ANOVA) analizini kullanarak verileri analizS ve farklı tedaviler arasında iki ortalama arasındaki farkları analiz en az önemli fark (LSD) testi kullanılır. P-değeri &lt;0.05 istatistiksel olarak anlamlı tanımlar. tablonun aynı sütunda değişik harfler istatistiksel analizi ile belirgin bir etki gösterdi. </li></ol></li></ol> Arı Kovanı koloni Düzeyde PPN 3. Toksisite <ol><li> bal arısı grupları oluşturun. <ol><li> 4 kare (Kısım A) ve 5 kare (Bölüm B) 4 kare bölümüne kraliçe sınırlandırırken, sağlıklı bir koloni 9 kare bölmek için dikey bir kraliçe dışlayıcı yerleştirin. yumurtalar için 4-çerçeve bölümünde en az bir adet boş çerçeve bırakın. Not: Başka bir kraliçe parçaları arasındaki hareket etmesini kraliçe önlemek için kraliçe kısmının üstüne Excluder koyun. </li><li> Queen 1 gün için yumurtlamaya sonra, yumurtaların görünüm için çerçeveler edin. elle bölüm A&#39;dan yumurta dışarı içeren uygun kareleri alın ve bal arısı işçileri çıkarmakArı fırça ile çerçeveden. </li><li> Şeffaf slayt ile çerçeve kapağı ve Raptiye ile çerçeve kenarına şeffaf slayt tırnak. </li><li> Rasgele yumurta içeren 100 kuluçka hücreleri seçmek ve şeffaf slayt üzerinde kalıcı bir kalem kullanarak her kuluçka hücreyi işaretleyin. Grup 1. farklı tedaviler arasında karışıklığı önlemek için kalıcı bir kalem kullanarak çerçeve ve şeffaf slaytta her tedavinin bilgi yaz olarak bu 100 yavru gözlerini atayın. </li><li> 3 gün boyunca bölüm A&#39;ya etiketli çerçeve dönün ve daha sonra yumurta bırakmaya bölüm B için kraliçe aktarın. </li><li> 1 gün sonra, B kısmının çerçeve kontrol bir uygun çerçeve belirledikten aşama 3.1.4 tarif edildiği gibi yumurta içeren 100 kuluçka hücreleri etiketlemek. Grup 2 olarak bu 100 yavru gözlerini atayın. </li><li> 3 gün boyunca kısım B etiketli çerçeve dönün ve yumurta bırakılması için bir bölüm için kraliçe aktarın. </li><li> 6 kez daha Kısım A ve Kısım B arasındaki kraliçe alışverişini tekrarlayın ve sayısal olarak gruplarını atamak,sırasıyla (Şekil 2). 9 gruplarının toplam olmalıdır. </li></ol></li><li> 13. günde, PPN şeker şurubu T reat bal arısı kolonileri (Şekil 2). <ol><li> 1 L&#39;lik bir plastik arı besleyici kutusunda 10 ya da 100 ppm (G x L x Y = 20 cm x 30 cm x 3.5 cm) içeren% 50 PPN şeker şurubu ekleyin ve deneysel koloniler çerçevelerin üstünde kutu koydu. Not: kuluçka hücreleri mühürlü edilmiş olarak Grup 1 13. günde PPN almaz. </li><li> aşama 2.2 &#39;de tarif edildiği gibi 1 L% 50 şeker şurubu (bir PPN) ile kontrol doyur. </li></ol></li><li> 5. günde (Şekil 2) her bir grup için, 100 etiketli kuluçka hücrelerinin yumurta kuluçka oranı olarak 1 günlük bir larva ve kayıt sayısı. Bal arısı yumurtaları genellikle 0 gün larva haline yumurtadan 3 gün sürebilir; Bu nedenle, kontrol ve 100 yumurta içeren 1. günde kuluçka hücreleri etiketlemek ve oranı kuluçka yüzdesi elde etmek için 5 gün Her grup için, 1 günlük bir larva sayısı. </li><li> C11. günde (Şekil 2) her bir grup için, 100 etiketli kuluçka hücrelerinin larva kapatma hızı başlıklı kuluçka hücreleri ve kayıt İzahname&#39;de. 6 ila 7 günlük bir larva kuluçka hücreleri larva, pupa haline gelmesi için bal arısı işçiler tarafından arı mumu ile sınırlandırılır. </li><li> Pupa olgunlaşmasını gözlemleyin ve 17. günde (Şekil 2) her bir grup için, 100 etiketli kuluçka hücrelerinin eclosion oranı kaydetmek. <ol><li> şapkalı kuluçka hücrelerinin arı balmumu çıkarın ve yavaşça yumuşak uçlu cımbız ile pupa çıkar. her bir altındaki laboratuar dokuların çift tabaka ile bir 24 oyuklu doku kültürü plakalarına pupa aktarın. </li><li> ortaya çıkıncaya kadar 34 ° C&#39;de ve% 70 bağıl nemde bir kuluçka makinesi içinde 24 oyuklu doku kültürü plakaları tutun. </li><li> Gözlemleyin ve pupa kaydetmek ve Grup 9 kadar her gruptan (49 deneysel gün) bal arıları ortaya çıktı. Not: Her bir işlem dört biyolojik tekrarları vardır. </li></ol></li><li> istatistik <ol><li> Kaydedilen veri hesaplama ve ortalama olarak mevcut± SD. </li><li> Tedaviler çiftleri arasındaki önemli farklar analiz (örneğin, 0 ppm / 10 ppm, 10 ppm / 100 ppm ve 0 ppm / 100 ppm) Student iki-kuyruklu-testi kullanılarak her bir grupta. P-değeri &lt;0.05 ise istatistiksel olarak anlamlı tanımlayın. </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>

Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

kraliçe sınırlı yumurtlama yöntemi ve kraliçe değişimi yöntemi bu protokol dahilinde saha testleri için bal arısı gruplarının oluşturulması kritik adımlardır. kraliçe sınırlı yumurtlama yöntemi bal arılarının yaşam döngüsünün senkronizasyonunu sağlar. Sonuç olarak, araştırmacılar pestisit farklı dozlarda tedavi için aynı yaşta 1 günlük bir larvaları seçebilir. kraliçe değiştirme metodu için, kraliçe bölüm A (4 kare) ve pestisit ve pestisit artıklarının etkilerini de?...

Çevrede pestisit varlığı en ciddi sorunlardan biri olduğunu etkileri bir bal arısı 1, 2, 3 yaşamı. Çeşitli çalışmalar, bal arısı kolonilerine ve arı ürünlerde pestisit artıklarının ortak varlığını göstermiştir. Tayvan&#39;da, pestisitlerin ortalama uygulama 11-12 kg / (2005 2013) her yıl ha oldu. Tayvan&#39;da kullanılan pestisitlerin miktarı, 5 AB ülkesinin daha yüksek olması ve Latin Amerika ülkeleri 4. Başka bir deyişle, arıcılık ortam özellikle Tayvan ve muhtemelen diğer ülkelerde, ciddi pestisit stresi çekiyoruz. Bal arısı Apis mellifera tarımsal sistemlerde 6 önemli tozlayıcı biridir ve aynı zamanda bal gibi değerli ürünler üretmektedir. Ancak, bal arıları fuarlara olanÇeşitli pestisit ve bu pestisitlere ed nektar ve polen 7, 8 toplarken pestisitler ile püskürtüldüğü çiçekler kazarak sonra kovan geri getirilebilir. Onlar da kendilerini kovan 9, 10, 11 iç haşere kontrol problemlerine yönelik arıcılar tarafından pestisit maruz kalabilir. Bal arısı larva gelişim, larva, uçaklar için hemşire arıların beslenir ve çünkü daha kraliçe bu pestisit kirlenen nektarı ve polen 12 maruz kalabilir. Bal arıları için çeşitli pestisitlerin toksisite 13 ele alınması gerekmektedir. Birçok çabalar çevresel pestisit kalıntı sorunları değerlendirmek için yapılmıştır. Yang ve diğ. bal arısı larvalarının gelişimi üzerine nörotoksik insektisit imidakloprid etkisini testarı kovanı ve imidakloprid bir subletal doz yetişkin arı 14 koku birleştirici davranışı ile sonuçlandığını bildirmiştir. Ayrıca, Urlacher ve diğ. laboratuvar koşullarında 15 altında bir bal arısı işçinin öğrenme performansı üzerinde organofosfat böcek ilacı, klorpirifos, alt öldürücü etkilerini inceledik. Daha önceki çalışmada, biz larva bal arılarına 16 (PPN) piriproksifen bir böcek büyümesi düzenleyici etkisini (IGR), değerlendirdi. Bu makalede, bal arıları gelişimine kimyasal etkileri değerlendirmek için yöntemler sunuyoruz. Bal arısı besleme yöntemleri tarif ve ya tek tek ya da bal arıları için bir koloni uygulanmıştır. İlk başta, in vivo olarak, bireysel bal arıları üzerinde pestisit etkisini değerlendirmek için kolonilerde larva üzerinde pestisit kirlenmiş temel larva diyet (BLD) farklı konsantrasyonlarda test edilmiştir. Daha sonra doğal condit simüle devamkovan içinde pestisit kirlenmiş şurup kullanılarak pestisit iyonları. Bu yöntemde, yaygın zararlı böceklere karşı kullanılan 17 ve bal arısı larva ve pupa 16, 18, 19 gelişimine zararlı olduğu PPN, sahada pestisitin olumsuz etki temsil edecek bir göstergesi olacaktır.

<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.

bal arısı alan testinde, bir kraliçe yumurtalar için 4-çerçeve bölümünün sınırlıydı. Bu aşama, bir çerçevede kuluçka yoğunluğunu artırmak ve sonraki gözlemlerin kolaylaştırabilir. Her tedavi etiketlendi ve bal arıların gelişim açıkça şeffaf slayt aracılığıyla gözlendi. Arı kovanı bal arısı larvası PPN&#39;i-BLD vivo besleme olarak kesin kolonideki bal arılarına gelişimi üzerinde PPN&#39;nin etkisini değerlendirmek amacıyla ...

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

Burada sunduğumuz Bir metot, bireysel bir bal arısı ve arı koloni hem pestisit kirlenmiş gıda yem için. Yöntem basit larva diyet in vivo besleme yoluyla ve aynı zamanda arı kovanı koloni doğal koşuluyla bağımsız bal arıları üzerinde pestisit etkisi değerlendirilmektedir.

Koloni Düzeyde Bireysel olan Bal Arısı Kalkınma Çevre Kimyasalların Etkisinin Değerlendirilmesi

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 Görüntüleme.  National Ilan University.  Burada sunduğumuz Bir metot, bireysel bir bal arısı ve arı koloni hem pestisit kirlenmiş gıda yem için. Yöntem basit larva diyet in vivo besleme yoluyla ve aynı zamanda arı kovanı koloni doğal koşuluyla bağımsız bal arıları üzerinde pestisit etkisi değerlendirilmektedir. 

Video: 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.

Otomatik Jel Boyut Seçimi Çevre Su Örneklerinden hazırlanmıştır nesil Sıralama Kütüphaneler Kalitesi Geliştirmek için

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

Çevre

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.

Ampirik, Metagenomic ve bilgisayar teknikleri mekanizma hangi mantar ilaçları uzlaşma arı sağlık ile aydınlatmak

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.

Vitro Yalnız arı yetiştirme: larva Risk faktörleri değerlendirmek için bir araç

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.

Bal Arısı RNAi-aracılı Çift Gen demonte ve tat Algı Ölçüm (<em&gt; Apis mellifera</em&gt;)

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

Nörobilim

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.

Pestisitlerin yalnız arıların larvaları üzerindeki etkisinin değerlendirilmesi

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

Biyokimya

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.

Tek kohort Kolonileri ve İşçi arıların Hormon Tedavisi hazırlanması Rolü ve / veya Endokrin Sistemi Fizyolojisi İlişkili Analiz

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: Gerçek zamanlı izleme böcek anten hareketleri ve hortum uzantısı refleks bir ortak hazırlık ve geleneksel donanım kullanarak

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.

Bal arıları üzerinde Sublethal pestisit pozlama koloni düzeyi etkilerinin izlenmesi

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

Biyoloji

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.

ÇiftLeşme Bal Arısı Kraliçeleri için Tarımsal Kimyasal Riskin Değerlendirilmesi

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.

Bal Arısı Dokusunda Histolojinin Temelleri ve Hücre Ölümü Tespiti

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