This work was supported by the National Institutes of Health grant 5R21AI081041-02 and Georgetown University.

İki A. 1. Larva Yetiştirme albopictus Grupları Erişkinliğe <ol><li> İki optimum diapause ifadesi 16 21 ° C&#39;de programlanabilir aydınlatma ile fotoperiyot dolapları ve yaklaşık% 80 bağıl nem ayarlayın. <ol><li> Bir 16L Program bir kabine: 8K ışık: karanlık döngüsü (non-diapause LD fotoperiod uyaran). Bir 8L ikinci kabine ayarlayın: 16D ışığı: karanlık döngüsü (uyarıcı diapause) 13. </li><li> Her iki dolapları aynı zamanda Programın &#39;ışıklar&#39; fotoperiyodların arasındaki sirkadiyen zaman senkronize etmek. </li></ol></li><li> Deney gerçekleştirmek için gerekli yumurta miktarını hesaplayın. Kafes başına 300-500 yumurta hedefleyin. En azından üç diapause kafesleri çoğaltmak ve olmayan üç-diapause kafesler RNA üretimi için gerekli olan çoğaltmak. Bu deney başına 1,800-3,000 yumurta Takriben için toplamları. <ol><li> Yaklaşık içine deiyonize H 2 O 500 ml yumurta kağıtları daldırarak tarafından Hatch yumurta </li><li> Ekle &lt;em&gt; ca. Zemin köpek maması ve tuzlu su karidesi oluşan 1 ml gıda bulamaç olarak daha önce 24 nitelendirdi. Kafes ile konteyner örtün ve bir lastik bant ile yerine örgü tutmak. </li><li> Yaklaşık 24 saat LD fotoperiyodun kabine yerleştirin. </li></ol></li><li> Aktarım yumurtadan larva için 10 x 10 x 2 cm&#39;lik Petri yaklaşık 90 ml ile doldurulmuş kaplar deiyonize H2O <ol><li> Tabak başına yaklaşık 30 larva koruyun. Örneğin, her Pazartesi, Çarşamba, Cuma ve (MWF) 24 yemeklere her 48-72 saat temizlemek için larva aktarın. </li><li> Daha önce olduğu gibi, iyonu giderilmiş su içinde ezilmesi köpek maması ve tuzlu su karidesi her MWF oluşan besleme yaklaşık 1 mi gıda bulamaç 24 tanımladı. </li></ol></li><li> Her kafes biyolojik çoğaltmak içermektedir her fotoperiyod tedavisi için 3-4 yetişkin kafesleri ayarlayın. <ol><li> 9.5 L kovalar ters taraftan, bir 10-by-14 cm delik kesip, ve 15 cm çapında bir delik. Comesh ile ilk Sür. Ortopedik çorap yaklaşık bir ayak uzunluğu kesin, ve diğer deliğin içine etrafında bir ucunu tutkal. </li><li> Çorap kapalı ve düğüm kapatma ayak ucunu kesin - kafesin iç erişim gerektiğinde yalnızca açık. Kafes kapak için, sadece jant bırakarak, tüm iç kesip, ve örgü 24 ile iç plastik değiştirin. </li><li> Kafesin tarafında kalıcı bir kalem ile deney ile ilgili numarayı, kafes başlangıç ​​tarihini ve diğer bilgileri çoğaltmak, fotoperiod unutmayın. </li></ol></li><li> Islak filtre kağıdı ile yetişkin kafesleri alt Hat. Filtre kağıdı 24 ovipozisyon teşvik, hangi kafeste yerel nem oranı yükselir, ancak suya durmaktan kaçınmak için yeterli deiyonize H 2 O ile filtre kağıdı nemlendirin. Gerektiğinde kurutulması için günlük olarak tekrar nemli filtre kağıdı edin. </li><li> En az 100 kadın dahil, bir RNA kütüphanesi için yeterli yumurta üretmek için / 9.5 L cage, kafes başına en fazla 500 sivrisinek ile. </li><li> En fazla 50 pupa başına 25 mi H2O yoğunluğunda, temiz H2O küçük bir kap içinde pupa MWF yeri ve toplamak Bir yetişkin kafesine pupa fincan aktarın. İlgili fotoperiyodun kabine yerleştirin kafesleri - A. albopictus pupa 15 fotosensitiftir. </li><li> Ölü pupa oluşumu kitle mortaliteye neden olabilir, çünkü, bardak H 2 O temiz ve berrak olduğunu günlük olun ve ölü pupa çıkarın. Tüm pupa ortaya Ey bardak sonra H 2 çıkarın. </li><li> Ortaya yetişkinler için şeker sağlamak için kafesin üst örgü organik üzüm yerleştirin. Üzüm Monitör ve küf birikmesini önlemek için her 3-5 gün onları değiştirmek. </li></ol> Yetişkin 2. Bakım Çiftleşme ve Yumurta Üretim izin ver <ol><li> (SECTI nemli bir filtre kağıdı ile kafes alt astar yüksek nemde (yakl.% 80) en kafesleri bakımı, ve yukarıda tarif edildiği gibi, küflü üzüm erişim sağlamakons 1.5 ve 1.9). </li></ol> 3. Kan-besleme <ol><li> Yumurtlama yaklaşık 100% diapause yumurta 14 başlamadan önce kadın en az sekiz net kısa gün maruz emin olmak için eclosion sonra 2-6 gün arasında kan besleme kadınlarda hazırlanın. </li><li> Hemotek Membran Besleme sistemi hazırlayın. Güç kaynağı besleyen birimleri takın. Ayar vidası kullanarak 37 ° C her biriminin sıcaklığını ayarlayın. Kalibrasyon sırasında besleme ünitesinin sıcaklığını ölçmek için bir elektronik termometre ve probu kullanın. </li><li> Yemek haznesini hazırlayın. Yemek rezervuar diyafram üzerinde kollajen besleme membranın bir kare gerin ve bir O-ring ile sabitleyin. Dikkatle kırışıklıkları kaldırmak için köşeleri çekin; makas ile aşırı membran düzeltin. NOT: Kollajen membran tüm sivrisinek türleri için de işe yaramayabilir ve bir çalışma varsa optimum membran bulmak için çeşitli denemek için gerekli olabilirA dışında başka türler albopictus. Parafilm pipiens&#39;in ile iyi çalışır. <ol><li> Kan kullanmadan önce en az 1 saat süre ile, oda sıcaklığında, dondurulmuş çözülme saklanır. </li><li> Membran aşağıya bakacak şekilde tutun rezervuar, desteksiz ve dolum portları yukarı bakacak. Bir anti-pıhtılaştırıcı olarak sodyum sitrat olan tavuk tam kan, yaklaşık 3 ml ile doldurulması için bir transfer pipeti veya şırınga kullanın. Plastik fişler ile dolum noktalarını Seal. </li></ol></li><li> Besleyici altındaki ısı transfer plaka üzerinde damızlık üzerine vidalanarak besleyici hazırlanan rezervuar takın. Sivrisinekler kafesin örgü ile besler, böylece besleyici ters çevirin ve kafesin üst, aşağı membran tarafına yerleştirin. Besleme en üst düzeye çıkarmak için yaklaşık 45 dakika boyunca kafes üzerine besleyici tutun. </li></ol> 4. yumurtlama teşvik <ol><li> Dört beş gün renkli bir karanlık her kafes donatmak, kan yemek sonrası 50 ml cağartılmamış tohum çimlenme kağıdı (yumurta kağıt) veya dokulu olmayan ağartılmış kağıt havlu ile kaplı ve deiyonize su ile 9 yarıya doldurun kadar. 250&#39;den fazla sivrisinek bir kafes içinde iseniz, iki bardak kullanın. NOT: Küçük kafeslerde veya tek dişi şişeleri için, yumurtlama kaplarda &quot;hay infüzyon&quot; nedeniyle mikrobiyal floranın 25 koku ovipozisyon artabilir. </li></ol> 5. toplayın ve Mağaza Yumurta <ol><li> Yumurta üretimi genellikle önümüzdeki hafta boyunca azalır, sonra beş gün kan beslenme sonra yaklaşık zirveleri, çünkü kan besleme 4-5 gün içinde yumurta toplama başlar. </li><li> Deneyin ihtiyaçları yumurta toplama sıklığını Vary. Genel amaçlar için, bir MWF programa yumurta kağıtları toplamak. Her kafes yumurta kağıtları çıkarın ve taze kağıt ile değiştirin. Yumurta depolama karıştırıcı etkilerini önlemek için SD fotoperiyodun kabinesinde Petri kapları ve mağaza son zamanlarda kaldırıldı kağıtları yerleştirin. </li><li> Yumurta kağıtları yeniden izin<html> Yumurta kuruma direncini artırır 26 serozal manikür oluşumuna, izin sonrası yumurtlama 2 gün boyunca ıslak ana. </li><li> Yaklaşık 48 saat sonra toplama, açık havada kuru yumurta. Kağıt H 2 O koyu veya yumurta yumurtadan uyaran o kadar ıslak gevşek ve hafifçe nemli, ama öyle ki kağıt kurutun. NOT: Bir 6,5 &quot;x 4&quot; Kağıt kuruması yaklaşık 3.5 saat sürebilir. Bu yumurta kuruma 27 neden olacak gibi değil, aşırı kuru yumurta kağıtları dikkatli olun. </li><li> LD ve SD hem fotoperiyodların rezerv ek yumurta diapause insidansını değerlendirmek ve (Diapause Ölçme Bölüm 6) photoperiodic etkisini yorumlamak. </li><li> Uzun süreli depolama için, 21 ° C&#39;de yumurta kağıtları tutmak ve Petri kutularına yaklaşık% 80 nem. Embriyo gelişimi, 21 ° C&#39;de 4-5 gün sürer, yerel nem korumak için bir su şişesi ile bir Saklama kabı depolama kabı içinde Petri kapları tutun. </li></ol>ove_title &quot;&gt; 6. Diapause İnsidans Tedbir <ol><li> Ek saklıdır embriyolar diapause yanıtı ölçmek için 7-20 gün eski (yumurtlama, Bölüm 4 Canlandıracak bakınız) kullanın. </li><li> Her yumurta kağıt üzerinde bulunan yumurta sayısını kaydedin. </li><li> Tamamen yaklaşık 80 ml ​​deiyonize H 2 O ile 90 ml&#39;lik Petri kabındaki bireysel yumurta kağıtları daldırarak yumurtadan yumurta teşvik Yaklaşık 0.25 mi gıda bulamaç ekleyin. </li><li> 24 saat sonra ince çizgiler birinci değişim safhasındaki larva sayısı kalın. Larvaları görselleştirmek ve çanağın bir tarafında bir ışık kaynağı yerleştirmek için siyah bir yüzey Petri tabağına yerleştirin. Larva bireysel larva net bir taksitli için izin ışık kaynağından uzak hareket edecek. Bireysel larva anlatırken önlemek için sayım sırasında bir pipet ile bireysel larva çıkarın. </li><li> Yeni bir Petri kabı ve yeniden-kuru yerleştirin yumurta kağıtları. Yeniden yumurtadan yumurta yine ~ 1 hafta ve sonra yukarıdaki yöntemi kullanarak yumurtadan yumurta taksitli. </li><li> Kalan u Yeri yumurta kağıtları<html> Yaklaşık 80 ml ​​beyazlatma solüsyonu 28 ml 90 yeni Petri kapları yumurta n-yumurtadan. Yumurta kağıtları tamamen ağartma çözeltisine batırılmış emin olun ve çamaşır suyu kokusunu önlemek için bir davlumbaz gecede altında bırakın. NOT: Bu maddeler, 4 ° C&#39;de yaklaşık 1 hafta boyunca muhafaza edilebilir, ancak aksi takdirde taze yapılmalıdır. </li><li> Koryon temizlemek ve embriyo, un-yumurtadan yumurta görselleştirme sağlayacak beyazlatma gibi bir ışık mikroskobu kullanılarak yumurta kontrol edin. Yumurta embriyolu ise yumurta sırt tarafında birbirine zıt iki küçük siyah noktalar şeklinde görünen gözlü grimsi beyaz bir renge sahip olur. Un-taranmış, embriyonlu yumurta 13 sayısını taksitli. </li><li> Hayır =% diapause: Aşağıdaki formül ile diapause sıklığını belirler. embriyone un-yumurtadan yumurta / (hayır. yumurtadan yumurta + hayır. embriyone un-yumurtadan yumurta) 100 13 x. </li></ol> Yumurta / pharate Larvalarına 7. RNA Ekstraksiyon &quot;ontent&gt; NOT: Bir laminer akış kaputu kullanın Trizol. <ol><li> Bir deve tüyü fırça kullanarak cam taşlama için yumurta kağıtları farklı gelişim zaman noktalarında gelişen embriyolar ya pharate larva içeren Fırça sivrisinek yumurtaları. Tamamen toz haline gelene kadar Trizol yumurta (doku 50-100 mg başına 1 mi) öğütün. Yeterli RNA elde kütüphanede başına en az 400 yumurta kullanın. <ol><li> Seçenek olarak ise, sıvı azot içinde dondurularak yumurta bileşenini ve Trizol taşlama önce bir ay kadar için mikrosantrifüj tüpleri içinde -80 ° C&#39;de saklandı. </li></ol></li><li> Trizol RNA ekstraksiyonu yapın, üreticinin talimatlarına uygun olarak, izopropanol çökeltme eklenmiştir. </li><li> RNA, bağlı bozulmayı önlemek için herhangi bir kalıntı nükleazlar kaldırmak için RNaz dekontaminasyon çözelti ya da diğer maddeler ile birlikte tezgah tedavi edin. <ol><li> DNase ile ekstre RNA tedavi edin. Üreticinin talimatlarına göre, 37 ° C&#39;de 30 dakika boyunca DNaz ile RNA örnekleri inkübe edin. 1 &amp; # kullanın181; 50 ml&#39;lik bir reaksiyon içinde RNA kadar 10 ug l DNaz. Bir reaksiyonda RNA fazla 10 ug varsa DNase miktarını artırır. </li><li> 5 ul askıya DNaz inaktivasyon reaktif ekleyerek DNase inaktive. Kuluçka döneminin (hafif girdaplama) sırasında üç kez karıştırma, oda sıcaklığında 5 dakika inkübe edin. </li><li> 1.5 dakika için 10,000 x g&#39;de santrifüjleyin. Sonraki adımlar için yeni tüplere tedavi RNA örnekleri ihtiva eden üst sıvıyı aktarın. </li></ol></li><li> Fluorometri toplam RNA örneklerinin kalitesini değerlendirmek. Bu görev için uygun alet ile bir özel tesisine örnekleri gönderin. Tesis çip aşılanan floresan boya ile görüntülendi numune RNA türlerinin, boyutlarını belirlemek için çip jel elektroforezi gerçekleştirecektir. Sonuçlar bir electropherogram olarak iade edilecektir. <ol><li> Varlığı o tarafından kanıtlandığı gibi, varlığında veya bozunma ürünlerinin yokluğunda toplam RNA numunelerinin bütünlüğünü belirlemekElde electropherogram (Şekil 1B) üzerinde 18S ve 5S ribozomal RNA zirveleri arasında f zirveleri. </li></ol></li></ol> 8. RNA Sıralama <ol><li> Standart protokolleri takip zenginleştirilmiş eşleştirilmiş-uç mRNA kütüphaneleri ve mRNA sıralama, yapımı için ticari bir sıralama merkezi yeterince yüksek kalitede (Şekil 1A) ve miktarı (kütüphane başına genellikle&gt; 3 mg) ile toplam RNA örnekleri gönderin. </li><li> Birden fazla şeritli bir tek deney sıralanması için kullanılan ediliyorsa, sıralama sırasında şerit arasında teknik varyasyon için hesap sıralama için iki şerit halinde bireysel kütüphaneler bölünmüş. </li></ol> 9. lllumina Oku Temizlik NOT: Şekil 2, bu protokolün biyoinformatik bölümünü özetlemektedir. Bu protokolün biyoinformatik bölümünde kullanılan tüm programlar ve kaynakların tam listesi için, Tablo 1 bakınız. InAyrıca, Ek Dosya 1 Aşağıdaki biyoinformatik protokol adımların her biri için komut satırı örneklerini içerir. <ol><li> NCBI UniVec Çekirdek veritabanına% 95 kimlik ya da daha yüksek bir eşleşme için ssaha2 29 (Tablo 1) kullanarak (Tablo 1), C. albopictus rRNA dizi (Gen Bankası # L22060.1), ve sıralama adaptörleri (Ek Dosya 1&#39;de verilmiştir Ayrıntılı komut satırı örnekleri). Sağlanan Perl script (Ek Dosya 2) adapte örneğin Perl kullanarak maçlarda veya benzeri bir komut dosyası aracı ile okuma çiftleri çıkarın. </li><li> Kalan Temiz SolexaQA paketiyle 30 okur (Tablo 1; Ek Dosya 1): DynamicTrim.pl varsayılan ayarları kullanarak 20&#39;den daha az bir phred puan eşdeğeri olan bölgeleri kırpın. <ol><li> Kaldır ileri hem LengthSort.pl ile daha kısa 25 bp okur ve ters aynı anda okur. FastQC (Tablo 1 ile fastq temizlenen dosyaların kalitesini değerlendirmek </stRong&gt;) - Özellikle, başına tabanı dizisi kalitesi ve başına dizisi kalitesi puanları 20&#39;nin üzerinde olduğunu doğrulayın. </li></ol></li></ol> 10. Dijital Normalleştirme <ol><li> K-mer boyutu 20, 20 bir kapsama kesme, ve x kullanarak (özellikle normalize-by-median.py; (Ek Dosya 1 Tablo 1) khmer aracını kullanarak 31 okur temizlenmiş dijital normalleşme bir tur yapın = 1E10). </li><li> Yüksek RAM ile bir makine mevcut (GBS yüzlerce) ise Alternatif, Trinity normalize_by_kmer_coverage.pl komut (Tablo 1) kullanın. </li></ol> 11. De Novo Transkriptom Meclisi <ol><li> Montaj büyüklüğüne bağlı olarak, RAM kadar 256 Gb ve 24 işlemcilerde olan bir bilgisayar veya bilgisayar kümeye erişimi edinin. </li><li> Komşularla içine yerleştirilen dijital normalize okuma monte etmek, Trinity 32 (Ek Dosya 1 Tablo 1) kullanın. Azaltmak için,bellek kullanımı, --min_kmer_cov 2 kullanın. </li></ol> 12. Meclis Değerlendirme <ol><li> Trinity kontig çıkış Assemblathon2 projesi 33 assemblathon_stats.pl çalıştırın. (; Ek Dosya 1 Tablo 1) Bu script gibi iskelelerinin sayısı, N50, montaj kompozisyon, ve daha fazlası gibi değerlendirilmesi montaj kalitesi ile ilgili temel hesaplamaları gerçekleştirir. </li></ol> Montajlı transcriptome 13. Ek Açıklama <ol><li> Bir referans protein kümesi karşı toplanma BLASTX (Tablo 1) gerçekleştirin; sivrisinekler için, Drosophila melanogaster, Anopheles gambiae, Culex pipiens ve Aedes aegypti uygun referanslar vardır. Özellikle, BLASTX (Ek Dosya 1) takip patlamanın referans protein Fasta dosyasını, biçimlendirmek. </li></ol> 14. Harita RSEM 34 Kullanma Meclise okur (Tablo 1) <ol><li> Bir &#39;transkript-to-gen haritasının&#39; dosyası oluşturun hangiİlk sütun referans gen kimlikleri ve ikinci sütun kontigi kimlikleri içeriyor. Bir elektronik tablo editörü olarak, BLASTX çıkışından birinci ve ikinci sütun takas ve bir .txt dosyasına bu sütunları yazın. Unix formatına sonuçlanan .txt dosyasında satır sonlarını dönüştürmek için lineBreak programını kullanın. </li><li> RSEM paketinin (Ek Dosya 1) sağlanan rsem-hazırlamak-referans komut kullanarak transcriptome Fasta dosyasından bir başvuru dataset oluşturun. </li><li> RSEM paketinin (Ek Dosya 1) sağlanan rsem-hesaplamak ifade komutunu kullanarak her kütüphane için ayrı ayrı ifade değerlerini hesaplayın. Okur gibi, okuma-temizleme adım (adım 9.2) kaynaklanan fastq eşleştirilmiş dosyaları kullanın. <ol><li> Biyolojik tekrarında RNA dizileme için iki şerit bölünmüş ise, tek bir dosya oluşturmak için ifade hesabında hem fastq dosyaları içerir. </li></ol></li><li> Kolayca diğer p tarafından işlenen bir matris her kütüphaneden ifade sonuçlarını dönüştürmeRSEM paketinin (Ek Dosya 1) sağlanan sağlanan komut rsem-oluşturmak-veri-matris kullanarak rograms. </li></ol> 15. Diferansiyel İfade Analizi <ol><li> R ve Edger (Tablo 1) takın. </li><li> Adım 14.3 (Ek Dosya 1) den RSEM sonuçlarını yüklemek için read.delim kullanın. Gerekirse, en yakın tam sayıya sayıları yuvarlak. NOT: Kenar düzeninin kılavuzu önemini algılamak için yeterince yüksek bir ifade ile genlere veri kümesi sınırlayan önerir. </li><li> Edger için verileri biçimlendirmek için, yüklenen veri dosyası (Ek Dosya 1) bir DGEList nesnesi oluşturur. Ardından, TMM normalleştirme (Ek Dosya 1) kullanarak veri normalleştirmek. Veri (Ek Dosya 1) ortak ve tagwise dağılımlarını tahmin. </li><li> Bir Benjamini-Hochberg ile farklı ifade genleri tanımlamak p-değeri &lt;0.05 (Ek Dosya 1) düzeltildi. Bereket vs log-kat-değişim (Ek Dosya 1) dağılımını çiziniz. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptional+element+of+the+diapause+program+in+the+Asian+tiger+mosquito,+Aedes+albopictus,+identified+by+suppressive+subtractive+hybridization.">A transcriptional element of the diapause program in the Asian tiger mosquito, Aedes albopictus, identified by suppressive subtractive hybridization.</a> J. Insect Physiol. 56, 1147-1154 (2010).</li><li>Huang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+duck+genome+and+transcriptome+provide+insight+into+an+avian+influenza+virus+reservoir+species.">The duck genome and transcriptome provide insight into an avian influenza virus reservoir species.</a> Nat. Genet. 45, 776-783 (2013).</li><li>Sessions, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+cell+transcriptome+profile+during+wild-type+and+attenuated+dengue+virus+infection.">Host cell transcriptome profile during wild-type and attenuated dengue virus infection.</a> PLoS Negl. Trop. Dis. 7 (3), 2107 (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Elsik, C. G., Denlinger, D. L., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+sequencing+reveals+complex+mechanisms+of+diapause+preparation+in+the+invasive+mosquito,+Aedes+albopictus.">Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus.</a> P. R. Soc B. 280, (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Elsik, C. G., Denlinger, D. L., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+sequencing+as+a+platform+to+elucidate+molecular+components+of+the+diapause+response+in+Aedes+albopictus.">Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in Aedes albopictus.</a> Physiol. Entomol. 38, 173-181 (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Denlinger, D. L., Elsik, C. G., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+de+novo+transcriptome+of+the+Asian+tiger+mosquito,+Aedes+albopictus,+to+identify+candidate+transcripts+for+diapause+preparation.">A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation.</a> BMC Genomics. 12, 619 (2011).</li><li>Benedict, M. Q., Levine, R. S., Hawley, W. A., Lounibos, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spread+of+the+tiger:+Global+risk+of+invasion+by+the+mosquito+Aedes+albopictus.">Spread of the tiger: Global risk of invasion by the mosquito Aedes albopictus.</a> Vector-Borne Zoonot. 7, 76-85 (2007).</li><li>Lounibos, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasions+by+insect+vectors+of+human+disease.">Invasions by insect vectors of human disease.</a> Annu. Rev. Entomol. 47, 233-266 (2002).</li><li>Urbanski, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+adaptive+evolution+of+photoperiodic+response+during+invasion+and+range+expansion+across+a+climatic+gradient.">Rapid adaptive evolution of photoperiodic response during invasion and range expansion across a climatic gradient.</a> Am. Nat. 179, 490-500 (2012).</li><li>Lounibos, L. P., Escher, R. L., Lourenco-de-Oliveria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+evolution+of+photoperiodic+diapause+in+temperate+and+tropical+invasive+populations+of+Aedes+albopictus+(Diptera+Culicidae).">Asymmetric evolution of photoperiodic diapause in temperate and tropical invasive populations of Aedes albopictus (Diptera Culicidae).</a> Ann. Entomol. Soc. Am. 96, 512-518 (2003).</li><li>Mori, A., Oda, T., Wada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studies on the egg diapause and overwintering of Aedes albopictus in Nagasaki. Trop. Med. 23, 79-90 (1981).</li><li>Pumpuni, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors influencing photoperiodic control of egg diapause in Aedes albopictus [dissertation]. , (1989).</li><li>Wang, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+influence+of+photoperiod+on+egg+diapause+in+Aedes+albopictus+Skuse.">Observations on the influence of photoperiod on egg diapause in Aedes albopictus Skuse.</a> Acta Entomol. Sinica. 15, 75-77 (1966).</li><li>Sota, T., Mogi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival-time+and+resistance+to+desiccation+of+diapause+and+non-diapause+eggs+of+temperate+Aedes+(Stegomyia)+mosquitoes.">Survival-time and resistance to desiccation of diapause and non-diapause eggs of temperate Aedes (Stegomyia) mosquitoes.</a> Entomol. Exp. Appl. 63, 155-161 (1992).</li><li>Reynolds, J. A., Poelchau, M. F., Rahman, Z., Armbruster, P. A., Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+profiling+reveals+mechanisms+for+lipid+conservation+during+diapause+in+the+mosquito,+Aedes+albopictus.">Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus.</a> J. Insect Physiol. 58, 966-973 (2012).</li><li>Andrewartha, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diapause+in+relation+to+the+ecology+of+insects.">Diapause in relation to the ecology of insects.</a> Biol. Rev. 27, 50-107 (1952).</li><li>Danks, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+Dormancy:+An+Ecological+Perspective.">Insect Dormancy: An Ecological Perspective.</a> Biological Survey of Canada (Terrestrial Arthropods). , (1987).</li><li>Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+diapause.">Regulation of diapause.</a> Ann. Rev. Entomol. 47, 93-122 (2002).</li><li>Rev Entomol, A. n. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 59, 93-122 (2014).</li><li>Armbruster, P. A., Conn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+of+larval+growth+in+North+American+Aedes+albopictus+(Diptera).">Geographic variation of larval growth in North American Aedes albopictus (Diptera).</a> Culicidae). Ann. Entomol. Soc. Am. 99, 1234-1243 (2006).</li><li>Reiter, P., Amador, M. A., Colon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+CDC+ovitrap+with+hay+infusions+for+daily+monitoring+of+Aedes+aegypti+populations.">Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations.</a> J. Am. Mosquito Contr. Association. 7 (1), 52 (1991).</li><li>Rezende, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+desiccation+resistance+in+Aedes+aegypti:+presumptive+role+of+the+chitinized+serosal+cuticle.">Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle.</a> BMC Dev. Biol. 8, 182 (2008).</li><li>Munstermann, L., Crampton, J., Beard, C., C, L. o. u. i. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care+and+maintenance+of+Aedes+mosquito+colonies.">Care and maintenance of Aedes mosquito colonies.</a> The Molecular Biology of Insect Disease Vectors. , 13-20 (1997).</li><li>Trpis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+bleaching+and+decalcifying+method+for+general+use+in+zoology.">A new bleaching and decalcifying method for general use in zoology.</a> Can. J. Zoolog. 48, 892-893 (1970).</li><li>Ning, Z. M., Cox, A. J., Mullikin, J. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nat. Biotechnol. 29 (7), 644-652 (2011).</li><li>Bradnam, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assemblathon+2:+evaluating+de+novo+methods+of+genome+assembly+in+three+vertebrate+species.">Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species.</a> GigaScience. 2, 10 (2013).</li><li>Li, B., Dewey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSEM:+accurate+transcript+quantification+from+RNA-Seq+data+with+or+without+a+reference+genome.">RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.</a> BMC Bioinformatics. 12, 323 (2011).</li><li>White, B. N., De Lucca, F. L., Turner, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+analysis+of+RNA.">Preparation and analysis of RNA.</a> Analytical Biochemistry of Insects. , (1977).</li><li>Hawley, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+Aedes+albopictus.">The biology of Aedes albopictus.</a> J. Am. Mosq. Contr. Assoc. 4, 1-39 (1988).</li><li>Dowling, Z., Ladeau, S. L., Armbruster, P., Biehler, D., Leisnham, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Socioeconomic+status+affect+mosquito+(Diptera:Culicidae)+larval+habitat+type+availability+and+infestation+level.">Socioeconomic status affect mosquito (Diptera:Culicidae) larval habitat type availability and infestation level.</a> J. Med Entomol. 50, 764-772 (2013).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2,4,10.+Bloodfeeding:+Membrane+apparatuses+and+animals.">Chapter 2,4,10. Bloodfeeding: Membrane apparatuses and animals.</a> Methods in Anopheles Research. , (2010).</li><li>Das, S., Garver, S., Ramirez, J. R., Xi, Z., Dimopolous, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dengue+infections+in+mosquitoes+(A.+aegypti)+and+infection+phenotype+determination.">Protocol for dengue infections in mosquitoes (A. aegypti) and infection phenotype determination.</a> J. Vis. Exp. 5 (220), (2007).</li><li>Goff, S. 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The authors have nothing to disclose.

Bu protokol A indüklenen diapause photoperiodically nedeniyle farklı şekilde ifade edilen genleri bulmak için yöntemler sunulur albopictus. photoperiodic diapause yanıt odaklanan kişiler için özellikle - protokolü benzersiz acemi kullanıcılar için erişilebilir bir moleküler fizyoloji programın tüm deneysel yönlerini yapmak için sivrisinek yetiştirme ve biyoinformatik teknikleri birleştiren önemli olduğunu. Yetiştiricilik hataları tespit etmek genellikle gereklidir - - Mevcut y?...

Rapid advances in next-generation sequencing (NGS) technologies are providing exciting opportunities to probe the molecular underpinnings of a wide range of genetically complex ecological adaptations in a broad diversity of non-model organisms1&#8211;3. This approach is extremely powerful because it establishes a basis for population and functional genomics studies of organisms with an especially interesting and/or well-described ecology or evolutionary history, as well as organisms of practical concern, such as agricultural pests and disease vectors. Thus, NGS technologies are leading to rapid advances in the fields of ecology and have the potential to address problems such as understanding the mechanistic bases of biological responses to rapid contemporary climate change4, the spread of invasive species5, and host-pathogen interactions6,7.
The extraordinary potential of NGS technologies for addressing basic and applied questions in ecology and evolutionary biology is in part due to the fact that these approaches can be applied to any organism at a moderate cost that is feasible for most research laboratories. Furthermore, these approaches provide genome-wide information without the requirement of a priori genetic resources such as a microarray chip or complete genome sequence. Nevertheless, to maximize the productivity of NGS experiments requires careful consideration of experimental design including issues such as the developmental timing and tissue-specificity of RNA sampling. Furthermore, the technical skills required to analyze the massive amounts of data produced by these experiments, often up to several hundred million DNA sequence reads, has been a particular challenge and has limited the widespread implementation of NGS approaches.
Recent RNA-Seq studies on the transcriptional bases of diapause in the invasive and medically important mosquito Aedes albopictus provide a useful example of some of the experimental protocols that can be employed to successfully apply NGS technology to studying the molecular basis of a complex ecological adaptation in a non-model organism8&#8211;10. A. albopictus is a highly invasive species that is native to Asia but has recently invaded North America, South America, Europe, and Africa11,12. Like many temperate insects, temperate populations of A. albopictus survive through winter by entering a type of dormancy referred to as photoperiodic diapause. In A. albopictus, exposure of pupal and adult females to short (autumnal) day lengths leads to the production of diapause eggs in which embryological development is completed, but the pharate larva inside the chorion of the egg enters a developmental arrest that renders the egg refractory to hatching stimulus15&#8211;17. Diapause eggs are more desiccation resistant5,18 and contain more total lipids19 than non-diapause eggs. Photoperiodic diapause in A. albopictus is thus a maternally controlled, adaptive phenotypic plasticity that is essential for surviving the harsh conditions of winter in temperate environments. Despite the well-understood ecological significance of photoperiodic diapause in a wide range of insects20,21, the molecular basis of this crucial adaptation is not well characterized in any insect22. In organisms such as A. albopictus that undergo an embryonic diapause at the pharate larval stage, it remains a particularly compelling challenge to understand how the photoperiodic signal received by the mother is passed to the offspring and persists through the course of embryonic development to cause arrest at the pharate larval stage.
This protocol describes mosquito rearing, experimental design and bioinformatics analyses for NGS experiments (transcriptome sequencing) performed to elucidate transcriptional components of photoperiodic diapause in A. albopictus. This protocol can be used for additional studies of diapause in A. albopictus, can be adapted to investigate diapause in other closely related species such as other aedine mosquitoes that undergo egg diapause23, and is also more generally relevant to employing NGS approaches to study the transcriptional bases of any complex adaptation in any insect.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Incubator - Model 818</td>
			<td style="width:116px;">Thermo-Scientific</td>
			<td align="right" style="width:109px;">3751</td>
			<td style="width:351px;">120V</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:209px;">Controlled environment room</td>
			<td>Thermax Scientific</td>
			<td style="width:109px;">N/A</td>
			<td style="width:351px;">Walk-in controlled environment room built to custom specifications by Thermax Scientific Products. A larger alternative to an incubator. http://thermmax.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Cool Fluorescent bulb</td>
			<td style="width:116px;">Philips</td>
			<td align="right">392183</td>
			<td style="width:351px;">4 Watt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Petri Dish 100mm x 20mm</td>
			<td style="width:116px;">Fisher</td>
			<td>08-772-E</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Filter Paper 20.5cm</td>
			<td style="width:116px;">Fisher</td>
			<td>09-803-6J</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">9.5L Bucket</td>
			<td style="width:116px;">Plastican</td>
			<td style="width:109px;">Bway Products</td>
			<td style="width:351px;">http://www.bwayproducts.com/sites/portal/plastic-products/plastic-open-head-pails/117</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Utility Fabric-Mosquito Netting White</td>
			<td style="width:116px;">Joann</td>
			<td>10173292</td>
			<td style="width:351px;">http://www.joann.com/utility-fabric-mosquito-netting-white/10173292.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Orthopedic stockings</td>
			<td>Albahealth</td>
			<td style="width:109px;">23650-040</td>
			<td style="width:351px;">product no. 081420</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Organic Raisins</td>
			<td style="width:116px;">Newman&#39;s Own</td>
			<td style="width:109px;">UPC: 884284040255</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Oviposition cups (brown)</td>
			<td>Fisher Scientific</td>
			<td>03-007-52</td>
			<td style="width:351px;">The product is actually an amber 125 mL bottle that we saw the top off of.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Recycled Paper Towels</td>
			<td style="width:116px;">Seventh Generation</td>
			<td style="width:109px;">30BPT120</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Modular Mates Square Tupperware Set</td>
			<td style="width:116px;">Tupperware</td>
			<td style="width:109px;"></td>
			<td style="width:351px;">http://order.tupperware.com/pls/htprod_www/coe$www.add_items</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Glass Grinder</td>
			<td style="width:116px;">Corning Incorporated</td>
			<td style="width:109px;">7727-2</td>
			<td style="width:351px;">These Tenbroeck tissue grinders break the eggs and release RNA into the TRI Reagent.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">TRI Reagent</td>
			<td style="width:116px;">Sigma Aldrich</td>
			<td style="width:109px;">T9424</td>
			<td style="width:351px;">Apply 1ml TRI Reagent per 50-100mg of tissue. Caution - this reagent is toxic.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:209px;">TURBO DNA-free</td>
			<td style="width:116px;">Ambion/Life Technologies</td>
			<td style="width:109px;">AM1907</td>
			<td style="width:351px;">This kit generates greater yield than traditional DNase treatment followed by phenol/chloroform cleanup, and it is simpler to use.</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:209px;">RNaseZap</td>
			<td style="width:116px;">Ambion/Life Technologies</td>
			<td style="width:109px;">AM9782</td>
			<td style="width:351px;">Apply liberally on the bench surfaces and any equipment that might be in contact with the RNA samples. The solution is slightly alkaline/corrosive, can cause irritation and is harmful when swallowed.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">2100 Bioanalyzer</td>
			<td style="width:116px;">Agilent Technologies</td>
			<td style="width:109px;">G2939AA</td>
			<td style="width:351px;">Place up to 12 RNA samples on one chip.</td>
		</tr>
		<tr height="187">
			<td height="187" style="height:187px;width:209px;">Hemotek Membrane Feeder</td>
			<td style="width:116px;">Hemotek&#160;</td>
			<td style="width:109px;">5W1</td>
			<td style="width:351px;">This system&#160; provides 5 feeding stations that can be used simultaneously. Includes PS5 Power Unit and Power cord; 5 FUI Feeders + Meal Reservoirs and O-rings; Plastic Plugs, Hemotek collagen feeding membrane; Temperature setting tool; and Plug extracting tool. The company&#39;s mailing address is: Hemotek Ltd; Unit 5 Union Court; Alan Ramsbottom Way; Great Harwood; Lancashire, UK; BB6 7FD; tel: +44 1254 889 307.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Digital Thermometer and Probe</td>
			<td style="width:116px;">Hemotek&#160;</td>
			<td style="width:109px;">MT3KFU</td>
			<td style="width:351px;">MicroT3 thermometer and KFU probe. This is used to set the temperature of each FUI feeding unit.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:209px;">Chicken Whole Blood, non-sterile with Sodium Citrate</td>
			<td style="width:116px;">Pel-Freez Biologicals</td>
			<td>33130-1</td>
			<td style="width:351px;">The 500 ml of blood were frozen and stored in 20 ml aliquots at -80 degrees C for up to 1 year.&#160; Thaw blood at room temperature for at least 1 h before using.</td>
		</tr>
	</tbody>
</table>

an experimental and bioinformatics protocol for rna-seq analyses of photoperiodic diapause in the asian tiger mosquito, aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

İki temsili RNA örneklerinin Florimetri (Şekil 1A, B) nt yaklaşık 2.000 iki bant gösterdi. böcek 28S ribozomal RNA, kolayca kısa ısıtma veya hidrojen bağları 35 bozan maddeler ile parçalanmıştır hidrojen bağları ile bir arada tutulan iki polinükleotid zincirlerinden meydana gelmektedir. Elde edilen iki bileşen yaklaşık 18S ribozomal RNA gibi aynı boyutta. ikinci RNA numunesi bozulması yüksek seviyelerde (Şekil 1B) göstermiştir. <p class="jove_c...

Watch this Scientific Journal Video about An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus at JoVE.com

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Asya Tiger Sivrisinek içinde Photoperiodic Diapause RNA-seq Analizler Deneysel ve Biyoinformatik Protokolü,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

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Research

JoVE Journal

Biology

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

13.2K Görüntüleme.  Georgetown University. RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data. 

Video: An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Sivrisinek Yetiştirme Protokolü (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Biyoloji

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

Bilgisayar Tabanlı Protein Yapısı ve İşlevi Tahmin Protokolü

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Trombin-tedavi içinde Transcriptomes Analizi ve Kontrol İnsan Pulmoner mikrovasküler endotel hücrelerinde

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

Sivrisinek Gut Metagenomic RNA-seq Ribozomal RNA tükenmesi

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Fizyolojik Kayıtlar ve Sarı-ateş Sivrisinek Tat Çıkıntılara RNA Sıralama<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Laboratuvar konut, turkuaz Killifish (Nothobranchius furzeri) için bir iletişim kuralı

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Drosophila toksisite laboratuvarda test etmek için bir omurgasız Model sistemi olarak kullanmak için deneysel protokol

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

RNA-seq Kullanarak Moleküler Evrimi ve Gen ekspresyonını Araştırmak için Biyoinformatik Boru Hattı

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Sivrisinek elektroantennografisi için adım adım kılavuz

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...