Name: methods for the extraction of endosymbionts from the whitefly bemisia tabaci
Uploaded: 2017-06-19T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

이 연구에 대한 재정 지원은 국가 핵심 연구 및 개발 프로그램 (2016YFC1200601)과 중국 국립 자연 과학 재단 (31390421)에 의해 제공되었습니다.

1. Whitefly 양육과 비밀 종 식별 <ol><li> 27 ± 1 ° C, 습도 70 ± 10 %, 빛 14 시간 : 어두운 곳에서 10 시간 동안 표준 조건에서 우리 속의 Gossypium hirsutum (Malvaceae) (cv. Zhe-Mian 1973)에 흰 반점 종을 유지한다. </li><li> 개별 성인 흰 반점을 수집하고 30 μl의 용해 버퍼 (10 MM 트리스, pH 8.4, 50 MM KCl, 0.45 % [wt / vol] Tween - 20, 0.2 % [wt / vol] 젤라틴, 0.45 % [vol / vol] Nonidet P 40, 60 g / mL 프로테이나 제 K). </li><li> 65 ° C에서 1 시간 동안 그리고 100 ° C에서 10 분 동안 균질 물을 품어 낸다. 참고 : 필요할 경우 65 ° C에서 배양 시간을 늘릴 수 있습니다. 100 ℃에서의 배양 시간은 Proteinase K를 충분히 비활성화시키고 DNA 손상을 피하기 위해 비판적으로 조절되어야합니다. </li><li> 미토콘드리아 시토크롬 옥시 다제 I 프라이머를 기반으로 한 whitefly DNA (1.3 절에서 획득)를 사용하여 중합 효소 연쇄 반응 (PCR)을 수행하십시오. COI-F : 5&#39;-TTGATTTTTTGGTCATCCAGAAGT-3 &#39; COI-R : 5&#39;-TAATATGGCAGATTAGTGCATTGGA-3 &#39; <ol><li> 1 U의 Taq DNA 중합 효소, 2.5 μL 10X 버퍼, 0.2 MM dNTP, 각 프라이머의 0.2 MM 및 whitefly DNA의 2 μL를 포함하는 25 μL의 최종 볼륨에서 PCR 반응을 수행하십시오. </li><li> 다음의 PCR 절차 조건을 사용하십시오 : 95 ℃에서 3 분간 변성 후 95 ℃에서 45 초 (변성), 50 ℃에서 45 초 (어닐링) 및 72 ℃에서 1 분간 (연장) 35 사이클 추가 확장을 위해 72 ℃에서 10 분. </li></ol></li><li> 권장 된 프로토콜로 DNA 겔 추출 키트를 사용하여 PCR 증폭 제품을 청소하십시오. </li><li> DNA 샘플 시퀀싱 시스템으로 제조사의 지시에 따라 정제 된 DNA 샘플을 시퀀싱합니다. </li><li> BLAST (Basic Local Alignment Search Tool)를 사용하여 시퀀스를 분석하여 가자미 종의 식별 및 다른 특정 종의 오염 방지es. </li></ol> 2. 내분 해 식별 및 지역화 <ol><li> 흰 둥지 내의 각 endosymbiont의 특정 유전자를 증폭하기 위해 whitefly DNA (1.3 단계에서 얻음)에서 PCR을 수행하십시오 (PCR 처방은 1.4 절에 설명되어 있으며 PCR 절차와 프라이머는 표 1 에 나와 있습니다). </li><li> 이전에 발표 된 프로토콜 20 (1 % 아가로 오스 겔, 실행 버퍼로 트리스 - 아세테이트 - EDTA, 전압 5V / cm)에 따라 증폭 된 샘플을 아가로 오스 겔 전기 영동으로하여 각 세균의 특정 유전자를 확인하십시오. </li><li> 흰 반점 내에서 박테리아 종을 결정하기 위해 각 밴드를 복구하고 순서를 정하십시오 (1.5-1.7 절 참조). </li><li> 이전에 발표 된 프로토콜 19 에 따라 endosymbionts의 위치를 ​​식별하기 위해 whiteflies에 형광 in situ hybridizations (FISH)를 수행하십시오. 참고 : 필요한 경우 형광 탐침의 농도를 높이십시오. </li></ol&gt; 3. 투과 전자 현미경 (Transmission Electron Microscopy, TEM) <ol><li> 2.5 % [vol / vol] 글루 타르 알데히드를 함유 한 인산염 완충액 (0.1 M, pH 7.0)에서 흰 반점을 4 시간 동안 담그고 고정시킨다. </li><li> 인산염 완충액 (0.1 M, pH 7.0)에서 시료를 3 회, 매회 15 분 동안 씻으십시오. </li><li> 1 % [wt / vol] OsO 4 를 함유 한 인산염 완충액 (0.1 M, pH 7.0)에 시료를 2 시간 동안 담근다. </li><li> 매번 15 분씩 일련의 에탄올 (30 %, 50 %, 70 %, 80 %, 90 %, 95 % 및 100 %)로 샘플을 탈수하십시오. </li><li> 샘플을 100 % 아세톤에 20 분 동안 담그고 담그십시오. </li><li> 실온에서 1 시간 동안 100 % 아세톤과 100 % Spurr 수지 (1 : 1)의 혼합물에 샘플을 놓습니다. </li><li> 실온에서 3 시간 동안 100 % 아세톤 및 100 % 스퍼 르 수지 (1 : 3)의 혼합물에 샘플을 옮긴다. </li><li> 전달하고 샘플을 100 % Spurr 수지 (70 ° C에서 잠복)에 9 시간 이상 담그십시오. </li><li> 마이크로를 사용하여 흰 반점 샘플 섹션나에게. </li><li> 5 분에서 10 분 동안 절대 우라 닐 아세테이트에 담금으로써 초박막 (섹션 3.9에서 획득)에 얼룩을지게하고, 5 ~ 10 분 동안 50 % 알칼리성 납 시트 레이트에 담그십시오. 참고 : 섹션 3.1-3.10에서 사용 된 시약의 부피는 샘플에 따라 다릅니다. 샘플이 시약에 담겨 있는지 확인하십시오. </li><li> 투과 전자 현미경 (15,000X)에서 샘플을 관찰하여 추가로 사용할 박테리아의 위치, 모양 및 크기를 결정합니다 (4.6 절 참조). </li></ol> 4. 흰 파리 박테리아의 해부 및 정제 <ol><li> 현미경 슬라이드에 1x PBS 용액 100 μL를 추가합니다. 면화 잎에서 3, 4 th instar nymph를 골라 PBS 용액에 담그십시오. </li><li> 실체 현미경으로 미세 곤충 바늘을 사용하고 흰 반점 몸체에서 세균들을 꺼내십시오. <ol><li> 요정의 한쪽에있는 구멍을 부드럽게 자르고, 다른 쪽을 살짝 누릅니다.bacteriomes를 내보내는 쪽. </li></ol></li><li> 0.5-10 μL 피펫으로 20 μL 마이크로 로더를 장착하십시오 (박테리아의 크기에 맞게 끝 부분을 자릅니다). PBS 용액에서 개별 세균을 추출하여 마이크로 로더에 넣습니다. </li><li> bacteriome을 PBS 용액에 pipetting하고 박테리아를 추출하여 다른 흰 반점 조직의 오염을 제거하십시오. 3 번 반복하십시오. </li><li> 즉시 1x PBS 용액 60 μL가 들어있는 원심 튜브에 세균을 세척합니다. </li><li> 5 μm 필터 멤브레인을 통해 조립 된 박테리아를 주사기로 걸러냅니다 (박테리아의 크기와 흰 반점이있는 박테리아 세포의 핵으로 결정됨). 혼합 된 액체를 필터 멤브레인으로 완전히 이동 시키려면 여러 번 반복하십시오. 참고 : 더 나은 증폭 및 시퀀싱 결과를 얻으려면 100 개가 넘는 박테리아를 조립하십시오. 박테리아의 손실을 줄이기 위해 1x PBS 용액을 사용하여 필터 멤브레인을 먼저 적시십시오.막에 결합. </li></ol> 5. Endosymbiont 게놈의 증폭 <ol><li> 일부 수정과 함께 권장 프로토콜에 의해 DNA 증폭 키트를 사용하여 여액 (주로 흰 반점의 endosymbionts을 포함)을 증폭. <ol><li> 혈액 또는 세포에서 게놈 DNA의 증폭 프로토콜을 선택하고 이후 실험을 위해 버퍼 D2를 준비하십시오. </li><li> 원심 분리 튜브에 여과 액 1.5 μL (4.6 절 참조)을 직접 넣은 다음 1.5 μL의 Buffer D2를 넣고 잘 섞는다. </li><li> 얼음 위에서 10 분 동안 인큐베이션하고 1.5 ㎕의 정지 용액을 첨가한다. </li><li> 반응 완충액 15 μL와 DNA 중합 효소 1 μL를 첨가하고 부드럽게 섞는다. </li><li> 혼합물을 30 ° C에서 16 시간 동안 배양하고, 65 ° C에서 3 분간 교반한다. </li></ol></li><li> 박테리아를 위해 고안된 특정 프라이머를 사용하여 증폭 된 여과 액에서 PCR을 직접 수행하십시오 (필요한 경우 증폭 된 시료를 희석하십시오).박테리아 종을 확인하기 위해 테리아 (2.1 절 참조). </li><li> 이전에 발표 된 방법 21 (PCR 처방은 1.4 절에서 설명 됨)에 따라 whitefly 유전자 베타 - 액틴 및 EF1 프라이머를 사용하여 숙주 게놈의 오염 여부를 확인하기 위해 PCR을 실시하십시오. </li></ol> 6. Endosymbiont Metagenome 시퀀싱 <ol><li> 샘플의 품질을 테스트하고 시퀀싱을위한 기준을 충족하는지 여부를 확인하기 위해 시퀀싱 전에 증폭 된 여액을 분광 광도계 및 형광 측정기 (제조사의 지침에 따라)에 부탁하십시오. </li><li> 제작사의 지시에 따라 게놈 시퀀서에 증폭시킨다. </li></ol>

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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=Differential+temporal+changes+of+primary+and+secondary+bacterial+symbionts+and+whitefly+host+fitness+following+antibiotic+treatments.">Differential temporal changes of primary and secondary bacterial symbionts and whitefly host fitness following antibiotic treatments.</a> Sci. Rep. 5, 15898 (2015).</li><li>Shan, H. W., 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=Temporal+changes+of+symbiont+density+and+host+fitness+after+rifampicin+treatment+in+a+whitefly+of+the+Bemisia+tabaci+species+complex.">Temporal changes of symbiont density and host fitness after rifampicin treatment in a whitefly of the Bemisia tabaci species complex.</a> Insect Sci. 23 (2), 200-214 (2016).</li><li>Zchori-Fein, E., Brown, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+prokaryotes+asscociated+with+Bemisia+tabaci+(Gennadius)+(Hemiptera:+Aleyrodidae).">Diversity of prokaryotes asscociated with Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae).</a> Ann. Entomol. Soc. Am. 95 (6), 711-718 (2002).</li><li>Thao, M. L., Baumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+relationships+of+primary+prokaryotic+endosymbionts+of+whiteflies+and+their+hosts.">Evolutionary relationships of primary prokaryotic endosymbionts of whiteflies and their hosts.</a> Appl. Environ. Microbiol. 70 (6), 3401-3406 (2004).</li><li>Zchori-Fein, E., Perlman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+the+bacterial+symbiont+Cardinium+in+arthropods.">Distribution of the bacterial symbiont Cardinium in arthropods.</a> Mol. Ecol. 13 (7), 2009-2016 (2004).</li><li>O'Neill, S. L., Giordano, R., Colbert, A. M., Karr, T. L., Robertson, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=116S+rRNA+phylogenetic+analysis+of+the+bacterial+endosymbionts+associated+with+cytoplasmic+incompatibility+in+insects.">116S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects.</a> Proc. Natl. Acad. Sci. U. S. A. 89 (7), 2699-2702 (1992).</li><li>Nirgianaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+infections+of+the+whitefly+Bemisia+tabaci.">Wolbachia infections of the whitefly Bemisia tabaci.</a> Curr. Microbiol. 47 (2), 93-101 (2003).</li><li>Everett, K. D., Thao, M. L., Horn, M., Dyszynski, G. E., Baumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+chlamydiae+in+whiteflies+and+scale+insects:+endosymbionts+'Candidatus+Fritschea+bemisiae'+strain+Falk+and+'Candidatus+Fritschea+eriococci'+strain+Elm.">Novel chlamydiae in whiteflies and scale insects: endosymbionts 'Candidatus Fritschea bemisiae' strain Falk and 'Candidatus Fritschea eriococci' strain Elm.</a> Int. J. Syst. Evol. Micr. 55, 1581-1587 (2005).</li></ol>

저자는 공개 할 것이 없습니다.

Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga...

상대적 숙주와 친밀한 공생 관계를 형성하는 박테리아는 절지 동물에서 널리 퍼져있다 1 . endosymbionts는 거의 모든 발달 단계 5 에서 영양 대사, 번식, 환경 스트레스에 대한 반응 2 , 3 , 4 등과 같은 숙주의 양상에 영향을 미치는 것으로 입증되었습니다. 그러나 협회를 뒷받침하는 메커니즘은 아직 많이 알려져 있지 않습니다. 유전체학은 박테리아의 잠재적 인 기능과 역할을 연구 할 때 우선 순위이며 중요합니다. 분류 학적 상태, 기능 유전자, 대사 경로, 분비 시스템과 같은 몇 가지 기본 정보는 공생에서 공생 공생체의 잠재적 역할에 대한 조명을 밝히는 게놈 서열로부터 추론 할 수 있습니다. 높은 처리량 시퀀싱의 개발로 방대한 세균 게놈이 시퀀싱되었습니다여러 가지 기능이 밝혀졌다. 내배체는 진딧물 7 , 벌레 8 , 유문 9 , 갈색 planthoppers 10 및 매미 11 과 같은 반월에서 매우 중요합니다. 예를 들어, 진딧물의 Buchnera 는 필수적인 공생체 로서 진딧물 유전체 12 의 유전자와 함께 필수 아미노산 생합성에 관여하는 것으로 입증되었습니다. 또한 Buchnera의 전사 조절 또한 밝혀졌다. psyllids에서는 Carsonella 가 서열화되어 가장 작은 박테리아 게놈을 14 번 발견했습니다. endosymbionts의 이러한 모든 특징은 게놈 서열을 기반으로하고 유추됩니다. 이러한 endosymbionts가 시험 관내 에서 배양 될 수 없기 때문에, seq에 적합한 박테리아를 분리하기 위해 여러 가지 접근법이 적용되었습니다어젠 싱. 진딧물에서, endosymbionts는 원심 분리 및 여과를 통해 추출하고 추가 게놈 및 transcriptomic 분석 5에 적용 됩니다. 갈색 딱정벌레에서는 곤충 게놈 전체와 함께 내분비물이 서열화됩니다 10 . Whitefly B. tabaci 는 35 종 이상의 형태 학적으로 구분할 수없는 종 (비밀 종)을 포함하고있는 종 복합체이며, 그 중 2 종의 침입 종이 전 세계에 침입하여 농산물에 막대한 피해를 입혔습니다 15 . 주목할 점은 B. tabaci 종의 내분비는 해충의 발생에 중요성을 보여 주었다. 현재까지, 8 종의 endosymbiont가 흰 반딧불에서 확인되었다. 그 중 하나는 obligent symbiont, Candidatus Portiera aleyrodidarum, 7 명의 보조 공생 자인 Hamiltonella , Rickettsia , Arsenophonus , Cardinium ,em&gt; Wolbachia, Fritschea 및 Hemipteriphilus 는 17 , 18을 정의했다. 이전에 기술 된 반 형제와 달리 흰 반점 B. tabaci 는 길이가 1mm에 불과한 매우 작은 벌레입니다. 대부분의 endosymbionts는 bacteriocytes 19 ( B. tabaci 에서 세균을 더 형성하는 symbionts를 포함하는 특수 세포)에 국한되어 있습니다. 또한, 이러한 내재화는 시험 관내에서 배양 할 수 없다. B. tabaci 에서 endosymbionts를 얻는 유일한 방법은 박테리아를 해부하는 것입니다. 그러나 해부학에 어려움이 있습니다. 첫째, 깨지기 쉬운 세균은 흰 반점의 다른 조직과 항상 연결되어 분리되기 어렵습니다. 두 번째로, 흰 반점의 작은 크기는 충분한 박테리아의 고립을 제한합니다. 세 번째로, 세균 내에서 내재물 (endosymbion)이 번식하여 한 종의 세균을 얻기가 매우 복잡합니다.

<table><tbody><tr><td>&lt;em&gt;Taq&lt;/em&gt; DNA polymerase</td><td>Takara</td><td>R001A</td><td>including rTaq, 10&amp;times;Buffer and dNTP</td></tr><tr><td>Gel DNA extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>DNA sample sequencing system</td><td>ABI</td><td>ABI-3730XL</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>EM UC7</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7650 TEM</td><td></td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>20 &amp;mu;L microloader</td><td>Eppendorf</td><td>F2771951</td><td></td></tr><tr><td>Filter holder</td><td>Millipore</td><td>SX0001300</td><td></td></tr><tr><td>Filter membrane filter</td><td>Millipore</td><td>SMWP001300</td><td>5.0 &amp;mu;m SMWP</td></tr><tr><td>REPLI-g UltraFast Mini Kit</td><td>Qiagen</td><td>150033</td><td>DNA amlification kit</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Genome Sequencer</td><td>Illumina</td><td>Hiseq 2000</td><td></td></tr></tbody></table>

methods for the extraction of endosymbionts from the whitefly bemisia tabaci

박테리아 공동체는 그들의 숙주와 친밀한 관계를 형성하고 대부분의 경우 숙주에게 이점을줍니다. 유전체 정보는 숙주에서 세균 공생체의 기능과 진화를 연구하는 데 중요합니다. 대부분의 공생 균 은 시험 관내 에서 배양 될 수 없으므로, 게놈 시퀀싱을위한 박테리아의 적절한 양을 분리하는 방법은 매우 중요합니다. 흰 반점 Bemisia tabaci 에서 여러 가지 내인성이 확인되었고 여러 가지 접근법을 통해 해충의 발생과 번식에 중요 할 것으로 예측됩니다. 그러나 협회를 뒷받침하는 메커니즘은 아직 많이 알려져 있지 않습니다. 장애물은 부분적으로 세균 증식 억제 물질 인 흰가루병에있는 내막 곰팡이가 숙주 세포에서 분리되기 어렵다는 사실에서 기인합니다. 여기에서 우리는 주로 해면체 B. tabaci 에서 endosymbionts의 동정, 추출 및 정제를위한 단계별 프로토콜을보고한다 .켜고 여과하십시오. 다른 endosymbiont 종의 혼합물이긴하지만,이 방법에 의해 준비된 endosymbiont 샘플은 B. tabaci 에서 endosymbionts의 가능한 역할의 후속 게놈 시퀀싱 및 분석에 적합합니다. 이 방법은 다른 곤충에서 내재물을 분리하는 데 사용될 수도 있습니다.

B. tabaci 단지의 중동 아시아 소아 1 (MEAM1) 종은 여기서 설명을 위해 예로 취했다. 흰가루병을 키우기위한 면화와 흰가루병의 여러 발달 단계가 면화 식물, 성충과 흰빛 반딧불이의 1 , 2, 4 번 족장 약충을 포함하여 그림 1에 나와 있습니다 (3 번 황충은 4 번 황후 요정과 유사하게 보입니다. ). 4 th instar 님?...

Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

여기, 우리는 해부 및 여과를 통해 흰 반점 Bemisia tabaci 에서 endosymbionts를 분리하는 프로토콜을 제시한다. 증폭 후, DNA 샘플은 endosymbionts와 whitefly 사이의 mutualism의 후속 시퀀싱 및 연구에 적합합니다.

Whitefly에서 Endosymbionts의 추출을위한 방법<em&gt; 베미 시아 타바시</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

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Biology

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

9.8K 조회수.  Zhejiang University.  여기, 우리는 해부 및 여과를 통해 흰 반점 Bemisia tabaci 에서 endosymbionts를 분리하는 프로토콜을 제시한다. 증폭 후, DNA 샘플은 endosymbionts와 whitefly 사이의 mutualism의 후속 시퀀싱 및 연구에 적합합니다. 

비디오: Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

연구

환경과학

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

생물 학적 문화와 진딧물 nerii에 유전자 발현 측정 유지: 식물 곤충 상호 작용에 대 한 비-모델 시스템

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

유전학

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

Bemisia tabaci Cytogenetic 기술을 사용 하 여 계란 수정 속도 결정

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

루프-중재 등온 증폭 (램프) 분석 결과 Bemisia tabaci 의 빠른 식별

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

형광<em&gt; 현장에서</em바이러스 및 식물과 곤충 조직에서 Endosymbiotic 박테리아의 지역화를위한&gt; 혼성화 (FISH)

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

면역학 및 감염병학

Trichogramma dendrolimi 및 Venom Extraction에 의한 인공 숙주 기생

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

저자 스포트라이트: Trichogramma Parasitoid Wasps의 효율적인 독 추출 방법 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

인공 숙주를 사용하여 기생 말벌 Trichogramma dendrolimi 에서 독 추출

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

생화학

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented.

가루이을 사용하여 전송하는 식물 바이러스

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

생물학

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

면역형광 및 정량적 PCR에 의한 화이트플라이 조직에서 베고모바이러스의 국소화 및 정량화

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

Whitefly에서 Endosymbionts의 추출을위한 방법

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Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

생물 학적 문화와 진딧물 nerii에 유전자 발현 측정 유지: 식물 곤충 상호 작용에 대 한 비-모델 시스템

Bemisia tabaci Cytogenetic 기술을 사용 하 여 계란 수정 속도 결정

루프-중재 등온 증폭 (램프) 분석 결과 Bemisia tabaci 의 빠른 식별

형광

인공 숙주를 사용하여 기생 말벌 Trichogramma dendrolimi 에서 독 추출

인공 숙주를 사용하여 기생 말벌 Trichogramma dendrolimi 에서 독 추출

인공 숙주를 사용하여 기생 말벌 Trichogramma dendrolimi 에서 독 추출

가루이을 사용하여 전송하는 식물 바이러스

면역형광 및 정량적 PCR에 의한 화이트플라이 조직에서 베고모바이러스의 국소화 및 정량화