gametocyte 문화의 A. 준비 : <ol><li> 신선한 인간의 혈액과 물 목욕 37 O C에 혈청을 가져와. </li><li> 무균 조건, pipet eppendorf 튜브에 따뜻한 혈액 1 ML 아래 퓸 후드에. 2,200 rpm으로 5 분간 원심 분리기에서 혈액을 돌려. 적혈구는 pelleted되어야하며 맑은 혈청을 제거하고 폐기. gametocytes에 대한 전체 혈액을 얻기 위해 새로운 혈청 3 회 중 동일한 볼륨 erythocytes 씻으십시오. </li><li> CO 2 배양기 또는 캔들 병에서 gametocyte 문화를 검색할 수 있습니다. 이 문화는 문화 일 16 일 모기에 RPMI 미디어 및 방송에서 유지 관리해야합니다. gametocytemia %를 (문화의 날 16 일)를 확인하려면 한방울의 피가있는 유리 슬라이드에 얇게 얼룩을 만들고 Geimsa 얼룩도 수행합니다. 기생충 문화의 분야에서 gametes의 수를 결정합니다. 그것은 2의 범위에서 일반적으로있다 - 4 %. 당신이 원하는 마지막 %의 gametocytemia (0.3 -1 %)를 달성해야합니다 전체 혈액의 볼륨을 계산합니다. </li><li> , pipet을 사용하여 gametocyte 문화 미디어의 대부분을 제거 방해하거나 기생충을 빨아 (미디어가 빨강 / 분홍 빛이 될 동안 기생충과 같이 검정색 나타납니다)하지 돌보는. 15 ML 팔콘 튜브에 미디어와 기생충의 나머지 Pipet. 2,200 rpm으로 5 분간 원심 분리기에 돌려. 기생충과 적혈구는 펠렛은, 제거 및 방해 기생충없이 뜨는 미디어 삭제됩니다. </li><li> 당신에게 이전 계산에 따르면 원하는 최종 gametocytemia 줄 준비가 전체 혈액의 볼륨 (단계 A2에서)와 기생충을 결합합니다. 철저하게 섞어 Pipet. (~ 1 분) </li></ol> 이것은 지금 당신이 모기에 피드됩니다 예제입니다. 물이 욕조에서 37 O C에서 보관. B. 먹이 모기 : <ol><li> 메쉬가 고무 밴드 또는 컵와 함께 제공된 판지 뚜껑을 확보할 수 그물로 덮여 왁스 늘어선 종이 컵에 아노 펠 레스 모기 (~ 30-50) 넣어. 우리는이 단계의 배터리로 작동하는 흡인기를 사용합니다. </li><li> 고무 튜브에 유리 모이통의 양쪽에 노즐을 삽입하여 순환 물 목욕을 준비합니다. 당신이 일련의 필요로 많은 모이통으로 연결하며 일련의 한쪽 끝을은 물 목욕 (피더를 통해 물들을 못살게 굴지되는 부분)의 유통자에 튜브로 연결되어 있어야합니다,이 시리즈의 다른 쪽 끝을는 고무 튜브로 구성해야 순환 물을 욕조에 다시 빈 수 있도록 욕조에 빠져들. 물 37에 대해 O C와 물을 욕조에 다시 피더를 통해 순환하고있다 때까지 목욕을 켭니다. 이것은 혈액 따뜻하게 유지하고 기생충이 살아있게하는 것이 필수적입니다. </li><li> 피더 오프닝에 단단히 밀봉 압력을 사용하여 유리 막 피더 주변 스트레칭하여 세포막을 (우리가 늘어 parafilm 작은 사각형을 사용하는) 준비. 이 막이 피부 역할을하게 될 것입니다. </li><li> 플레이스 각 모기 컵 및 클램프 또는 테이프를 사용하여 보안 모이통에 한 피더 막 측 다운. 모기는 컵 내부에서 액세스할 수 있도록 막이 그물을 잔뜩 앉아해야합니다. </li><li> 피더의 목에 조심스럽게 피펫 기생충 - 혈액 식사 (단계 A5에서), 혈액이 모기가 피를에 액세스할 수 있도록 멤브레인 문의, 저수지에 목을 처음부터 끝까지 간다 확실히 하려구요. 우리는 피더 당 혈액 200 μl에 대해 사용할 수 있지만이 다를 수 있습니다. </li><li> 모기가 먹이를 허용합니다. 우리는 그들이 ≈ 30 분 동안 먹을 수있게 (theexperiment에 따라 변수를, 대부분의 실험을 위해, 때로는 얼마나 많은가 이미 복부에있는 혈액의 콘텐츠를보고 식사를 데리고 모니터링.) </li><li> 수유 후, 모기 컵에서 피더를 제거합니다. 물 목욕을 끄고 튜브에서 모이통 연결을 끊습니다. </li><li> 멤브레인를 제거하고 오염을 제거하다와 물로 씻어 10 %의 표백제에 모이통 만끽해보세요. 이곳은 많은 생물 학적 소켓에있는 작은 생물 학적 가방 보증금으로 (세포막, 종이 수건, 장갑, 등) 자료를 오염. </li><li> 냉장고 또는 차가운 방에서 컵을 배치하여 모기를 마취. 일단 모기가 완전히 anesthetized 있으며, 얼음 양동이에 포함된 배양 접시에서 그들을 전송합니다. 정렬 모기는 혈액 식사의 기호에 대한 abdomens에서 신중하게보고하고, 피드 않았들을 폐기. 다른 사람들이 섭취 혈액의 좁은 밴드를 표시합니다 반면 어떤 모기가 완전히 부풀어 오른 것입니다, 모두는 &quot;먹이&quot;로 가능합니다. 종이 컵에 다시 먹은 모기 놔. </li><li> 모기에 설탕 식사 (10 % 자당에 배어 목화의 조각)를 제공하고, 7 일 동안 정상 insectary 조건에서 알을 품다. 매일 모기가 사망률에 대한 모니터링해야합니다. 또한, 면화는 그들이 음식을 박탈하지 않도록, 자당와 젖은하여야한다. 그것은 모기가 습한 상태로 유지하려면 증류수에 담가 종이 타월을 추가하려면 아직 더 있습니다. 이러한 모기 피 아르 falcipraum 감염된 모든 관리가 더 모기가 escap 수 없을 정도로 이동해야하므로컵에서 전자. 작은 cagefor 이중 보호에 얹어하는 것이 좋습니다. </li></ol> C. 모기가 midguts 분석 해 봅시다하고 oocyst의 하중을 추정 : <ol style="margin-top: 0in"><li style="tab-stops: list .5in"> 배터리로 작동하는 흡인기를 사용하여 종이 컵에서 대기음 모기 (단계 B에서). 이동식 흡인기 저수지 안에 마취 모기, 차가운 방에 이동하거나 적어도 5 분 또는 모기 중단 이동까지 얼음에있는 저수지를 압도하여. </li><li style="tab-stops: list .5in"> 저수지에서 얼음에 포함된 유리 페트리 접시에 anesthetized 모기를 전송합니다. 단일 레이어로 모기를 확산 플라스틱 페트리 접시의 뚜껑이있는 표지. 접시에 숙박하실 수 없습니다 모기 추운 방 또는 얼음에 반환되는 저수지에 남아 있습니다. </li><li style="tab-stops: list .5in"> 세부적으로 밀고 집게를 사용하여 해부 현미경 아래에 탑재 유리 슬라이드에 포함된 100 UL PBS로 요리에서 단일 모기를 전송합니다. 냉장 페트리 접시에 나와 다른 둡니다. </li><li style="tab-stops: list .5in"> 세부적으로 밀고 집게를 사용하여, 흉부 및 복부 뒷부분에서 모기를 개최. midgut 노출 때까지 분리 시체를 당겨. gut.The 뱃속에서 초과 조직을 제거하는 집게를 사용하여이 흰색처럼 몸 색처럼 보인다. 0.2 %가 mercurochrome의 5-10 μl의 12 잘 유리 슬라이드 및 담가 잘 하나 gut.Mount 창자에서 여분의 조직을 제거합니다. 호박은 12 자 슬라이드 때까지 다른 모기를 위해 젖은 종이 towel.Continue에 시체와 폐기가 가득 찼습니다. 유리 coverslip으로 탑재 용기를 커버. </li><li style="tab-stops: list .5in"> 희미한 / 맑은 분홍색 굿 조직에 대한 밝은 분홍색 얼룩 원형 oocysts를 찾고, 가벼운 현미경으로 배짱을 검사합니다. , oocysts 백작 기록하고 다음 예제를 계속합니다. 모든 실험 치료에서 모기 모든 계속합니다. </li><li style="tab-stops: list .5in"> 완료되면, 종이 타월로 깨끗이 닦아 슬라이드. 작은 생물 학적 가방으로, 용기와 종이 타월, 그리고 시체와 함께 종이 타월을 놓고 쓰레기통에 폐기합니다. 장갑이 절차 전반에 걸쳐 착용합니다. 치료는 감염된 모기 탈출하게하지 않도록합니다. </li></ol>

<ol>
	<li>Avila, S. L., Ferreira, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+diagnosis:+a+review.">Malaria diagnosis: a review.</a> Braz J Med Biol Res. 29 (4), 431-443 (1996).</li><li>Bell, A. S., Ranford-Cartwright, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+PCR+assay+for+quantifying+Plasmodium+falciparum+infections+in+the+mosquito+vector.">A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.</a> Int J Parasitol. 34 (7), 795-802 (2004).</li><li>Collins, K. M., Hochberg, L. P., Ryan, P. R., Collins, W. E., Wirtz, R. A., Ryan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Plasmodium+malariae+infection+in+mosquito+vectors.">Quantification of Plasmodium malariae infection in mosquito vectors.</a> Ann Trop Med Parasitol. 98 (5), 469-472 (2004).</li><li>Czeher, C., Labbo, R., Djibrilla, A., Here, L., Arzika, I., Duchemin, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA+study+of+oocyst-sporozoite+transition+in+malaria+vectors.">ELISA study of oocyst-sporozoite transition in malaria vectors.</a> Parasite. 13 (3), 257-261 (2006).</li><li>Haghdoost, A. A., Mazhari, S., Bahadini, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+results+of+light+microscopy+with+the+results+of+PCR+method+in+the+diagnosis+of+Plasmodium+vivax.">Comparing the results of light microscopy with the results of PCR method in the diagnosis of Plasmodium vivax.</a> J Vector Borne Dis. 43 (2), 53-57 (2006).</li><li>Patsoula, E., Spanakos, G., Sofianatou, D., Parara, M., Vakalis, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-step,+PCR-based+method+for+the+detection+and+differentiation+of+Plasmodium+vivax.">A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax.</a> Ann Trop Med Parasitol. 97 (1), 15-21 (2003).</li><li>Osta, M. A., Christophides, G. K., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mosquito+genes+on+Plasmodium+development.">Effects of mosquito genes on Plasmodium development.</a> Science. 303 (5666), 2030-2032 (2004).</li><li>Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Dimopoulos, G., Kafatos, F. C., Adams, J. H., Fujioka, H., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Plasmodium+and+Anopheles+transcriptomes+during+oocyst+differentiation.">Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation.</a> J Biol Chem. 279 (7), 5581-5587 (2004).</li></ol>

이 Plasmodium 감염 및 감염 표현형 결정에서 일부 차이를 보일 수 있으며, 다른 연구소는 다른 기술을 따라 수 있습니다.

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

유전자가 말라리아에 대한 잠재적 내화물로 식별되면, 그것은 모기 이내 Plasmodium 감염을 예방의 역할에 대해 평가해야합니다. 이 프로토콜은 모기의 plasmodium 감염의 범위가 assayed 수있는 방법을 보여줍니다. gametocyte 문화, 멤브레인 수유 모기 인간의 혈액을 준비하고 모기 midgut에서 바이러스 titers을 시금에 대한 기술은 증명됩니다.

Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

모기와 감염 표현형 결정에서 Plasmodium falciparum 감염에 대한 프로토콜

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

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17.8K 조회수.  Johns Hopkins University. 유전자가 말라리아에 대한 잠재적 내화물로 식별되면, 그것은 모기 이내 Plasmodium 감염을 예방의 역할에 대해 평가해야합니다. 이 프로토콜은 모기의 plasmodium 감염의 범위가 assayed 수있는 방법을 보여줍니다. gametocyte 문화, 멤브레인 수유 모기 인간의 혈액을 준비하고 모기 midgut에서 바이러스 titers을 시금에 대한 기술은 증명됩니다.

비디오: Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

모기 (A. aegypti)와 감염 표현형 결정에 뎅그열 감염 프로토콜

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

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Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

성인 모기 (A. gambiae)에서 RNAi의 Assays을위한 프로토콜

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

버딩 효모에서 단백질 농도의 함수로 형태학의 표현형의 발전 분석

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

차 소장 Organoid 문화에서 레트로 바이러스 감염의 비디오 프로토콜

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

말라리아 모기 전송하기위한 다중 탐지 분석

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

이미지 분석을위한 클록 스캔 프로토콜 : ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

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.

실험실 주택의 청록색 송사리 (Nothobranchius furzeri)에 대 한 프로토콜

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

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

세균성 감염 및 Shedding 초파리 melanogaster 에

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

모기에 소설 Insecticidal 화학 물질의 독성을 테스트 하기 위한 프로토콜

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

모기의 벡터 역량을 분석 하는 방법으로 타 액 분 비를 강제

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...