<meta charset="utf8"></head><body>用于微生物组分析的高精度蚊子器官分离

<ol>
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<table><tbody><tr><td>Alcohol 96&amp;deg;</td><td>Fisher scientific</td><td>10332562</td><td></td></tr><tr><td>Binocular magnifier&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Bleach</td><td>RAJA</td><td>145517</td><td>150 tablets of 1.5 g</td></tr><tr><td>DNA prep kit</td><td>Illumina</td><td>Provided by MGX sequencing platform</td><td>Previously known as Nextera DNA Flex</td></tr><tr><td>DNA-RNA Shield (50 mL)</td><td>Zymo research</td><td>ZR1100-50</td><td>Preservation buffer</td></tr><tr><td>DNeasy Blood and Tissue Kit&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>DNA extraction kit</td></tr><tr><td>Filter tips 20 &amp;micro;L</td><td>Starlab</td><td>S1120-3810</td><td></td></tr><tr><td>Filter tips 200 &amp;micro;L</td><td>Starlab</td><td>S1120-8710</td><td></td></tr><tr><td>Filter tips 1000 &amp;micro;L</td><td>Starlab</td><td>S1122-1730-C</td><td></td></tr><tr><td>Forceps</td><td>FST (Fine Science Tools)</td><td>11252-20</td><td>Dumont Forceps #5</td></tr><tr><td>Library quantification kit</td><td>Roche</td><td>Provided by MGX sequencing platform</td><td>KAPA Library Quanitification Kits</td></tr><tr><td>Micropipettes 2-20 &amp;micro;L</td><td>Eppendorf</td><td>6.291704</td><td></td></tr><tr><td>Micropipettes 20-200 &amp;micro;L</td><td>Eppendorf</td><td>6.291703</td><td></td></tr><tr><td>Micropipettes 100-1000 &amp;micro;L</td><td>Eppendorf</td><td>7.648488</td><td></td></tr><tr><td>Microscope slides&amp;nbsp;</td><td>Epredia</td><td>J1800BMNZ</td><td>dimension : 75 mm x 50 mm</td></tr><tr><td>Needles</td><td>Terumo</td><td>AN*2719R1</td><td></td></tr><tr><td>NGS kit&amp;nbsp;</td><td>Agilent</td><td>Provided by MGX sequencing platform</td><td>Fragment Analyzer Systems HS Genomic DNA 50kb Kit</td></tr><tr><td>NovaSeq 6000</td><td>Illumina</td><td>Provided by MGX sequencing platform</td><td>Sequencer</td></tr><tr><td>PBS Phosphate Buffered Saline (Sterile)</td><td>Fisher scientific</td><td>10212990</td><td></td></tr><tr><td>Permanent black marker</td><td></td><td></td><td></td></tr><tr><td>Sterile Eppendorf&amp;nbsp;</td><td>Dutscher</td><td>33871</td><td>1.5 mL</td></tr><tr><td>Support for needles</td><td>FST (Fine Science Tools)</td><td>26016-12</td><td>Moria MC1 Pin Holder 12 cm</td></tr></tbody></table>

dissection of mosquito ovaries, midgut, and salivary glands for microbiome analyses at the organ level

<meta charset="utf8"></head><body>作者聚焦：优化蚊子器官解剖以研究共生体和媒介微生物组

解剖蚊子卵巢、中肠和唾液腺，用于器官水平的微生物组分析

The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a ...

dissection-mosquito-ovaries-midgut-salivary-glands-for-microbiome

Research

JoVE Journal

Biology

Dissection of Mosquito Ovaries, Midgut, and Salivary Glands for Microbiome Analyses at the Organ Level

1.4K 次观看.  University of Montpellier, INRAe, CNRS, IRD. 我们的研究重点是使用宏基因组分析在单个器官规模上蚊子中的微生物组群落，我们研究卵巢、中肠和唾液腺中的共生体。通过防止解剖过程中的交叉污染，我们的目标是研究这些微生物如何在前沿尺度上影响蚊子生物学。以及添加有针对性的控制方法。处理单个器官真的很复杂，因为我们需要完整地处理它们，而不会在器官之间造成任何损坏...

视频: 作者聚焦：优化蚊子器官解剖以研究共生体和媒介微生物组

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

从AE肠和唾液腺的解剖。埃及伊蚊蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

科研

生物学

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

免疫与感染

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

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.

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

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

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

荧光 原位 杂交研究蚊子精子发生

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

作者聚焦:全安装荧光原位杂交研究按蚊精子发生  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

全安装荧光 原 位杂交研究 按 蚊精子发生

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

抗生素处理蚊子的细菌口服喂养测定

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

解剖蚊子卵巢、中肠和唾液腺，用于器官水平的微生物组分析

副本

摘要

探索更多视频

此视频中的章节

从AE肠和唾液腺的解剖。埃及伊蚊蚊

杂交

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

经口喂养和显微注射对成年蚊子进行实验性病毒感染

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生

抗生素处理蚊子的细菌口服喂养测定

恶性疟原虫通过人工膜喂养进行配子体培养和蚊子感染

解剖蚊子卵巢、中肠和唾液腺，用于器官水平的微生物组分析

副本

摘要

探索更多视频

此视频中的章节

从AE肠和唾液腺的解剖。埃及伊蚊蚊

杂交

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

经口喂养和显微注射对成年蚊子进行实验性病毒感染

全安装荧光 原 位杂交研究 按 蚊精子发生

全安装荧光 原 位杂交研究 按 蚊精子发生

全安装荧光 原 位杂交研究 按 蚊精子发生

抗生素处理蚊子的细菌口服喂养测定

恶性疟原虫 通过人工膜喂养进行配子体培养和蚊子感染

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生

恶性疟原虫通过人工膜喂养进行配子体培养和蚊子感染