تشريح المعي المتوسط ​​وأنسجة الغدة اللعابية من البعوض ويتطلب التحضير المسبق للفوسفات 1X التخزين المؤقت المالحة (1X PBS) الحل والتخدير من البعوض عن طريق إخضاع لدرجة حرارة 4 درجات مئوية ، وحتى وظائفها. البعوض لا تزال تخدير عبر وضعها في طبق بيتري أن يتم الاحتفاظ الباردة على الجليد. وتشمل غيرها من المواد المطلوبة : مجهر الضوء مزودة الهدف 10X ، pipettor ، ملقط الجميلة ذات الرؤوس ، شريحة زجاجية ، تحقيقات إبرة معلومات سرية. المعي المتوسط ​​تشريح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع قطرة من 1X PBS على شريحة زجاجية شنت تحت المجهر الخفيفة. </li><li style=";text-align:right;direction:rtl"> نقل البعوض على الشريحة التي أعدتها طعن في الصدر مع التحقيق الذي تجريه البعوض إبرة معلومات سرية. </li><li style=";text-align:right;direction:rtl"> أثناء الضغط باستمرار على البعوض مع التحقيق ، واستخدام الملقط لفهم الجزء الثاني الماضي ، وعلى البطن برفق خارج البطن البعوض في حركة واحدة. ينبغي أن تظل المعي المتوسط ​​تعلق على الصدر وظائفها. </li><li style=";text-align:right;direction:rtl"> تجاهل البطن. استخدام ملقط لفصل المعي المتوسط ​​من القفص الصدري. </li></ol> تشريح الغدة اللعابية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع قطرة من 1X PBS على شريحة زجاجية شنت تحت المجهر الخفيفة. </li><li style=";text-align:right;direction:rtl"> تلتقط بواسطة البعوض stabbling القفص الصدري مع التحقيق الذي تجريه إبرة معلومات سرية. </li><li style=";text-align:right;direction:rtl"> سحب قبالة أرجل البعوض باستخدام أصابعك. </li><li style=";text-align:right;direction:rtl"> نقل البعوض على الشريحة. </li><li style=";text-align:right;direction:rtl"> إزالة رأس البعوض باستخدام ملقط. </li><li style=";text-align:right;direction:rtl"> أثناء الضغط باستمرار على الصدر البعوض مع التحقيق ، واستخدام مسبار آخر لدفع برفق لأسفل على القفص الصدري. </li><li style=";text-align:right;direction:rtl"> تقع الغدد اللعابية في الجزء الأمامي من الصدر ، ويمكن أن تكون معزولة عن طريق استخدام معلومات سرية التحقيق الإبرة ، وقطع المرفقات التي تربط الغدة إلى القفص الصدري. وتتكون الغدد اللعابية سليمة من ثلاثة فصوص : اثنان فصوص الفص الجانبي وسطي. </li></ol>

The authors have nothing to disclose.

dissection of midgut and salivary glands from ae. aegypti mosquitoes

المعى المتوسط ​​في البعوض والغدد اللعابية هي مفتاح الدخول والخروج لمسببات الأمراض مثل الطفيليات المتصورة وفيروسات حمى الضنك. هذا البروتوكول يوضح تقنيات الفيديو تشريح لإزالة الغدد اللعابية والمعي المتوسط ​​من الزاعجة المصرية البعوض.

Watch this Scientific Journal Video about Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes at JoVE.com

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

المعى المتوسط ​​في البعوض والغدد اللعابية هي مفتاح الدخول والخروج لمسببات الأمراض مثل الملاريا المنجلية ناقلات وفيروس حمى الضنك. هذا الفيديو يوضح تشريح تقنيات لإزالة الغدد اللعابية والمعي المتوسط ​​من الزاعجة المصرية البعوض.

تشريح الغدد اللعابية والمعي المتوسط ​​من AE. بعوض البعوض

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

dissection-of-midgut-and-salivary-glands-from-ae-aegypti-mosquitoes

Research

JoVE Journal

Biology

34.8K Views.  University of California, Irvine (UCI). المعى المتوسط ​​في البعوض والغدد اللعابية هي مفتاح الدخول والخروج لمسببات الأمراض مثل الملاريا المنجلية ناقلات وفيروس حمى الضنك. هذا الفيديو يوضح تشريح تقنيات لإزالة الغدد اللعابية والمعي المتوسط ​​من الزاعجة المصرية البعوض.

Video: Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

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.

بروتوكول للعدوى حمى الضنك في البعوض (بعوض ألف) ، وتحديد النمط الظاهري العدوى

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 ...

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.

تهجين<em&gt; في الموقع</em&gt; من الغدد اللعابية والمبيض والأجنة من البعوض الحامل للمرض

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

Immunology and Infection

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.

التحليل الفينوتي لطفيليات الملاريا القوارض غير الجنسية ومراحل الدم الجنسي ومراحل البعوض

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

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

انتشار الطفيلي Microsporidian Edhazardia aedis في Aedes aegypti البعوض

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

تغذية وقياس الدم المشتق من الحيوانات والوجبات الاصطناعية في البعوض Aedes aegypti

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

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.

العدوى الفيروسية التجريبية في البعوض البالغ عن طريق التغذية الفموية والحقن المجهري

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

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.

بلازموديوم فالسيباروم ثقافة الخلايا الجامائية والعدوى البعوض من خلال تغذية الغشاء الاصطناعي

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

دراسة نشاط الببتيدات العصبية وغيرها من منظمات نظام الإخراج في البعوض البالغ

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

تشريح الغدد اللعابية اليرقات من البعوض الغامبي Anopheles و المناعة

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

تشريح الغدد اللعابية والمعي المتوسط من AE. بعوض البعوض

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بروتوكول للعدوى حمى الضنك في البعوض (بعوض ألف) ، وتحديد النمط الظاهري العدوى

تهجين

التحليل الفينوتي لطفيليات الملاريا القوارض غير الجنسية ومراحل الدم الجنسي ومراحل البعوض

انتشار الطفيلي Microsporidian Edhazardia aedis في Aedes aegypti البعوض

تغذية وقياس الدم المشتق من الحيوانات والوجبات الاصطناعية في البعوض Aedes aegypti

العدوى الفيروسية التجريبية في البعوض البالغ عن طريق التغذية الفموية والحقن المجهري

بلازموديوم فالسيباروم ثقافة الخلايا الجامائية والعدوى البعوض من خلال تغذية الغشاء الاصطناعي

دراسة نشاط الببتيدات العصبية وغيرها من منظمات نظام الإخراج في البعوض البالغ

تشريح الغدد اللعابية اليرقات من البعوض الغامبي Anopheles و المناعة

الإدارة الفعالة لتداخل الحمض النووي الريبي عن طريق الفم (RNAi) للبعوض الأنوفيلة الغامبية البالغة

تشريح الغدد اللعابية والمعي المتوسط ​​من AE. بعوض البعوض

Summary

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Chapters in this video

بروتوكول للعدوى حمى الضنك في البعوض (بعوض ألف) ، وتحديد النمط الظاهري العدوى

تهجين

التحليل الفينوتي لطفيليات الملاريا القوارض غير الجنسية ومراحل الدم الجنسي ومراحل البعوض

انتشار الطفيلي Microsporidian Edhazardia aedis في Aedes aegypti البعوض

تغذية وقياس الدم المشتق من الحيوانات والوجبات الاصطناعية في البعوض Aedes aegypti

العدوى الفيروسية التجريبية في البعوض البالغ عن طريق التغذية الفموية والحقن المجهري

بلازموديوم فالسيباروم ثقافة الخلايا الجامائية والعدوى البعوض من خلال تغذية الغشاء الاصطناعي

دراسة نشاط الببتيدات العصبية وغيرها من منظمات نظام الإخراج في البعوض البالغ

تشريح الغدد اللعابية اليرقات من البعوض الغامبي Anopheles و المناعة

الإدارة الفعالة لتداخل الحمض النووي الريبي عن طريق الفم (RNAi) للبعوض الأنوفيلة الغامبية البالغة

تشريح الغدد اللعابية والمعي المتوسط من AE. بعوض البعوض