<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לבדוק אם כנימה מסוגל וקטור וירוס, אנו להאכיל את הכנימות על רקמות נגועים עבור 2 ימים. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, אנו מאפשרים את זה כדי להאכיל במפעל בריאה למשך 5 ימים. המפעל חיטוי אז אחרי 3 שבועות אנחנו מתחילים לחפש את התסמינים. </li><li style=";text-align:right;direction:rtl"> כאשר אנו מוצאים כי הכנימה לא יכול וקטור וירוס, אנחנו יכולים להעריך מי מבין 2 מחסומים היא לעצור את הנגיף באמצעות הליך microinjection. </li><li style=";text-align:right;direction:rtl"> הווירוס מוזרק ישירות לתוך hemocoel, ובכך לעקוף את מחסום המעיים הילוכים. </li></ol> בקר <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.arplant.59.032607.092825">ביקורות שנתי של ביולוגיה הצמח</a> לקבלת מידע נוסף על חסינות פרוטוקול צמח זה herbivory חרקים.

The authors have nothing to disclose.

testing the physiological barriers to viral transmission in aphids using microinjection

תפוחי אדמה loafroll וירוס (PLRV), מן Luteoviridae משפחה מדביק צמחים solanaceous. זה מועבר על ידי כנימות, בעיקר, את הכנימה ירוק אפרסק. כאשר כנימה נגוע ניזון הצמח נגוע זה החוזים וירוס דרך השיפה המפעל. לאחר ingested, הנגיף חייב לעבור מהבטן חרקים hemolymph (דם החרקים) ולאחר מכן צריך לעבור דרך בלוטת הרוק, על מנת להיות מועבר בחזרה למפעל חדש. כנימה עשוי להימשך עד וירוסים שונים כאשר לועס צמח, אולם רק חלק קטן יעבור דרך המעי בלוטת הרוק, שני המחסומים העיקריים להעברת להדביק צמחים ועוד. במעבדה, נשתמש בצמחים physalis ללמוד הילוכים PLRV. ב לארח זו, התסמינים מתאפיינים ירקון המגבילה ו interveinal (הצהבה של העלים בין הוורידים עם הוורידים הנותרים ירוק). וידאו אנו מציגים מדגים שיטה לביצוע microinjection כנימה על חרקים שאינם PLVR וקטור וירוסים בדיקות אם במעיים מונעת העברת נגיפי. וידאו אנו מציגים מדגים שיטה לביצוע microinjection כנימה על חרקים שאינם PLVR וקטור וירוסים בדיקות אם הבטן או בלוטות הרוק שמונע העברת ויראלי.

Watch this Scientific Journal Video about Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection at JoVE.com

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

כנימות הם משדרים יעיל של וירוסים הצמח. Microinjection כנימה של הנגיף, ההליך נוכל להראות לכם היום, היא טכניקה המאפשרת לחוקרים להזרים וירוס ישירות hemocoel של הכנימה, לעקוף את המעיים, אחד 2 המחסומים העיקריים להעברת הנגיף באופן circulative. באותה טכניקה משמשת גם להזריק dsRNA עבור RNAi.

בדיקת חסמים פיזיולוגיים להעברת נגיפי ב כנימות שימוש Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

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Research

JoVE Journal

Biology

11.9K Views.  Cornell University. כנימות הם משדרים יעיל של וירוסים הצמח. Microinjection כנימה של הנגיף, ההליך נוכל להראות לכם היום, היא טכניקה המאפשרת לחוקרים להזרים וירוס ישירות hemocoel של הכנימה, לעקוף את המעיים, אחד 2 המחסומים העיקריים להעברת הנגיף באופן circulative. באותה טכניקה משמשת גם להזריק dsRNA עבור RNAi.

Video: Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

משלוח חומצות גרעין דרך Microinjection העובר חרקי ההדברה החקלאיים ברחבי העולם,<em&gt; זבוב הפירות הים תיכוני</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Genetics

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

זיהוי של תנאים קריטיים לImmunostaining באפונה בכנימות עוברים: הגדלת רקמות חדירות והפחתת מכתים רקע

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Developmental Biology

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

בדיקת סובלנות ניקוטין ב כנימות באמצעות ניסוי דיאט מלאכותית

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

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.

שמירה על תרבויות ביולוגי מדידה ביטוי גנים ב Aphis nerii: מערכת Non-מודל עבור אינטראקציות צמח-חרק

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

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

DsRNA לשחזור Microinjection, ההטלה Bioassay של יתושים וזבובים הבית

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Environment

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

הקמתה של זיהום נגיפי וניתוח של התערבות מארח-וירוס דרוזופילה Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Immunology and Infection

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.

זיהום ויראלי ניסיוני ביתושים בוגרים על ידי האכלה אוראלית ומיקרו-הזרקה

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

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

Here we describe two protocols: first to propagate, extract, purify, and quantify large quantities of honey bee non-enveloped virus particles, including a method for removing honey bee pupae and second to test the effects of viral infection using a highly repeatable, high-throughput cage bioassay.

הכנת אינוקולום מועשר בנגיף לזיהום אוראלי של דבורי דבש (Apis mellifera)

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee ...

איסוף המולימפה מפרוקי רגליים קטנים

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus transmission. Hemolymph collection is the basic technology for studying virus-vector interactions. Here, we describe a novel and simple method for the quantitative collection of hemolymph from small arthropods using Laodelphax striatellus (the small brown planthopper, SBPH) as a research model, as this arthropod is the main vector of rice stripe virus (RSV). In this protocol, the process begins by gently pinching off one leg of the frozen arthropod with fine-tipped tweezers and pressing the hemolymph out of the wound. Then, a simple micropipette consisting of a capillary and a pipette bulb is used to collect the transudative hemolymph from the wound according to the principle of capillary forces. Finally, the collected hemolymph can be dissolved into a specific buffer for further study. This new method for collecting hemolymph from small arthropods is a useful and efficient tool for further research on arboviruses and vector-virus interactions.

מחבר זרקור: שיטה מדויקת וניתנת לכימות לאיסוף המולימפה מפרוקי רגליים קטנים  

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

בדיקת חסמים פיזיולוגיים להעברת נגיפי ב כנימות שימוש Microinjection

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משלוח חומצות גרעין דרך Microinjection העובר חרקי ההדברה החקלאיים ברחבי העולם,