בניסוי זה, 4 כנימות מבוגר מוקפים כוס פלסטיק קטנה. הם ניזונים מהתזונה נוזלי pipetted בין 2 שכבות של parafilm נמתח מעל הכוס. אחרי 3 ימים האכלה, הרעילות של תזונה המכילה ריכוזים שונים של ניקוטין לעומת שליטה דיאטות ידי ספירת מבוגרים כנימות הזחל בכוס אחד. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך את פתרונות המכיל סוכרוז 20 חומצות אמינו. כנימות יכול להתקיים על דיאטה זו, להתרבות על רמות נורמליות, במשך שבוע לפחות 1. אנא ראה קובץ מצורף על הדיאטה מלאכותית. </li><li style=";text-align:right;direction:rtl"> Aliquot 1 מ&quot;ל של דיאטה זו תוך 6 צינורות שונים Eppendorf המכיל ניקוטין. לייבל אליהם בכבוד לריכוזים שונים של ניקוטין המשמש, החל 0-500μM. </li><li style=";text-align:right;direction:rtl"> בחר 4 כנימות, אדום או ירוק, והכניסו אלה כוסות ניסיוני. </li><li style=";text-align:right;direction:rtl"> בעת בחירת כנימות, חשוב שהם יהיו בגיל עקבי. בדרך כלל, לחפש כנימות בגודל דומה ובצבע (את הכנימות לקבל אדום כהה כאשר הם ישנים). </li><li style=";text-align:right;direction:rtl"> מכסים את הכוס עם חתיכת parafilm. הקף את הדיאטה בין 2 חתיכות parafilm. חלקית למתוח את רמת parafilm שנייה על שולי הכוס. Aliquot דיאטת נוזלים בין השכבות, ואת החותם את השכבה העליונה כלפי מטה. </li><li style=";text-align:right;direction:rtl"> שמור אלה כוסות בחדר חרק תחת האורות photoperiod ארוכת יום במשך 3 ימים, לאחר זמן את הנתונים עבור הניסוי ניתן לאסוף. </li></ol> בקר <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.arplant.59.032607.092825" target="_blank">ביקורות שנתי של ביולוגיה הצמח</a> לקבלת מידע נוסף על חסינות פרוטוקול צמח זה herbivory חרקים.

The authors have nothing to disclose.

testing nicotine tolerance in aphids using an artificial diet experiment

צמחים עשויים upregulate הייצור של מטבוליטים רבים seconday שונה בתגובה האכלה חרקים. אחד מהם מטבוליטים, ניקוטין, הוא יודע היטב יש תכונות חרקים. תגובה אחת של צמחי טבק herbivory, או להיות מכורסם על ידי חרקים, היא להגדיל את הייצור של אלקלואיד זה neurotoxic. כאן, נדגים כיצד להגדיר ניסוי לכתובת זו השאלה האם טבק מותאם זן של הכנימה אפרסק ירוק, persicae Myzus, יכולים לסבול רמות גבוהות של ניקוטין מאשר זן של חרק זה לא טבק שורצים ב השדה.

Watch this Scientific Journal Video about Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment at JoVE.com

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

בסרטון זה assay הוא הוכיח כי הבדיקות סובלנות של ניקוטין שני סוגים של כנימות אחד פושט צמחי טבק בתחום ואחד לא.

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 ...

testing-nicotine-tolerance-aphids-using-an-artificial-diet

Research

JoVE Journal

Biology

10.9K Views.  Cornell University. בסרטון זה assay הוא הוכיח כי הבדיקות סובלנות של ניקוטין שני סוגים של כנימות אחד פושט צמחי טבק בתחום ואחד לא.

Video: Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

בחירה אין ברירה מבחני לבחינות ההתנגדות של thaliana א &#39;חרקים לעיסת

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for 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 ...

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 ...

Genetics

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Assay רומן ההתנהגות לחקור תגובות Gustatory של הפרט, חופשי-נע דבורים Bumble (<em&gt; בומבוס האדמה</em&gt;)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Neuroscience

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Assay העדפות טעם למבוגרים<em&gt; תסיסנית</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

דרך פשוטה למדוד שינויים בהתנהגות שוחרת זכה שימוש<em&gt; תסיסנית</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 &#176;C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

The present protocol provides instructional information for using tobacco hornworm Manduca sexta in cannabinoid research. The method described here includes all necessary supplies and protocols to monitor physiological and behavioral changes of the insect model in response to cannabidiol (CBD) treatment.

קרן טבק כמערכת מודל חרקים למחקרים פרה-קליניים קנבינואידים

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical ...

הערכת השפעות פיטוכימיות על Helicoverpa armigera באמצעות בדיקת האכלה

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

מחבר זרקור: התפתחות ואתגרים של בדיקות האכלה להדברת חרקים 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

פיתוח מערכת בדיקת האכלה להערכת ההשפעה קוטלת החרקים של פיטוכימיקלים על Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

בדיקת סובלנות ניקוטין ב כנימות באמצעות ניסוי דיאט מלאכותית

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בחירה אין ברירה מבחני לבחינות ההתנגדות של thaliana א 'חרקים לעיסת

בדיקת חסמים פיזיולוגיים להעברת נגיפי ב כנימות שימוש Microinjection

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

Assay רומן ההתנהגות לחקור תגובות Gustatory של הפרט, חופשי-נע דבורים Bumble (

Assay העדפות טעם למבוגרים

דרך פשוטה למדוד שינויים בהתנהגות שוחרת זכה שימוש

קרן טבק כמערכת מודל חרקים למחקרים פרה-קליניים קנבינואידים

פיתוח מערכת בדיקת האכלה להערכת ההשפעה קוטלת החרקים של פיטוכימיקלים על Helicoverpa armigera

פיתוח מערכת בדיקת האכלה להערכת ההשפעה קוטלת החרקים של פיטוכימיקלים על Helicoverpa armigera

פיתוח מערכת בדיקת האכלה להערכת ההשפעה קוטלת החרקים של פיטוכימיקלים על Helicoverpa armigera