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במהלך העשורים האחרונים, מערכות ביטוי heterologous היה בשימוש נרחב כדי לבטא ולבודד כמויות גדולות של חלבונים, ובכך מאפשר נחישות הביוכימי פונקציונלי ואפיון של אנזימים במבחנה. נכון להיום, מספר מערכות מודל שונה לרבות Escherichia coli, Pichia pastoris, Sacccharomyces cerevisiae frugiperda זחל כנימה הותאמו ע...

קוטל חרקים ההתנגדות היא כיום נושא מרכזי של הבית הדברת הזבוב ברחבי העולם1,2. המאמצים כדי לקבוע המנגנון של עמידות חרקים מאפשר הבנה טובה יותר של בעיה זו, ובכך לספק אסטרטגיות הרומן ביעילות למנוע או לצמצם את התפשטות ההתנגדות פיתוח3. Carboxylesterases, בתור אחד של אנזימים דטוקסיפיקציה הגדולות, משכו המון תשומת לב על השתתפותם ב sequestering, חילוף חומרים קוטלי חרקים שונים חרקים4,5,6. המחקר הקודם שלנו זיהה carboxylesterases מרובים בזבובים הבית ואת רמות הביטוי שלהם היו לא רק צורונים למעלה מוסדר בנימה ALHF עמידים, אבל גם יכול להיות רמות המושרה כדי גבוה יותר בתגובה בפרמתרין7 . עם זאת, אפיוני תפקודי הגנים האלה carboxylesterase ' בחילוף חומרים קוטלי חרקים נשארים כדי להבטיח את הזמנתכם.
מאז הדוח הראשון ב שנות ה-80 המוקדמות8, מערכת ביטוי baculovirus בתיווך גנים זרים כבר מועסקים נרחב עקב שלה יעילות ייצור חלבון וחלבון האיקריוטים עיבוד יכולות9. זו מערכת בינארית מורכב שני מרכיבים חיוניים: baculovirus רקומביננטי נבנה העברת גנים זרים לתוך התאים מארח, ואת הביטוי בקנה מידה גדול של חלבונים גם על ידי תאים נגועים baculovirus רקומביננטי. במהלך העשורים האחרונים, מערכת ביטוי תא baculovirus מתווכת כבר בשימוש נרחב כדי לייצר אלפי חומרים, החל cytosolic אנזימים מאוגד-קרום חלבונים בחרקים, יונקים תאים10. המחקר הקודם שלנו הביעה בהצלחה מספר אנזימים CYP450 בתאים Sf9 חרקים עם המערכת הזו11. במחקר זה, אנו נבנה baculovirus carboxylesterase-רקומביננטי להדביק תאים Sf9 חרקים, חקר הסובלנות תא בפרמתרין שונים, ולאחר בקנה מידה גדול carboxylesterase ביטוי חלבונים במבחנה עבור פונקציונלי חקר. במקום חוקרים מרובים איזואנזים carboxylesterase תערובות של חרקים homogenates כפי שאומצה על ידי מחקרים קודמים12,13, בתיווך baculovirus תא חרקים ביטוי המערכת מאפשרת ביטוי ספציפי, בידוד של חלבונים ממוקד יותר פלואורסנציה תכונותיהם הביוכימי ומבניים.
Tetrazolium מבוססי מלח וזמינותו (MTT) היא שיטה ערכי צבע מוחלטים תפוקה גבוהה פיתח, ממוטב כדי למדוד את הכדאיות תא. Assay זו מבוססת על מנגנון זה רק חיים תאים המסוגלים חילוף חומרים הכימית MTT בגוון צהוב חום התמיסה formazan בצבע סגול כהה, אשר יכול להיות מנותח colorimetrically לאחר מומס ממיסים אורגניים14, 15. כמה מדויק יותר אבל שיטות זמן רב, כמו אי-כלילה Trypan blue תימידין טיטור assay16,17, פותחו בשנים האחרונות. עם זאת, וזמינותו MTT מבוססת תא עדיין כיום מזוהה כמספר השיטה המהירה ביותר המופעל בקלות לזהות במהירות את הכדאיות תא. כאן, אנו משתמשים וזמינותו MTT כדי לחקור את הסובלנות תא נגד חרקים טיפולים. הסובלנות משופרת של תאים כאשר נגוע baculovirus רקומביננטי carboxylesterase חריפה תומך את התפקידים מטבולית של carboxylesterases הדברה, אשר בתורו מרמז על מעורבותם בהתנגדות בקוטלי חרקים.
בנוסף, במבחנה מטבולית assay נערך גם במחקר זה. לעומת מבחני carboxylesterase כללי המשתמשות סובסטרטים נפוצות כגון α-napthyl אצטט (α-נה) וβ-naphthyl אצטט (β-נה) כדי לשקף פעילויות hydrolytic של carboxylesterases, המחקר במבחנה מטבולית נחשבת כדרך מדויקת למדוד ישירות פעילויות של carboxylesterases לקראת הדברה18. שיטה זו כבר מועסקים בהצלחה לאפיין ציטוכרום מרובים P450s בשיתוף עם קוטל חרקים ההתנגדות11,19,20בחרקים שונים. עם זאת, שיטה זו לא טרם הוחל במחקרים carboxylesterase. עם הזמינות של חלבונים carboxylesterase המיוצר על ידי המערכת ביטוי בתיווך baculovirus, נוכל לבצע על במבחנה מטבולית המחקר של carboxylesterases כלפי פרמתרין, אשר יכול לספק עוד עדויות חזקות של מעורבות של carboxylesterases משוחח pyrethroid ההתנגדות בבית זבובים.

<table><tbody><tr><td>Q5 High-Fidelity DNA Polymerase</td><td>New England Biolabs inc.</td><td>M0491L</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli</td><td>Invitrogen by life technology</td><td>K240020</td><td>S.O.C medium and universal M13 sequence primers were included in this kit.</td></tr><tr><td>PureLink HiPure Plasmid Miniprep Kit</td><td>Invitrogen by life technology</td><td>K210002</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme mix for BaculoDirect&lt;sup&gt;TM&lt;/sup&gt; Kits</td><td>Invitrogen by life technology</td><td>11791-023</td><td></td></tr><tr><td>BaculoDirect C-Term Linear DNA Transfection Kit</td><td>Invitrogen by life technology</td><td>12562-019</td><td>Cellfectin transfection reagent and ganciclovir were included in this kit</td></tr><tr><td>pENTR-CAT plasmid</td><td>Invitrogen by life technology</td><td></td><td>Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL</td></tr><tr><td>Heat inactivated Fetal Bovine Serum, Certified</td><td>Gibco by Life Technologies</td><td>10082-139</td><td></td></tr><tr><td>Sf9 cells in Sf-900 III SFM</td><td>Gibco by Life Technologies</td><td>12659017</td><td></td></tr><tr><td>Insect Cell-PE LB Insect Cell Protein Extraction &amp;amp; Lysis Buffer</td><td>G Biosciences by A Geno Technology Inc</td><td>786-411</td><td></td></tr><tr><td>Sf-900 III SFM (1&amp;times;) Serum Free Medium Complete</td><td>Gibco by Life Technologies</td><td>12658-019</td><td></td></tr><tr><td>Grace&#039;s Insect Medium, unsupplemented</td><td>Gibco by Life Technologies</td><td>11595030</td><td></td></tr><tr><td>Permethrin (isomers) analytical standard</td><td>SUPELCO by Solutions Within&lt;sup&gt;TM&lt;/sup&gt;</td><td>442748</td><td></td></tr><tr><td>Methanol (analytical graded)</td><td>Sigma-Aldrich</td><td>67-56-1</td><td></td></tr><tr><td>Acetonitrile (analytical graded)</td><td>Sigma-Aldrich</td><td>75-05-8</td><td></td></tr><tr><td>GHP Acrodisc 25 mm Syringe Filters with 0.45 &amp;mu;m GHP Membrane (HPLC Certified)</td><td>Pall Life Sciences</td><td>21890388</td><td></td></tr><tr><td>Alliance Waters 2695 HPLC System</td><td>Waters</td><td></td><td></td></tr><tr><td>T100 Thermal Cycle</td><td>Bio-Rad Laboratories Inc.</td><td>1861096</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophotometers</td><td>ThermoFisher Scientific</td><td>ND2000CLAPTOP</td><td></td></tr><tr><td>Cytation 5 Cell Imaging Multi-Mode Reader</td><td>BioTek</td><td></td><td></td></tr></tbody></table>

functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica

בתיווך Carboxylesterase חילוף החומרים הוא חשב לשחק תפקיד מרכזי בהתנגדות בקוטלי חרקים, חרקים שונים. מספר גנים carboxylesterase נמצאו למעלה מוסדר בנימה זבוב הבית עמיד, ואילו התפקידים שלהם משוחח עמידות חרקים נשארו כדי להבטיח את הזמנתכם. . תיכננו פרוטוקול עבור אפיון פונקציונלי carboxylesterases. 3 דוגמה ניסויים מוצגים: (1) בביטוי ובידוד של חלבונים carboxylesterase דרך חרק בתיווך baculovirus זחל כנימה frugiperda (Sf9) תא ביטוי מערכת; (2) MTT מבוססת-תא (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium ברומיד) cytotoxicity assay כדי למדוד את רגישות התאים חרקים כדי הטיפול בפרמתרין שונים; ומחקרים (3) במבחנה חילוף החומרים כדי לבדוק את היכולות מטבולית של carboxylesterases כלפי פרמתרין. הגן carboxylesterase MdαE7 היה שוכפלה מ בית עמיד לטוס זן ALHF, נהגו לבנות baculovirus רקומביננטי לדלקת תאים Sf9. Viabilities תא נגד שונה הטיפול בפרמתרין נמדדו עם MTT וזמינותו. הסובלנות תא משופרת של קבוצת הניסוי (MdαE7-רקומביננטי baculovirus נגוע תאים) לעומת אלו של קבוצות הבקרה (החתול-רקומביננטי baculovirus נגוע תאים ותאים GFP-רקומביננטי baculovirus נגוע) כדי פרמתרין טיפולים הציע את היכולות של MdαE7 של חילוף חומרים קוטלי חרקים, ובכך להגן על תאים מפני נזקים כימיים. חוץ מזה, חלבונים carboxylesterase היו הביע בתאים Sf9 חרקים, מבודד לנהל במבחנה מטבולית המחקר. התוצאות שלנו המצוין של משמעותי במבחנה מטבולית היעילות של MdαE7 כלפי פרמתרין, ישירות המציין את מעורבותם של carboxylesterases של חילוף חומרים קוטלי חרקים וזבובים ולכן היוועצות עמידות חרקים בבית.

הכדאיות תא לכיוון שונה הטיפול בפרמתרין (MTT assay)
Cytotoxicity של פרמתרין נבדק ב MdαE7-רקומביננטי baculovirus נגוע Sf9 תאים (קבוצת הניסוי) ותאים baculovirus (שסופק על-ידי ערכת baculovirus נגוע) נגוע חתול-רקומביננטי (קבוצות הבקרה). הסטייה תא משופרת כדי פרמתרין ב MdαE7 לבטא ת...

Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

כאן, אנו מציגים פרוטוקול לייצר בית carboxylesterase לעוף חלבונים במבחנה עם מערכת ביטוי תא חרקים baculovirus מתווכת, מאוחר יותר פונקציונלית לאפיין את תפקידיהם פרמתרין חילוף חומרים, ובכך, היוועצות pyrethroid התנגדותם מבוססת תא MTT assay, במבחנה מטבולית מחקרים.

אפיון פונקציונלי Carboxylesterases הבית בקוטלי חרקים עמידים בזבובים, דומסטיקה זבוב

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

functional-characterization-carboxylesterases-insecticide-resistant

Research

JoVE Journal

Biochemistry

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

7.4K Views.  Auburn University. כאן, אנו מציגים פרוטוקול לייצר בית carboxylesterase לעוף חלבונים במבחנה עם מערכת ביטוי תא חרקים baculovirus מתווכת, מאוחר יותר פונקציונלית לאפיין את תפקידיהם פרמתרין חילוף חומרים, ובכך, היוועצות pyrethroid התנגדותם מבוססת תא MTT assay, במבחנה מטבולית מחקרים.

Video: Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

פליטת אור סידן Assay עבור ניתוח פונקציונלי של יתוש ( Aedes aegypti) ו טיק ( Rhipicephalus microplus) G-חלבון בשילוב רצפטורים

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Immunology and Infection

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

שיטה להערכת יעילות הדברה נגד פשפש מיטה,<em&gt; Cimex lectularius</em&gt;, ביצים ראשית Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

Environment

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

מבנה גבישי של N-מסוף תחום של Ryanodine הקולטן של Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

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 ...

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

זיהוי מוטציות מיקרו-חדיר ונוקאאוט של CRISPR/Cas9 גנום ערוך Helicoverpa Armigera (Hübner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

Behavior

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong preference for human hosts over other warm-blooded animals for blood meals. Olfactory cues play a critical role for mosquitoes as they explore their environment and seek a human host to obtain blood meals, thus transmitting human diseases. Odorant receptors (ORs) expressed in the olfactory sensory neurons are known to be responsible for the interaction of mosquito vectors with human odors. To gain deeper insights into Ae. aegypti&#8217;s olfactory physiology and investigate their interactions with humans at the molecular level, we used an optimized protocol of Xenopus Oocytes heterologous expression to functionally analyze Ae. aegypti odorant receptors in response to human odors. Three example experiments are presented: 1) Cloning and synthesizing cRNAs of ORs and odorant receptor co-receptor&#160;(Orco) from four to six days old Ae. aegypti antennae; 2) Microinjection and expression of ORs and Orco in Xenopus oocytes; and 3) Whole-cell current recording from Xenopus oocytes expressing mosquito ORs/Orco with a two-electrode voltage-clamp. These optimized procedures provide a new way for researchers to investigate human odor reception in Aedes mosquitoes and reveal the underlying mechanisms governing their host-seeking activity at a molecular level.

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong ...

Neuroscience

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

הקלטות פיסיולוגיות וRNA רצף של האיברים התעוררו אצל של יתוש הקדחת הצהובה<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Biology

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in agronomic practices resulted in serious outbreaks of stable flies in several countries. These outbreaks disrupted livestock production and human recreation resulting in public demands for increasing research and management efforts for this pest. A simple and inexpensive procedure for rearing stable flies for laboratory studies is presented. The procedure uses locally available diet components, equipment and supplies. The procedure can be adapted for rearing other muscoid flies including face fly (Musca autumnalis), horn fly (Haematobia irritans), and house fly (Musca domestica). The procedure produces stable fly puparia averaging 12.5 mg and ~35% egg to adult survival. Approximately 3000 flies are produced in each pan.

A procedure for rearing stable flies (Stomoxys calcitrans) is presented. The procedure uses locally available materials for diet components, equipment and supplies.

מעבדה לגידול של אחרים Muscoid Diptera והזבובים יציב

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

יישום אקטואלי Bioassay כדי לכמת רעילות קוטל חרקים עבור יתושים וזבובי פירות

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

פרוטוקולים עבור CRISPR/Cas9 Mutagenesis של זבוב הפירות המזרחי Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

אפיון פונקציונלי Carboxylesterases הבית בקוטלי חרקים עמידים בזבובים, דומסטיקה זבוב

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פליטת אור סידן Assay עבור ניתוח פונקציונלי של יתוש ( Aedes aegypti) ו טיק ( Rhipicephalus microplus) G-חלבון בשילוב רצפטורים

שיטה להערכת יעילות הדברה נגד פשפש מיטה,

מבנה גבישי של N-מסוף תחום של Ryanodine הקולטן של Plutella xylostella

DsRNA לשחזור Microinjection, ההטלה Bioassay של יתושים וזבובים הבית

זיהוי מוטציות מיקרו-חדיר ונוקאאוט של CRISPR/Cas9 גנום ערוך Helicoverpa Armigera (Hübner)

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

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