Name: RNA Interference in Ticks (Video) | JoVE Journal
Uploaded: 2011-01-20T19:00:00.000+00:00
Duration: 9 min 6 s
Description: Learn RNA interference in ticks through dsRNA injection to study gene silencing and tick biology. This method enables researchers to evaluate gene function and develop strategies for controlling tick-borne diseases.

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5 minutes

We thank members of our laboratories for fruitful discussions and technical assistance. This video presentation was supported by the Associate Dean for Research and the Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University. The research was funded by the Ministerio de Ciencia e Innovaci&oacute;n, Spain (project BFU2008-01244/BMC), the CSIC intramural project PA1002451 to JF, the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, CVHS 2009 RAC grant, OAES Animal Health Funds and USDA, National Research Initiative Competitive Grant, No. 2007-04613.

1. Generation of dsRNA.
<ol>
<li>Synthesize oligonucleotide primers containing T7 promoter sequences for in vitro transcription and synthesis of dsRNA (for example, for Dermacentor variabilis subolesin use oligonucleotide primers D8AAT75: 
5'-TAATACGACTCACTATAGGGTACTGACTGGGATCCCCTGCACAGT-3' and D8DVT73: 5'-TAATACGACTCACTATAGGGTACTCGAGCTTGGTGGAAAGGACG-3').</li>
<li>Amplify target gene by RT-PCR using 10 pmol of each oligonucleotide primer and 1-10 ng of tick total RNA.</li>
<li>Purify the PCR product.</li>
<li>Synthesize dsRNA using 8 μL of the purified PCR product.</li>
<li>Quantify dsRNA by spectrometry.</li>
</ol>
2. Injection of Ticks with dsRNA.
2.1. Preparation of ticks for injection.
<ol>
<li>First, wash the ticks in a series of solutions by shaking them in each solution in a 50 mL disposable centrifuge tube, decanting the solution through a fine mesh wire screen over the tube top to retain the ticks. The sequence of the solutions for washing ticks is tap water, 3% hydrogen peroxide, two washes of distilled water, 70% ethanol and two more washes with distilled water.</li>
<li>Blot-dry the ticks on paper towels.</li>
<li>Count the ticks into groups of 20 to 50, depending upon the experiment, place the ticks from each group in a 1.25 oz plastic cup with a tightly fitted lid and label with the experimental group number.</li>
</ol>
2.2. Tick injection team.
The RNAi team consists of three people: (1) one person who positions each tick on double sticky tape affixed to a sheet of red dental wax, (2) one person who injects the ticks and (3) one person who monitors the ticks after injection, breathes CO2 on the ticks to activate them and counts the living ticks into cups labeled with the experimental group number. All team members must wear disposable gloves.
2.3. Placement of ticks for injection.
<ol>
<li>Capture a tick using Dumont fine forceps and place it ventral side up on double sticky tape affixed to a 3" x 6" sheet of red dental wax. The ticks are closely positioned together in groups of 5 ticks.</li>
<li>Place a small strip of masking tape over the mouthparts of all 5 ticks in order to further restrain them but while leaving most of the body exposed so that the injection process can be observed by the tick injector (Figure 1).</li>
</ol>
2.4. Injection of ticks.
<ol>
<li>The ticks will be injected in the lower right quadrant of the ventral surface of the exoskeleton.</li>
<li>First, pierce a hole in the exoskeleton using a Monoject insulin syringe fitted with a ½", 29 gauge needle (Figure 2a).</li>
<li>Inject ticks immediately with 0.2-0.5 μL of dsRNA solution (5 x 1010 - 5 x 1011 molecules per μL) using a custom-made Hamilton syringe with a 1 inch, 33 gauge needle with a 45° beveled point (Figure 2b). The needle should be placed well into the tick cavity to insure the placement and retention of the dsRNA. Some fluid is likely to escape from the injection site (Figure 2c). Care should be taken not to over inject the ticks, which would cause loss of hemolymph and could cause the death of the tick.</li>
<li>Clean the Hamilton syringe after completing the injections in each experimental group. before using for another experimental group. Fill the syringe first from a beaker containing 3% hydrogen peroxide and then expel into a waste container, and repeat 15 times. Fill the syringe from a beaker containing sterile water and then expel into a waste container, and repeat 15 times. Take care not to bend the plunger of the Hamilton syringe because, if bent, the plunger will not move smoothly and respond to the gentle touch required for injection of ticks.</li>
</ol>
2.5. Treatment of ticks after injection.
<ol>
<li>Pick up the injected tick immediately from the double sticky tape with the fine forceps and place it in a plastic recovery container (approximately 6" x 6" and ringed with masking tape to prevent escape of the ticks). The ticks will be briefly inactive after injection but should soon begin to crawl around the dish.</li>
<li>Breath CO2 onto the ticks immediately after placing them in the recovery container to help activate the ticks. Once the ticks are crawling and active, the injection wound will heal rapidly and they will most likely survive.</li>
<li>Count the ticks according to the number in each experimental group and place them in a labeled plastic cup with a tightly fitted lid. Replacement ticks should be injected to replace any that any died before injecting the next experimental group.</li>
</ol>
2.6. Tick holding.
<ol>
<li>Place the ticks in a humidity chamber (12hr light: 12 hr dark photoperiod at 22-25°C and 95% relative humidity) and hold for 1 day.</li>
<li>Place ticks in tick-feeding cells, one per experimental group, glued to a sheep and allow them to feed with an equal number of uninjected male or female ticks (whichever sex was not injected). Female ticks that feed to repletion, those that are removed from the sheep after 10 days of feeding or when the control females have dropped off the host are collected and weighed.</li>
<li>Place the ticks in cartons, and hold in the humidity chamber until completion of oviposition. Evaluate oviposition by weighting the egg mass produced by all ticks in the group.</li>
</ol>
2.7. Analysis of tick phenotype after RNAi.
<ol>
<li>Evaluate tick phenotype after feeding by determining the number of ticks that survived, tick weight, oviposition and egg fertility. However, other analyses may be performed depending on the targeted gene and objectives of the study.</li>
</ol>
3. Analysis to Confirm Gene Silencing by RT-PCR. 
<ol>
<li>Dissect salivary glands and guts from individual ticks from control-injected and dsRNA-injected groups after feeding.</li>
<li>Extract total RNA from individual tissue samples.</li>
<li>Analyze target gene transcripts in individual tissues by real-time RT-PCR and normalize RNA levels against tick 16S rRNA using the genNorm method (ddCT method as implemented by Bio-Rad iQ5 Standard Edition, Version 2.0).</li>
<li>Run dissociation curves at the end of the reaction to ensure that only one amplicon is formed and that the amplicons denature consistently in the same temperature range for every sample.</li>
<li>Compare mRNA levels (normalized Ct values) between control-injected and dsRNA-injected ticks using the Student`s t-test (P=0.05).</li>
</ol>
4. Representative Results:
The protocol described herein has been used in our laboratory for RNAi in many different ixodid tick species (Table 1). The amount of dsRNA injected into the ticks varies with the size of the tick; larger tick species can accommodate a larger volume. Negative control ticks should be injected with an unrelated dsRNA. Several dsRNAs such as subolesin14-19,22-25,27-32,34 and beta-actin20,21 could be used as positive controls. Note that it is important to wash the syringe between treatments to avoid mixing dsRNA solutions. If the protocol is done correctly, less than 5% mortality should be obtained from the injection procedure after 24 hours. A typical phenotype after gene knockdown in ticks is shown in Figure 3 with a panel of ticks injected with pools of dsRNA in order to screen for tick protective antigens.
<table border="1">
 <tbody>
 <tr>
 <td>Tick species</td>
 <td>dsRNA injected</td>
 <td>References</td>
 </tr>
 <tr>
 <td>Ixodes scapularis</td>
 <td>cDNA library, subolesin, actin, nucleotidase, NF-kB, akirin</td>
 <td>21, 22, 29, 30</td>
 </tr>
 <tr>
 <td>Dermacentor variabilis</td>
 <td>subolesin, GST, ubiquitin, vATPase, selenoproteins M and W2a, hematopoietic stem/progenitor cells protein-like, actin Proteasome 26S subunit, ferritin1, varisin, akirin </td>
 <td>15, 19, 22, 24, 26, 30-32</td>
 </tr>
 <tr>
 <td>Dermacentor marginatus</td>
 <td>subolesin</td>
 <td>22</td>
 </tr>
 <tr>
 <td>Amblyomma americanum</td>
 <td>cDNA library, subolesin, akirin</td>
 <td>17, 22, 30</td>
 </tr>
 <tr>
 <td>Amblyomma hebraeum</td>
 <td>subolesin, voraxin</td>
 <td>28</td>
 </tr>
 <tr>
 <td>Rhipicephalus sanguineus</td>
 <td>Rs86, subolesin</td>
 <td>22, 23</td>
 </tr>
 <tr>
 <td>Rhipicephalus microplus</td>
 <td>GST, ubiquitin, selenoprotein, Bm86, Bm91, subolesin, GI, GIII, EF1a, flagelliform silk protein, von Willebrand 
 factor </td>
 <td>16, 18, 25, 27</td>
 </tr>
 <tr>
 <td>Rhipicephalus annulatus</td>
 <td>ubiquitin, subolesin, EF1a, GIII</td>
 <td>16</td>
 </tr>
 </tbody>
</table>
Table 1.Tick species in which the RNAi protocol has been used.
 <img src="/files/ftp_upload/2474/2474fig1.jpg" alt="Ticks feeding on skin, close-up image, parasitology study, blood-feeding behavior analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Placement of ticks, ventral side up, on double sticky tape adhered to a sheet of red dental wax. The ticks are placed in groups of 5, after which a small strip of masking tape is placed over the mouthparts in order to further secure the ticks while allowing the injector to observe the body of the tick during injection.
 <img src="/files/ftp_upload/2474/2474fig2.jpg" alt="Tick removal process sequence, diagram showing steps with tweezers for safe extraction." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. The injection procedure includes (a) piercing the lower right quadrant of the tick exoskeleton with a insulin syringe fitted with a 29 gauge needle in order to create an injection site, (b) immediate injection of the dsRNA at this site using a Hamilton syringe with a 33 gauge needle which (c) most likely will result in some leakage of tick hemolymph/fluids.
 <img src="/files/ftp_upload/2474/2474fig3.jpg" alt="Tick infestation treatment test on animal skin with negative and positive controls for subolesin efficacy." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. A panel of tick six groups in which RNAi was used to screen for tick protective antigens in Amblyomma americanum. The phenotypic changes in ticks can be seen when compared with the positive subolesin RNAi control and the negative unrelated dsRNA control. In this experiment the effect of RNAi on tick mortality, weights, and oviposition of each group was statistically analyzed.

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<li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transovarial+silencing+of+the+subolesin+gene+in+three-host+ixodid+tick+species+after+injection+of+replete+females+with+subolesin+dsRNA%5BTitle%5D)+AND+%22Parasitol.+Res%22%5BJournal%5D)">Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA.</a> Parasitol. Res. 100, 1411-1415 (2007).</li>
<li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+tick-pathogen+interface+for+novel+control+strategies%5BTitle%5D)+AND+%22Front.+Biosci%22%5BJournal%5D)">Targeting the tick-pathogen interface for novel control strategies.</a> Front. Biosci. 13, 6947-6956 (2008).</li>
<li>Kurscheid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+of+a+tick+RNAi+pathway+by+comparative+genomics+and+reverse+genetics+screen+of+targets+with+known+loss-of-function+phenotypes+in+Drosophila%5BTitle%5D)+AND+%22BMC+Mol.+Biol%22%5BJournal%5D)">Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila.</a> BMC Mol. Biol. 10, 26-26 (2009).</li>
</ol>

No conflicts of interest declared.

Although other methods have been described for RNAi in ticks14, 33, the injection of dsRNA described here is the most widely used in both unfed (Table 1) and fed ticks16,25,34. RNAi has been shown to be a valuable tool for the study of tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14,35. In particular, RNAi has become the most valuable tool for functional analyses in ticks35.
Methodologically, RNAi will likely evolve into more efficient methods that may allow gene knockdown in a large number of individuals. The mechanism of dsRNA-induced RNAi in ticks should be refined to contribute to a better understanding and utilization of this genetic approach in this species35,36. The extent of off-target effects of RNAi in ticks is also an important question that needs to be fully addressed14,27. Finally, RNAi will most likely provide comprehensive contributions to the study of tick gene regulation and systems biology and the tick-pathogen interface and may have an impact on the development of vaccines to control tick infestations and the transmission of tick-borne pathogens.

<table><tbody><tr><td>Access RT-PCR system</td><td>Promega</td><td>A1250</td><td></td></tr><tr><td>Purelink PCR purification kit</td><td>Invitrogen</td><td>K3100-02</td><td></td></tr><tr><td>Megascript RNAi kit</td><td>Ambion</td><td>AM1626M</td><td></td></tr><tr><td>Red dental wax</td><td>Electron Microscopy Sciences</td><td>72674</td><td></td></tr><tr><td>Plastic cups, 1.25 oz and lids</td><td>Solo Cup Company, Urbana Ill.</td><td></td><td></td></tr><tr><td>Fine forceps</td><td>Electron Microscopy Sciences</td><td>Various</td><td></td></tr><tr><td>Insulin syringe</td><td>Monoject</td><td></td><td>Fitted with a &amp;#189;&quot;, 29 gauge needle</td></tr><tr><td>Hamilton syringe</td><td>Hamilton</td><td>701SN,33/.375&amp;#8221;/45DGR</td><td>Custom made</td></tr><tr><td>TriReagent</td><td>Sigma</td><td>93289</td><td></td></tr><tr><td>iScript One-Step RT-PCR Kit with SYBR Green</td><td>Bio-Rad</td><td>170-8892</td><td></td></tr><tr><td>Real-time PCR detection system</td><td>Bio-Rad</td><td>Several</td><td>Please refer to &lt;a href=&quot;http://www.bio-rad.com/&quot;&gt;http://www.bio-rad.com/&lt;/a&gt;</td></tr></tbody></table>

rna interference in ticks

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

The overall goal of this procedure is to demonstrate the technique of RNA interference in ticks by injection. This is accomplished by first synthesizing double stranded RNA of the gene or genes of interest. The RNA is then injected into ticks, after which they're held in a humidity chamber for 24 hours.After this holding period, the ticks are allowed to feed on an animal such as a sheep finally tick. Feeding parameters such as weight, molting and ova position, and the expression of the targeted gene or genes of interest by real-time R-T-P-C-R are determined. Ultimately, results can be obtained that demonstrate the effect of silencing targeted genes on tick biology by evaluation of tick biological parameters and gene expression by real-time R-T-P-C-R.Our group works with ticks trying to characterize molecular events at the tick pathogen interface to use this basic information to develop methods for control of tick infestation and the transmission of pathogens to humans and animals. RNA interference has emerged as an essential tool in this research because it is the only method available for genetic manipulation of ticks. The function of many tick genes is unknown.RNA interference allows us to define the role of tick genes and gene expression in many aspects of tick biology, including mating, reproduction, feeding, digestion, salivary gland function, and the tick vector competency for the transmission of pathogens. Demonstrating the procedure will be the tick RNA interference team from our laboratory, Dr.Ed Lewin, graduate student and Busby and myself. To generate double stranded RNA first synthesize oligonucleotide primers containing T seven promoter sequences for in vitro transcription of RNA.For example, derma center vari lesin. Use the oligonucleotide primers D eight A a T 75, and D eight DVT 73 shown here. Amplify the target gene by R-T-P-C-R using 10 picomoles of each oligonucleotide primer and one to 10 nanograms of tick total RNA.Next, purify the PCR product. Then use eight microliters or approximately 100 nanograms to synthesize double stranded RNA. To prepare the ticks for injection first, wash the ticks in the following series of solutions, tap water 3%hydrogen peroxide distilled water twice, 70%ethanol and two final washes with distilled water.Perform each wash in a 50 milliliter disposable centrifuge tube by shaking. Then decanting the solution through a fine mesh wire screen. Block the ticks dry on paper towels, then aliquot the ticks into groups of 20 to 50 depending upon the experiment.Place each group into a 1.25 ounce plastic cup with a tight fitting lid and label each cup with the experimental group. Number next, assemble an RNAi team consisting of three people that will carry out the injection process. The first person positions each tick on double sticky tape affixed to a three by six inch sheet of red dental wax.The second person injects the ticks and the third monitors the ticks after injection breathes carbon dioxide on the ticks to activate them and counts and transfers the living ticks into cups labeled with the experimental group number. All team members must wear disposable gloves To begin the tick injection process, capture a tick using dumont fine forceps and place it ventral side up on double sticky tape affixed to the sheet of red dental Wax closely Position the ticks into groups of five. Place a small strip of masking tape over the mouth parts of all five ticks.In order to further restrain them, leave most of the body exposed so that the injection process can be observed by the tick injector. To inject the tick pierce a hole in the lower right quadrant of the ventral surface of the exoskeleton using a mono eject insulin syringe fitted with a 29 gauge half inch needle. Next, immediately inject the ticks with 0.2 to 0.5 microliters of double stranded RNA solution using a custom made Hamilton syringe and a 33 gauge one inch needle with a 45 degree beveled point, place the needle well into the tick cavity to ensure the placement and retention of the double stranded RNA.Even though fluid is released after injection, deep placement of the needle for injection will ensure that sufficient double stranded RNA is deposited into the tick cavity to cause gene silencing care should be taken not to overin. Inject the ticks, which will cause loss of hemolymph and death of the tick. Immediately after injecting the ticks, pick them up from the double sticky tape with the fine forceps and place them in a plastic recovery container.The ticks will briefly remain inactive after injection, but should soon begin to crawl around the dish. Activate the ticks by breathing carbon dioxide onto them. Once the ticks are crawling and active, the injection wound will heal rapidly and they will most likely survive.Count the ticks in each experimental group and place each group in a labeled plastic cup with a tightly fitted lid. Replace any ticks that die in the current group before injecting the next experimental group. Clean the Hamilton syringe before injecting the next experimental group by filling and then expelling the syringe with fresh 3%hydrogen peroxide.15 times followed by 15 washes with sterile water. Take care not to bend the plunger of the Hamilton syringe, otherwise the plunger will not move smoothly and respond to the gentle touch required for tick injection. After the injecting, place the ticks in a chamber with a container filled with water and potassium sulfate, which maintains high humidity and them for one day.Next place individual ticks into tick feeding cells glued to a sheep and allow them to feed with an equal number of unin injected male or female ticks, whichever sex was not injected. Collect and wave female ticks from the feeding cell after they complete feeding for approximately 10 days or when the control females have dropped off, the host incubate each group of ticks in the humidity chamber until the completion of ova position. Evaluate ova position by group by weighing the eggs mass produced by all the ticks in a group to evaluate the tick phenotype after feeding.Determine the number of ticks that survived and calculate the tick weight as well as the ova position and the fertility of the eggs. Depending on the targeted gene and objectives of the study, other analyses may be performed to confirm gene silencing by R-T-P-C-R. After feeding, dissect the salivary glands and guts from individual ticks from the control injected and double stranded RNA injected groups, and then extract total RNA from the individual tissue samples.Analyze the target gene transcripts in individual tissues by real-time R-T-P-C-R and normalize the RNA levels against tick 16 s ribosomal RNA. Using the gene norm method, run dissociation curves at the end of the reaction to ensure that only one amplicon is formed and that the amplicons consistently denature in the same temperature range for every sample, compare the normalized CT values for mRNA levels between control injected and double stranded. RNA injected ticks using the student's T-test.The protocol described here has been used in our laboratory for RNAi in many different exot tick species. The amount of double stranded RNA injected into the ticks varies with the size of the tick. Larger tick species can accommodate a larger volume.The references can be found in the accompanying written portion of the protocol. If the protocol is done correctly, less than 5%mortality should be obtained from the injection procedure after 24 hours. A typical phenotype after gene knockdown in ticks is shown here where a panel of ticks were injected with pools of double stranded RNA that were used to screen for tick protective antigens.In ticks that are examined by light microscopy, cellular degeneration in tick tissues may be observed. Phenotypic changes may also be accompanied by loss of function. For example, RNAI of SUBIN results in sterile males that cannot mate successfully with the females Following RNA interference and ticks.Other methods can be used to determine the impact of gene silencing on gene and protein expression and on tick biology, including real-time R-T-P-C-R, light electron or confocal microscopy. Genomics or proteomics. RNA interference can also be done in tick cell cultures and provides an in vitro method for the study of tick cell pathogen interactions.

Research

JoVE Journal

Immunology and Infection

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks.  Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours.  We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is the most commonly-reported vector-borne disease in North America.  The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks.  Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Presented here is a method to blood feed ticks in vitro via an artificial membrane system to allow for partial or full engorgement of a variety of tick life stages.

Tick Artificial Membrane Feeding for Ixodes scapularis

This article presents the methodology for exposing humans to larval Ixodes scapularis for clinical research. The technique is relatively simple, tolerable by the research volunteers, and can be modified according to experimental needs. Such research involving human subjects must be conducted under clinical study protocols approved by the appropriate regulatory authorities.

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

Here, we present a protocol to collect saliva, haemolymph, salivary glands, and midgut, following the dissection of an individual fed tick, to study tissue-specific localization of tick-borne pathogens to advance understanding of vector competence and pathogen-microbiome interactions.

Extraction of Saliva, Haemolymph, Salivary Glands, and Midgut from Individual Ticks (Acari: Ixodidae)

This method outlines the development of an in vitro tick blood-feeding system incorporating a 3D-printed chamber and a laboratory-fabricated silicone membrane. The system enables artificial feeding of ticks, allowing for easy sampling at various feeding stages and utilizing low volumes of blood, providing an invaluable tool for studying ixodid ticks.

Tick Blood Feeding through an Artificial Membrane on a 3D-Printed Chamber in Ixodid Tick Research

Learn RNA interference in ticks through dsRNA injection to study gene silencing and tick biology. This method enables researchers to evaluate gene function and develop strategies for controlling tick-borne diseases.

RNA Interference in Ticks

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