This study was financed in part by the Coordenac&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) from Brazil, finance code 001. CAPES provided Ph.D. scholarship for A.F. Marciano. We thank National Council for Scientific and Technological Development (CNPq) of Brazil for providing Ph.D. scholarship for J. Fiorotti. This research was also supported by grants of Carlos Chagas Filho Foundation for Research of the State of Rio de Janeiro (FAPERJ) and CNPq. V.R.E.P. Bittencourt is a CNPq researcher.

Ticks used in the present study were obtained from an artificial colony, mantained at Federal Rural University of Rio de Janeiro, which methods have been approved by the Committee on Ethics for the Use of Vertebrate Animals (CEUA-IV/UFRRJ #037/2014).
1. Tick engorged females
<ol>
	<li>After tick gathering, wash engorged females using tap water and immerse them in 0.5% (v/v) sodium hypochlorite solution for 3 min in a 500 mL glass beaker recipient, for cuticle hygiene (Figure 1), then dry all females using sterile paper towels (Figure 2).</li>
	<li>Divide females in homogeneous weighted groups (with 20 females each): one group without any treatment, one control group inoculated with 5 &#181;L of 0.1% polyoxyethylene sorbitan monooleate aqueous solution (v/v), and one infected-group inoculated with 5 &#181;L at 1.0 x 107 blastospores mL-1. 
	NOTE: Only untreated ticks were used for volume and hemocyte concentration. Three replicates of each group were performed.</li>
</ol>
2. Pathogens inoculation between the scutum and capitulum
NOTE: In the present study, entomopathogenic fungal suspensions were used as an example.
<ol>
	<li>Suspend fungal blastospores in 1 mL of 0.1% polyoxyethylene sorbitan monooleate aqueous solution (v/v) and adjust to a final concentration of 1.0 x 107 blastospores mL-1. Add an aliquot of 5 &#181;L Metarhizium blastospores (Figure 3) suspension to the surface of a plastic paraffin film. 
	NOTE: Metarhizium blastospores suspension was adjusted using a Neubauer&#180;s chamber. To speed the inoculation process, multiple bubbles can be added to the plastic paraffin film surface, each bubble corresponding to 5 &#181;L.</li>
	<li>Use a 1 mL ultra-fine insulin syringe and a 0.3 mm needle to pull the suspension and inoculate it into the tick. Remember to take all the air out of the syringe before using it.</li>
	<li>Inoculate 5 &#181;L of fungal suspension into the tick female between the scutum and capitulum. Inoculate females from the control group with 5 &#181;L of 0.1% polyoxyethylene sorbitan monooleate aqueous solution (v/v) with no fungus. 
	NOTE: A small volume of hemolymph may be present on the foramen after the needle insertion. Be careful to not inoculate air.</li>
</ol>
3. Inoculation of entomopathogenic fungi between the leg thigh and the tick female&#39;s body
<ol>
	<li>Inoculate females with fungal suspension (5 &#181;L at 1.0 x 107 blastospores mL-1) between the leg tigh and the female&#39;s body, using a 1 mL ultra-fine insulin syringe coupled to a 0.3 mm needle. Inoculate females from the control group with 5 &#181;L of 0.1% polyoxyethylene sorbitan monooleate aqueous solution (v/v). 
	NOTE: When Metarhizium blastospores inoculation is performed between the leg tigh and the tick female&#39;s body, a small volume of hemolymph can be present on the tigh after the needle insertion. Be careful to not inoculate air.</li>
</ol>
4. Dorsal hemolymph collection
<ol>
	<li>Use the rubber part of a winged infusion set, a 0.3 mm capillary tube, and a filtered tip to perform the hemolymph collection.</li>
	<li>Disrupt the female dorsal cuticle using a 0.3 mm needle.</li>
	<li>After disruption, apply gentle pressure on the anterior part of the tick body. Observe an almost transparent liquid pulling out of the disruption site. Collect the hemolymph by sucking the liquid through the filter tip coupled to the rubber part of a winged infusion set, and a 0.3 mm capillary tube. 
	NOTE: Do not press the tick&#39;s body hardly during its immobilization because this may disrupt the midgut and contaminate the hemolymph. Wait until gentle pressure can expel the fluid without contamination.</li>
</ol>
5. Hemolymph collection from the tick leg
<ol>
	<li>Immobilize the tick, cut a piece of a front leg with a pair of scissors. 
	NOTE: One or more legs can be cut, as well as, the same leg can be cut more than one time.</li>
	<li>Apply gentle pressure on the tick&#39;s posterior body part. Observe a transparent liquid bubble that shows up on the cut site and collect it with the capillary tube, as described in step 4.3. 
	NOTE: Do not press the tick&#39;s body hardly during its immobilization because this may disrupt the midgut and contaminate the hemolymph. Wait until gentle pressure can expel the fluid without contamination.</li>
</ol>
6. Hemolymph processing
<ol>
	<li>After hemolymph collection, deposit it in 1.5 mL microtubes previously filled with 30 &#181;L protease cocktail and 82 &#181;L saline buffer. Keep the microtubes on ice throughout the hemolymph collection.</li>
	<li>Centrifuge samples (500 x g for 3 min at 4 &#176;C). A soft pellet of hemocytes will be formed after hemolymph centrifugation. 
	NOTE: For hemolymph quantification, quantify the hemolymph volume obtained by counting the total volume inside the microtube and discounting the volume of protease cocktail and saline buffer.</li>
	<li>Carefully remove the supernatant (cell-free hemolymph). Gently resuspend the cells in, for example, Leibovitz&#39;s L-15 culture adjusted to pH 7.0-7.2. Quantify hemocytes by placing 10 &#181;L of resuspended hemocytes in a Neubauer chamber.</li>
</ol>
7. Hemocyte sampling slide preparation
<ol>
	<li>Cut the tick&#39;s front leg with a pair of scissors.</li>
	<li>Apply gentle pressure on the tick&#39;s posterior body part. Observe a transparent liquid bubble that shows up on the cut site.</li>
	<li>Apply the hemolymph drops directly on clean microscope slides, after that, use appropriated methods to stain the cells.
	<ol>
		<li>To stain hemocytes using Giemsa, air-dry the hemolymph on the slide for 30 min, fix it at room temperature with methanol for 3 min, and stain in Giemsa solution (1:9 ratio of Sorensen&#39;s buffer solution, pH 7.2) for 30 min at room temperature. Wash the slides with running water to remove the excess of dye, air-dry the slide and evaluate the cells at 400x in an optical microscope.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of ticks. , (2014).</li><li>Tan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+proliferation+in+larval+silkworm+Bombyx+mori.">Characterization of hemocytes proliferation in larval silkworm Bombyx mori.</a> Journal of Insect Physiology. 59 (6), 595-603 (2013).</li><li>Bowden, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+humoral+immune+systems+of+the+American+lobster+(Homarus+americanus)+and+the+European+lobster+(Homarus+gammarus).">The humoral immune systems of the American lobster (Homarus americanus) and the European lobster (Homarus gammarus).</a> Fish Research. 186, 367-371 (2017).</li><li>Sonenshine, D. E., Hynes, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+and+related+aspects+of+the+innate+immune+response+in+ticks.">Molecular characterization and related aspects of the innate immune response in ticks.</a> Frontiers in Bioscience. 13, 7046-7063 (2008).</li><li>Tsakas, S., Marmaras, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+immunity+and+its+signaling:+an+overview.">Insect immunity and its signaling: an overview.</a> Invertebrate Survival Journal. 7, 228-238 (2010).</li><li>Burgdorfer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph+Test.+A+technique+for+detection+of+Rickettsiae+in+ticks.">Hemolymph Test. A technique for detection of Rickettsiae in ticks.</a> American Journal of Tropical Medicine and Hygiene. 19, 1010-1014 (1970).</li><li>Dunham-Ems, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+reveals+a+biphasic+mode+of+dissemination+of+Borrelia+burgdorferi+within+ticks.">Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks.</a> Journal of Clinical Investigation. 119, 3652-3665 (2009).</li><li>Patton, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva,+salivary+gland,+and+hemolymph+collection+from+Ixodes+scapularis+ticks.">Saliva, salivary gland, and hemolymph collection from Ixodes scapularis ticks.</a> Journal of Visualized Experiments. 60, e3894 (2012).</li></ol>

The authors have nothing to disclose.

Inoculation of pathogens is useful when the study aims to investigate the in vivo action of microorganisms in experimental arthropod models because it assures that the pathogen is inside the host. The technique can also be applied to inoculate molecules such as RNA interference (RNAi). Inoculation between the scutum and capitulum is considered easier to perform but frequently damages Gen&#233;&#39;s organ, impairing the eggs viability12,13. Gen&#233;&#39;s organ is anatomically located close to the anterior part of capitum, and it is an important organ for the tick oviposition14. Accordingly, inoculation between the leg tigh and the female&#39;s body is more appropriate if the study requires the observation of females&#39; biological parameters since the Gen&#233;&#39;s organ will not be injured. Despite the inoculation method between the leg thigh and the tick&#39;s body is considered more difficult and easily damage or expose the tick internal organs, such as the midgut and the Malpighian tubules, when it is well performed, it will not disrupt these organs.
Hemolymph analysis is essential to understand the arthropod immune system as well as to understand pathogenesis15,16. Accordingly tick hemolymph can be used to unveil tick physiology, assure tick infectivity, understand tick-pathogen interactions, and the cellular and humoral immune responses9,17,18.
Tick hemolymph collection through the leg cutting is reported in several studies19,20,21. Despite this method being simple with no or very low hemolymph contamination, it is considered counter productive for studies that require high concentrations of hemocytes or large volumes of hemolymph. On the other hand, the correct execution of hemolymph collection through the dorsal region of engorged females is not easy to achieve since the gut occupies almost the entire tick body and rupturing it with the needle causes hemolymph contamination. Contamination due to gut disruption can also be observed when the tick is naturally infected with high loads of hemoparasites (viz., Babesia spp.), or in the final steps of the death process caused by entomopathogens8. In these cases, hemolymph shall be discarded since hemocytes and plasma analyzes can be affected.
The centrifugation speed of hemolymph samples for hemocytes harvesting is also important and directly influence the hemocytes concentration, since high relative centrifugal field (RCF) speeds contribute to: i) cell pellet difficult to resuspend, ii) cells disruption, and iii) hemocytes degranulation. The ideal cell pellet is a soft pellet easy to resuspend. For this reason, the centrifugation at 500 x g for three minutes at 4 &#176;C8 was used. In the present study, hemocytes were resuspended in a culture medium and not in phosphate-buffered saline (PBS) because the culture medium supports better cell viability.
The methods presented here may face limitations when applied to immatures stages (larva and nymph) of ticks or unfed adults. The inoculation method, for example, is only applied for adult engorged females because the needle size will damage immature stages and possibly unfed adult ticks. To inoculate these stages, a microinjector shall be used. Similarly, hemolymph collection through the dorsal region is more effective when applied to engorged ticks, while hemolymph collection by cutting the legs can be used in studies with unfed adult ticks or immature stages. Despite this, our goal here was to show methods that require very low technological resources. In addition, centrifugation technique has to be performed at low centrifugation force because high force or extended spin time can damage cells.
The methods disclosed here can be used as guidelines for studies involving inoculation of entomopathogens into ticks and hemolymph/hemocytes harvesting. The techniques presented here require very low technological resources and are classical steps for studies about tick physiology, pathology, and tick&#39;s immune response.

The cattle tick, Rhipicephalus microplus, is an hematophagus ectoparasite with an enormous negative impact on livestock in tropical areas. This tick is the vector of pathogenic agents such as Babesia bovis, Babesia bigemina, and Anaplasma marginale that, combined with the direct hemofeeding damage, can reduce milk and meat production, cause anemia and ultimately death. Losses caused by this ectoparasite were estimated in 3.24 billion dollars annually in Brazil1. Sustainable methods are demanded and the use of entomopathogenic agents is considered a promising alternative to reduce the use of chemical&#160;acaricides2,3,4.
Entomopathogenic fungi, such as Metarhizium spp., are natural enemies of arthropods including ticks, and some isolates can be used as biocontrollers. These pathogens actively infect the host through the cuticle and colonize their body2,5,6. As the infection develops, cellular and humoral responses are mediated by the tick immune system. Analysis of the tick hemolymph is reported as a useful tool to evaluate the immune responses after the challenging with pathogens7,8.
Arthropods&#39; immune response is divided into humoral and cellular responses. The humoral response involves hemagglutination processes and production of antimicrobial proteins/peptides, whereas the cellular immune response is performed through the hemocytes. These cells are present in the hemolymph from all arthropods and are reported to develop an expressive role in studies involving innate immune response9, as it is directly related to the phagocytosis and encapsulation processes. Accordingly, studies about hemocytes can help to investigate death pathway and understand processes such as autophagy, apoptosis, and necrosis. In some invertebrates as bivalves, the hemocytes&#39; collection faces limitations like cell disruption, low hemolymph volume obtained, and low concentration of recovered cells10. Very frequently, depending on the methodology applied, reduced concentration of cells is obtained, impacting directly on cells quantification and analysis.
The number of hemocytes circulating in the hemolymph is variable among different arthropods and it can change in the same species due to different physiological stages such as sex, age, and the arthropod&#39;s developmental stage11. Hemocytes can also be found adhered to some organs and be released into circulation just after the infection process11. Nevertheless, most studies reported use insects, while ticks remain less explored regarding their physiology and pathology. Despite pathogen inoculation and hemolymph collection in ticks are less used techniques, establishing standard methods helps the development of more accurate studies.
The aim of the present study was to compare the most used methods for hemolymph collection and inoculation of pathogens into R. microplus ticks, evaluating the efficacy in the hemolymph acquisition and hemocytes concentration.

<table><tbody><tr><td>Alkaline Hypochlorite solution</td><td>Sigma-Aldrich</td><td>A1727</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270-1KG</td><td></td></tr><tr><td>EDTA</td><td>Synth</td><td>2706</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000036</td><td></td></tr><tr><td>Flexible rubber</td><td>BD</td><td></td><td></td></tr><tr><td>Giemsa stain</td><td>Sigma-Aldrich</td><td>48900-500ML-F</td><td></td></tr><tr><td>Glass capillary</td><td>CTechGlass</td><td>CT95-02</td><td></td></tr><tr><td>Insulin syringe (needle)</td><td>BD</td><td>SKU:&amp;nbsp;324910</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Vetec</td><td>60REAVET014512</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 culture medium&amp;nbsp;</td><td>Gibco</td><td>11415-064</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860-1L-R</td><td></td></tr><tr><td>Microscope slides</td><td>Kasvi</td><td>K5-7105</td><td></td></tr><tr><td>Microtubes</td><td>BRAND</td><td>Z336769-1PAK</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Vetec</td><td>60REAVET014593</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653-1KG</td><td></td></tr><tr><td>Neubauer chamber&amp;nbsp;</td><td>Kasvi</td><td>K5-0111</td><td></td></tr><tr><td>Penicillin</td><td>Gibco</td><td>15140163</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P2714</td><td></td></tr><tr><td>Tween 80</td><td>Vetec</td><td>60REAVET003662</td><td></td></tr></tbody></table>

disclosing hemolymph collection and inoculation of metarhizium blastospores into rhipicephalus microplus ticks towards invertebrate pathology studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

This article approaches inoculation and hemolymph collection methods applied to ticks. After the inoculation between the leg thigh and the tick female&#39;s body, some fluid (hemolymph) can be secreted during the process; however, it is important to note that when the inoculation is finished, no liquid or tissues were present in the needle tip or at the inoculation site, ensuring that the fungal suspension was completely inoculated. When the inoculation process was correctly performed, the needle insertion did not cause tick females&#39; death. On the other hand, ticks died approximately 48 h after inoculation of the entomopathogenic fungus. Inoculation between the leg thigh and tick female&#39;s body can damage tick intern organs such as midgut and the Malpighian tubules, while damage to Gene&#39;s organ can occur during inoculation between the scutum and capitulum.
When the tick midgut is disrupted by the needle during the dorsal hemolymph collection, the fluid obtained is red, not colorless. This indicates an incorrect hemolymph collection. In these cases, hemolymph shall be discarded since it is contaminated with intestinal content.
The correct hemolymph collection is crucial to properly conduct the experiments and obtain reliable results. When hemolymph was collected from cut tick legs (n = 20 ticks homogenously weighed), the volume obtained was lower than the total hemolymph obtained from the dorsal collection (n = 20 ticks homogenously weighed) (Figure 4). It is suggested that, due to the low volume of each drop that pulls out of the cut leg, hemolymph clotting can be frequently present during this process of hemolymph collection. This may negatively interfere in the hemocytes acquisition and classification (Figure 5 and Figure 6). Despite the higher volume achievement of hemolymph, the dorsal collection is considered more difficult to be performed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59899/59899fig1.jpg" /> 
Figure 1: Ticks&#39;cuticle hygienization. Rhipicephalus microplus fully engorged females were washed using tap water and immersed in 0.5% sodium hypochlorite solution (v/v) for 3 min in a 500 mL glass beaker recipient. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59899/59899fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59899/59899fig2.jpg" /> 
Figure 2:&#160;Ticks&#39;cuticle drying. Rhipicephalus microplus fully engorged females were dried using sterile paper towels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59899/59899fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59899/59899fig3.jpg" /> 
Figure 3:&#160;Metarhizium blastospores. Fungal propaluges used in tick inoculation. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59899/59899fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59899/59899fig4.jpg" /> 
Figure 4:&#160;Representative graph demonstrating hemolymph volume obtained with each collecton method. Rhipicephalus microplus hemolymph volume obtained after dorsal or leg collection. A pool of 20 homogenously weighed ticks were used for each method. These ticks were not previously inoculated. Mean values &#177; standard deviation followed for the same letter do not differ statistically after analysis of variance (ANOVA) test (P&#160;&#8805; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59899/59899fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59899/59899fig5.jpg" /> 
Figure 5:&#160;Hemocytes concentration obtained with each hemolymph collection method. Rhipicephalus microplus hemocytes concentration obtained after resuspension in Leibovitz&#39;s L-15 culture medium after dorsal or leg collection. Mean values &#177; standard deviation followed for the same letter do not differ statistically after Kruskal-Wallis test (P&#160;&#8805; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59899/59899fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59899/59899fig6.jpg" /> 
Figure 6:&#160;Hemocytes found in Rhipicephalus microplus hemolymph smear. R. microplus hemocytes were stained by Giemsa. Black arrows indicate the different cells in the tick hemolymph. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59899/59899fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies at JoVE.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.8K Views.  Federal Rural University of Rio de Janeiro. Teken zijn hetolfachtige ectoparasieten, Rhipicephalus microplus, in de volksmond bekend als de runderen teek heeft een enorme negatieve economische impact op het vee in tropische gebieden. Bovendien stimuleren toenemende bezorgdheid over aangetaste milieuomstandigheden en zorgen over de volksgezondheid als gevolg van oneigenlijk gebruik van chemische acariciden en toenemende weerstand van tekenpopulaties tegen traditionele parasieten onderzoek naar alternatieve bestrijdingsmethoden, zoals he...

Video: Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

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

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

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.

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

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

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

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.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

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

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

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

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.

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and&#160;Borreliella spirochetes&#160;are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including&#160;B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto&#160;(s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick- or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

In vitro culture is a direct detection method for the presence of living bacteria. This protocol describes methods for the culture of diverse Borrelia spirochetes,&#160;including those of the Borrelia burgdorferi sensu lato complex, relapsing fever Borrelia species, and Borrelia miyamotoi. These species are fastidious and slow growing but can be cultured.

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently ...

Human Exposure to Larval Ticks for Tickborne Disease Research

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

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick must remain attached to the skin of the host and complete its multi-day feeding process to acquire its blood meal. Exposing animals to ticks is a common practice for studying host responses to tick bites and tickborne diseases. We developed the procedure, conducted the first human research study, and published the findings on exposing human volunteers to uninfected larval Ixodes scapularis ticks. This article describes the methodology used to construct the containment dressing, how to apply and secure the ticks to the host, how to maintain the dressing, and how to remove the ticks from the host. Exposing volunteers to tick bites is an experimental procedure and must be performed under a clinical research protocol approved by the appropriate regulatory authorities. This method allows for translational research to better understand the human response to tick bites and foster the development of diagnostics, prevention, and therapies for tickborne diseases.

Author Spotlight: Controlled Human Exposure Model for Tick Research and Lyme Disease Studies

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.

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick ...

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

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

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.

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...

Biology

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

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

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

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.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

Tick Artificial Membrane Feeding for Ixodes scapularis

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ticks during both study and rearing can limit experimental questions or the ability of labs to research ticks and their associated pathogens. An artificial membrane feeding system can reduce these problems and open up new avenues of research that may not have been possible with traditional animal feeding systems. This study describes an artificial membrane feeding system that has been refined for feeding and engorgement success for all Ixodes scapularis life stages. Moreover, the artificial membrane feeding system described in this study can be modified for use with other tick species through simple refinement of the desired membrane thickness. The benefits of an artificial membrane feeding system are counterbalanced by the labor intensiveness of the system, the additional environmental factors that may impact feeding success, and the need to refine the technique for each new species and life stage of ticks.

Tick Artificial Membrane Feeding for Ixodes scapularis

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.

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

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