Name: disclosing hemolymph collection and inoculation of metarhizium blastospores into rhipicephalus microplus ticks towards invertebrate pathology studies
Uploaded: 2019-06-01T15:00:00
Description: Watch this Scientific Journal Video about Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies at JoVE.

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 (Fi...

<ol>
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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>

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 ...

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Alkaline Hypochlorite solution</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:158px;">A1727</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">D-(+)-Glucose</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:158px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">EDTA</td>
			<td style="width:116px;">Synth</td>
			<td style="width:158px;">2706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal Bovine Serum</td>
			<td style="width:116px;">Gibco</td>
			<td style="width:158px;">16000036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flexible rubber</td>
			<td style="width:116px;">BD</td>
			<td style="width:158px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Giemsa stain</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:158px;">48900-500ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glass capillary</td>
			<td style="width:116px;">CTechGlass</td>
			<td style="width:158px;">CT95-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Insulin syringe (needle)</td>
			<td style="width:116px;">BD</td>
			<td style="width:158px;">SKU:&#160;324910</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td style="width:116px;">Vetec</td>
			<td style="width:158px;">60REAVET014512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Leibovitz&#39;s L-15 culture medium&#160;</td>
			<td style="width:116px;">Gibco</td>
			<td style="width:158px;">11415-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Methanol</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:158px;">34860-1L-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microscope slides</td>
			<td style="width:116px;">Kasvi</td>
			<td style="width:158px;">K5-7105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microtubes</td>
			<td style="width:116px;">BRAND</td>
			<td style="width:158px;">Z336769-1PAK</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Na2HPO4</td>
			<td style="width:116px;">Vetec</td>
			<td style="width:158px;">60REAVET014593</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:158px;">S7653-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Neubauer chamber&#160;</td>
			<td style="width:116px;">Kasvi</td>
			<td style="width:158px;">K5-0111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Penicillin</td>
			<td style="width:116px;">Gibco</td>
			<td style="width:158px;">15140163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Protease inhibitor cocktail</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:158px;">P2714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tween 80</td>
			<td style="width:116px;">Vetec</td>
			<td style="width:158px;">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, th...

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 ...

Ticks are hematophagous ectoparasites, Rhipicephalus microplus, popularly known as the cattle tick has enormous negative economical impact on livestock in tropical areas. In addition, increasing concerns about degradated environmental conditions and public health worries caused by inappropriate use of chemical acaricides and increasing resistance of tick populations to traditional parasites, are stimulating research on alternative control methods, such as the use of entomopathogenic fungi. These entomopathogens actively infect arthropod hosts, through their cuticle and colonize their body.The arthropod may have different responses to the fungal infection. Arthropod immune system is divided into humoral and cellular responses. The humoral response involves hemagglutination process and production of antimicrobial molecules, whereas hemocytes mediate the cellular immune response.Most studies on arthropod immune response are reported using insects while tick responses remain less explored. Also, pathogen inoculation and tick hemolymph collection are poorly widespread techniques necessary to the better understand of the arthropod's immune response. Accordingly, this video address techniques\used to film the inoculation, hemolymph collection and processing.Since tick hemocytes harvesting face some limitations, such as cells disruption, low hemolymph value obtaining, and low concentration of recovered cells. Directly infecting on cells configuration and analysis. After tick gathering, wash engorged females.Use tap water and immerse them in 0.5%sodium hypochlorite solution for three minutes in a 500 milliliters glass beaker recipient. For cuticle hygiene, divide females in homogeneous groups with 20 females each. Three replicates of each group were performed.Metarhizium robertsii blastospores were used in the inoculation process. Add on a liquid of five microliters metarhizium blastospores suspension to the surface of a plastic paraffin film. To speed the inoculation process, multiple bubbles can be added to the plastic paraffin film surface, each bubble corresponding to five microliters.Use an one-millimeter ultra-fine insulin syringe and a 0.3 millimeters needle to put a suspension and inoculate it into the tick. Remember to take all the air out of the syringe before using it. Inoculate 5 microliters of fungus suspension into the tick female between the scutum and capitulum.A small volume of hemolymph may be present on the framing after the needle incision. Be careful to do not inoculate air. Inoculate females with fungus suspension between the leg thigh and the female's body using a one-milliliter ultra-fine insulin syringe coupled to a 0.3 millimeter needle.When the metarhizium blastospores inoculation is performed between the leg thigh and the tick female's body, a small volume of hemolymph can be present on the thigh after the needle insertion. Be careful to do not inoculate air. Use the rubber part of a winged infusion set a 0.3 miliimeters capillary tube and a filter tip to perform the hemolymph collection.Disrupt the female dorsal cuticle using a 0.3 millimeters needle. After disruption apply gentle pressure on the anterial part of the tick body. Observe an almost transparent liquid pooling out of the disruption site.Collect the hemolymph by sucking the liquid through the filter tip coupled to a rubber part of a winged infusion set and a 0.3 millimeters capillary tube. Do not press the tick's body hardly during it's immobilization because this may disrupt the midgut and contaminate the hemolymph. Wait until gentle pressure can expel the fluid without contamination.Immobilize the tick. Cut a piece of a front leg. One or more legs can be cut as well as the same leg can be cut more than one time.Apply gentle pressure on the tick's posterior body part. Observe a transparent liquid bubble show up on the cut side and collect it with the capillary tube. Do not press the tick's body hardly, during it's mobilization because this may disrupt the midgut and contaminate the hemolymph.Wait until gentle pressure can expel the fluid without contamination. After hemolymph collection, deposit it in 1.5 mililiters microtube previously filled with 30 microlitres protease cocktail and 82 microlitres saline buffer. Keep the microtubes on ice throughout the hemolymph collection.Centrifuge samples and the soft pellet of hemocytes will be formed after hemolymph centrifugation. 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. Carefully remove the supernatant cell-free hemolymph, and gently re-suspend the cells.Quantify the hemocytes by placing 10 microlitres of re-suspended hemocytes in the Neubauer chamber. For cells re-suspension a culture media was used. Cut the tick's front leg, apply gentle pressure on the tick's posterior body part, observe a transparent liquid bubble show up on the cut site.Apply the hemolymph drops directly on clean microscope slides. After that use appropriate methods to stain the cells. Inoculation between the leg thigh and tick female body can also damage tick internal organs such as midgut and the malpighian tubules.When the tick midgut is disrupted by the needle at the dorsal hemolymph collection the fluid obtained is red, not transparent. This indicates an incorrect hemolymph collection. In this case, hemolymph shall be discarded as it is contaminated.Hemolymph contamination can also be observed when the tick is naturally infected with high loads of pathogens, or in the final steps after death process caused by inoculated pathogens. The hemolymph volume obtained after the dorsal collection is higher than the volume obtained at the collection through the leg. Despite easiness of contamination is higher in the first collection method.The hemocytes concentration was also significantly higher at the dorsal collection. When correct hemocytes collection is performed a good smear can be obtained and it's possible to distinguish different hemocytes after staining. This video provided information on methods to perform inoculation of pathogen, specifically entomopathogenic fungi, into ticks and collect their hemolymph.The techniques here presented require very low technological resources in our classical steps for studies about tick physiology, tick pathology, and tick's immune response. The correct harvesting of the arthropod cells, and cell-free hemolymph is crucial to afford reliable information for subsequent studies. For that reason, it is also important to be aware of the most common mistakes observed in the execution of these techniques.Additionally, the proposals of this study shall determine the more appropriate site for hemolymph collection through deforming or cutting the legs, considering the difference in the volume obtained and the probability of contamination.

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Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

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

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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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 ...

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...