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

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.