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

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 acaricides^2,3,4.

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 body^2,5,6. 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 pathogens^7,8.

Arthropods' 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' 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'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.

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

1. 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).
2. Divide females in homogeneous weighted groups (with 20 females each): one group without any treatment, one control group inoculated with 5 µL of 0.1% polyoxyethylene sorbitan monooleate aqueous solution (v/v), and one infected-group inoculated with 5 µL 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.

2. Pathogens inoculation between the scutum and capitulum

NOTE: In the present study, entomopathogenic fungal suspensions were used as an example.

1. 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 µL Metarhizium blastospores (Figure 3) suspension to the surface of a plastic paraffin film.
   NOTE: Metarhizium blastospores suspension was adjusted using a Neubauer´s chamber. To speed the inoculation process, multiple bubbles can be added to the plastic paraffin film surface, each bubble corresponding to 5 µL.
2. 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.
3. Inoculate 5 µL of fungal suspension into the tick female between the scutum and capitulum. Inoculate females from the control group with 5 µL 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.

3. Inoculation of entomopathogenic fungi between the leg thigh and the tick female's body

1. Inoculate females with fungal suspension (5 µL at 1.0 x 10⁷ blastospores mL^-1) between the leg tigh and the female'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 µL 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's body, a small volume of hemolymph can be present on the tigh after the needle insertion. Be careful to not inoculate air.

4. Dorsal hemolymph collection

1. Use the rubber part of a winged infusion set, a 0.3 mm capillary tube, and a filtered tip to perform the hemolymph collection.
2. Disrupt the female dorsal cuticle using a 0.3 mm needle.
3. 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'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.

5. Hemolymph collection from the tick leg

1. 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.
2. Apply gentle pressure on the tick'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'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.

6. Hemolymph processing

1. After hemolymph collection, deposit it in 1.5 mL microtubes previously filled with 30 µL protease cocktail and 82 µL saline buffer. Keep the microtubes on ice throughout the hemolymph collection.
2. Centrifuge samples (500 x g for 3 min at 4 °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.
3. Carefully remove the supernatant (cell-free hemolymph). Gently resuspend the cells in, for example, Leibovitz's L-15 culture adjusted to pH 7.0-7.2. Quantify hemocytes by placing 10 µL of resuspended hemocytes in a Neubauer chamber.

7. Hemocyte sampling slide preparation

1. Cut the tick's front leg with a pair of scissors.
2. Apply gentle pressure on the tick's posterior body part. Observe a transparent liquid bubble that shows up on the cut site.
3. Apply the hemolymph drops directly on clean microscope slides, after that, use appropriated methods to stain the cells.
   1. 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'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.

This article approaches inoculation and hemolymph collection methods applied to ticks. After the inoculation between the leg thigh and the tick female'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' death. On the other hand, ticks died approximately 48 h after inoculation of the entomopathogenic fungus. Inoculation between the leg thigh and tick female's body can damage tick intern organs such as midgut and the Malpighian tubules, while damage to Gene'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.

Figure 1: Ticks'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. Please click here to view a larger version of this figure.

Figure 2: Ticks'cuticle drying. Rhipicephalus microplus fully engorged females were dried using sterile paper towels. Please click here to view a larger version of this figure.

Figure 3: Metarhizium blastospores. Fungal propaluges used in tick inoculation. Scale bar = 50 µm. Please click here to view a larger version of this figure.

Figure 4: 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 ± standard deviation followed for the same letter do not differ statistically after analysis of variance (ANOVA) test (P ≥ 0.05). Please click here to view a larger version of this figure.

Figure 5: Hemocytes concentration obtained with each hemolymph collection method. Rhipicephalus microplus hemocytes concentration obtained after resuspension in Leibovitz's L-15 culture medium after dorsal or leg collection. Mean values ± standard deviation followed for the same letter do not differ statistically after Kruskal-Wallis test (P ≥ 0.05). Please click here to view a larger version of this figure.

Figure 6: 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 µm. Please click here to view a larger version of this figure.

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 Gené's organ, impairing the eggs viability^12,13. Gené'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's body is more appropriate if the study requires the observation of females' biological parameters since the Gené's organ will not be injured. Despite the inoculation method between the leg thigh and the tick'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 pathogenesis^15,16. Accordingly tick hemolymph can be used to unveil tick physiology, assure tick infectivity, understand tick-pathogen interactions, and the cellular and humoral immune responses^9,17,18.

Tick hemolymph collection through the leg cutting is reported in several studies^19,20,21. 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 °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's immune response.