Attachment Structure of Wood Ticks; a Fine Structure Study

Heckmann Pakistan Journal of Parasitology 65; June 2018

ATTACHMENT STRUCTURE OF WOOD TICKS; A FINE STRUCTURE STUDY

Richard Heckmann Department of Biology - 1114 MLBM, Brigham Young University, Provo,Utah 84602 USA

Abstract: The mouthparts of four species of ticks (Rhipicephalus, Amblyomma, Dermacenter and Haemaphysalis) were viewed with SEM and compared to one species of mite (Varrao). The ticks have the characteristic pedipalps, chelicera and hypostome to attach and feed on hosts. The appendage modification for host attachment was viewed including the pulvilli and Haller’s organ. Specific mouthparts were scanned with x-ray (XEDS) and the barbs of the hypostome were cut with a gallium ion beam (LIMS). For comparison, the mouthparts of a mite (Varrao) were included in the study. Those organisms studied belonged to the Acarina.

Keywords: Ticks, Mites, Mouthparts, Hypostome, Acarina.

INTRODUCTION

The mouthparts of a tick are designed for puncture of a host skin and then feed on the host body fluids, especially blood. During the feeding process the eight leg Acarinid can transmit many diseases may acquired by human hosts.

Birds, mammals and reptiles are infected with blood feeding ticks. (Macnair, 2016) reported that approximately 850 species have been described worldwide (Wikipedia). Tick, 2018 and Sonenshine, 1991 had suggested that ticks are important vector of disease in humans and animals.

Mouthparts of ticks have three visible components for mouthparts. The two outside parts which are joined are highly mobile palps also called (pedipalps) in between these are paired chelicerae which protect the hypostome. While ticks are deeding the palps more laterally. Beak-like projections are present or the bared hyperstome, this structure plunges while feeding on host skin.

Moreover, most hard ticks are capable of secreting a cement like substance which while feeding glues the tick. This substance once feeding is complete is dissolved. (Vredevoe, 2017; Sonenshine, 1991; Bishop et al., 2002 and Betz, 1996). Parts of the gnathostome represent other parts of the tick body.

The objectives of this paper were to demonstrate using scanning electron microscopy (SEM) the mouthparts, or feeding mechanisms of four species of Ixodidae namely, Haemaphysalis, Rhipicephalus, Dermacenter, and Amblyomma and comment on the mouthparts of other hard- body ticks. The second goal was to show the structures critical for initial attachment and

7 Heckmann Pakistan Journal of Parasitology 65; June 2018 sensing of a tick, with subsequent feeding of the ectoparasite. These tick mouthparts will be compared to a mite (Varrao) which is closely related to the ticks. The Varrao is destructive to honeybees (Apis).

Description of the four ectoparasites is as follows; (Sonenshine, 1991).

Rhipicephalus, is a common tick throughout the world. One species is known as the dog tick (R. sanguineus). It is more common in warmer climates. It can complete its lifecycle indoors. There are numerous species of this genus, which are cosmopolitan in distribution.

Amblyomma is the third largest tick in the number of species and distribution for the Ixodidae. There are many species, which are widely distributed and account for anaplasmosis and in Brazil Rocky Mountain spotted fever.

Haemaphysalis a common hard-body tick (Ixodidae) found on many mammalian hosts both domestic and wild, worldwide. Also on birds and reptiles. Vector of many diseases including Lyme disease, tick paralysis, tularemia, Q fever, etc.

Varrao is a mite that infects honeybees and feeds on the endolymph of the host. Like ticks, it has 8 appendages and biting mouthparts with prominent chelicerae. They are effective vectors of virus and bacteria diseases found in honey bees.

Dermacenter: A hard body tick with 3 life stages (larvae, nymph and adult). Most species such as D. andersoni generally are found in North West United States and South West Canada along the Rocky Mountains. It is a vector for many diseases (Colorado Tick fever, Tularemia, Rocky Mountain spotted fever). The female can feed for up to 5-15 days, thus ticks must be immediately removed (Manyarubuga, 2012).

Ticks are small acarinids, part of the order Parasitiformes. Along with mites, they constitute the subclass Acari. Ticks are ectoparasites living by feeding on the blood of mammals, birds, and sometimes reptiles and amphibians. Moreover, the ticks belong to a family Argasidae and Ixodidae. Ticks and mites have lost abdomen segmentation which their ancestors possessed leading to fusion of abdomen with the cephalothorax. They lack a head with eyes and brain. The tagmata typical of other Chelicerae have been replaced by two new body sections, the anterior capitulum (or gnathosoma), which is retractable and contains the mouthparts, and the posterior idiosoma which contains the legs, digestive tract, and reproductive organs. For piercing skin and blood sucking capitulum is used; it is only in the front of the head and contains neither the brain nor the eyes (Wikipedia, 2018; and Sonenshine, 1991). This study emphasized the mouthparts of Ticks.

Major modifications are present on the appendages of the Tick including pulvilli and Haller’s organ, along with a questing activity aiding in the host attachment and detection.

8 Heckmann Pakistan Journal of Parasitology 65; June 2018

MATERIALS AND METHODS

Specimens of both ticks (Haemaphysalis, Rhipicephalus, Dermacenter and Amblyomma) and mites (Varrao, honeybee mite) were obtained and fixed in 70% ethyl alcohol.

SEM (Scanning Electron Microscopy)

The ticks were fixed in 70 percent ethanol and processed according to Lee, 1992, which included critical point drying (CPD) is sample baskets and subsequently mounting on SEM sample mounts (stubs) with the help of double-sided carbon tape. Samples were coated with gold and palladium for 3 min using a Polaron #3500 sputter coater (Quorum (Q150 TES) www.quorumtech.com) establishing an approximate thickness of 20 nm. Samples were placed and observed in an FEI Helios 660 NanoLab DualBeam (FEI, Hillsboro, OR) scanning electron microscope, with digital images obtained in the Nanolab software system (FEI, Hillsboro, OR), and later transferred to a USB for future reference. Moreover, the images were taken at various magnifications. The samples were received under low vacuum conditions with 10 KV, spot size 2, 0.7 Torr using a GSE detector.

RESULTS

Figures 1 to 12 (Plate 1 and 2) represent the SEM results for the wood ticks mainly Rhipicephalus and Dermacenter while figures 13-16 (Plate 3) are SEM scans of the honeybee mite, Varrao destructor. The gallium cuts of the barbs of the hypostome (Fig. 6A and 6B) are represented by Fig. 10. The gallium cut results will be part of another article.

Pedipalps, chelicerae, and hypostome are visible (Figs. 1 to 5, 8, 9). The hypostome with its barbs is very prominent (Figs. 1 and 2). The initial cutting appendage, chelicerae, shows prominently for Figs. 4, 5 and 6. The intact mouthparts show up for Fig. 8 for another tick species. The cut barb of a hypostome (Fig. 9) is displayed by Figure 10. Part of the host tissue surrounded by the tick mouthparts is given with Fig. 7. Figs. 11A, 11B and 12 display the unique pulvillus near Haller’s Organ. Note the claw-like structure on the terminal segment of the appendage (Figs. 11A, 11B and 12). Plate 3 (Figs. 13-16) are SEM micrographs of the Varrao mite. Figs. 13 and 14 the ventral side with the mouth parts while Figs. 15 and 16 are the chelicerae of the mite, the cutting organ of the parasite.

DISCUSSION

One of the unique activity for host attachment for the ticks is “questing”. The first walking appendage has a unique structure called Haller’s organ used for chemical sensing along with terminal claws and pads on the appendages called pulvilli (Ray, 2012). On the stems of grass or edges of leaves the ticks crow in order to come closer to the host. Heat, movement along with carbon dioxide act as stimuli for questing behavior with the help of extended front legs the ticks climb on the potential host (Macnair, 2016).

9 Heckmann Pakistan Journal of Parasitology 65; June 2018

The chemicals released by the host could also be detected by Haller’s organ. The adult ticks have 8 appendages extending from their body (idiosome). Each appendage has a series of joints and terminates with hook-like fingers, called claws, which aid attachment, especially for questing.

The Ixode ticks release a number of substances which anticoagulant anti-inflammatory and immunosuppressive, thus the host is unaware of the tick feeding and pathogen establish themselves in the infected host (Francischetti et al., 2009 and John and Ryan, 2017).

There have been many studies on the saliva of ticks and the anticoagulants (Jordan et al., 1990; Simo et al., 2017; Kazimirova, 2008 and Bowman et al., 1997). For attachment to a new host, ticks have many anatomical modifications as well as the questing activity. The tip of each appendage has well-developed claws and a pulvillus. Note on Figs. 11A and 11B the pulvillus is well-developed at the top of the first appendage in relation to the same structure on the second appendage (Fig. 12). This structure along with the Haller’s organ can be used for chemical sensing of potential hosts. The pulvilli also have adhesive properties with a spine (empodium) extending between them, there are two pulvilli on each appendage.

Pulvilli are lobes or pads that are located between the tarsal claws of many insects (Niederegger et al., 2002 and Sukontason et al., 2006). The pads have adhesive properties, including the use of an adhesive fluid, and this helps the insect or tick stick to the surface on which it is standing. Between the pulvilli there is often a spine called the empodium. The appendages of Arthropods that become ectoparasites have unique modifications on the terminal end of the walking leg for attachment. These are often used for taxonomy and identifying species (Sonenshine, 1991) of ticks is a complex sensory organ. The organ detects hosts which the tick, being an obligate parasite, must find in order to survive. The organ is important for olfaction and the sensing of humidity, temperature and carbon dioxide. Haller’s organ mite first legs of ticks are minute cavity at the terminal segment (not the pedipalps) (Fig. 12). Each one is composed of a pit and a capsule, and contain sensory setae (Gorb, 2004).

The ticks encounter problem of the host hemostasis immunity and inflammation while attempting to find on their host. To overcome these barriers, ticks evolved a complex and sophisticated pharmacological armamentarium, comprising of bioactive lipids and proteins, to assist blood feeding. Recent progress in transcriptome research has uncovered that hard ticks have hundreds of different proteins expressed in their salivary glands, the majority of which have no known function, and include many novel protein families, these could be important for human pharmaceuticals (Kuthejlova et al., 2001). There are 3,500 putative salivary proteins from different tick species, which may assist the scientific community in the process of functional identification of these unique proteins (Kazimirova, 2008 and Waxman et al., 1998). There may be a valuable use for many in human medicine (Slamova et al., 2011 and Kuthejlova et al., 2001).

Hypostome (also called the maxilla, radula, or labium) is a calcified harpoon-like structure near the mouth area of certain parasitic arthropods including ticks, that allows them to anchor

10 Heckmann Pakistan Journal of Parasitology 65; June 2018 themselves firmly in place on a host mammal for sucking the blood (Bishop et al., 2002). This mechanism is normally strong enough for removal of a lodged tick requires two actions: One to get rid of the tick, and the other to remove the remaining head section of the tick (John et al., 2017 and Manyarubuga, 2012). If the head of the Tick remains on the host after forced detachment it could cause secondary infection.

Chelicerae are located next to the hypostome. When a tick wants to bite, it starts by gently coursing its chelicerae over the skin of its host. Each one ends in a tooth that’s tapered to an especially sharp point, which scrapes and punctures the skin using very little force. The chelicerae tips look like harpoons. This also releases smells and other chemical information that the tick can weigh up (Vredevoe, 2017).

Diseases Transmitted by Ticks

The tick has the widest variety of pathogens of any blood sucking arthropod, including bacteria, rickettsiae, protozoa, and viruses. Presently some important human diseases in the United States caused by tick-borne pathogens include Lyme disease, ehrlichiosis, babesiosis, Rocky Mountain spotted fever, tularemia, and tick-borne relapsing fever (John et al., 2017).

Recently, ticks have been crediting with transmitting a new form of Anaplasmosis. For disease transmission ticks are excellent vectors. The blood feeding trait of ticks is one reasons for successful disease transmission. They are second only to mosquitos as vectors of human disease, both infectious and toxic. More than 800 species of these obligate blood-sucking creatures have been recorded (John et al., 2017 and Ray, 2012).

Ticks can carry and transmit a number of pathogens, including bacteria, spirochetes, rickettsiae, protozoa, viruses, nematodes, and toxins. A single tick bite can transmit multiple pathogens, a phenomenon that has led to atypical presentations of some classic tick-borne diseases. In the United States, ticks are the most common vectors of vector-borne diseases (Vredevoe, 2017).

In North America, the following diseases are caused by tick bites:

l Lyme disease l Human granulocytic and monocytic ehrlichiosis l Babesiosis l Relapsing fever l Rocky Mountain spotted fever l Colorado tick fever l Tularemia l Q fever l Tick paralysis l Red meat allergy (Sanson, 2017)

11 Heckmann Pakistan Journal of Parasitology 65; June 2018

Mites, a close relative to ticks, have some of the same mouthparts and characteristics as ticks. An example is the Varrao mite, found in the honeybees (Apis) especially in the area of the host spiracle. Like ticks, the mites are vectors for an array of diseases (Vredevoe, 2017).

Mites are small arthropods belonging to the class Acarinids and the subclass Acari (also known as Acarina). The term “mite” refers to the members of several groups in Acari but it is not a clade, and excludes the ticks, order Ixodida. The mites and ticks are characterized by the body being divided into two regions, the cephalothorax or prosoma (there is no separate head), and an opisthosoma. According to scientific discipline to study ticks and mites is called acarology. Unique to mites and ticks is the lack of a well-defined head with eyes and brain. Both depend on sensing and tactile structures for host attachment.

At the front of the body is the gnathosoma or capitulum. This is not a head and does not contain the eyes or the brain, but is a retractable feeding apparatus consisting of the chelicerae, the pedipalps and the oral cavity. It is covered above by an extension of the body carapace and is connected to the body by a flexible section of cuticle. The mouthparts differ between taxa depending on diet; in some species the appendages resemble legs while in others they are modified into chelicerae-like structures. The oral cavity connect posteriorly to the mouth and pharynx (Wikipedia, 2018).

Parasitic mites (such as the Varrao mite on honeybees) use their hosts to subsequently spread from host to host by direct contact. Another strategy is phoresy; the mite, often equipped with suitable claspers or suckers, grips onto an insect or other animal, and is transported to another place (Figs. 13 to 16). A phoretic mite is a hitchhiker and does not feed during the time it is carried by its temporary host. These travelling mites are mainly species that reproduce rapidly and are quick to colonize different environment (Wikipedia, 2018). Again the highly modified terminal segment of the walking leg is critical.

Both the Varrao mites from the honeybee and ticks (Rhipicephalus, Haemaphysalis, Dermacenter, and Amblyomma) have similar mouthparts with prominent chelicerae which are used to make the initial entry into the host tissue.

Scanning Electron Microscopy is critical for a study like these and others involving the technique with EDXA accessories (Amin et al., 2018; Heckmann, 2006 and Heckmann et al., 2013, 2012a, 2012b, 2007, 2010; Standing and Heckmann, 2014).

12 Heckmann Pakistan Journal of Parasitology 65; June 2018

Fig. 1. Mouth region of the Tick (Rhipicephalus); H is the hypostome (H) with barbs, (S) a sensory organ, (P) the pedipalp surrounding the hypostome and chelicerae (Top of Hypostome). Arrows Fig. 2. Mouth region of Dermacenter andersoni. Prominent Chelicerae above the barbed hypostome. Pedipalps surround the biting and piercing mechanism of the tick. Fig. 3. Enface view of the mouthparts of Rhipicephalus. Note opening (arrows), Hypostome (H), Pedipalps (P), and Chelicera (C). Fig. 4. Hypostome (H) critical for host attachment, note many scales or barbs on hypostome. Fig. 5. Chelicera (C) with harpoon like tip. Cutting organ of the wood tick. Fig. 6A. Intact barb from the surface of the hypostome. Fig. 6B. Cut barb (Gallium beam) of a tick exposing internal parts of the barb.

13 Heckmann Pakistan Journal of Parasitology 65; June 2018

Fig. 7. Host tissue (H) surrounded by the tick pedipalps. Host tissue inside of the mouth of the tick. Fig. 8. Mouthparts of another tick species showing barbed hypostome (H), Chelicera (C), sensory organ (S) on pedipalp (P). Good representation of functional parts of a tick mouth. Fig. 9. Tick hypostome (H) with attached chelicera (C). Barbs (B) of hypostome allow tick to remain attached. Fig. 10. Gallium cut barb (cross section) depicting the solid center of the barb, high in chlorine ions. Fig. 11A. Pulvillus or pad for the tick appendage, note the terminal claw. First appendage. Fig. 11B. Pulvillus (P) or pad in the joint area of the tick second appendage. Fig. 12. Top view of the terminal area of the appendage with the terminal claw (C) and pulvillus (P). The area for Haller’s organ.

14 Heckmann Pakistan Journal of Parasitology 65; June 2018

Fig. 13. Ventral side of the Varrao mite. Note the highly modified appendages with a bell shaped terminal end. Aid to holding on to the host. Fig. 14. Ventral side of the mite with prominent mouthparts, no hypostome. Fig. 15. Chelicerae of the mite, used to puncture the hosts tegument. Fig. 16. Gallium cut mite Chelicera internal area exposed.

15 Heckmann Pakistan Journal of Parasitology 65; June 2018

LITERATURE CITED

Amin, O.M. Heckmann, R.A. and Bannai, M.A. 2018. Cavisoma magnum (Cavidomidae), a unique Pacific Acanthocephalan redescribed from an unusual host, Mugil cephalus (Mugilidae), in the Arabian Gulf, with notes on histopathology and metal analysis. Parasite., 25: 1-13

Betz, O. 1996. Function and evolution of the adhesion-capture apparatus of Stenus species (Coleoptera, Staphylinidae). Zoomorphology., 116: 15-34.

Bishop, R., Lambson, B., Wells, C., Pandit, P., Osaso, J. and Nkonge, C. 2002. A cement protein of the tick Rhipicephalus appendiculatus located in the secretory e cell granules of the type III salving gland acini, induces strong antibody response in cattle. Int. J. Parasitology., 32: 833-842.

Bowman, A.S., Coons, L.B., Needham, G.R. and Sauaer, J.R. 1997. Tick Saliva: recent advances and implications for vector acceptances. Medical and Veterinary Entomology., 11: 277-285.

Francischetti, I.M., Sa-Nunes, A., Mans, B.J., Santos, I.M. and Ribeiro, J.M. 2009. ‘The role of saliva in tick feeding. Front Biosci (Landmark Ed)., 14: 2051-2088.

Gorb, S.N. 2004. Walking on the ceiling: structures, functional principles, and ecological implications. Arthropof Strct Dev., 33: 1-2.

Heckmann, R.A., Amin, O.A. and El-Naggar, A.M. 2013. Micropores of Acantheocephala, a scanning electron microscopy study. Scientia Parasitologica., 14: 105-113.

Heckmann, R.A., Van Ha, N. and El-Naggar, A.M. 2012b. Electron Optics Study (SEM, EDXA) of Diplozoon paradoxum (Nordmann, 1832) (Diplozoidae, Trematoda) from the common carp. Cyprinus carpio L. (Cyprindae, Osteoichthyes) in Vietnam with comments on potential host fish. Sci. Parasitologica., 13: 109-117.

Heckmann, R.A. 2006. Energy Dispersive X-ray Analysis (EDXA) in conjunction write electron optics, a tool for analyzing Aquatic animal parasitic diseases and deaths, an update. Proc. Parasitol., 41: 01-18.

Heckmann, R.A., Naggar, A.E., Radwan, N.A.E., Standing, M.D. and Eggett, D.L. 2012a. Comparative Chemical Element Analysis using Energy Dispersive X-ray Microanalyss (EDXA) for four species of Acanthocephla. Scientia Parasitologia., 13: 27-35.

Heckmann, R.A., Amin, O.M. and Standing, M.D. 2007. Chemical analysis of metals in Acanthocephalans using energy dispersive x-ray analysis (EDXA) in conjunction with a scanning electron microscope (SEM). Com. Parasitology., 74: 388-391.

16 Heckmann Pakistan Journal of Parasitology 65; June 2018

Heckmann, R., El-Naggar, A., Radwan, N. and Standing, M. 2010. Fine structure and chemical analysis of Neoechinorhynchus idahoensis (Acanthocephala: Neoechinorhynchidae) in the bridgelip sucker Catostomus columbianus. Proc. Parasitol., 50: 63-71.

Jordan, S.P., Waxman, G., Smith, D.E. and Viasuk, G.P. 1990. Tick anticoagulant peptide. Kinetic analysis of the recombinant inhibitor with blood coagulation factor Xa. Biochemistry., 11095-11100.

John and Ryan, K. 2017. “A Tiny Tick Can Cause a Big Health Problem.” Indian Journal of Ophthalmology., 65.11: 1228-1232. PMC.

Kazimirova, M. 2008. Pharmacologically active compounds in Tick salivary glands. Book. Advances in Arachnology and Developmental Biology, Monographs, 281-296.

Kuthejlova, M., Kopecky, J., Stepanova, G. and Macela, A. 2001. Tick salivary gland extract inhibits killing of Borrelia afzelli spirochaetes by mouse macrophages. Infect. Immunology., 69: 575-578.

Lee, R.E. 1992. Scanning Electron Microscopy and X-Ray Microanalysis. Prentice Hall Englewood Cliffs, New Jersey, 458.

Macnair, P., 2016. “All about ticks: why it’s so important to remove a tick.”www.netdoctor.co. uk/conditions/infections/a5 5 59/tick-removal/. Netdoctor.

Manyarubuga, J. 2012. “Dermacentor andersoni”.(http://animaldiversity.org/accounts/ Dermacentor andersoni/) Animal Diversity Web. Retrieved April 19, 2017.

Niederegger, S., Gorb, S. and Jiao, Y. 2002. Contact behaviour of tenant setae in attachment pads of the blowfly Calliphora vicina (Diptera, Calliphoridae). Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology., 187(12): 961-70.

Ray, C. C. 2012. “The Mighty Tick.” (www.nytimes.com/2012/05/29/science/would- eradicating- deer-ticks-hurt-the-ecosystem.html). New York Times.

Sanson, T.G. 2017. “Tick-Borne Diseases.” Overview, Biology and Life Cycle of Ticks, Guidelines on the Diagnosis and Management of Tickborne Rickettsial Diseases, Medscape.

Simo, L., Kazimirova, M., Richardson, J. and Bonnet, S. 2017. The essential role of Tick salivary Glands and saliva in tick feeding and pathogen transmission. Frontiers in Cellular and Infection Microbiology., 7: 281-330.

Slamova, M., Skallova, A., Palenikora, J. and Kopecky, J. 2011. Effect of Tick saliva on immune interactions between Borrelia afzelli and marine dendritic cells. Parasite Immunology., 33: 654-660.

17 Heckmann Pakistan Journal of Parasitology 65; June 2018

Sonenshine. E.D. 1991. Biology of Ticks. New York City, N.Y. Oxford University Press.

Standing, M.D. and Heckmann, R.A. 2014. Features of Acanthocephalan Hooks Using Dual Beam Preparation and XEDS Phase Maps. Poster. Submission Number 0383-00501. 2014 Microscopy & Microanalysis Meeting. Hartford, CT.

Sukontason, K.L., Bunchu, N., Methanitikorn, R., Tarinee, C. and Kuntalue, B. 2006. Ultrastructure of adhesive device in fly in families Calliphoridae, Muscidae and Sarcophagidae, and their implication as mechanical carriers of pathogens. Parasitoloy research. 98. 477-81. 10.1007/s00436-005-0100-0.

Vredevoe, L. 2017. “Tick Biology.” UC Davis Department of Entomology and Nematology, University of California, Davis. entomology.ucdavis.edu/FacultyRovert_B_ Kimsey/ Kimsey_Research/Tick_Biology/.

Waxman, L., Smith, D.E., Arcuri, K.E. and Vlasuk, G.P. 1990. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science., 248: 593-596.

Wikipedia, Tick. 2018. Tick Wikimedia Foundation, 23 Jan. 2018, en.wikipedia.org/wiki/Tick

Wikipedia. 2018. Mite. 22 Jan. 2018, en.wikipedia.org/wiki/mite

(Received on 2nd of May, 2018 and accepted for publication on 28th of May, 2018)