ISSN 0013-8738, Entomological Review, 2010, Vol. 90, No. 8, pp. 1095–1100. © Pleiades Publishing, Inc., 2010. Original Russian Text © S.A. Leonovich, 2010, published in Parazitologiya, 2010, Vol. 44, No. 1, pp. 70–76.

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The Tarsal Gland of Ixodid Ticks Ixodes persulcatus and Ixodes ricinus (Mesostigmata, Ixodidae)

S. A. Leonovich Zoological Institute, Russian Academy of Sciences, St. Petersburg, 199034 Russia

e-mail: leonssa@mail.ru Received October 22, 2009

Abstract—A complicated multicellular gland is situated in all the leg tarsi, occupying from one third to half the segment. The glandular cells form a single-layer sack; the inner surface of the gland cavity is covered with the multi-layer membrane. Cuticular rods (“sinews”) of muscles moving the claw pass inside the gland cavity. The glandular cells are characterized by the presence of numerous microvilli on their apical surfaces and by the pres-ence of secretory vacuoles. The basal part of each secretory cell is characterized by accumulations of lipid vacuoles and glycogen granules. Problems concerning the possible role of tarsal gland in the production of the trace phero-mone are discussed. DOI: 10.1134/S0013873810080130

Ixodid ticks Ixodes ricinus and I. persulcatus are known as vectors of many dangerous transmissive infections such as the tick-borne encephalitis and the Lyme disease. Modern methods of protection of hu-mans from tick attacks mainly include the use of pro-tective cloths or chemical repellents. Such methods do not appear to influence tick population density in the wild and cannot limit their distribution to the new territories observed at present (Korotkov et al., 2006). The use of pheromones, and, in particular, of phero-mones providing the meeting of sexes in the wild be-fore contact with hosts seems to be a new innovatory approach to the control of the distribution of these dangerous vectors (the presence of fertilized hungry females of I. persulcatus is described in detail in: Leonovich, 1985). The presence of sex pheromones determining sex recognition and female attractiveness is demonstrated in several publications (see a review by Sonenshine, 2006). Recently it was demonstrated that females of the European forest tick Ixodes ricinus produced a trace pheromone, allowing males to pursue unfertilized females (Zemek et al., 2002); attractive-ness of traces (marks) left by engorged females was higher than that left by hungry females. Glands secret-ing products that can leave traces (marks) on the sur-face of the substrate include tarsal glands providing the work of leg pulvilli (Leonovich, 1976), idiosomal glands, and also, probably, glands of the area porosa. No other exosecreting glands are present in I. ricinus and I. persulcatus. Taking into account this fact, the

author examined the structure of the tarsal gland in hungry and engorged females of I. ricinus and also in hungry males and females of the closely related spe-cies, the taiga tick I. persulcatus by electron micros-copy methods. This study is important for two reasons. First, it describes in detail the structure of the gland which is poorly known (in fact, virtually unknown) to the majority of acarologists (ixodologists), in practi-cally important species, vectors of the tick-borne en-cephalitis and the Lyme disease. Secondly, any changes in the gland structure during feeding can serve as indirect evidence of the presence of the above men-tioned male-attracting trace pheromone in the tarsal gland structure. The role of tarsal glands in the pro-duction of trace pheromones is not anything unusual; it is known in some insects, in particular, in ants (Hölldobler and Palmer, 1989).

The first mention of a complicated gland found it tarsi of all the legs in ixodid ticks can be found in a brief communication by Leonovich (1976), where this gland was revealed in several occasionally se-lected species belonging to different genera and sub-families of ixodid ticks (Leonovich, 1976). In this work, the gland was named the tarsal gland, according to its location place. Later, this gland was examined by the author in the tick Hyalomma asiaticum, belonging to the subfamily Amblyomminae. Materials of this study were published in the Atlas of ixodid ticks ultra-structure edited by Yu.S. Balashov (Atlas, 1979). Ac-tually, all the data on the tarsal gland of ixodid ticks

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are limited to these two small publications. No data on the structure of the tarsal gland in representatives of the subfamily Ixodinae which includes both the spe-cies examined (I. ricinus and I. persulcatus) are avail-able in the literature.

MATERIALS AND METHODS

Ticks Ixodes ricinus (L.) were collected by the au-thor in the environs of Ceske Budejovice (Czech Re-public) in the summer of 2007 and fixed for studying under the electron microscope. Some ticks for fixation were taken from the laboratory culture of the Institute of Parasitology, Czech Academy of Sciences. Taking into account the fact that data on the attraction of males to traces left by engorged females were per-formed at this institute (Zemek et al., 2002), it was the morphology of glands of these ticks that we decided to examine. Engorged females of the European forest tick I. ricinus were collected in Leningrad Province (Mer-evo Vill., Luga district) from stray dogs in the summer of 2008. The taiga ticks I. persulcatus were collected near Lisii Nos Station (Kurortnyi District, Leningrad Province).

For studying under the transmissive electron micro-scope, live ticks were dissected under a layer of fixing fluid (leg tarsi were cut off) and fixed in 1% glutharal-dehyde solution in the 0.1 M phosphate buffer. After washing in the same buffer, the material was postfixed in the 1% solution of osmium tetroxide in the same buffer. The material was embedded in Araldite resin mixture, fine cuts were made with the LKB-3 ultrami-crotome (Sweden). The material was examined under the Tesla-500 transmitting electron microscope.

The material collected in the Czech Republic was fixed directly in the 1% solution of osmium tetroxide in the phosphate buffer.

Semi-fine cuts were stained with toluidine and ex-amined under the Leica optical microscope.

Ticks fixed in 70% alcohol were used for examina-tion under the scanning electron microscope. The fixed material was dehydrated in a series of alcohols, then transmitted into acetone and dried in a HCP-2 critical point dryer (Hitachi, Japan) using liquid car-bon dioxide as a working agent. All the material had been previously cleaned in a D-300 ultrasonic cleaner (Russia). Dried ticks were mounted on alumina stubs with double-sided sticky film, coated with platinum in an Eiko-5 vacuum coater (Japan) and examined under the Hitachi-S570 scanning electron microscope. In

order to examine the three-dimensional structure of the gland, some preparations were made of specimens embedded into the Araldite resin mixture; the resin was removed with KOH according to our method de-scribed earlier (Balashov and Leonovich, 1984). The data was recorded in the digital form.

The present study is illustrated with images ob-tained under scanning and transmitting electron micro-scopes (Figs. 1, 2, 7–9), with images of semi-fine cuts under the optical microscope (Figs. 3–6), and also with a figure (Fig. 10).

RESULTS AND DISCUSSION

Tarsal gland is situated in all leg tarsi, occupying from one third to half the segment length in its distal part (the area occupied by the gland depends on the length of the tarsus. For example, in the long fore tar-sus, where the Haller’s organ is situated (Fig. 1), the gland occupies approximately the distal third of the segment; in other tarsi, approximately half of the seg-ment). In the glandular area, the segment is virtually entirely occupied by glandular cells (Figs. 2, 3), form-ing a single-layer “sack” (Fig. 3), separating the cavity of the gland from the cavity of the tarsus, filled with haemolymph (Figs. 3, 4). The muscles are observed only in the proximal part of the segment (Fig. 5); more distally, they are connected with cuticular rods (“sin-ews”), passing into the gland cavity (Figs. 3, 7). The leg nerve passes outside the gland cavity, shifting lat-erally to the hypodermal cells situated closer to the inner surface of the tarsus (Figs. 3, 5). In the most distal part of the gland no glandular cells are observed, and the central cavity of the gland, identified by its multilayer cover, continues as a duct going to the pulvillus (Fig. 6).

Glandular cells form a single-layer sack (Figs. 3, 4); the inner surface of this sack is covered with a mem-brane. In cross-cuts, this membrane possesses a fibril-lar structure, with more electron-dense fibrils inter-laced with less electron-dense ones (Fig. 7). In the pro-ximal-distal direction, fibrils of the membrane become denser, turning into the solid electron-dense layer (Fig. 6). The apical parts of glandular cells form nu-merous microvilli; accumulations of mitochondria are found under these microvilli (Fig. 7). The combination of these two characters is commonly known as a fea-ture characterizing cells with active ion transport. Numerous secretory vacuoles (granules) are accumu-lated in the apical part of each glandular cell (Figs. 3, 4, 7). In the cuts examined under the optical micro-

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scope (Figs. 3, 4) and in the fine cuts examined under the transmissive electron microscope (Fig. 7), the se-cret in these vacuoles is transparent; either it is washed out during preparation procedures, or it possesses a decreased electron density. It is interesting that lipid

granules found in the basal area of glandular cells are well-preserved after preparation procedures (Fig. 8). The rough endoplasmic reticulum in glandular cells is absent, testifying to the non-protein nature of the ma-terial secreted into the gland cavity. The structures of

Figs. 1–6. Tarsal gland in a scanning electron microscope (1, 2) and in an optical microscope (3–6). (1, 5, 6) I. persulcatus; (2, 3, 4) I. ricinus [(1) anterior tarsus, × 90; (2) tarsal gland in the anterior tarsus, × 400; (3) transverse cut of tarsus III, × 500; (4) secre-tory cells in an optical microscope, × 1200; (5) cross cut of tarsus II in proximal region (leg muscles and nerve and the most proximal part of the gland), × 480; (6) cross cut of tarsus III in the distal region, × 600]. (c) cavity of the gland; (cu) cuticle; (ho) Haller’s organ; (m) muscle fibers; (p) pulvillus; (sc) secretory cells; (sg) secretory granules; (r) cuticular rods attaching muscle fibers to the claw.

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the Golgi complex are also poorly developed (Fig. 7). The author failed to see places of fusion of vacuole membranes with the external cell membrane in the apical area of glandular cells. We assume that in this case, merocrine secretion occurs, and vacuoles serve only as reservoirs for secret accumulation, probably, in order to provide continuous secretion. The basal parts

of glandular cells contain an accumulation of glycogen granules (Fig. 9). The anatomy and histology of the gland is schematically represented in Fig. 10.

No basic differences in the gland structure between males and females were revealed. The same is true for hungry and engorged females. The main differences

Figs. 7–9. Fine structural details of secretory cells (electron microscopy): (7) apical part of secretory cells in the region of contact with membranous layer (cs) separating central cavity of the gland from glandular cells, × 18000; (8) accumulation of lipid granules (lg) in basal part of the cell, ×20000; (9) glycogen granules (gl) in basal part of glandular cells, × 24000. (mv) microvilli; (r) cuticular rods attaching muscle fibers to the claw; (sg) secretory granules (vacuoles).

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are as follows. In engorged females, the number of glycogen granules and lipid vacuoles in the basal parts of glandular cells significantly decreases in compari-son with hungry females. At the same time, the aver-age density and number of secretory vacuoles remain approximately at the same level. This phenomenon can be explained by the fact that the secret of the gland is produced from deposits (glycogen granules and lipid drops) consumed by engorged nymphs; on the whole, engorged individuals are older than ticks collected by the author in the wild (newly emerged individuals after wintering) and newly emerged ticks from the labora-tory culture. In older individuals, this resource is evi-dently decreased. Thus, we can rather reasonably as-sume that feeding does not affect the functioning of the tarsal gland.

The question of the functional significance of the tarsal glands remains open. Its role in the functioning of the pulvillus is undoubted, because the duct of the

gland terminates in the pulvillus (Fig. 6). In many insects, e.g., in flies, sticky secret of tarsal glands, being spread on the surface of the pulvillus, provides moving of these insects on smooth surfaces (Sukon-tason et al., 2006). However, another mechanism also occurs. In some hymenopterans (e.g., bees and ants), the tarsal gland provides the functioning of the arolium, a king of a small “pad” between claws. The gland secrets liquid into the central cavity and the whole gland looks like a closed resilient vesicle. Claw-moving muscles press on the gland, pumping the fluid inside the arolium, or weaken, decreasing the pressure. Biomechanics of this process, providing the moving of insects on smooth surfaces, is examined in detail (Fe-derle et al., 2001). It is interesting that, in this case, the secret of the gland does not go outside, and the arolium functions only due to the hydraulics of the specific structure of the cuticle of the arolium. Re-cently, a similar mechanism of the work of the pad

Fig. 10. The structure of the tarsal gland in Ixodes ticks, according to scanning and transmitting electron microscopy, and to optical microscopy. (cl) claw; (gl) glycogen granules; (pl) pulvillus; (lv) lipid vacuoles; (m) tarsal muscles; (mv) microvilli; (sv) secretory vacu-oles; (tg) tarsal gland.

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between the claws was revealed in stick insects (Scholz et al., 2008). In our case, such a mechanism of functioning of the pulvillus seems doubtful, because contraction of muscles will not result in pumping of the fluid into the pulvillus, but will rather change the shape of the gland inside the tarsal segment. (Figs. 3, 10).

Thus, we may rather reasonably assume that the tar-sal gland produces a non-protein secret providing the functioning of the pulvillus as a sticky pad. Some traces of these secretions are inevitably left on the substrate and can be detected by contact chemorecep-tors (Grenacher et al., 2001; Leonovich, 2005). Unfor-tunately, electron microscopy methods do not allow us to judge of the differences in the chemical nature of the secret produced by males and females. We can assume that, in the wild, detection of traces left by ticks of both sexes makes contact between them easier; at the same time, recognition of sexes can occur after the contact of individuals belonging to one or different sexes owing to other factors that are not associated with functioning of the tarsal gland.

ACKNOWLEDGMENTS

This work was financially supported by the Russian Foundation for Basic Research, project no. 09-04-00390-a, and a grant of the President of Russian Fed-eration for the support of scientific schools NSh-5563.2006.4. The author is also grateful to Edwin Bouman (Institute of Parasitology, Czech Academy of Sciences, Ceske Budejovice, Czech Republic) for the assistance in tick collecting and also to A. Tennison and T. Tsogoev for their assistance in electron micros-copy studies.

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