J. Acarol. Soc. Jpn., 22(1): 1-23. May 25, 2013 © The Acarological Society of Japan http://www.acarology-japan.org/ 1

[REVIEW]

Reproduction in a Metastriata Tick, Haemaphysalis longicornis (Acari: Ixodidae)

1 2 3 4 Tomohide MATSUO *, Nobuhiko OKURA , Hiroyuki KAKUDA and Yasuhiro YANO

1Laboratory of Parasitology, Department of Pathogenetic and Preventive Veterinary Science, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan 2Department of Anatomy, School of Medicine, University of the Ryukyu, Nishihara, Okinawa 903-0215, Japan 3Fukuoka Joyo High School, Chikushi 901, Chikushino, Fukuoka 818-0025, Japan 4Division of Immunology and Parasitology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan (Received 20 June 2012; Accepted 29 December 2012)

ABSTRACT

The superfamily Ixodoidea includes two major families: the Ixodidae called “hard tick” and Argasidae called “soft tick”. Furthermore, Ixodidae is classified into Prostriata (Ixodidae: Ixodes), and Metastriata (Ixodidae except for Ixodes) based on their reproductive strategies. That is, species in each group have characteristic reproductive organs and systems. Ticks are important as vectors of various pathogens. Haemaphysalis longicornis belonging to the Metastriata is characterized by having both the parthenogenetic and bisexual races, and is widely distributed in Australia, New Zealand, New Caledonia, the Fiji Islands, Japan, the Korean Peninsula and northeastern areas of both China and Russia. This species is known as a vector of rickettsiae causing Q fever, viruses causing Russian spring-summer encephalitis, and protozoa causing theileriosis and babesiosis. H. longicornis, the most dominant tick in Japanese pastures, is very important in agricultural and veterinary sciences because this species also transmits piroplasmosis caused by Theileria and Babesia parasites among grazing cattle. We present here an overview reproduction in the bisexual race of H. longicornis. Key words: Metastriata, tick, reproduction, bisexual race, vector

INTRODUCTION

A taxonomic group referred to as “tick” in the subclass Acari, which includes over 35,000 species (Oliver, 1989) is classified as Ixodoidea of the Metastigmata (Ixodida). The superfamily Ixodoidea includes two major families: the Ixodidae called “hard tick” and Argasidae called “soft * Corresponding author: e-mail: [email protected] DOI: 10.2300/acari.22.1 2 Tomohide MATSUO et al. tick”. A third family Nuttalliellidae is minor and consists of only a single species for which reproductive strategies are unknown. Furthermore, Ixodidae is classified into Prostriata (Ixodidae: Ixodes) and Metastriata (Ixodidae except for Ixodes) based on their reproductive strategies. It is known that the valid genus and species names of ticks are more than 850 (Baker and Murrell, 2004; Guglielmone et al., 2009). Ticks cause severe toxic conditions, e.g. tick paralysis, various tick toxicoses, irritation, tick bite allergies, immune responses and economic losses due to blood sucking (Sonenshine, 1991). Haemaphysalis longicornis Neumann, 1901 belonging to the Metastriata is widely distributed in Australia, New Zealand, New Caledonia, the Fiji Islands, Japan, the Korean Peninsula and northeastern areas of both China and Russia. This species is known as a vector of the rickettsiae causing Q fever, viruses causing Russian spring-summer encephalitis, and protozoa causing theileriosis and babesiosis, respectively (Hoogstraal et al., 1968). H. longicornis is the most dominant tick in Japanese pastures and transmits piroplasmosis by Theileria sergenti / buffeli / orientalis group (Fujisaki, 1992; Fujisaki et al., 1994) and Babesia ovata among grazing cattle (Ishihara 1968). Additionally, reproduction of H. longicornis is very interesting because the species includes both pathenogenetic and bisexual races (Kitaoka, 1961). Our research group has studied reproduction in the bisexual race for more than 10 years. Recently, numerous studies on vector ticks have reported and elucidated roles of reproduction in the transmission of pathogens such as the transovarial transmission of protozoans. Therefore, studies on the reproduction of this species need to be summarized in order to not only understand their life cycles for control but also to assist in investigations on the relationship between ticks and pathogens. Therefore, in this review we describe reproduction in the bisexual race of H. longicornis in hopes the information will assist the progress of research on ticks as vectors of various pathogens.

MALE REPRODUCTION

Reproduction in male H. longicornis is described: 1) the development of the testes during feeding (Matsuo et al., 1997a) and 2) the turnover of the spermatogenic cells (Matsuo and Mori, 2000), 3) the external shape and histological changes of the male accessory genital glands during a feeding (Matsuo and Mori, 2000; Matsuo et al., 1997b) and after a copulation (Matsuo, 2000), 4) ultrastructure of the spermatophore which play an important role in the transfer of male germ cells from a male to a female and action of the spermatophore in vitro (Matsuo et al., 1998) and 5) the derivation of the spermatophore components from the accessory glands (Matsuo, 2000; Matsuo et al., 1997b). 1) Testes and Spermatogenesis The adult male H. longicornis has a paired tubular testes similar to other ticks, and the testes increased approximately twice in length and five times in thickness during feeding (Fig. 1). The testes are fused posteriorly (Argasidae), broadly joined (most Prostriata), or connected only by an extremely thin filamentous strand of tissue (Metastriata) (Oliver, 1982). At each feeding stage the testes contain spermatogenic cells in various stages of spermatogenesis with more advanced spermatogenesis in the posterior region of the testes than the anterior region connected to the vas deferens. The spermatogenic cells are arranged in reverse order as usually found in the tubular Reproduction in Haemaphysalis longicornis 3 testes of most invertebrates (Oliver, 1982). Spermatogonia and early primary spermatocytes are contained in the anterior region of the testes of unfed males, and slightly enlarged primary spermatocytes in the posterior region. Spermatogonia, primary spermatocytes during their growth phase, and early spermatids just after maturation division are found in the testes of 3-day fed males. Spermatogenesis is completed within the testes, and spermatogenic cells at all stages from spermatogonia to elongated spermatids were contained in the testes of completely (5-day) fed males. Furthermore, the spermatogenic cells are packed in cysts composed of cyst cells as seen in other ticks (Oliver, 1982) (Fig. 1). Cysts arranged radially form the central lumen as a passage extending the length of testes for elongated spermatids to move to the vas deferens. The cyst cells face the lumen and contain microvilli, so are also called ‘nutritive cells’ (Reger, 1961) or ‘interstitial cells’ (Raikhel, 1983). The development of all cells within a particular cyst is synchronous, and the number of spermatogenic cells appears to be species specific (Khalil, 1969, 1970). Spermatogonia and early primary spermatocytes are contained in the anterior part of the testes of unfed males. These cells are small and have a large nucleus. Polysomes and mitochondria are found in the cytoplasm, and intercellular bridges have already formed. Subsequently, the main growth phase of primary spermatocytes has begun, and these cells gradually increase in size. The polysomes disappear and the Golgi complex emerges in the primary spermatocytes after the beginning of this phase. The subplasmalemmal cisternae (SC) that become cellular processes on the surface of spermatozoa begin to be formed, and are also called ‘subsurface cisternae’ (Reger, 1962) or ‘cortical alveoli’ (Raikhel, 1983). Although the origin of the SC is uncertain, the endoplasmic reticulum (Reger, 1961, 1962, 1963), Golgi complex (Reger, 1974) and plasma membrane (Oliver and Brinton, 1972; Suleiman and Brown, 1978) are suggested as origins. In H. longicornis, the timing of the emergence and disappearance of the Golgi complex is synchronized with the addition of SC initiation and completion. Therefore, we conclude the SC originates from the Golgi complex. The size of the cells reduced by meiosis after the main growth phase is completed at this time. Early spermatids that formed the largest SC are found immediately after the reduction in size. Thereafter, the cells in which the relocation of the nucleus and SC, and the formation of the cisternal cavity have occurred are early rounded spermatids. However, the second reduction of cell size is also observed as reduction in area of sections with a change in shape during the formation of the cisternal cavity in H. longicornis. Oliver (1982) also indicated that the spermatogenic cell where the nucleus is located centrally and the periphery remains surrounded by a cup-shaped SC is the early spermatid. These cells in which the cisternal cavity has formed begin to elongate, and finally the elongated spermatids or prospermia (Till, 1961) consisting of an outer sheath and inner cord are completed. The cellular processes originating from the SC are arranged on the outer surface of the inner cord and the inner surface of the outer sheath. Detailed studies on tick spermatogenesis have particularly observed the structure of the elongated spermatids, cellular processes called ‘motile processes’, and the specialization of the sperm membrane (El Said and Swiderski, 1980; Oliver and Stone, 1983; Wüest et al., 1978). The SC changes into the cellular processes with formation of the cisternal cavity by membrane fusion (Reger, 1962), and the presence of the electron-dense fibrillar materials suggests that a contractile system is involved in sperm movements (Rothchild, 1961). Therefore, it is generally known that 4 Tomohide MATSUO et al.

Fig. 1. Diagram of the dorsal view of genital glands in an adult male. Relative size of testes (TE) is almost the same as that of a feeding male. Paired testes are connected by a strand of connective tissue at their posterior ends. The vas deferens (VD) forms the seminal vesicle (SV) after it turns twice, and the seminal vesicle opens to the dorsal surface of the ejaculatory duct (EJ). Spermatogenic cells in the area III are at a more advanced stage than those in the area I. AG, male accessory genital glands. After Matsuo et al. (1997a) with permission from the Journal of Faculty of Agriculture, Kyushu University.

these fibrillar structures participate in the movement of the sperm. However, tick spermatogenic cells undergo remarkable morphological changes, that is, elongation from rounded spermatids to elongated spermatids and spermateleosis, the processing from elongated spermatids to spermatozoa that occurs in the female genital tract. In addition, the fibrillar materials found in the cellular processes are thought to take part in formation of the spermatozoa morphology. Most Metastriata males require a blood meal during the adult stage to copulate, while elongated spermatids are completed without feeding in the adult stage of Aponomma concolor, Aponomma hydrosauri, and Amblyomma triguttatum in Australia as well as the Argasidae and Prostriata (Guglielmone and Moorhouse, 1983; Oliver and Stone, 1983). That is, most Metastriata male spermatogenesis is stimulated by a blood meal, and some genes essential for sperm development have been identified recently (Guo and Kaufman, 2008). The division of new spermatogenic cells at the anterior ends of the testes is synchronized with the progress of spermatogenesis in the posterior region of the testes. Oliver (1986) also described that a pool of pre-spermatogenetic germ cells is retained during spermatogenesis, and this pool provides a continuous source for repeated waves of spermatogenesis following each new blood meal, enabling the ticks to copulate many times. 2) Turnover of Spermatogenic Cells The spermatogenic cells at the posterior region of the testes shrink and look like granules in the testes at 2 days after complete feeding. The cysts contract as a result of the shrinkage of the spermatogenic cells in each cyst, so the lumen enlarges, and then the lumen is filled with elongated spermatids that have been completed in the posterior end of the testes. The Reproduction in Haemaphysalis longicornis 5 degenerating area contracts more in the testes 4 days after complete feeding, and almost all of the posterior region of the testes appears to be filled with elongated spermatids. This state is retained up to 10 days after complete feeding. The testes become considerably thinner, but their length hardly changes. The degenerating cells shrink, and become ultrastructurally electron-dense. The shape of the nucleus become irregular, and the chromatin condenses into uniformly dense masses. The plasma membrane contains some blebs, but no loss of integrity. Ultrastructural observations also demonstrate that the degeneration occurring in the spermatogenic cells begins at feeding in the adult stage without complete elongated spermatids, that is, from primary spermatocytes at the middle of the main growth phase to spermatids just before the completion of elongation. This suggests the spermatids are stable morphologically and physiologically. There appears to be enough elongated spermatids in the testes and seminal vesicle of a completely fed male so the male can copulate a few times, although their longevity of the spermatids is uncertain. Males may be able to copulate until elongated spermatids die even if degeneration of the spermatogenic cells occurs. As H. longicornis (most Metastriata) copulate on the host, males can re-attach to the host soon if they cannot find a female. Therefore, the developing spermatogenic cells are expected to degenerate rapidly, and the testes develop again after re-feeding if necessary. However, male Argasidae and Prostriata, which complete spermatogenesis within the testes at the engorged nymphal stage and mainly copulate off the host, may have to maintain developed testes longer than Metastriata. As mentioned above, the degeneration and turnover of spermatogenic cells may play an important role in making multi-copulation of a male tick possible in H. longicornis. 3) Male Accessory Genital Glands The male accessory genital glands consist of two single and five paired lobes opening to a collecting duct (CD)(Fig. 2). The CD lying on the floor of the body cavity is connected to the ejaculatory duct (EJ) anteriorly, and the vas deferens (the seminal vesicle) opened to the dorsal surface of the EJ. The single dorso-median lobe (DML) opens to the posterior end of the CD and extends in an antero-dorsal direction. The single MVL opens to the antero-ventral region of the CD. Five paired lobes are on both sides of the CD; the antero-ventral lobe (AVL), the latero- ventral lobe (LVL), the dorso-lateral lobe (DLL), the postero-lateral lobe (PLL) and the postero- ventral lobe (PVL) from anterior to posterior. The DLL is incorporated with the PLL before opening to the CD, and they are also histologically identical. The external shape of the accessory glands is different between Ixodidae and Argasidae (El Shoura, 1987a; Mulmule and Thakare, 1985; Sonenshine, 1991; Tatchell, 1962), and we defer to Chinery (1965) for names of lobes because the external shape in H. longicornis is similar to that in other ixodid tick species. In the unfed males, all lobes of the accessory glands consist of undeveloped cells with no secretory granules. While, in completely fed males, cells of all lobes are filled with many granules of ultrastructurally various sizes and shapes. Some lobes morphologically has several types of granules; 10 types in the DML, 3 types in the MVL, 4 types in the AVL, 4 types in the LVL, 1 type in the DLL and the PLL, and 2 types in the PVL. The epithelial cells of the DLL and PLL are highly similar, and wedge-like cells with homogenous cytoplasm and no secretory granules scattered between glandular cells in the two lobes. The secretory granules found in all lobes after feeding (just before copulation) are almost completely released just after copulation. 6 Tomohide MATSUO et al.

Fig. 2. Diagrams of the dorsal (a) and lateral (b) views of the male accessory genital glands. AVL, antero-ventral lobe; CD, connecting duct; DLL, dorso-lateral lobe; DML, dorso-median lobe; EJ, ejaculatory duct; GA, genital aperture; LVL, latero-ventral lobe; MVL, medio-ventral lobe; PLL, postero-lateral lobe; PVL, postero-ventral lobe. After Matsuo et al. (1997b) with permission from the Journal of Acarological Society of Japan.

Although adult Prostriata and Argasidae males are able to copulate without feeding, adult Metastriata males require feeding to copulate. This also indicates that the latter accessory glands develop during feeding in the adult stage. In H. longicornis, the epithelial cells of each lobe of the accessory glands undergo various histological changes synchronized with feeding and copulation. Particularly, dramatic histological changes occur during copulation (approximately for 10–15 min). These changes suggest that secretions of the accessory glands form the spermatophore. 4) Spermatophore The completed spermatophore is pear-shaped, and consists of an ectospermatophore, contents, an endospermatophore, a cord-like structure, and a plug on the tip of the spermatophore in H. longicornis, and some of these structures are different from those in other ixodids (Feldman- Muhsam and Borut, 1983) and argasids (Feldman-Muhsam, 1967; Feldman-Muhsam and Borut, 1978) (Fig. 3). The ectospermatophore wall is composed of 4 layers, namely layer I, layer II, layer III and layer IV, as seen by electron microscopy. The ectospermatophore wall is described to consist of three layers, the outermost thin acid mucopolysaccharide layer, the thick proteinaceous layer and a thin mucopolysaccharide layer (Feldman-Muhsam and Borut, 1984). Layer I of the ectospermatophore in H. longicornis is thought to come from salivary secretions (Feldman-Muhsam et al., 1970) or mucin (Tatchell, 1962), whereas layers II to IV are equivalent to the three layers in other ixodid ticks. However, we considered layer I to be a component of the ectospermatophore so we carried out experiments to confirm this hypothesis. In this experiment the spermatophores were collected immediately after formation at the genital aperture and the Reproduction in Haemaphysalis longicornis 7 male mouthparts never touched the spermatophore, so we showed it was impossible for salivary secretions to produce this outermost layer. While, the endospermatophore wall was composed of an inner layer consisting of a few layers and a coarse sponge-like outer layer. The contents of the spermatophore consist of spermatogenic cells and secretions from the male accessory glands, and they clearly divide in the spermatophore before extrusion of the endospermatophore as mentioned below. The spermatogenic cells in the spermatophore are only ‘elongated spermatids’ before and after extrusion of the endospermatophore as found in the seminal vesicle of feeding males. Three types of secretions from the male accessory glands are morphologically distinguishable. As described in other ixodid ticks, the contents of the spermatophore consist of seminal fluid, large granules, sperm and adlerocysts (Feldman-Muhsam and Borut, 1983), or granular materials, spermatids and fluid (Oliver et al., 1974). The only large protein granules or granular materials observed in the spermatophore are materials from the male accessory glands. Shepherd et al. (1982a, b) investigated these secretions in the spermatophore and showed they trigger ‘spermateleosis’ (Borut and Feldman-Muhsam, 1976) in the female genital tract. Although it is difficult to specify the function of each granule, the three types of granules appear to have different functions, for example the destruction of the endospermatophore wall in the female receptaculum seminis and the chemical stimulus of copulation. Furthermore, extrusion of the spermatophore also was observed in vitro because extrusion occurs during copulation and only the endospermatophore enters into the female genital tract (Fig. 3). All spermatophores immersed in 1–5% NaCl solutions started extrusion of the endospermatophore, and concentrations of the solutions affected the timing of the onset and the speed of the extrusion. At first a cord-like structure protruded from the tip of the spermatophore, and then a sac expands from the base of the cord-like structure. The contents flow into the sac through a neck connecting the cord-like structure to the sac, and the sac expands with the inflow. The tip of the cord-like structure is attached to the surface of the sac as a supporting structure and together they form a loop. The extrusion of the endospermatophore is attributed to CO2 bubbles secreted by symbiotic yeast in the spermatophore (Feldman-Muhsam et al., 1973) or the contraction of the elastic protein resilin in the spermatophore wall (Singaravelu, 1991). As the extrusion in vitro occurred in NaCl solutions, which does not occur in vivo, these experiments do not completely emulate the extrusion in the female genital tract. However, CO2 bubbles are not observed in H. longicornis. 5) Derivation of the Spermatophore Substances forming the ectospermatophore may be released by exocytosis, so the AVL shows features of exocytosis and the DLL and the PLL contain an identical type of large number of electron-dense granules. The formation of a thick and electron-dense layer of the ectospermatophore may require the largest quantity of secretion because a thick layer envelopes the entire spermatophore. In addition, the coil-like membrane in the lumen of the DLL and the PLL just after copulation may be shed from a type of granules after the apocrine-like secretion because the large number of granules must be released within a short time. Therefore, we conclude that the thin outer layer (layer II) is derived from granules in the AVL and the thick inner layer (layer III) from granules in the DLL and PLL. A gland enveloping the ejaculatory duct 8 Tomohide MATSUO et al.

Fig. 3. Diagrams of the spermatophore before (a) and after (b) extrusion of the endospermatophore (EN). a, Contents consist of spermatids and granules, endospermatophore and cord like structure (CS) are packed within the ectospermatophore (EC). The plug (P) is found on the tip of the spermatophore. b, The cord-like structure attaches to the surface of the endospermatophore to form a loop. Arrow shows the course through which contents flow into the endospermatophore. Ultimately, all contents flow into the endospermatophore. The ectospermatophore is empty. NE, neck of the endospermatophore. After Matsuo et al. (1998) with permission from Acta Zoologica. in H. longicornis, which has not been previously identified in ticks, was confirmed (unpublished data) and its secretion may form the outermost layer similar to the lobular accessory glands of female ticks that secrete coating for eggs. However, the derivations of the outermost and innermost layers of the ectospermatophore are not ascertained. The DML is the largest lobe in the accessory glands and ultrastructurally contains 10 types of granules. The formation of the spermatophore occurs through formation of the ectospermatophore, and injection of the male germ cells and granular contents before the formation of the endospermatophore. Different types of secretory granules appear to be randomly scattered in the DML, so it may be difficult for only Reproduction in Haemaphysalis longicornis 9 a specific type of granule to be secreted at specific times. The endospermatophore consists of the endospermatophore sac, the cord-like structure and the plug, and overall ultrastructural and histochemical analyses indicate these structures are derived from secretions of the DML. Findings of the granular contents of the spermatophore were also obtained by comparison with the ultrastructure of the spermatophore and then supported by histochemical data. The accessory gland secretions resembling the granular contents are as follows; two types of granules in the PVL, that differ in electron density and shape, as well as one with an annulate lamellae-like structure, and electron-dense and irregularly shaped granules found in the proximal region of the LVL, isolated mitochondria also found in the lumen just after copulation. Moreover, the third granules in the spermatophore are histochemically shown to be derived from the MVL. Ultrastructurally, they are enveloped by a plasma membrane with mitochondria. Therefore, the apocrine is expected to occur in the MVL. Small granules similar to the third granules are only observed in the lumen of the MVL just after copulation.

FEMALE REPRODUCTION

Reproduction in female H. longicornis will be described (Fig. 4): The female genital system consists of the vestibular vagina, cervical vagina, receptaculum seminis, connecting tube, oviducts, a looped ovary (Kakuda et al., 1995a, 1997a), which function in oogenesis (Yano et al., 1989b) and oviposition (Yano et al., 1989a). Additionally, the lobular accessory glands (Kakuda et al., 1997b) and the tubular accessory glands (Kakuda et al., 1994, 1995a) open into the female genital tract. In addition, the Gené’s organ and the porose area with their accessory glands are (Kakuda et al., 1992, 1995b, c) specialized for oviposition. 1) Vestibular Vagina The vestibular vagina leads to the genital aperture and has a thick cuticle which continues along the ventral body wall. A layer of squamous or cuboidal epithelium lines the vestibular cuticle in the unfed stage, then increases in volume during feeding to become the lobular accessory glands as mentioned later. The epithelial cells have a nucleus situated in the basal region (haemocoelic side) and the cytoplasm contains numerous small vesicles. During oviposition, in the lobular accessory glands as mentioned below the apical region of the epithelium detaches from the cuticle and the large lumen to form the vestibular sinus. 2) Cervical Vagina The cervical vagina has a folded thin cuticle lined with epithelial cells, externally surrounded by a muscle layer. The cuticle unfolds with longitudinal enlargement of the cervical vagina during feeding. The lumen (genital tract) is filled with material derived from the spermatophore inserted by the male after copulation. The cervical vagina only connects the receptaculum seminis to the vestibular vagina, the inserted endospermatophore and ascending spermatozoa pass through it, but no eggs pass through the cervical vagina during oviposition because the connecting tube leading to the oviduct opens into the junction between the vestibular vigina and cervical vagina. The cytoplasm of the epithelial cells increases in volume during feeding and muscles immediately external to the tract also develop and become stratified. During oviposition, no structural changes are observed in either the epithelium or the cuticle, but the muscle layers 10 Tomohide MATSUO et al.

Fig. 4. Diagrams of the dorsal (a) and lateral (b) views of the genital system in the unfed female H. longicornis. CT, connecting tube; CV, cervical vagina; GA, genital aperture; OD, oviduct; OV, ovary; RS, receptaculum seminis; TAG, tubular accessory gland; VV, vestibular vagina. After Kakuda et al. (1995a) with permission from the Journal of Faculty of Agriculture, Kyushu University.

become thinner and the material in the lumen disappears. Developed muscle layers that externally surround the cervical vagina probably facilitate the passage of the endospermatophore into the receptaculum seminis, and spermatozoa to the connecting tube. 3) Receptaculum Seminis The receptaculum seminis is a structure peculiar to the Metastriata in the family Ixodidae (Diehl et al., 1982). The Prostriata and the Argasidae do not possess such a separate and blind sac. The highly stained secretion-like substances within the cytoplasm of epithelial cells and the lumen observed just after copulation appear to be involved in destruction of the inserted endospermatophore (Khalil, 1970), or completion of spermiogenesis (Suleiman and Brown, Reproduction in Haemaphysalis longicornis 11

1978), after which sperm ascent commences. Spermiogenesis of ixodid ticks starts in the male body and continues also in the female tract after copulation (Oliver, 1982). In Am. hebraeum, spermiogenesis is completed during storage in the receptaculum seminis when spermatozoa first appear (El Said et al., 1981). Morphological changes at late spermiogenesis from elongated spermatids to spermatozoa in the female genital tract is called ‘spermateleosis’ (Borut and Feldman-Muhsam, 1976). Migration of male germ cells in the female genital tract is also reported in Argasidae and Prostriata (Resler et al., 2009; Yamauchi at al., 2000). The depressed receptaculum seminis in the unfed stage has greatly folded cuticle. The cuticle consists of a thin epicuticle and thicker fibrillar procuticle, which surround a collapsed lumen. A single squamous epithelium lining the cuticle exhibits a complicated shape with the folded cuticle. As feeding proceeds, epithelial cells of the receptaculum seminis become active in secretion. The lumen has still collapsed but the space between the epithelium and the cuticle is filled with electron-dense material in 5-day fed sexually active virgin females. Enlarged epithelial cells that are characterized by extremely abundant thick microtubules project well-developed microvilli on the apical surface and numerous mitochondria are present below them. Abundant microtubules traversing the cytoplasm have presumably a cell supporting role as cytoskeleton in the receptaculum seminis largely distending by the insertion of the endospermatophore. In feeding H. flava, the same genus as H. longicornis, a receptaculum seminis contains over 10 endospermatophores (unpublished data), and also in Hyalomma sp. several ones are observed (Feldman-Muhsam, 1986). Therefore, the receptaculum seminis of H. longicornis may be able to receive more endospermatophores and enlarge more. In just copulated females, the receptaculum seminis distends by the insertion of the endospermatophore, the cuticle unfolds considerably and the large lumen is filled with highly stained material. In the apical region of epithelial cells, electron-dense secretory products are discharged by intense secretion. Secretory products discharged are released into the lumen through the cuticle. This releasing process is very similar to that of the lobular accessory glands lining the vestibular vagina. The wall of the �������������endospermato- phore just after the insertion consists of an electron-opaque multilayered inner layer and an electron-dense outer layer with rough surface. These secretions attach to the wall of the endospermatophore and may act chemically on the wall. Resumption of spermiogenesis is observed in spermatids in the endospermatophore. In engorged females on the third day after copulation, male germ cells yet remain in the receptaculum seminis. Electron-dense material that has filled the lumen just after copulation disappears, but a small quantity of secretory products float in the lumen and attach to the wall of the endospermatophore. Although epithelial cells become sparse, they have many mitochondria and basal infoldings, and electron-dense secretory products are still discharged from the cells. No dissolution of the endospermatophore wall occurs in this stage. In the endospermatophore, spermatozoa that have completed spermiogenesis are waiting for the onset of ascent. The receptaculum seminis found in the metastriate ticks does not function as a sperm-storage site where spermatozoa are maintained in a viable state for insemination like the receptaculum seminis in spiders (Kraus, 1978) and the spermatheca in insects (Gillott, 1988). In ticks, male germ cells are stored only for several days, and when spermatozoa emerge, sperm ascent to the ovary begins. In the prostriate ixodids, Ixodes, and argasids, the common oviduct and the 12 Tomohide MATSUO et al.

“uterus”, play the role of the metastriate receptaculum seminis (Diehl et al., 1982). Spermiogenesis of ticks is interrupted in the stage of the elongated spermatids in the male body and it resumes at copulation (Oliver 1982). In Dermacentor variabilis, secretions from the receptaculum seminis trigger the resumption of spermiogenesis (Suleiman and Brown, 1978). In H. longicornis, the fact that spermateleosis is already in progress in undissolved endospermatophores just after copulation suggests that secretions from epithelial cells of the receptaculum seminis act as a destructive agent of endospermatophores as described by Khalil (1970) for Hy. anatolicum excavatum. In addition, the increase in length and volume of spermatids during spermateleosis may a role in rupture of the endospermatophore. 4) Connecting Tube The connecting tube opens ventrally at the junction between the vestibular vagina and the cervical vagina, and so links the vagina to the common oviduct. In the unfed stage, the folded cuticle lined with an epithelial layer is surrounded by thick muscle layers similar to those of the cervical vagina. During feeding, the epithelial cells become irregularly shaped and the cuticle unfolds as the cells enlarge. Well-developed muscle layers surrounding the connecting tube overlap each other. No further significant structural changes are observed during oviposition. 5) Oviduct The regularly arranged oviductal epithelium consists of simple cuboidal epithelial cells surrounded by an undeveloped muscle layer. The cells become columnar and their cytoplasm now contains numerous vesicles and the nucleus is rounded during feeding. During egg passage, the oviductal epithelium is stretched and flattened. Numerous vesicles observed in the oviductal epithelial cells just after copulation presumably contain secretory products that, when released, may exert some influence upon the passing spermatozoa and eggs. In D. andersoni, the oviductal epithelia have well-developed microvilli and cell processes on the apical surface. Brinton et al. (1974) suggests that both may help to protect them against the intracellular invasion of spermatozoa. These structures are present also in the oviduct of H. longicornis. In Hy. dromedarii (El Shoura, 1989) and D. andersoni (Brinton et al., 1974), oviductal epithelial cells display granular material that we presume is involved in sclerotization of the egg shell during passage. Muscle layers surrounding the oviduct affect peristaltic contractions during the passage of both spermatozoa and eggs. 6) Ovary (Oogenesis and Oviposition) In the unfed stage, the ovary consists of ovarian epithelial cells with a small nucleus, oocytes with a larger, round nucleus containing a distinct nucleolus, and external connecting tissue. Additionally, meiotic divisions have not been observed during any period of ovarian development in adult D. andersoni females (Brinton and Oliver, 1971a), and it is thought that only oocytes are present in the females, because of additional enlargement and organellar differentiation of germinal cells, and especially the presence of their well-organized nucleoli (Brinton and Oliver, 1971b). On the other hand, in parthenogenetic H. longicornis, both few oogonia and many primary oocytes occupy the ovary before the female feeds, and no further development occurs until female feeding (Khalil, 1972). Oocytes contain free ribosomes throughout the cytoplasm and massive round mitochondria in parts. Both the oocyte and the ovarian epithelial cell face the haemocoel, and are connected to the ovarian surface by funicle cells derived from the ovarian Reproduction in Haemaphysalis longicornis 13 epithelial cells. The oocyte cytoplasm contains numerous mitochondria, well-developed rough endoplasmic reticulum and the Golgi complex, and the eggshell does not appeared before copulation. The cuboidal funicle cells contain a large nucleus and scanty cytoplasm with massive glycogen particles. The oocyte and the funicle cells attach to one another by microvilli, both of which have numerous vesicles in their boundary zone. The ovarian luminal surface of the funicle cells is in contact with the flattened ovarian epithelial cells, and does not directly face the ovarian lumen. The oocytes develop further until the detachment from the host 2 days after copulation, the large nucleus moves to the side of the funicle cells, and numerous yolk granules appear. The nuclear-nuleolar complexes, Golgi body, mitochondria and plasmalemma brush border contribute to yolk formation as shown by an electron microscopic study on oogenesis in D. andersoni (Brinton and Oliver, 1971). The conspicuous development of microvilli and active micropinocytosis in the oocyte plasma membrane suggests intake of exogenous yolk protein precursors in H. longicornis. Eggshell precursors begin to be produced in the gap between the oocyte and the basement membrane. Then, eggshell synthesis and vitellogenesis are almost completed at 4 days after detachment. The nuclear envelope is concaved together with the oocyte plasma membrane at the side of the funicle cells, and forms a large nuclear crypt beneath the eggshell that may be associated with the mechanism of fertilization. The microvilli are retracted through eggshell slits. As a result, an interchorionic space is formed between the oocyte and eggshell. The mature oocyte just before ovulation is sustained by 20–30 columnar funicle cells that are arranged in a monolayer and one end is exposed to the eggshell on the opposite side of the ovarian lumen. The ovary just before oviposition contains oocytes in all developmental stages because there is no synchrony of oocyte development and some mature oocytes are ovulated into ovarian lumen. Tick oogenesis has been classified into four (Recaldo et al., 2007 for Am. triste), five (Balashov, 1972 for Argas persicus, Hy. asiaticum and I. ricinus) and six (Saito et al., 2005 for Boophilus microplus) stages. Numerous spermatozoa ascending to the ovarian lumen are in close contact with microvilli of the ovarian epithelial cells just before ovulation. The site of fertilization in ticks is still a point of contention. Balashov (1972) concluded that oocytes are fertilized in the anterior part of oviducts. Whereas, spermatozoa exist in the ovarian lumen at the beginning of oviposition in H. longicornis and D. andersoni (Brinton and Oliver, 1971). Furthermore, some spermatozoa are wedged into an indentation of the funicle plasma membrane in H. longicornis. These facts imply that fertilization occurs within the tick ovary. However, it remains unclear whether the actual site of fertilization is the ovarian lumen or the intercellular space of funicle cells, or whether fertilization occurs as a result of the penetration of the spermatozoon through the eggshell just before ovulation. Normally copulated and engorged H. longicornis females can oviposit more than 3,000 eggs but their oviposition ability is affected by their body weight. Balashov (1972) showed that minimum engorgement weight required for ixodid ticks to oviposit is about 10% of their normal engorgement weight. In the bisexual H. longicornis, a minimum weight of 28.4 mg for oviposition corresponds to 10.4% of the mean engorgement weight. On the other hand, although the proportion of oviposition threshold engorgement weight to the unfed weight seems to vary within species, the proportion for H. longicornis is 17.8 times. Such differences may reflect 14 Tomohide MATSUO et al. disparity in the efficiency of ixodid egg production. A linear relationship exists between the weight of fully engorged ticks and the number of deposited eggs, and the efficiency of egg production is expressed as the number of deposited eggs per mg of female fully engorged weight (Diehl et al., 1982). In H. longicornis, complete oviposition (about 12.5 eggs/mg) occurs in the detatched females (142–235mg). Moreover, females of the bisexual race of H. longicornis appear to not always need mating for egg production and they have the ability to oviposit eggs with feeding stimuli alone, although most Metastriata females needs copulation for normal engorgement. Such an ability may have become a factor in the establishment of parthenogenesis. In the absence of mating, however, incompletely engorged females are inferior to normally mated and engorged females in oviposition performance and of course no larva emerges from the eggs. Therefore, the bisexual pattern is markedly influenced by the presence or absence of mating and its timing, and oocyte growth and egg production are under the control of the quantity of the blood meal. 7) Lobular Accessory Glands In the unfed stage, the vestibular cuticle is lined with a single layer of squamous or cuboidal epithelium. This epithelium continues along the epithelium of the ventral body wall, and no morphological difference is observed between them in this stage. However, epithelial cells of the vestibular vagina are not stained with haematoxylin more strongly than those of the body wall. During feeding, the vestibular epithelium develops lobularly. The lobular accessory glands surrounding the vestibular cuticle consist of glandular epithelial cells with basally situated nuclei, and their cytoplasms contains granular structures. In 10-day feeding virgin females, no remarkable changes in the glands are observed. Hypertrophy of the glands occurs only in detached females on the first day after copulation. During oviposition, the apical region in the lobular accessory glands protruding into the haemocoel detaches from the vestibular cuticle and the large space formed is called the vestibular sinus. The sinus lumen is filled with eosinophilic secretions. Glandular epithelial cells containing euchromatin-rich nuclei and numerous secretory granules project well-developed microvilli on the apical surface. In the microvillar surface, small vesicles appear to be undergoing exocytosis into the sinus lumen. Secretions from the lobular accessory glands are once accumulated in the vestibular sinus formed between the epithelium and the cuticle. Later, the secretions permeate into the loose fibrillar procuticle and are released into the genital tract of the vestibular area through the epicuticle. The ultrastructure of the lobular accessory glands in H. longicornis exhibits characteristic protein synthesis, so secretions accumulated in the vestibular sinus and the procuticle during oviposition may be proteinaceous materials. Simultaneously, the glands of this tick exhibit the typical characteristics of transporting epithelia, i.e. infoldings of basal plasma membrane associated with mitochondria, intercellular spaces and well-developed microvilli (Berridge and Oschman, 1972). Low molecular compounds like fatty acids are probably released by transmembrane transport, if the glands discharge lipid- rich secretions as described by Lees and Beament (1948). Furthermore, secretions from the glands act as a genital sex pheromone in D. variabilis (Sonenshine et al., 1985). However, a peak of release of sex pheromones attracting males should come just before copulation. The facts that secretory activity of the lobular accessory glands is observed during oviposition and no activity in fed virgin females in H. longicornis, and that the glands develop during feeding in most Ixodes Reproduction in Haemaphysalis longicornis 15 females (Lees and Beament, 1948) that copulate in the unfed stage just after moulting suggests that the glands are involved in the passage of eggs through the vestibular vagina. Secretions from the glands also partially waterproof the egg surface (Lees and Beament, 1948), but they act as a lubricant of the vestibular vagina so several thousand eggs can pass through. 8) Tubular Accessory Gland A pair of tubular accessory glands opens into the vagina at the junction of the vestibular and cervical area. The glands are small and club-shaped in the unfed stage. The glandular epithelial cells are undeveloped with few cell organelles and most of the cell volume is occupied by the irregularly-shaped nucleus. Two kinds of cells are distinguishable, namely dark cells containing abundant free ribosomes and pale cells located between the dark cells. Both cells have poorly developed microvilli in the apical portion constituting a simple columnar epithelium. The secretory cells of the glands in Ornithodoros erraticus have already contained secretory granules before feeding in adult stage (El Shoura, 1988a). This is probably due to facultative autogeny described in several species of Ornithodoros (Balashov, 1972; Feldman-Muhsam, 1973; Feldman-Muhsam and Havivi, 1973). The dark cells increase in volume and increase threefold 5 days after attachment. They possess large, round nuclei with distinct nucleoli and increased euchromatin, membrane-bound granules, numerous free ribosomes and mitochondria, and developing microvilli, indicating a secretory function. While, the pale cells become very slender from the pressure of the enlarged dark cells and appear to thread through the dark cells with only a narrow portion attached to the basement membrane. Abundant parallel microtubules traversing the entire cell cytoplasm indicate a supporting role of the pale cells. In D. variabilis the supporting cells of the tubular accessory glands have been described as “stellate supporting cells”, but they are not illustrated (Sonenshine et al., 1985). In H. longicornis, it is relatively difficult to distinguish the supporting cells from the secretory cells in the unfed stage, and then the supporting cells become more slender between the secretory cells in ovipositing females. Therefore, supporting cells of the tubular accessory glands may not be noticed. For this reason these cells may have not been described in Hyalomma (Raikhel, 1983; El Shoura, 1989) and Ornithodoros (El Shoura, 1988a). The supporting cells maintain the shape of the glands during the release of secretions into the genital tract by contraction of the well-developed musculature surrounding them. Muscle layers with striated muscle fibers also develop during feeding. After rapid feeding and engorgement following copulation, the secretory cells show well-developed microvilli, conspicuously abundant free ribosomes, developing rough endoplasmic reticulum, incomplete secretory granules and accumulation of glycogen particles. During oviposition the glands assume a large “sausage-like” shape and are surrounded by reticulate musculature. Eosinophilic granules occupy the gland and similar secretions are apparent in the central lumen and the duct. Granular contents are actively discharged by exocytosis and the lumen is ultrastructurally filled with dense material. In 10-day feeding virgin females, the secretory cells contain dense granules, but the cells are smaller than those of ovipositing females. No secretory activity, abundant free ribosomes, and rough endoplasmic reticulum are observed. These characteristics observed in long feeding virgin females suggest that copulatory stimuli and full engorgement induce absorption and accumulation from the period of synthesis and discharge. A pair of tubular accessory glands is a common feature in the Ixodoidea. The ultrastructure of the 16 Tomohide MATSUO et al. secretory cells in ovipositing females also suggests that the cells are involved in protein synthesis. These proteinaceous secretions may act as a waterproofing agent for the egg surface and/or as a lubricant to facilitate egg passage (Robinson and Davidson, 1914; Douglas, 1943), as do those of the lobular accessory glands. 9) Gené’s Organ and Tubular Glands Gené’s organ of H. longicornis is basically similar to that of the other Ixodidae (Arthur, 1953; Balashov, 1972; Booth et al., 1984b; El Shoura, 1987b; Schöl et al., 2001a, b). In the unfed stage, Gené’s organ located immediately beneath the anterior part of the scutum is small in size and covers the chelicerae. This organ has an epithelial layer, a thicker inner cuticle lined by this layer, and a thinner and highly folded bellows-like outer cuticle. Gené’s organ in H. longicornis also has the same structures and the tubular glands that develop during feeding. Although there are interspecific variations in the morphology of Gené’s organ, that of H. longicornis is very similar to H. flava as described by Saito (1960). The unfed female possesses fairly flattened body, but increases in volume during feeding, and expands remarkably until engorgement. In 1-day ovipositing females, this glandular epithelium increases in size and the tubular glands become large. When oviposition is about to take place, the capitulum is tucked firmly against the body wall. At the same time the gap between the posterior edge of the basis capituli and the anterior margin of the scutum is widened and a pair of horns of the Gené’s organ emerge. The emerged horns have a wrinkled surface and the median region where a deposited egg attaches is covered with fine and thorn-like papillae. The vestibular vagina also prolapses and the deposited egg is manipulated by the horns. The horns grasping the egg are completely retracted within the body cavity, and placed on the anterior part of the scutum by upswing of the capitulum. As previously reported in I. ricinus (Lees and Beament, 1948) and B. microplus (Booth et al., 1984b), the oviposition movements in H. longicornis are composed of movements of the capitulum, Gené’s organ and vestibular vagina, and these movements are accurately synchronized. Since this organ has no intrinsic musculature for eversion, the eversion is caused by an increase in haemolymph pressure and contraction of dorso-ventral muscles (Arthur, 1953; Booth et al., 1984b; El Shoura, 1987b). When finished depositing an egg, Gené’s organ is retracted into the body cavity by the retractor muscle, which arises in the hard cuticular scutum and inserts in the outer cuticle of this organ. Therefore, eversion and retraction of Gené’s organ may be performed by the integrated action of muscles. Tubular glands associated with the Gene’s organ and glandular epithelia distributed in the peripheral region of the horns conspicuously develops during feeding and becomes four bunches of large simple branched tubular glands connected with a stalk by two pairs of ducts. In engorged females, the glandular epithelial cells increase in volume and small vesicles are actively fusing with the apical plasma membrane that projects well-developed microvilli. The basal plasma membrane has infoldings and the basement membrane becomes thick. In addition, the cytoplasmic inclusions (lipid droplets and glycogen particles) increase. The glandular epithelial cells with domed apical region separated by the deep intercellular crypts become higher and have large oval nuclei in the mid-region of the cytoplasm of ovipositing female. The lumen is filled with a little dense material. Ultrastructure of the tubular glands of H. longicornis changes dramatically in accordance with events the adult females undergo. Especially, intercellular crypts Reproduction in Haemaphysalis longicornis 17 formed after copulation enable the microvillar free surface to increase, which may contribute to great expansion of the cell surface. This large increase in the surface area of the plasma membrane suggests that secretions from the tubular glands are released by transmembrane transport. The glandular epithelial cells change their function during and after feeding; formation of the intercellular crypts, intake and accumulation of precursors occur for a few days from copulation to the onset of oviposition, and secretion occurs during oviposition. The egg wax is composed of complicated constituents such as branched and non-branched chain alkanes, steroids, fatty acids and alcohol (Gilby, 1957; Cherry, 1969a, b; McCamish et al., 1977), so the secretions from the tubular glands may be resynthesized into the egg wax after release into the lumen of the tubular glands. The secretions pass through the pore canals that penetrate the outer cuticle and are transported to the egg surface (Booth et al., 1984b; El Shoura, 1987; Booth, 1989). Normally oviposited eggs adhere together in a cluster unless ovipositing females are disturbed. In contrast, Yano et al. (1987, 1988) showed eggs oviposited after blockade of the Gene’s organ fail to adhere together like normal and disperse on the bottom of a laboratory dish. These eggs have a glossy surface with no visible egg wax, and exhibit much lower hatching ratios (average 4.9%) than eggs before the blockade (average 94.5%) at 30℃, 100% RH. In the case of eggs soaked in liquid paraffin, hatching ratios after blockade are lower (average 71.8%) than those of normal eggs in liquid paraffin (average 99.3%), but are considerably higher than eggs deposited after blockade only. This experiment reveals that coating of Gené’s organ secretions is indispensable for egg survival even at optimal conditions of 30℃ and 100% RH for development and growth of H. longicornis. Also the results of the present hatching tests in eggs soaked in liquid paraffin suggest that the egg wax has a waterproofing function and enables the deposited eggs to carry out gas exchange. 10) Porose Areas and their Accessory Glands The porose areas of H. longicornis are shown as two circles symmetrically located on the dorsal surface of the basis capituli. In each circle many pores of about 3 µm in diameter are irregularly arranged like a riddle. The number of pores is about the same in both the right and left circles but range from 50 to 70 with individual variations. The valve-like structure in the mid- region of the duct penetrating the thick cuticle separates the lumen consisting of the duct cells from the extraintegumental space. A sagittal section of the duct shows a unique shape like an orchid flower. The region above the valve-like structure is the cuticular duct lined with the epicuticle that continues along the body surface. However, duct cells form the cellular duct beneath the valve-like structure, and the distal part extends to the haemocoel of the basis capituli where secretory cells are present. Sheath cells are interposed between the duct cell and the procuticle of the basis capituli. Microtubule-rich and electron-lucent duct cells extend downward during feeding, and the lumen of the duct is lined with sparse microvilli. During oviposition, this long cellular duct surrounded by sheath cells associates with each pore of the porose areas. Duct cells forming the cellular duct correspond to the proximal cells of Rhipicephalus evertsi evertsi (Gothe et al., 1987). In the duct cells just beneath the valve, microtubules extending downward from the cuticular structures (the flap-like structure and the cuticular projection) may play a mechanical role in closing and opening the valve. The cavity of the basis capituli is too small for distal accessory glands to remarkably develop during oviposition, so extension of the cellular 18 Tomohide MATSUO et al. duct up to the anterior idiosomal cavity enables the glands to develop fully. Abundant microtubules traversing the cytoplasm of the duct cells presumably play a mechanical role to maintain endurance for the cells during extreme extension. External sheath cells support the lengthened duct and their microtubules may support themselves. In the unfed stage, the small accessory gland beneath the dorsal cuticle of the basis capituli consists of duct cells surrounded by sheath cells and some undeveloped secretory cells without lumen. The small lumen formed by duct cells is filled with amorphous material. In 10-day feeding virgin females, duct cells elongate considerably, but no remarkable changes in secretory cells are observed. During oviposition, secretory cells extremely increase in volume and become a simple branched acinar gland consisting of 6–8 acini associated with a pore of the porose areas via a long non-branched cellular duct. All acini are located in the anterior idiosomal cavity. The lumenal surface of the canaliculi projects long, well-developed microvilli beneath which a large number of mitochondria accumulates. Secretions in the cytoplasm are discharged into the secretory canaliculi by exocytosis, secretions in the canaliculi are released into the duct lumen via the narrow part of the junction of the secretory and duct cells, and are released from pores of the porose areas. Secretory cells of accessory glands correspond to the distal cells of R. evertsi evertsi (Gothe et al., 1987). In B. microplus a single acinus connects with each pore of the porose areas through a duct (Booth et al., 1984a), but in H. longicornis 6–8 acini are connected with each pore. Ultrastructure of secretory cells of the acini is basically similar to that of B. microplus (Booth et al., 1984a), indicating their ability to synthesize proteins. However, the contents of most secretory granules in the cytoplasm are lost during preparation suggesting the granules contain lipid components. Secretory and absorption activities of the cells during oviposition indicates the accessory glands of the porose areas are deeply involved in oviposition. The porose areas and their accessory glands have a very similar structure to the dermal gland and opening of ticks (El Shoura, 1988b). The dermal gland is involved in formation of constituents of the cuticle wax layer (Balashov, 1972; Amosova, 1983). While, the porose areas are present only in females, and I. kopsteini exhibiting an unusual oviposition process in which larvae directly emerge from the female body cavity not from eggs, lacks the porose areas (Anastos et al., 1973). These two facts imply that the porose areas are involved in oviposition. Additionally, the location of the porose areas suggests that secretions from them are applied to the horns of the Gené’s organ moving repeatedly above and have specific functions for the egg wax (Booth et al., 1984a). However, there is no significant difference in the number of deposited eggs, hatching ratio (Atkinson and Binnington, 1973; Booth et al., 1984a) and rate of oviposition (Gothe and Nadler, 1986) between females with blocked and unblocked (normal) porose areas. Therefore, it is clear that secretions from the porose areas do not function as a lubricant for horns of Gené’s organ repeating eversion and retraction during oviposition as described by Feldman- Muhsam and Havivi (1960) and Feldman-Muhsam (1963).

ACKNOWLEDGEMENTS

This study was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science. We also thank Dr. T. A. Uchida and Dr. S. Shiraishi for their Reproduction in Haemaphysalis longicornis 19 guidance and encouragement during the course of these studies.

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摘要 フタトゲチマダニの繁殖学 松尾 智英（鹿児島大・獣医）・大倉 信彦（琉球大・医）・角田 浩之（福岡常葉高）・ 矢野 泰弘（福井大・医） マダニ上科は主に 2 つの科，すなわちマダニ科（Hard tick）とヒメダニ科（Soft tick）とに分類され，マダニ科はさらにそれらの繁殖の違いによって Prostriata（マダ ニ科マダニ属）および Metastriata（マダニ属を除くマダニ科の属）に分けられる．す なわち，各グループに属する種はそれぞれ特徴的な繁殖システムや器官を有している． 加えて，マダニ類は様々な病原体のベクターとしても重要な生物群である．Metastriata グループに属するフタトゲチマダニは単為生殖系統と両性生殖系統が存在するという特 徴をもち，オーストラリア，ニュージーランド，ニューカレドニア，フィジー諸島，日 本，朝鮮半島および中国・ロシア北東部に広く分布している．本種はまた Q 熱リケッ チア，ロシア春夏脳炎ウィルス，タイレリアおよびバベシア原虫のベクターとしても知 られており，我が国における牧野の最優占種であるフタトゲチマダニは放牧牛にピロプ ラズマ病を媒介するという点で農学・獣医学上重要であると考えられている．そこで， 我々がこれまでに明らかにしてきたフタトゲチマダニ両性生殖系統の繁殖に関する知見 をここにまとめる．