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2008 Observations on : small Nymphs feed on mammalian hosts and have a salivary gland structure similar to Ixodid ticks

Journal of Parasitology, Lancaster, v. 94, n. 4, p. 953-955, 2008 http://producao.usp.br/handle/BDPI/2098

Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo J. Parasitol., 94(4), 2008, pp. 953–955 ᭧ American Society of Parasitologists 2008

Observations on Antricola Ticks: Small Nymphs Feed on Mammalian Hosts and Have a Salivary Gland Structure Similar to Ixodid Ticks

A. Estrada-Pen˜ a, J. M. Venzal*, Katherine M. Kocan†, C. Tramuta‡, L. Tomassone‡, J. de la Fuente†§, and M. Labruna࿣ Department of Parasitology, Veterinary Faculty, Miguel Servet 177, 50013 Zaragoza, Spain; *Department of Parasitology, Veterinary Faculty, Av. Alberto Lasplaces 1620, CP 11600 Montevideo, Uruguay; †Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74078 U.S.A.; ‡Dipartimento di Produzioni Animali, Epidemiologia, Ecologia, Facolta` di Medicina Veterinaria, Universita` degli Studi di Torino, Via Leonardo da Vinci, 44, 10095 Grugliasco (TO), Italy; §Instituto de Investigacio´n en Recursos Cinege´ticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13071 Ciudad Real, Spain; ࿣Department of Preventive Veterinary Medicine and Animal Health, Veterinary Faculty, University of Sao Paulo, Sao Paulo, SP, Brazil. e-mail: [email protected]

ABSTRACT: Ticks use bloodmeals as a source of nutrients and energy has been identified as the nutrient that supports survival until the to molt and survive until the next meal and to oviposit, in the case of parasite obtains a blood meal (Chinzei and Yano, 1985). Nymphal An- females. However, only the larvae of some tick species are known to tricola spp. have never been found attached to and, therefore, it is feed upon bats; females are obligatorily autogenous, and nymphal stages not understood how a single blood meal of the larva could provide are believed to not feed. We investigated the presence of blood in a energy for several nymphal molts as well as for oviposition by females. natural population of nymphal ticks collected from Some have speculated that these stages could feed upon an unknown guano; their ability to feed upon laboratory hosts; and the micro- food source in bat guano (De la Cruz and Estrada-Pen˜a, 1995). scopic structure of both salivary glands and gut. DNA amplification of In this paper, we report detection of blood in freshly collected early gut contents of freshly collected material was positive for a mammal in nymphal stages of Antricola spp., as well as the experimental feeding 4 of 11 first instar nymphs, but we were unsuccesful in the amplification of these stages on laboratory-housed rabbits. We also present a brief of host bloodmeal DNA from late instar nymphs. All early nymphal light microscopic description of the salivary glands and gut of all post- stages (n ϭ 10) fed on rabbits, and host DNA was detected and se- larval stages of Antricola spp., thus providing a comparison of these quenced from gut contents. However, all the large nymphs (n ϭ 10) organs with both a South American Carios species and with morpho- rejected feeding, and host DNA remained undetected in these ticks. All logic studies of Argas vulgaris published previously. Specimens of An- stages of A. delacruzi have salivary glands similar in morphology to tricola delacruzi Estrada-Pen˜a, Venzal, and Battesti were collected in a the ixodid agranular Type I salivary gland acini and to granular Type cave located within the Porto Velho municipality (Rondonia State, Bra- II or Type B acini. All stages of A. delacruzi had a similar gut structure, zil) in November 2005. Until used for these studies, the ticks were kept consisting of digestive cells in the basal portion that contained hematin alive at 30 C, in complete darkness, in guano collected from the cave. granules. Neither regenerative nor secretory cell traces were observed Eleven early, and 6 late, nymphal specimens were used for detection of in the sections of gut. host blood by DNA amplification. Ten specimens of early and late nymphs were dissected, using a stereomicroscope, and fixed and pro- cessed for microscopy studies. In addition, another group of 10 early Cooley and Kohls (1944) erected Antricola to include Antricola co- and 10 late nymphs were allowed to feed in a capsule over a shaved prophilus (McIntosh), a species described originally within Ornitho- area of skin on the back of a white giant New Zealand rabbit. The doros. Antricola is a homogeneous assemblage of 17 tick species sup- rabbit was maintained and housed in accordance with the Institutional posedly restricted to the United States and Caribbean Islands. Some Animal Care and Use Committee guidelines. recently described species, however, greatly expanded its distribution For DNA extraction, individual ticks were homogenized with a pestle range into Brazil (Estrada-Pen˜a et al., 2004). in a microcentrifuge tube. DNA was extracted using the DNeasy tissue The systematic position of Antricola species is still doubtful. Some au- thors conducted phylogenetic analyses at major generic and subgeneric lev- els within the and proposed a number of modifications to the classification of the family (Klompen, 1992; Klompen and Oliver, 1993). The Carios Latreille, 1796, was raised from Argas (Carios) and included in the Ornithodorinae Pospelova-Shtrom, 1946. Several of the recognized groupings within Carios (Reticulinasus, Nothoaspis, Antricola, Carios) appeared to be monophyletic, but recognition of the groupings as subgenera would create a proliferation of poorly supported subgenera or genera. Klompen and Oliver (1993) concluded that more detailed studies were required to evaluate the relationships within Carios spp. These phy- logenetic analyses, together with partial 16S rRNA sequences, provided valuable insight into the evolution of Argasidae. While we adhere to the existence of Carios as a taxonomic assemblage, we prefer to retain the denomination of Antricola as separate from that genus until more detailed data becomes available, data that would allow for greater confidence about its classification within this group. Of relevance to these concerns is the feeding pattern of some species. Larvae of some Ornithodoros species do not feed, while the larva is the only stage of Antricola spp. collected on hosts. Moreover, Antricola spp. adults have non-functional mouthparts resembling primitive me- sostigmatid-type chelicera and, therefore, most likely do not feed (Ol- iver, 1989). These dentate chelicera also appear in late, but not in early, nymphal stages of Antricola spp. Antricola spp. adults and nymphs inhabit bat-occupied caves and live on bat guano on the cave floor. Ornithodorinae have a variable number of nymphal stages. As we still do not know how many nymphal stages exist in Antricola, we herein designate ‘‘early’’ (or small) nymphs to the first stages and ‘‘late’’ (or large), which are close to the adult morphology and size, to the final stages. As reported by Oliver (1989), species of Antricola, together with FIGURE 1. Longitudinal sections of early (A) and late (B) Antricola those of Otobius and probably the former Nothoaspis (now also con- delacruzi nymphal stages after feeding on laboratory rabbits. Blood is sidered within Carios), are truly obligatory autogenous ticks. Vitellin seen in the gut of early nymphs (arrows).

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FIGURE 2. Light photomicrographs of salivary glands of early (A) and late (B) nymphs and an adult female (C)ofAntricola delacruzi. Type I agranular cells (black arrow) and Type II granular cells (white FIGURE 3. Light photomicrographs of cross section of salivary arrow) are observed in the 3 stages. Mallory’s stain, ϫ100 (A and B) glands from a Carios puertoricensis nymph (A) that contains both Type and ϫ400 (C). I and Type II salivary gland acini. Salivary glands from a female tick contains (B) agranular Type I acinus and (C) granular Type II or B acini. Mallory’s stain, ϫ100. kit (QIAGEN GmbH, Hilden, Germany). Vertebrate DNA in the tick gut was identified by polymerase chain reaction (PCR) using the uni- versal primers 0033 and 0049 designed to amplify a 150 bp fragment guano. Sequence analysis of PCR products showed poor homology with in mammal species, and a 120 bp fragment in bird species of the 18S sequences in GenBank and, therefore, was tentatively identified as rDNA gene, as described by Pichon et al. (2003). As positive controls, mammalian because it was positive to the specific probe. No DNA was we used DNA from Mus musculus and Bos taurus. Amplicons were detected in late instars A. delacruzi nymphs collected on bat guano. The purified using the QIAquick PCR purification kit (QIAGEN GmbH). lack of detection of host DNA in 7 early nymphs may have resulted Sequencing of PCR products was performed using PCR-derived primers from the amount of time between tick feeding and DNA amplification. in an ABI Prism 310 Genetic Analyser (Applied Biosistems, Milan, The possibility remains that the DNA detected was from the larval Italy). The sequences obtained were analyzed with the software CHRO- bloodmeal. Studies conducted by Pichon et al. (2003), using I. ricinus MAS 2.0 (Technelysium, Helensvale, Australia) and submitted to nymphs kept under natural field conditions in Ireland (average temper- BLAST analysis (BLAST௡, NCBI, Bethesda, Maryland; Altschul et al., ature of 10 C), show that possibilities to detect host DNA drop to 40% 1997). Sequences were then aligned using ClustalW multiple-alignment after 7 mo. However, the temperature in the caves where A. delacruzi software provided in the BioEdit package, version 7.0.5.2 (BioEdit, were collected remains constant around 35 C. Under such conditions, Carlsbad, California) (Hall, 1999). host DNA will degrade more quickly than in published experiences. Ticks were cut in half for histological procedures, separating the right Furthermore, the lack of DNA detection in late nymphs contributes and left sides, and then fixed in 2% glutaraldehyde in 0.2 M sodium cac- support to the absence of inter-stage contamination. Our conclusion is odylate buffer (pH 7.4). Further treatment and staining with Mallory’s stain that, while we cannot totally exclude mammalian DNA coming from was done (Kocan et al., 1980; Richardson et al., 1960). The sections were larval blood meal detected in nymphs, it is most probable that DNA observed via light microscopy and photographed using a 3-chip digital detected in early nymphs came from the actual nymphal blood meal. camera attached to the microscope. A neotropical species, Carios puerto- Host DNA was detected in all the 10 specimens fed on laboratory- ricensis, was also allowed to feed on rabbits, both as a control and to housed hosts (Fig. 1). Sequence analysis of PCR products showed 98% provide further comparisons of salivary glands and gut structure. Nymphs homology (148/150 bp) with the corresponding sequence of Oryctola- and adults were fed on rabbits and fixed and processed as described pre- gus cuniculus in GenBank (AY150553). Late nymphs infesting rabbits viously. Further details were obtained from descriptions of salivary glands failed to feed, and remnants of mammalian DNA were not detected in as published for Argas arboreus (Roshdy and Coons, 1975; Coons and these nymphs. Therefore, the ability to feed on laboratory hosts and Roshdy, 1981). host-derived DNA content was confirmed only for early nymphs. All Host blood meal DNA was detected and identified as mammalian in stages of Antricola spp. have similar salivary glands that include acinar 4 of 11 specimens of early instars A. delacruzi nymphs collected on bat types similar in morphology to the ixodid agranular Type I salivary RESEARCH NOTES 955

glands of A. delacruzi had a unique morphology as seen via light mi- croscopy, they also retained some morphologic characteristics similar to ticks from species of Argas rather than those of Carios. The unique structure of the gut, together with the presence of hematin granules, suggests a life-cycle pattern for A. delacruzi different to that found in most argasid ticks (Oliver, 1989). Furthermore, the presence of a gran- ular (secretory) structure in the salivary glands, along with results from experimental infestations, provided evidence that A. delacruzi nymphs were able to feed. These features, together with the general structure of the body and the morphology of mouthparts, suggest that A. delacruzi may form a cluster of tick species that are related to the current Carios spp. and basal to the Ixodida as a first evolutionary step from the Hol- othyrida. Whether late nymphal instars are facultative blood feeders remains an open, unconfirmed matter. We are grateful for the help of Alessandro Mannelli (University of To- rino, Italy) and of Flavio Terassini and Luis Marcelo A. Camargo (ICB5, University of Sao Paulo, Brazil) in the development of this study.

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ALTSCHUL, S. F., T. L. MADDEN,A.A.SCHAFFER,J.ZHANG,Z.ZHANG, W. M ILLER, AND D. J. LIPMAN. 1997. Gapped BLAST and PSI- BLAST: A new generation of protein database search programs. Nucleic Acid Research 25: 3389–3402. CHINZEI,Y.,AND I. YANO. 1985. Vitellin is the nutrient reserve during starvation in the nymphal stage of a tick. Cellular and Molecular Life Sciences 41: 948–950. COOLEY,R.A.,AND G. M. KOHLS. 1944. The Argasidae of North Amer- ica, Central America and Cuba. The University Press, Notre Dame, Indiana, 152 p. COONS,L.B.,AND M. A. ROSHDY. 1981. Ultrastructure of granule se- cretion in salivary glands of Argas (Persicargas) arboreus during feeding. Zeitschrift fu¨r Parasitenkunde 65: 225–234. DELACRUZ, J., AND A. ESTRADA-PEN˜ A. 1995. Four new species of Antricola ticks (Argasidae: Antricolinae) from bat guano in Cuba and Curac¸ao. Acarologia 36: 277–286. EL-SHOURA, S. M. 1988. Ultrastructural studies on the midgut epithelium and digestion in the female tick Argas (Persicargas) arboreus (Ixo- FIGURE 4. Light photomicrographs of cross sections of gut in an doidea: Argasidae) Experimental and Applied Acarology 5: 121–136. early (A) and late (B) nymph of Antricola delacruzi and a nymph of ESTRADA-PEN˜ A, A., J. M. VENZAL,D.BATTESTI,V.ONOFRIO,E.TRAJANO, Carios puertoricensis (C). Mallory’s stain, ϫ100. AND J. V. L. FIRMINHO. 2004. Three new species of Antricola (Ac- ari: Argasidae) from Brazil, with a key to the known species in the genus. Journal of Parasitology 90: 490–498. gland acini and to granular Type II or Type B acini (Fig. 2). The type HALL, T. A. 1999. BioEdit: A user-friendly biological sequence align- B acinus contained alternating transport and granular secreting cells ment editor and analysis program for Windows 95/98/NT. Nucleic similar to the morphology of A. arboreus salivary glands (Roshdy and Acids Symposia Series 41: 95–98. Coons, 1975; Coons and Roshdy, 1981). Carios spp. nymphs and fe- KLOMPEN, J. S. H. 1992. Comparative morphology of argasid larvae males also have 2 salivary gland acinar types: Type I agranular and (: Ixodida: Argasidae), with notes on phylogenetic relation- Type II granular (Fig. 3). Unlike the Antricola spp., the Type II granular ships. Annals of Entomological Society of America 85: 541–560. acinus in Carios spp. contained only granulated cells, which are most ———, AND J. H. OLIVER,JR. 1993. Systematic relationships in the soft likely all secretory cells. All stages of A. delacruzi had a similar gut ticks (Acari: Ixodida: Argasidae). Systematic Entomology 18: 313–331. structure consisting of digestive cells in the basal portion that contained KOCAN, K. M., J. A. HAIR, AND S. A. EWING. 1980. Ultrastructure of hematin granules, presumably retained from a previous feeding (Fig. Anaplasma marginale Theiler in Dermacentor andersoni Stiles and 4). No traces of either regenerative or secretory cells were observed in Dermacentor variabilis (Say). American Journal of Veterinary Re- the sections of gut from the different stages of A. delacruzi ticks. Even search 41: 1966–1976. though different stages of ticks that fed on rabbits were processed, OLIVER,J.H.JR. 1989. Biology and systematics of ticks (Acari: Ixo- changes of this general digestive structure were not observed. A diges- dida). Annual Review of Ecology and Systematics 20: 397–430. tive cycle in argasid ticks, involving several changes in the structure of PICHON, B., D. EGAN,M.ROGERS, AND J. S. GRAY. 2003. Detection and digestive cells, has been reported for A. arboreus (El Shoura, 1988). identification of pathogens and host DNA in unfed host-seeking However, A. delacruzi tick guts in this study appeared only to have Ixodes ricinus L. (Acari: ). Journal of Medical Entomology digestive cells filled with hematin (probably DII type), although they 40: 723–731. lack the specialized surface for endocytosis characteristic of tick diges- RICHARDSON, K. C., L. JARRET, AND F. H . F INKE. 1960. Embedding in tive cells (El Shoura, 1988). This morphology was also different from epoxy resins for ultrathin sectioning in electron microscopy. Stain that found on fed Carios spp. ticks, which appeared to have the typical Technology 35: 313–323. tick gut cells with hematin granules, while the apical part sloughed into ROSHDY, M. A., AND L. B. COONS. 1975. The subgenus Persicargas the gut lumen as feeding progressed. Using light microscopy, secretory (Ixodoidea, Ixodidae, Argas). 23. Fine structure of the salivary and digestive DI type cells were also evident in the gut of Carios spp. glands of unfed A.(P.) arboreus Kaiser, Hoosgtraal, and Kohls. It is interesting to note from the present study that, while salivary Journal of Parasitology 61: 743–752.