Description of the nymph and larva and redescription ...

Systematic Parasitology 57: 211–219, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Description of the nymph and larva and redescription of the female of Ixodes neuquenensis Ringuelet, 1947 (Acari: Ixodidae), a parasite of the endangered Neotropical marsupial Dromiciops gliroides Thomas (Microbiotheria: Microbiotheriidae) Alberto A. Guglielmone1, Jos´e M. Venzal2 , Guillermo Amico3 , Atilio J. Mangold1 & James E. Keirans4 1 Instituto

Nacional de Tecnolog´ıa Agropecuaria, CC 22, CP 2300 Rafaela, Santa Fe, Argentina de Parasitolog´ıa Veterinaria, Facultad de Veterinaria, Universidad de la Rep´ublica, Av. Alberto Lasplaces 1550, Montevideo, Uruguay 3 Laboratorio Ecotono, Universidad Nacional de Comahue, Unidad Postal Universitaria, CP 8400 San Carlos de Bariloche, R´ıo Negro, Argentina 4 United States National Tick Collection, Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA 30460, USA 2 Departamento

Accepted for publication 1st October, 2003

Abstract The female of Ixodes neuquenensis Ringuelet, 1947 (Acari: Ixodidae) is redescribed and the nymph and larva are described from specimens collected from the endangered marsupial Dromiciops gliroides Thomas (Microbiotheria: Microbiotheriidae) in Argentina. At first sight the female of I. neuquenensis resembles a member of the subgenus Ixodes Latreille, 1795. However, the female of I. neuquenensis is peculiar in having the combination of two spurs on coxae II–IV and a pair of chitinous plaques internal to coxa I. Both the nymph and larva have an anterior and posterior process on palpal article I, characteristics of the subgenus Ixodiopsis Filippova, 1957 and some representatives of the subgenus Pholeoixodes Schulze, 1942. Analysis of 16S mitochondrial rDNA sequences showed no strong relationship with any known Ixodes subgenus.

Introduction Ixodes neuquenensis Ringuelet, 1947 has been known only from the holotype female collected from the marsupial Dromiciops gliroides Thomas (= Dromiciops australis Philippi) and a nymph presumably collected from a bat nest (Ringuelet, 1947). D. gliroides is peculiar for its exclusive mutualism with the loranthaceous mistletoe Tristerix corymbossus (Linnaeus), which has an aerial parasitic life-style (Amico & Aizen, 2000). The marsupial host of I. neuquenensis is endemic to the northern temperate forests of southern South America that extends along the Pacific rim in Argentina and Chile, home of an endemic and endangered flora and fauna (Armesto et al., 1998). Thus, the parasites of D. gliroides are also at risk of extinction.

I. neuquenensis is the only tick species ever found on D. gliroides. The type-specimens were first confused by Boero (1945) with I. brunneus Koch, 1844. Recently we had the opportunity to study new tick specimens obtained from D. gliroides close to the type-locality for I. neuquenensis. Therefore, a comparison with descriptions of the female of I. neuquenensis (see Ringuelet, 1947; Boero, 1957), and a comparison of DNA sequences of the larva, nymph and female of I. neuquenensis were carried out to confirm that all stages belong to the same species. Ringuelet (1947) also presented a brief description of the alleged nymph of I. neuquenensis. His figure of a ventral view of the basis capituli and hypostome of the nymph showed that palpal article I did not have the prominent processes that characterise our nymph

212 and larvae of I. neuquenensis (see below). In addition, his alleged nymph of I. neuquenensis was not collected from D. gliroides, indicating that Ringuelet’s nymph belongs to another species of Ixodes. Herein, we redescribe the female and describe the nymph and larva of I. neuquenensis. A comparison of 16S mitochondrial rDNA sequences of I. neuquenensis with sequences of other Ixodes species is also presented. The type-specimens of I. neuquenensis were deposited in the Museo La Plata, colección Ing. Havrilenko (La Plata, Buenos Aires, Argentina) (Ringuelet, 1947). However, a search for the type-specimens yielded negative results. Therefore, we designate as the neotype a female of I. neuquenensis (ex Dromiciops gliroides, Parque Municipal Llao Llao, Department Bariloche, Province of Río Negro, Argentina: 41◦ 02 S, 71◦ 32 W; March 8, 2000; collector G. Amico). It is deposited in the United States National Tick Collection under accession number RML 123262.

Materials and methods Live-trapping of D. gliroides was carried out with official permission of Argentinean authorities in a 2 ha plot of the Llao-Llao Reserve (41◦ 02 S, 71◦ 32 W), 25 km W of the city of Bariloche, Río Negro Province, Argentina, during the 1999–2000 summer season (December–March). The site supports an old-growth forest of Nothofagus dombeyi Oersted. The shrub Aristotelia chilensis Stuntz (c.80% of total shrub cover) dominates the understory. Live-trap small mammal single-door cage traps (30 × 15 × 15 cm) baited with pieces of apple, banana and peanut butter were used. Traps were attached to tree branches at 1.5–3 m above ground for a total of 360 trap nights. All animals were released after inspection for ticks. Tick specimens were preserved in 70% ethanol and deposited in the tick collection of Departamento de Parasitología, Facultad de Veterinaria, Montevideo, Uruguay (CDPFVU), the United States National Tick Collection (USNTC), currently in the Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, Georgia, and the tick collection of Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Santa Fe, Argentina (CINTAR). Females, larvae and nymphs were prepared for scanning electron microscopy following Corwin et al. (1979). Larvae, nymphs and female ticks preserved in

ethanol and stored at −20 ◦ C until they were used for DNA extraction and polymerase chain reaction (PCR) amplification as described by Mangold et al. (1998). PCR conditions included an initial denaturation step at 94 ◦ C for 2 min followed by 35 cycles for 45 s at 94 ◦ C, 45 s for primer annealing and 45 s for primer extension at 72 ◦ C. The annealing temperature of the first 7 cycles was increased by 0.3 ◦ C every second cycle from 47 to 48.8 ◦ C, followed by 28 cycles using an annealing temperature of 50 ◦ C. A final extension step was carried out for 7 min at 72 ◦ C. The primers used for the amplification and sequencing of c.460 bp fragment of the 16S rRNA were: forward, 5 -CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3 (16S + 1, Black & Piesman, 1994); reverse, 5 -CCG GTC TGA ACT CAG ATC AAG T-3 (16S − 1, Black & Piesman, 1994). All PCR reactions were performed in a 100 µl volume. Negative controls (no template) were always run simultaneously, and entire reaction mixtures were discarded if a product appeared in the negative control. A 10 µl volume of the reaction mixture was examined by 1% agarose-gel electrophoresis followed by staining with ethidium bromide. The amplified DNA was purified using ConcertTM Rapid PCR Purification System (Invitrogen Corp.) according to the manufacturer’s protocol. Purified PCR products were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit and an Applied Biosystems 373A gene sequencer. The 2 strands of DNA were assembled using a biosequence editor and analysis applications (SeqPup v.0.9e, © 1990–1997 by D.G. Gilbert, Biology Dept., Indiana University, Bloomington, IN 47405, USA). Multiple alignments were made by using Clustal W (Thompson et al., 1994). For all data-sets, to ensure that sequences would begin and end at the same position, all sequences were truncated. Aligned sequences were examined using the computer program MEGA 1.01 (Kumar et al., 1993) and a similarity matrix was constructed. The following mitochondrial 16S rDNA sequences of Ixodes spp. available in GenBank were also used for pairwise comparisons: I. (Pholeoixodes) cookei Packard, 1869 (GenBank accession No. U95883), I. (Ph.) hexagonus Leach, 1815 (L34298), I. (Ixodiopsis) angustus Neumann, 1899 (U14140), I. (Lepidixodes) kopsteini Oudemans, 1926 (U95890), I. (Partipalpiger) ovatus Neumann, 1899 (U95900), I. (Eschatocephalus) vespertillonis Koch, 1844 (U95910), I. (Ixodes) loricatus Neumann, 1899 (U95892), I. (I.) luciae Senevet, 1940 (U95894), I. (I.) acutitarsus (Karsch, 1880)

213 (U95877), I. (I.) affinis Neumann, 1899 (U95879), I. (I.) ricinus (Linnaeus, 1758) (Z97882), I. (I.) scapularis Say, 1821 (L34293), I. (I.) spinipalpis Hawden & Nuttall, 1916 (L34297), I. (I.) pacificus Cooley & Kohls, 1943 (L34296), I. (I.) muris Bishopp & Smith, 1943 (U95896), I. (I.) persulcatus Schulze, 1930 (L34295), I. (I.) jellisoni Cooley & Kohls, 1938 (U95888), I. (I.) granulatus Supino, 1897 (U95885), I. (Endopalpiger) tasmani Neumann, 1899 (U95906) and I. (Ceratixodes) uriae White, 1852 (U95908).

Ixodes neuquenensis Ringuelet, 1947 Material studied A total of 57 D. gliroides were captured during the summer of 2002. Only individuals that were captured in March, the last month of the season, were parasitised by I. neuquenensis. All individual hosts were males. Animals infested with I. neuquenensis were smaller than those without this parasite (Amico, in preparation). A total of 7 females, 14 nymphs and 321 larvae were collected from five D. gliroides during March 8–9, 2000 at Llao Llao Reserve. Two females, 3 nymphs and 8 larvae of I. neuquenensis were destroyed to obtain DNA; one female tick (neotype), 3 nymphs and 7 larvae are deposited in the USNTC; 1 female, 2 nymphs and 5 larvae (in poor conditions) were deposited in CINTAR; the remaining specimens are in CDPFVU. The DNA sequence of 16S rDNA were identical for the female, nymph and larva (GenBank accession numbers: AY254393 for female sequence, AY254394 for nymph sequence and AY254395 for larva sequence), supporting the finding that all stages belong to the same species. Redescription of the female and description of the nymph and larva Female (Figures 1–4, 13) Measurements (mm) from best preserved female, slightly engorged. Body (Figures 1–2). Length from apices of hypostome to posterior body margin 3.73, breadth 1.93. Outline oval, widest at level posterior to spiracular plates. Capitulum (Figures 3–4). Length from palpal apices to cornua apices 0.68. Basis capituli (Figure 3) dorsally broad, 0.51; shape broadly quadrangular; cornua small and triangular; posterior margin between cornua slighty sinuous; 2 large porose areas (0.12 diameter) separated by slightly less (0.10)

than diameter of porose areas. Ventrally (Figure 4) with posterior margin slightly curved and small triangular proccesses at each posterolateral angle; small triangular auriculae present laterally; transverse suture absent. Palpi broad, stout, 0.57 long, 0.20 wide; article II 0.30, less than 1/3 longer than article III, 0.23; articulation between 2 segments distinct. Article IV recessed within ventrolateral surface of article III. Article I with 2 setae. Hypostome stout, narrowly rounded apically; length of toothed portion 0.34; dental formula 3/3 for more than half of length, then 2/2 to base; c.9 teeth in files 1 and 2; c.6 in file 3. Scutum (Figure 1) elongate-oval in outline, slightly longer than broad; length 1.45, breadth 1.10. Lateral carinae faint; scapulae small, pointed; cervical grooves very shallow, converging and then diverging on anterior 1/3 of scutum; large punctations in lateral and central fields; each punctation contains single seta which is readily seen in scanning electron photomicrographs. Spiracular plate (Figure 2) subcircular in outline; goblets as illustrated. Legs (Figures 2, 13) rather long and slender. Coxa I with 2 well-developed spurs, both reaching coxa II; internal spur longer than external; coxae II-IV each with 2 triangular spurs decreasing in size from II to IV; each internal spur smaller than external. Trochanters lacking spurs. Alloscutum (Figures 1–2) with abundant, short, whitish, dorsal and ventral setae, ventral chitinous plaque present medial to coxa I; these plaques are more easily seen using light-microscopy than in scanning electron micrographs. Genital aperture situated between coxa III. Anal groove: an inverted U-shape reaching posterior margin of body. Anal valves with 3 pairs of setae. Nymph (Figures 5–8, 14) Measurements (average in mm) from 3 slide-mounted, slightly engorged specimens. Body (Figures 5–6). Length from palpal apices to posterior body margin 2.00, breadth 1.37. Outline oval, broadest at level of spiracular plate. Capitulum (Figures 7–8). Length from apex of hypostome to cornua apices 0.25, breadth 0.23. Basis capituli trapezoidal dorsally, with prominent triangular cornua; posterior margin between cornua slightly convex; small triangular auriculae present ventrally; posterior border convex. Palpi broad, stout, 0.20 long, 0.10 wide; suture between articles II and III indistinct; setae number 14 on articles II and III combined, c.12 on IV and 0 on I; palpal article I with large ventral anterior and posterior processes. Hypostome moderately elongate, rounded apically; length of toothed portion 0.12, width 0.06;

214

Figures 1–4. Ixodes neuquenensis female. 1. Dorsal view. 2. Ventral view; arrows indicate chitinous plaques beside each coxa I. 3. Basis capituli, dorsal view. 4. Basis capituli, ventral view; arrows indicate the triangular proccess at each side of the posterior margin. Scale-bars: 1, 1 mm; 2, 500 µm; 3, 100 µm; 4, 125 µm.

dental formula 2/2; 8–9 denticles in file 1, 7-8 in file 2. Scutum (Figure 5) slightly broader than long; length 0.57, breadth 0.54. Faint lateral carinae; cervical grooves shallow, converging then diverging to reach posterolateral scutal margin. Scutal surface with irregular depressions; punctations present in lateral and central fields; several short setae also present. Legs (Figure 8, 14). Coxa I with 2 well-developed spurs, both reaching coxa II, internal spur longer than ex-

ternal; coxa II-IV with two triangular spurs decreasing in size from II to IV, internal spurs smaller than externals. Trochanters lacking spurs. Tarsus I 0.31 long. Alloscutum (Figures 5–6) with small, ventral chitinous plaques located medially to coxa I; anal groove with slightly divergent branches; spiracular plate circular in outline.

215

Figures 5–8. Ixodes neuquenensis nymph. 5. Dorsal view. 6. Ventral view. 7. Basis capituli, dorsal view. 8. Basis capituli, ventral view. Scale-bars: 5–6, 500 µm; 7, 50 µm; 8, 100 µm.

Larva (Figures 9–12, 15) Measurements (average in mm) from 4 slide-mounted slightly engorged specimens. Body (Figures 9– 10) subcircular, 0.90 long (including capitulum), 0.65 broad. Sensilla sagittiformia absent. Dorsal setae 13 pairs; 4–5 pairs of central dorsals; 8 pairs of marginal dorsals. Ventral setae 13–14 pairs plus 1 pair on anal valves; 3 pairs of sternals; 2 pairs of pre-anals; 4–5 of pairs premarginals; 4 pairs of marginal ventrals. Anal groove as shallow diverging branches reaching posterior body margin, becoming deeper before reaching posterior margin. Capitulum (Figures 11–12). Length from apex of hypostome to

apices of cornua 0.14, breadth 0.13. Basis capituli dorsally trapezoidal, with small but obvious cornua; posterior margin almost straight. Auriculae absent ventrally; posterior margin slightly convex. Posthypostomal setae 2 pairs; distance between PH1 0.05, between PH2 0.04. Palpi short, stout, length 0.12, width 0.05; setae 0 on segment I; 8 dorsally, 3 ventrally on segments 2 and 3 combined (suture between 2 and 3 indistinct); segment IV with c.14 setae; palpal segment I with large anterior and posterior processes. Hypostome rounded anteriorly; 0.09 long, 0.04 wide; dentition 2/2 throughout length with corona of several small denticles apically; 7 denticles in file 1,

216

Figures 9–12. Ixodes neuquenensis larva. 9. Dorsal view. 10. Ventral view. 11. Basis capituli, dorsal view. 12. Basis capituli, ventral view. Scale-bars: 9–10, 100 µm; 11, 20 µm; 12, 50 µm.

6 denticles in file 2. Scutum (Figure 9) length 0.34, breadth 0.34; margin diverging for 1/3 of its length, then converging with rounded posterior margin. Cervical grooves shallow, converging and then diverging, almost reaching posterolateral margin. Setae 5 pairs. Legs (Figures 12, 15). Coxa I with 2 large spurs reaching coxa II, internal spur slightly larger than external; coxa II and III with triangular internal spur, lacking external spur. Coxal setae: 3 on I and 2 on II and III. Tarsus I 0.22 long.

bush monkey’). During the winter, when temperatures drop and food becomes scarce, D. gliroides hibernates. Before hibernation, the base of the tail becomes swollen with fat deposits (Marshall 1987). D. gliroides is the only extant representative of the family Microbiotheriidae, an ancient marsupial lineage. Recent morphological, chromosomal, and molecular evidence suggest that this living fossil shares greater phylogenetic affinities with Australian than other American marsupials (Spotorno et al., 1997, Springer et al., 1998).

Discussion

Species relationships

Hosts

Clifford et al. (1973) and Keirans (1992) listed the species of Ixodes Latreille, 1795. We consider that there are now 244 species of Ixodes worldwide, including the recently described Neotropical species, I. paran-

Dromiciops gliroides is a small, nocturnal, arboreal mammal, known locally as ‘monito del monte’ (‘litttle

217 Table 1. Matrix of sequence divergence (% nucleotide differences) on pairwise comparisons of the 16S mitochondrial rDNA sequences for 21 Ixodes species from eight subgenera (initials in parentheses) OTUs

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21

1 - ricinus (I.) 2 - affinis (I.) 3 - scapularis (I.) 4 - cookei (Ph.) 5 - vespertillonis (Es.) 6 - hexagonus (Ph.) 7 - kopsteini (L.) 8 - ovatus (Par.) 9 - loricatus (I.) 10 - luciae (I.) 11 - neuquenensis (?) 12 - acutitarsus (I.) 13 - spinipalpis (I.) 14 - pacificus (I.) 15 - muris (I.) 16 - persulcatus (I.) 17 - jellisoni (I.) 18 - granulatus (I.) 19 - tasmani (En.) 20 - uriae (C.) 21 - angustus (Ix.)

– 6 8 11 14 11 10 14 12 13 13 9 9 10 8 8 10 7 15 18 11

– 8 13 14 13 11 15 12 12 14 10 8 10 8 8 10 9 16 18 12

– 14 13 14 11 15 11 13 13 10 6 9 7 8 10 9 16 19 13

– 11 9 9 11 13 14 15 12 15 15 15 14 15 13 16 19 11

– 12 11 12 13 14 14 12 13 15 15 13 15 12 19 20 10

– 8 13 12 12 14 13 13 15 13 15 15 13 18 19 11

– 11 12 12 14 12 11 11 12 13 13 12 17 18 12

– 15 16 13 13 16 16 14 14 17 13 17 19 14

– 4 11 10 12 15 14 9 12 11 18 20 11

– 12 11 14 14 15 11 13 12 18 19 11

– 11 15 18 14 15 15 12 21 20 14

– 12 13 12 11 11 8 16 19 12

– 8 8 8 10 8 15 20 15

– 9 11 10 10 17 18 17

– 9 10 9 16 20 15

– 11 – 9 8 – 15 15 16 – 19 21 18 20 – 13 15 14 18 21

–

Abbreviations: OTUs, Operational taxonomic Units, I., Ixodes, Ph., Pholeoixodes, Es., Eschatocephalus, L., Lepidixodes, Par., Partipalpiger, E., Endopalpiger, C., Ceratixodes, Ix., Ixodiopsis; ?, I. neuquenensis with uncertain subgeneric status.

aensis Barros Battesti, Arzua, Pichorim & Keirans, 2003 and I. dicei Keirans & Ajohda, 2003. In constructing this list, I. neotomae Cooley, 1944, is treated as a synonym of I. spinipalpis Hadwen & Nuttall, 1916 (Norris et al., 1997) and I. dammini Spielman, Clifford, Piesman & Corwin, 1979 is treated as a synonym of I. scapularis Say, 1821 (Oliver et al., 1993). The female of I. neuquenensis appears to resemble a member of the subgenus Ixodes (see Clifford et al., 1973); however, it is peculiar in having two obvious spurs on coxae II to IV. The only species that we found with two spurs on coxae II to IV are the Ethiopian I. (I.) theilerae Arthur, 1953 and the Palaearctic I. (I.) turdus Nakatsuji, 1942. However, in I. theilerae the internal spurs on coxae II to IV are rounded thickenings of the coxal margins, and in I. turdus the internal spur on coxa IV is very small and widely separated from the external spur. In addition, the spurs on coxa I are almost of identical size, none of them reaching coxa II. Chitinous plaques on the ventral surface of the alloscutum of the female have been found in several representatives of the subgen-

era Ixodes and Afrixodes Morel, 1966 but never as distinctive as in I. neuquenensis. The small, ventral, triangular proccesses at the posterolateral angles of the basis capituli are not present in most species of Ixodes. The diagnostic characters unique to the female of I. neuquenensis are a combination of two spurs on coxa II to IV, and the chitinous plaques adjacent to the internal border of coxa I. The nymph and larva of I. neuquenensis have a greatly enlarged palpal article I with both anterior and posterior proccesses. This feature of palpal article I is also found in nymphs and larvae of the Ixodes subgenera Endopalpiger Schulze, 1935 (Australian), Exopalpiger Schulze, 1935 (Neotropical, Palaearctic, Afrotropical, Australian) and Partipalpiger Hoogstraal, Clifford, Saito & Keirans, 1973 (Palaearctic, Oriental), in most representatives of the Holarctic subgenus Ixodiopsis Filippova, 1957, in some members of the mostly Nearctic-Palaearctic subgenus Pholeoixodes Schulze, 1942, which includes three Neotropical species (Clifford et al., 1973; Robbins & Keirans, 1992), and in the Neotropical I. sigelos Keirans, Clifford & Corwin, 1976 (of uncertain subgeneric

Figures 13–15. Ixodes neuquenensis coxae. 13. Female; arrows indicate chitinous plaques beside each coxa I. 14. Nymphs; arrow indicates chitinous plaques beside each coxa I. 15. Larva. Scale-bars: 13, 200 µm; 14, 100 µm; 15, 50 µm.

218 status). The immature stages of one of the Neotropical species of Pholeoixodes, I. nuttalli Lahille, 1913 (see Nuttall, 1916) are known, but not the larvae and nymphs of the other two Neotropical members of this subgenus, I. chilensis Kohls, 1956 and I. taglei Kohls, 1969. The enlarged article I of the palpi of immature stages of species from the subgenera Endopalpiger, Exopalpiger and Partipalpiger are partly fused with the basis capituli, while that in Ixodiopsis, Pholeoixodes, I. sigelos and I. neuquenensis is not. The nymph of I. neuquenensis has two spurs on coxae II to IV, a feature not shared with any other species in Ixodiopsis or Pholeoixodes whose nymphs are known, or with I. sigelos. This characteristic, in combination with the presence of chitinous plaques medial to coxae I, is diagnostic for the nymph of I. neuquenensis. The diagnosis of the larva of I. neuquenensis is more difficult. However, it can be separated from species of the subgenus Ixodiopsis, and also from the Neotropical I. (Ph.) nuttalli and the Nearctic I. (Ph.) marmotae Cooley & Kohls, 1938, I. (Ph.) sculptus Neumann, 1904 (representatives of the subgenus Pholeoixodes bearing proccesses on palpal article I of the larva) and I. sigelos, because none of them bear triangular spurs on coxae II and III (Nuttall, 1916; Senevet & Ripert, 1967; Robbins & Keirans, 1992), which are characteristic of larvae of I. neuquenensis. The male of I. neuquenensis remains unknown. In this sense, I. neuquenensis is similar to many other species of Ixodes, especially those species with nidicolous habits and whose males are still undescribed. The comparison of sequences of 16S mitochondrial rDNA of I. neuquenensis with other species of the genus Ixodes showed no strong association with any of them (the lowest percent divergence was 11%, with two representatives of the subgenus Ixodes, the Neotropical I. loricatus and the Oriental I. acutitarsus, and the maximum was 21% with the Australian I. (E.) tasmani) (Table 1). On the whole, the divergence was less with ticks from the subgenus Ixodes than from species in the subgenera Pholeoixodes, Ixodiopsis, Lepidixodes, Partipalpiger, Endopalpiger and Ceratixodes. I. neuquenensis is a unique tick of the endangered D. gliroides. Some morphological features of the female appear to show that it may belong to the subgenus Ixodes, while the proccesses on palpi I of the nymph and larva are compatible with Ixodiopsis or Pholeoixodes. However, DNA analysis showed no strong

219 relationship with any known Ixodes subgenus. Consequently, we believe that is premature to assign this tick species to a particular subgenus.

Acknowledgements We are grateful to Alejandro Márquez and Jorge Troccoli from the Unidad de Microscopía Electrónica, Facultad de Ciencias, Montevideo (Uruguay), for their assistance with scanning electron microscopy. We acknowledge the support of INTA and Fundación ArgenINTA to AAG and CIDEC project 6383/01779/2002 for assistance to JMV. Portions of this work were supported by National Institutes of Allergy and Infectious Diseases grant AI 40729 to JEK.

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