Amblyomma Yucumense N. Sp. (Acari: Ixodidae), a Parasite of Wild Mammals in Southern Brazil

MORPHOLOGY,SYSTEMATICS,EVOLUTION Amblyomma yucumense n. sp. (Acari: Ixodidae), a Parasite of Wild Mammals in Southern Brazil

FELIPE S. KRAWCZAK,1 THIAGO F. MARTINS,1 CAROLINE S. OLIVEIRA,2 LINA C. BINDER,1 1 3 0 1 FRANCISCO B. COSTA, PABLO H. NUNES, FABIO GREGORI, AND MARCELO B. LABRUNA1,4

J. Med. Entomol. 52(1): 28–37 (2015); DOI: 10.1093/jme/tju007 ABSTRACT During 2013–2014, adult ticks were collected on the vegetation and subadult ticks were collected from small mammals [Didelphis aurita Wied-Neuwied, Sooretamys angouya (Fischer), Eur- yoryzomys russatus (Wagner), Akodon montensis Thomas, Oxymycterus judex Thomas] in an Atlantic rainforest reserve in southern Brazil. Analyses of the external morphology of the adult ticks revealed that they represent a new species, Amblyomma yucumense n. sp. Partial 16S rRNA sequences generated from males, females, and nymphs were identical to each other and closest (95% identity) to corresponding sequences of Amblyomma dubitatum Neumann. A. yucumense is morphologically and genetically closest related to A. dubitatum. Dorsally, male of these species can be separated by major longitudinal pale orange stripes associated with a pseudoscutum indicated by a pale stripe in A. yucumense, in contrast to pale creamy longitudinal stripes and absence of pseudoscutum in A. dubitatum. Ventrally, male coxal I spurs are separated by a space narrower than external spur width in A. yucumense, and wider than external spur width in A. dubitatum. Females of the two species can be separated by coxal I spurs, longer in A. yucumense than in A. dubitatum. In addition, the adult capitulum and ventral idiosoma of A. yucumense are generally dark brown colored, while A. dubitatum is yellowish or light brown colored. The nymph of A. yucumense differs from A. dubitatum by the scutal cervical groove length, slightly shorter in the former species. Currently, A. yucumense is restricted to southern Brazil.

KEY WORDS Amblyomma, new species, description, Brazil

Introduction Atlantic rainforest, are transmitted to humans by different Amblyomma species (reviewed by Parola et al. The Brazilian tick fauna is currently composed by 66 2013). Herein, we describe an additional new species, 45 Ixodidae, and 21 Argasidae (Martins et al. Amblyomma species from South America, based on 2014, Nava et al. 2014a). The genus Amblyomma is the specimens collected in southern Brazil. most numerous, with 31 species (Dantas-Torres et al. 2009, Nava et al. 2014a), comprising 46% of the Brazil- ian tick fauna. In the world, the genus Amblyomma is currently composed of 136 valid species, distributed in Materials and Methods all continents except for Europe and Antarctica From June 2013 to January 2014, ticks were col- (Guglielmone and Nava. 2014, Nava et al. 2014b). Dur- lected in the “Parque Estadual do Turvo”, an Atlantic ing the past 10 yr, eight new Amblyomma species were rainforest Reserve located in Derrubadas Municipality, described or resurrected, all from South America, state of Rio Grande do Sul, southern Brazil, as part of where nearly half of world Amblyomma species occur an ongoing study on the ecology of tick-borne spotted (Labruna et al. 2005; Nava et al. 2009, 2014a,b). From fever in Rio Grande do Sul. Questing ticks were col- the medical stand point, the genus Amblyomma is the lected in the vegetation by dragging or by visual search most important in South America, where human dis- in animal trails, as previously described (Terassini et al. eases caused by tick-borne agents, namely, Rickettsia 2010). Ticks were also collected on small mammals that rickettsii, Rickettsia parkeri,andRickettsia sp. strain were collected by Sherman and Tomahawk traps along- side the same animal trails. Collected ticks were brought alive or in absolute eth- 1 Department of Preventive Veterinary Medicine and Animal anol to the laboratory, where they were separated for Health, Faculty of Veterinary Medicine, University of Saõ Paulo, Saõ morphological and molecular analyses; engorged larvae Paulo, SP, Brazil. were kept alive in an incubator (25C, 95% relative hu- 2 Department of Preventive Veterinary Medicine, Federal Univer- midity) to molt to nymphs. Unfed specimens, compris- sity of Santa Maria, Santa Maria, RS, Brazil. 3 Institute of Biosciences, Saõ Paulo State University, Rio Claro, SP, ing 10 males, 9 females, and 10 nymphs were Brazil. measured using the Image-Pro Plus 5.1 program for 4 Corresponding author, e-mail: [email protected]. analysis of images and morphometry, fitted to an

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] January 2015 KRAWCZAK ET AL.: PARASITE OF WILD MAMMALS IN SOUTHERN BRAZIL 29

Olympus SZX stereoscope microscope (Olympus Cor- ex Hydrochoerus hydrochaeris (L.), Saõ Bernardo do poration, Tokyo, Japan). In the description that follows, Campo, state of Saõ Paulo, 29 November 1999 (CNC all measurement are in mm; a range is given, with a 263); 2 females ex vegetation, Araçariguama, state of mean 6 standard deviation in parentheses. Three speci- Saõ Paulo, 27 March 1998 (CNC 129); 5 males and 3 mens of each stage (male, female and nymph) were females (collected as nymphs) ex Myocastor coypus prepared for scanning electron microscopy (SEM) fol- (Molina), Jundiai, state of Saõ Paulo, 17 August 1999 lowing techniques described by Corwin et al. (1979). (CNC 288). Light-microscopy photographs of live adult ticks were Three males and three females of A. dubitatum prepared to show the natural scutal ornamentation collected ex vegetation, Intervales State Park, Ribei- pattern. raõ Grande Municipality, state of Saõ Paulo, Brazil, Representative tick specimens were used for molec- 18 September 2005 (CNC 927), were used for SEM. ular analysis. For this purpose, each of the 2 males and One live and 1 live female of A. dubitatum,collected 2 females had its leg segments collected in a sterile ex vegetation, Pirassununga, state of Saõ Paulo, microtube containing 50 ml of Tris-EDTA buffer, subse- Brazil, 10 March 2014, were used for light-micros- quently subjected to DNA extraction with DNeasy Tis- copy photographs to show the natural scutal orna- sue Kit (Qiagen, Valencia, CA), and processed by mentation pattern. polymerase chain reaction (PCR) with the use of primers targeting a 460-bp fragment of the tick 16S rDNA mitochondrial gene, as previously described Results (Mangold et al. 1998). The same procedure was per- formed with two nymphs, but in this case, the whole Analyses of the external morphology of the adult tick body was individually used for DNA extraction. ticks collected in the present study revealed that they PCR products of the expected size were sequenced in were morphologically distinct from any other known an ABI automated sequencer (Applied Biosystems/ Amblyomma species, justifying their description as a Thermo Fisher Scientific, model ABI 3500 Genetic new species. Partial 16S rRNA sequences (404 bp) gen- Analyser, Foster City, CA) with the same primers used erated from 2 males, 2 females, and 2 nymphs were for PCR. Generated sequences were compared to each 100% identical to each other, and by BLAST analysis, other and submitted to BLAST analyses (www.ncbi. were closest (95% identity) to several sequences of nlm.nih.gov/blast) to infer closest similarities available A. dubitatum from the states of Saõ Paulo (GU301914) in GenBank. The consensus sequence obtained was and Rio Grande do Sul (GU301913), Brazil, and from aligned with the corresponding 16S rRNA sequences Uruguay (DQ858954, DQ858955, and GU301912) and from 12 different sequences of the genera Amblyomma Argentina (GU301910 and GU301911). Because all retrieved from GenBank (including the sequences that nymphs examined in the present study had the same matched closest BLAST similarities to the present new external morphology, the above molecular results con- species) using Clustal/W v.1.8.1 (Thompson et al. firm that they represent the nymphal stage of the pre- 1994). Neighbor-joining and maximum likelihood phy- sent new Amblyomma species. logenetic trees using T92þG (Tamura 3-parameter with Gamma: 0.17) substitution model were generated using Mega 6.06 software (Tamura et al. 2013)with Amblyomma yucumense Krawczak, Martins & 1,000 bootstrap replicates. The substitution model was Labruna n. sp. (Figs. 1-4) selected using Mega 6.06 software (Tamura et al. 2013) according to the lowest Bayesian Information Criterion Male. (Figs. 1Aand2): Ten unfed specimens meas- (BIC) score. The sequence of Amblyomma latum Koch ured. Length from apices of scapulae to posterior body was used as outgroup. margin 4.13–5.36 (4.70 6 0.36), breadth 3.17–4.04 For morphological comparisons, the following speci- (3.56 6 0.29). Outline oval, broadest at level of spiracu- mens of Amblyomma dubitatum Neumann available at lar plates (Fig. 1A). Genital aperture broadly U-shaped the tick collection “Coleçaõ Nacional de Carrapatos” (Fig. 2D). Spiracular plate comma-shaped with elon- (CNC) at the Faculty of Veterinary Medicine of the gate macula and numerous minute globlets. Eyes flat. University of Saõ Paulo, Saõ Paulo, SP, Brazil, were ex- Basis capituli rectangular and triangular cornua on amined in the present study: 53 males, 87 females ex posterior margin; numerous small punctations dorsally vegetation, Itu Municipality, state of Saõ Paulo, Brazil, (Fig. 2A). Length of palpal apices to cornua apices 23 January to 07 February 2006 (accession number 0.95–1.32 (1.09 6 0.11), breadth 0.76–0.99 (0.83 6 CNC 958); 6 males, 8 females ex vegetation, Serra da 0.07). Length of palpal article I 0.03–0.11 (0.07 6 0.02); Canastra National Park, Saõ Roque de Minas Munici- length of palpal article II 0.32–0.54 (0.46 6 0.07); pality, state of Minas Gerais, Brazil, 14 August 2007 length of palpal article III 0.15–0.26 (0.21 6 0.04), and 08 March 2008 (CNC 1046, 1099); 16 males, 24 fe- suture between II and III distinct. Presence of marked males ex vegetation, Intervales State Park, Ribeiraõ ventral prolongation on palpal article I, as illustrated Grande Municipality, state of Saõ Paulo, Brazil, 18 Sep- (Fig. 2B). Hypostome elongate, broadly rounded api- tember 2005 (CNC 927). In addition, we also examined cally with corona of fine denticles. Total length of hypo- the following specimens collected in Brazil, which were stome 0.63–0.78 (0.71 6 0.04); length of toothed used by Estrada Penã et al. (2002) for the redescription portion 0.34–0.42 (0.38 6 0.02). Hypostomal dentition and revalidation of the species A. dubitatum:10males 3/3, with six to eight teeth per complete row (Fig. 2B); 30 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 1

Fig. 1. Dorsal view of the adult stages of A. yucumense [(A) Male. (B) Female] and A. dubitatum [(C) Male. (D) Female] by light microscopy.

Fig. 2. SEM of A. yucumense male. (A) Dorsal capitulum (scale bar: 200 mm). (B) Ventral capitulum (scale bar: 200 mm). (C) Scutum (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 400 mm). in some specimens hypostomal dentition were 4/4 dark brown elevated spots lacking punctations as fol- posteriorly. lows: median-lateral spots, postero-lateral spots, and a Scutum ornate with pale orange markings (stripes) postero-median spot; limits of pseudoscutum indicated over a dark brown background; major longitudinal by a pale stripe (Fig. 1A), clearly observed by naked stripes starting at the cervical area, diverging posteri- eyes. Cervical grooves short, deep, comma-shaped. orly at the level of first festoon; numerous large and Marginal groove complete, deep posteriorly, marked deep punctations uniformly distributed, interposed by with numerous deep punctations anteriorly (Fig. 2C). January 2015 KRAWCZAK ET AL.: PARASITE OF WILD MAMMALS IN SOUTHERN BRAZIL 31

Fig. 3. SEM of A. yucumense female. (A) Dorsal capitulum (scale bar: 200 mm). (B) Ventral capitulum (scale bar: 200 mm). (C) Dorsal idiosoma (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 200 mm).

Punctations present on all festoons (Fig. 2C), festoons surrounded by numerous minute globlets. Eyes flat. 4 and 6 with less ornamentation than remaining fes- Festoons without tubercles. toons (Fig. 1A). Short or indistinct ventral plates Basis capituli and palpi brown; if ornate, only spiked present on all festoons, length 0.05–0.08 (0.06 6 0.01). by rare pale yellow marking. Basis capituli rectangular; Coxa I with two moderately long, parallel, and robust length of palpal apices to cornua apices 0.92–1.40 spurs of similar length; external spur slightly longer (1.14 6 0.16), breadth 0.71–1.05 (0.93 6 0.12). Cornua than internal in some specimens; external thinner than faint or absent, posterior margin slightly concave (Fig. internal. Presence of an accessory spur situated ante- 3A). Porose areas deeply depressed, diameter of one rior to the two regular spurs, as illustrated (Fig. 2D). area 0.08–0.17 (0.12 6 0.03), interporose area Coxae II–III each with triangular rounded external 0.07–0.15 (0.11 6 0.03). Length of palpal article spur and fine salient concave ridge extending from I 0.08–0.13 (0.10 6 0.01); length of palpal article II spur to postero-internal angle of coxa, where a shorter 0.38–0.58 (0.48 6 0.07); length of palpal article III internal spur is evidenced in most specimens; a single 0.20–0.37 (0.27 6 0.05), suture between II and III long, stout spur on coxa IV, spur length similar to the distinct. Presence of marked ventral prolongation on coxa (Fig. 2D). Trochanters without spurs (Fig. 2D). palpal article I, as illustrated (Fig. 3B). Hypostome Tarsus I 0.73–1.06 (0.89 6 0.10) in length, 0.20–0.29 elongate, apically rounded with corona of fine den- (0.26 6 0.03) in breath. Tarsus IV 0.73–0.83 (0.79 6 ticles. Total length of hypostome 0.64–0.95 (0.84 6 0.03) in length, 0.23–0.31 (0.27 6 0.03) in breath. 0.11); length of toothed portion 0.34–0.50 (0.44 6 0.05). Female. (Figs. 1B and 3). Nine unfed specimens Hypostomal dentition 3/3 anteriorly, 4/4 posteriorly (in measured. Length from apices of scapulae to posterior one specimen it was 3/3 posteriorly) with 7 to 10 teeth body margin 3.18–4.79 (4.30 6 0.56), breadth per complete row. 2.65–3.74 (3.41 6 0.39). Outline elliptical, broadest just Scutum length 1.61–2.62 (2.20 6 0.30), breadth anterior to the level of spiracular plates (Fig. 1B). Geni- 1.84–2.74 (2.39 6 0.29), ornamentation consisting pri- tal aperture broadly V-shaped, with short and bulging marily of pale yellow large spots on cervical and post- postero-lateral flaps (Fig. 3D). Spiracular plate sub- ero-lateral fields, separated by dark brown central triangular with rounded angles, elongate macula stripe ending at the posterior margin (Fig. 1B). 32 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 1

Fig. 4. SEM of A. yucumense nymph. (A) Dorsal capitulum (scale bar: 30 mm). (B) Ventral capitulum (scale bar: 30 mm). (C) Scutum (scale bar: 100 mm). (D) Coxae I–IV (scale bar: 200 mm).

Numerous large and deep punctations concentrated in length 0.18–0.23 (0.21 6 0.02), breadth 0.11–0.16 the lateral fields. Cervical grooves deep, converging (0.13 6 0.02). Gnathosoma length from palpal apices to anteriorly then diverging as shallow depressions at scu- posterior margin 0.26–0.32 (0.28 6 0.02); breadth tal median third (Fig. 3C). 0.26–0.29 (0.28 6 0.01). Basis capituli slightly hexago- Coxa I with two moderately long and robust spurs of nal, posterior margin straight, without cornua (Fig. 4A); similar length, the external thinner; presence of an posterior margin convex ventrally, without auriculae. accessory spur situated anteriorly to the two regular Palpi length 0.21–0.25 (0.23 6 0.02), article I with ves- spurs, as illustrated (Fig. 3D). Coxae II–IV each with tigial ventral prolongation; article II 0.13–0.16 (0.14 6 small short external spur and fine salient ridge extend- 0.01) in length, article III 0.06–0.08 (0.07 6 0.01) in ing from spur to postero-internal angle of coxa, occupy- length. Hypostome rounded apically; length 0.22–0.25 ing position of missing internal spur. This external spur (0.23 6 0.01); length of toothed portion 0.11–0.13 broader than longer on coxae II–III; pointed, as long as (0.13 6 0.01); dentition 2/2 with seven to eight teeth broad on coxa IV. Trochanters without spurs (Fig. 3D). per row (Fig. 4B); row pairs diverge towards the ante- Tarsus I 0.78–1.28 (0.99 6 0.18) in length, 0.21–0.36 rior extremity. Coxa I with two pointed spurs, the exter- (0.28 6 0.05) in breath. Tarsus IV 0.57–0.86 (0.76 6 nal longer; coxae II-IV with a small triangular spur 0.11) in length, 0.17–0.27 (0.22 6 0.03) in breath. (Fig. 4D). Trochanters without spur; tarsus I 0.29–0.34 Nymph. (Fig. 4). Ten unfed specimens measured. (0.33 6 0.02) in length, 0.08–0.12 (0.09 6 0.01) in Length from apices of scapula to posterior body margin breath; tarsus IV 0.24–0.28 (0.26 6 0.02) in length, 1.18–1.37 (1.28 6 0.06), maximum breadth 0.93–1.09 0.06–0.09 (0.08 6 0.01) in breath. (1.02 6 0.05); outline oval, with 11 festoons without TYPES. Holotype male, Allotype female, nine Para- tubercles. Scutal length 0.61–0.66 (0.64 6 0.02), type males, eight Paratype females, all unfed speci- breadth 0.71–0.78 (0.75 6 0.02), breadth/length ratio mens, ex vegetation at “Parque Estadual do Turvo” 1.14–1.22 (1.18 6 0.03); inornate, deep punctations (27 1402600 S; 53 5801900 W; altitude 390 m), Derruba- evenly distributed, larger laterally, smaller centrally das Municipality, state of Rio Grande do Sul, Brazil, 11 (Fig. 4C). Eyes large, not orbited, at lateral scutal January 2014; collectors: M.B. Labruna, F. Krawczak. angles at the level of scutal midlength. Cervical grooves Legs of two Paratypes of each sex were processed for long and deep reaching the scutal posterior third; DNA extraction. Thirty-tree Paratype nymphs, all length 0.31–0.34 (0.33 6 0.01); cervical groove length/ molted from engorged larvae collected on Didelphis scutal length ratio: 0.50–0.52 (0.51 6 0.01). Spiracular aurita Wied-Neuwied, 12 January 2014. Holotype, plate rounded with an evident dorsal prolongation; Allotype and 32 Paratypes (5 males, 4 females, 25 January 2015 KRAWCZAK ET AL.: PARASITE OF WILD MAMMALS IN SOUTHERN BRAZIL 33 nymphs) deposited in the CNC tick collection under the “Parque Estadual do Turvo” Atlantic Forest accession numbers CNC 2784–2787. Two Paratypes of Reserve, where the new species was found. each sex from lot CNC 2785 and two Paratype nymphs Phylogenetic Analysis. In the phylogenetic tree from CNC 2787 were used for SEM. Paratypes depos- inferred by partial sequences of the mitochondrial 16S ited in other tick collections: 1 male, 1 female, 3 rRNA gene (Fig. 5), A. yucumense grouped in a branch nymphs deposited at the Acari Collection of the Insti- with Amblyomma coelebs Neumann and several tuto Butantan (Sa˜o Paulo, Brazil) under accession num- sequences of A. dubitatum from Brazil, Uruguay, and ber IBSP 11716–11717; 3 males, 3 females, 3 nymphs Argentina. This topology was observed either by deposited at the United States National Tick Collection neighbor-joining or maximum likelihood analysis. (Statesboro, Georgia) under accession number USN- Although these three species formed a group, their 16S MENT00861423, CEN/RML 125117 and USN- DNA sequences were relatively distantly to each other. MENT00861424, CEN/RML 125118; 1 male, 1 In this regard, the sequence of A. yucumense was 4.9 female, 2 nymphs deposited at the INTA tick collection to 5.4% divergent from the A. dubitatum sequences (Rafaela, Santa Fe, Argentina). (DQ858954, DQ858955, GU301910–GU301914), and Additional Material Collected. The following 6.6% divergent from A. coelebs (FJ424408). The additional specimens of A. yucumense were collected at genetic divergences between A. dubitatum and A. coe- the “Parque Estadual do Turvo” (27 1402600 S, 53 lebs were 8.6–9.4%. Finally, the intraspecific divergen- 5801900 W, 390 m; and 27 1305100 S, 53 5100400 W, ces among the A. dubitatum sequences, representing 430 m), Derrubadas Municipality, state of Rio Grande populations from geographically separated areas of Bra- do Sul, Brazil: 2 nymphs (1 molted to a male) ex D. zil, Uruguay, and Argentina were in all cases <1%. The aurita, 05 July 2013 (CNC 2791); 1 nymph ex Sooret- partial sequence of the mitochondrial 16S rRNA gene amys angouya (Fischer), 05 July 2013 (CNC 2789); 5 generated in this study for A. yucumense has been nymphs ex vegetation, 03 July 2013 (CNC-2790); 37 deposited in GenBank under the accession number nymphs (3 molted to 1 male and 2 females) ex D. KJ914670. aurita, 09 October 2013 (CNC 2792); 6 nymphs ex D. aurita, 10 October 2013 (CNC 2793); 8 nymphs ex Discussion D. aurita, 09 October 2013 (CNC 2794); 3 nymphs ex vegetation, 10 October 2013 (CNC 2797); 1 nymph A. yucumense is morphologically and genetically clos- ex Euryoryzomys russatus (Wagner), 11 October 2013 est related to A. dubitatum (Figs. 1, 6,and7). These (CNC 2796); 1 nymph ex Akodon montensis two species are unique in combining the following mor- Thomas, 09 October 2013 (CNC 2795); 8 nymphs (2 phological characters: males with basis capituli rectan- destroyed for DNA extraction in the present study) ex gular and triangular cornua on posterior margin, coxa I Oxymycterus judex Thomas, 13 January 2014 (CNC with two spurs of similar size, coxal IV spur much lon- 2798); 3 males ex vegetation, 11 January 2014 (CNC ger than coxal II–III spurs, marginal groove complete, 2788). scutum with major longitudinal stripes starting at the Hosts and Distribution. Nymphs (and engorged cervical area and numerous large and deep punctations larvae from at least one host species) of A. yucumense uniformly distributed, interposed by dark brown ele- were collected from five species of small mammals (D. vated spots lacking punctuations, festoon ventral aurita, S. angouya, E. russatus, A. montensis,andO. plates not incised; females with basis capituli rectangu- judex), suggesting that small mammals are important lar,coxaIwithtwospursofsimilarsize,coxaII–IVwith hosts for subadult ticks of A. yucumense. Because adult a single spur, scutum with a dark brown stripe at the ticks were found only on vegetation, hosts for the adult posterior field, and genital aperture broadly V-shaped, stage remain unknown. However, it is noteworthy that with short and bulging postero-lateral flaps. most of these adults were collected by the visual search Dorsally, male of these species can be separated by method. In this case, ticks were found questing at a sta- major longitudinal pale orange stripes associated with a tionary position on the vegetation (ambushing), at pseudoscutum indicated by a pale stripe in A. yucu- heights between 50 and 80 cm. Many of these ticks mense (Fig. 1A), in contrast to pale creamy longitudinal were found together with adults of Amblyomma inci- stripes and absence of pseudoscutum in A. dubitatum sum Neumann, a typical parasite of tapirs [Tapirus ter- (Fig. 1C). Ventrally, male coxal I spurs are separated by restris (L.)]. Because tapirs are large animals a space narrower than external spur width in A. yucu- (with heights over 80 cm), and are known to frequently mense (Fig. 2D), and wider than external spur width in use the trails where ticks were collected, it is possible A. dubitatum (Fig. 6D). Females of the two species can that tapirs are major hosts for adults of A. yucumense,a be separated by the scutal large and deep punctations condition yet to be demonstrated. Until now, the evenly distributed in A. dubitatum (Fig. 7C), concen- distribution of A. yucumense is restricted to its type trated in the lateral fields in A. yucumense (Fig. 3C). locality, an Atlantic forest Reserve in southern Brazil. Ventrally, female coxal I spurs are unequivocally longer Because this Reserve is contiguous on the west to a in A. yucumense (Fig. 3D) than in A. dubitatum much larger Atlantic forest Reserve in Argentina, it is (Fig. 7D), when the two species are observed side by possible that A. yucumense also occurs in this later side. Finally, the adult capitulum and ventral idiosoma country. of A. yucumense arewidelydarkbrowncolored(Fig Etymology. The new species is named yucumense 1A and B), while A. dubitatum is yellowish or light in reference to the Yucuma˜Falls,atUruguayRiver,in brown colored (Fig. 1C and D). 34 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 1

Fig. 5. Neighbor-joining phylogenetic tree of 16S rRNA partial sequences (393 nt) of A. yucumense and closely related Ixodidae. Numbers on the nodes indicate bootstrap values from 1,000 replicates. Only bootstrap values >50 are shown. Numbers in brackets are GenBank accession numbers. The corresponding 16S rRNA partial sequences of A. latum was used as outgroup. Scale bar: number of substitutions per site.

Fig. 6. SEM of A. dubitatum male. (A) Dorsal capitulum (scale bar: 100 mm). (B) Ventral capitulum (scale bar: 100 mm). (C) Scutum (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 200 mm). January 2015 KRAWCZAK ET AL.: PARASITE OF WILD MAMMALS IN SOUTHERN BRAZIL 35

Fig. 7. SEM of A. dubitatum female. (A) Dorsal capitulum (scale bar: 200 mm). (B) Ventral capitulum (scale bar: 200 mm). (C) Dorsal idiosoma (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 200 mm).

Recently in Argentina, Nava et al. (2014a,b) described by Martins et al. (2010), and determined described Amblyomma hadanii, a species closest their cervical groove length: 0.49–0.55 (0.52 6 0.03). related to A. dubitatum and A. coelebs. Based on the Considering the scutal length values of these A. dubita- examination of two paratypes (1 male and 1 female) of tum nymphs (Martins et al. 2010), the cervical groove A. hadanii (CNC 1435), we could verify that this spe- length/scutal length ratio is 0.63–0.67 (0.65 6 0.02) for cies is morphologically very closely related to A. yucu- A. dubitatum, higher than the ratio values here mense, although males could be distinguished by the reported for A. yucumense nymphs: 0.50–0.52 scutal dark brown elevated spots lacking punctuations, (0.51 6 0.01). In the dichotomous identification key larger in A. yucumense (Fig. 2C) than in A. hadanii proposed for taxonomic identification of the nymphal [Figure 1 of Nava et al. (2014a,b)]. Females of these stage of 27 Amblyomma species from Brazil (Martins two species can be separated by the genital aperture, et al. 2010), the nymph of A. yucumense would key broadly V-shaped with short and bulging postero-lateral with A. dubitatum; therefore, these two species should flaps in A. yucumense (Fig. 3D), and U-shaped without be separated through the cervical groove length/scutal bulging postero-lateral flaps in A. hadanii [Figure 9 of length ratio. In addition, these identifications should be Nava et al. (2014a,b)].A.coelebsare readily distin- preferably associated with the identification of the adult guished from A. yucumense though distinct scutal orna- stage from the same collection site, as A. yucumense mentation patterns, due to the presence of large bright and A. dubitatum seem to have distinct ecological red-orange patches in the scapular area and absence of preferences. The nymph of A. hadanii is distinguished pseudoscutum in males of A. coelebs, and absence of a from A. yucumense through the cervical groove, dark brown central stripe ending at the posterior mar- which ends as shallow depression at the posterior gin of the scutum in females of A. coelebs. divergent half of the former species, in contrast to the The nymphs of A. yucumense and A. dubitatum are narrower and deeper posterior half in the later. The morphologically very similar to each other. The only nymph of A. coelebs is readily distinguished from A. difference we could assign to separate them was the yucumense through scutal large and deep puncations, extension of cervical groove, which is slightly longer in present in both lateral and central fields of the former A. dubitatum. For the present study, we re-examined species, while concentrated in the lateral fields of the the same 10 A. dubitatum nymphal specimens recently later. 36 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 1

As recently reviewed by Guglielmone and Nava facts indicate that although A. yucumense and A. dubi- (2014), the list of synonyms of A. dubitatum include tatum are morphologically and genetically closely four names: Amblyomma lutzi Aragaõ (collected in the related, they are distinct regarding their ecological state of Minas Gerais, Brazil), Amblyomma cooperi preferences. Nuttall and Warburton (collected in Paraguay), Amblyomma ypsilophorum Schulze (collected in the state of Rio de Janeiro, Brazil) and Amblyomma cajen- Acknowledgments nense chacoensis Ivancovich (collected in Chaco, north- We are very grateful to M. Haase and V. Lohrmann ern Argentina). Although type specimens of A. lutzi, A. (U¨ bersee-Museum Bremen, Germany) for providing high- cooperi,andA. cajennense chacoensis were not avail- resolution pictures of the type specimens of A. ypsilophorum, able for examination in the present study, we discard to the staff of the “Parque Estadual do Turvo” for providing the possibility that any of them could represent A. invaluable logistic support for our field work, and to Gustavo yucumense because they were described as having typi- Fernochi for his technical support in editing photographs. cal features that separate A. dubitatum from A. This work was financially supported by the Fundaçaõ de yucumense, e.g., female scutum with large and deep Amparo a Pesquisa do Estado de Saõ Paulo (FAPESP), Con- selho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico punctations evenly distributed, female coxal I spurs (CNPq), and Coordenadoria de Apoio a Pesquisa e Desenvol- short, distance between male coxa I spurs wider than vimento (CAPES), and has been authorized by the “Instituto the external spur width (Aragaõ 1908, Nuttall and War- Chico Mendes de Conservaçaõ da Biodiversidade” (author- burton 1908, Ivancovich 1973). In the description of A. ization SISBIO 38502-1) and the Ethical Committee in Ani- ypsilophorum, the drawings provided by Schulze mal Research of the Faculty of Veterinary Medicine of the (1941) showed coxa I spurs compatible with A. yucu- University of Saõ Paulo (protocol 2908/2013). mense, namely, distance between male coxa I spurs narrower than the external spur width. Owing to this reason, we requested the U¨ bersee-Museum Bremen, References Cited where the types of A. ypsilophorum are deposited in Aragaõ,H.B.1908.Algumas novas especies de carrapatos bra- Germany, to provide us pictures of the type specimens. zileiros. Braz. Med. 22: 111–115. By examining high-resolution pictures of A. ypsilopho- Corwin, D. C., C. M. Clifford, and J. E. Keirans. 1979. rum provided to us, our conclusions on coxa I spurs An improved method for cleaning and preparing ticks for were inconclusive, as the space between coxa I spurs examination with the scanning electron microscope. J. Med. was nearly the same as the external spur width. On the Entomol. 16: 352–353. Dantas-Torres,F.,V.C.Onofrio,andD.M.Barros- other hand, other features that separate A. dubitatum Battesti. 2009. The ticks (Acari: Ixodida: Argasidae, from A. yucumense were observed, namely, absence of Ixodidae) of Brazil. Syst. Appl. Acarol. 14: 30–46. pseudoscutum in the male scutum, and adult capitulum Estrada Pen˜ a, A., J. M. Venzal, and A. A. Guglielmone. and ventral idiosoma yellowish colored (Supp Figure 1 2002. Amblyomma dubitatum Neumann: description of [online only]). nymph and redescription of adults, together with the descrip- Adults of A. yucumense were observed inside the tion of the immature stages of A. triste Koch. Acarologia 42: Atlantic forest, questing on the vegetation 50–80 cm 323–333. above the soil in animal trails. These trails were not Guglielmone, A. A., and S. Nava. 2014. Names for Ixodidae used by capybaras (H. hydrochaeris) (data not shown), (Acari: Ixodoidea): valid, synonyms, incertae sedis, nomina dubia, nomina nuda, lapsus, incorrect and suppressed which are the only known host species capable of sus- names - with notes on confusions and misidentifications. taining A. dubitatum populations within its distribution Zootaxa 3767: 001–256. area (Nava et al. 2010). 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