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Description of the larva of Amblyomma calcaratum Neumann, 1899 (Acari: Ixodidae) by light and scanning electron microscopy
Ticks and Tick-borne Diseases 4 (2013) 531–536
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Original article
Description of the larva of Amblyomma calcaratum Neumann, 1899 (Acari: Ixodidae) by light and scanning electron microscopy Fábio S. Barbieri a,∗ , Luciana G. Brito a , Marcelo B. Labruna b , Darci M. Barros-Battesti c , Luis Marcelo A. Camargo d , Kátia M. Famadas e a
Embrapa Rondônia, Rodovia BR 364, Km 5,5, caixa postal 127, 76815-800, Porto Velho, RO, Brazil Faculdade de Medicina Veterinária e Zootecnia/USP, São Paulo, SP, Brazil c Laboratório Especial de Colec¸ões Zoológicas/Instituto Butantan, São Paulo, SP, Brazil d Instituto de Ciências Biomédicas 5/USP, Monte Negro, RO, Brazil e Departamento de Parasitologia Animal/UFRRJ, Seropédica, RJ, Brazil b
a r t i c l e
i n f o
Article history: Received 19 March 2013 Received in revised form 10 June 2013 Accepted 11 July 2013 Available online 26 October 2013 Keywords: Amblyomma calcaratum Larva Description Morphology Chaetotaxy Integumentary structures
a b s t r a c t The larval stage of Amblyomma calcaratum Neumann is described using optical and scanning electron microscopy. Unfed larvae were obtained from a colony of A. calcaratum originating from engorged females collected on Tamandua tetradactyla in the Jaraguá Mountain (23◦ 40 S, 45◦ 44 W), São Paulo County, Brazil. Eleven larvae were prepared and mounted on slides and observed under a light microscope equipped with a drawing tube. Three specimens were prepared for SEM. Several morphological characters are described, including the chaetotaxy of the idiosoma, palpi, and Haller’s organ, as well as morphological features of the idiosoma, gnathosoma, and legs of A. calcaratum larvae. In addition, topographical and numerical patterns of integumentary structures on the larval idiosoma are described using a recently proposed nomenclature. On the idiosoma, setaes, lyrifissures, small glands, and large wax glands were found. These structures were observed isolated or associated over the entire idiosoma, except on the scutum, which lacks large wax glands. The topographical and numerical patterns of integumentary structures of the A. calcaratum larva showed only minor differences when compared with patterns of other Amblyomma larvae; however, a few key features can be used to differentiate A. calcaratum from other members of this genus. © 2013 Elsevier GmbH. All rights reserved.
Introduction Currently, there are 130 valid species belonging to the genus Amblyomma in the world (Guglielmone et al., 2010). In Brazil, there are 29 established species of Amblyomma (Dantas-Torres et al., 2009), from which the larval stage has been described for only 19 species (Barbieri et al., 2012). Indeed, this low number of descriptions is related to the difficulty of obtaining engorged females (and resultant larvae) of some species, since the majority of Brazilian ticks preferentially feed on wild animals and are hard to breed in the laboratory (Onofrio et al., 2006; Szabó et al., 2009). Amblyomma calcaratum Neumann, 1899, has been reported from Mexico to Argentina, where the adult stage is found feeding almost exclusively on anteaters, while nymphs feed preferably on passerine birds (Jones et al., 1972; Guglielmone et al., 2003; Onofrio et al., 2006; Guzmán-Cornejo et al., 2006; Ogrzewalska et al., 2011). Birds seem also to be preferred hosts for the larva,
∗ Corresponding author. Tel.: +55 69 3901 2520; fax: +55 69 3222 0409. E-mail address: [email protected] (F.S. Barbieri). 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.07.004
although there have been only a very limited number of host records (Ogrzewalska et al., 2010). A. calcaratum is of potential medical importance because a spotted fever group bacterium, identified as a Rickettsia parkeri-like agent, was recently reported infecting nymphs collected from birds in Brazil (Ogrzewalska et al., 2013). In the present study, the larva of A. calcaratum is described and illustrated by optical and scanning electron microscopy (SEM). Materials and methods Unfed larvae were obtained from engorged females of A. calcaratum collected on Tamandua tetradactyla at the Jaraguá Mountain (23◦ 40 S, 45◦ 44 W), São Paulo County, Brazil. Eggs laid by these females under laboratory conditions (27 ◦ C, 90 ± 5% RH) were pooled and placed in modified disposable syringes whose open ends were covered with hydrophilic cotton. Eleven larvae were individually prepared for light microscopy, as previously described (Barbieri et al., 2007) while another 5 specimens were processed for SEM according to Keirans et al. (1976). The A. calcaratum engorged females were determined to species using the keys and redescriptions of Guimarães et al. (2001). The
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Fig. 1. Amblyomma calcaratum, larva. Gnathosoma, dorsal (right) and ventral (left) views. Abbreviations: d, dorsal; v, ventral; a, antiaxial; p, paraxial; t, terminal; F, femur; G, genu; Tt, tibiotarsus.
chaetotaxy terminology of Clifford and Anastos (1960), Hess and Vlimant (1983), and Woolley (1988), and the porotaxy terminology of Barbieri et al. (2007) were used in our larval description. The frequency of integumentary structures was determined by independently analyzing each idiosomal side (left and right), according to Klompen et al. (1996). Thus, a total of 22 idiosomal sides were analyzed. All measurements were made with a Wild M11 light microscope and are reported in millimeters. The mean is followed by the standard error, with the range in parentheses. Four larval specimens were deposited in the Acari Collection of the Instituto Butantan (IBSP 5000), São Paulo, Brazil. Description Amblyomma calcaratum Neumann 1899, Larva (Figs. 1–10) Idiosoma: Dorsal surface (Figs. 4 and 7). Length from apices of scapulae to posterior margin of body 0.602 ± 0.015 (0.579–0.628); greatest width 0.585 ± 0.012 (0.559–0.598); outline oval, with 11 festoons. Setae: 2 central dorsal pairs (Cd1, Cd2); 8 marginal dorsal pairs (Md1–Md8), Md1 and Md2 before a large wax gland present in segment VIII (WdVIII1), and Md3 pair located in the inner side behind this gland, Md4–Md8 pairs posterior to large wax gland, each one in one subsequent festoon. Scutum (Figs. 4 and 7): outline subtriangular; length 0.273 ± 0.012 (0.255–0.296); breadth 0.430 ± 0.015 (0.408–0.455) at level of eyes. Scutum with few punctations. Eyes slightly bulging and shallow; cervical grooves slightly convergent, parallel to the proximities of setae Sc3. Setae: 3 scutal pairs (Sc1, Sc2, Sc3) (Fig. 4). Ventral surface (Figs. 4, 8 and 9): Anal aperture on central portion of opisthosoma. Setae: 3 sternal pairs (St1, St2, St3), 2 preanal pairs (Pa1, Pa2), 4 premarginal pairs (Pm1–Pm4), 5 marginal ventral pairs (Mv1–Mv5), and 1 anal pair (A1) (Figs. 4, 8 and 9). Gnathosoma: Dorsal (Figs. 1 and 5). Basis capituli triangular in outline; length from posterior margin of trochanter to posterior margin of capituli 0.073 ± 0.005 (0.065–0.079), width 0.157 ± 0.004 (0.151–0.163). Posterior margin straight, cornua absent. Palpal grooves on segments well-defined (Fig. 5). Palp length from apices of tibiotarsal segment to posterior margin of trochanter 0.122 ± 0.003 (0.116–0.125), width 0.055 ± 0.004 (0.051–0.061); trochanter length 0.019 ± 0.002 (0.016–0.022),
width 0.044 ± 0.003 (0.039–0.047); femur length 0.051 ± 0.003 (0.046–0.056), width 0.053 ± 0.002 (0.050–0.056); genu length 0.052 ± 0.002 (0.049–0.058), width 0.047 ± 0.003 (0.042–0.053). Femur with sensillum near seta Fa1 (Fig. 5). Ventral: Basis capituli as illustrated (Figs. 1 and 6). Hypostome compact, spatulate, length from apices to posthypostomal seta 0.092 ± 0.002 (0.087–0.096), dental formula 2/2 in file teeth, apical corona usually with 9 small denticles; 1 pair of posthypostomal setae (Ph1) (Fig. 6). Palpal setae (Fig. 1): 10 setae on tibiotarsus, 6 terminal (Ttt1–Ttt6), 2 paraxial (Ttp1, Ttt2), and 2 antiaxial (Tta1, Tta2) (Fig. 6); 6 genual setae, 1 paraxial (Gp1), 1 antiaxial (Ga1), 3 dorsal (Gd1, Gd2, Gd3), and 1 ventral (Gv1); 6 femoral setae, 1 paraxial (Fd1), 2 antiaxial (Fa1, Fa2), 1 dorsal (Fd1), and 2 ventral (Fv1, Fv2); trochanter 0.
Figs. 2 and 3. Amblyomma calcaratum, larva. Tarsus I: (Fig. 2) dorsal view; (Fig. 3) ventral view. Abbreviations: d, dorsal; v, ventral; a, antiaxial; p, paraxial; la, lateral anterior; lp, lateral posterior.
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Fig. 4. Amblyomma calcaratum, larva. Segmentation model of the idiosoma. Segments are indicated by Roman numerals (III–VI and VIII–XIV) and delimitated by dashed lines (- - -); series are indicated by Arabic numbers and delimitated by dotted lines (· · ·). Integumentary structures are illustrated.
Legs: Coxa I with 2 triangular spurs, the external slightly longer than the internal; coxae II and III each with a single, large spur. Setae: 3 on coxa I, 1 anterior (CIa), 1 posterior (CIp), and 1 paraxial (CIpa); coxae II and III each with 2 setae, 1 anterior (CIIa, CIIIa) and 1 posterior (CIIp, CIIIp) (Fig. 8). Trochanter lacking spur. Tarsus I length 0.220 ± 0.006 (0.210–0.227), width 0.085 ± 0.004 (0.076–0.090). Setae (Figs. 2, 3 and 10): dorsal, 2 in dorsal I group (dI1, dI2); 6 dorsal II (dII1–dII6) (Figs. 2 and 10); 2 dorsal III (dIII1, dIII2); 2 dorsal IV (dIV1, dIV2); 0 dorsal V; 2 dorsal VI (dVI1, dVI2); ventral, 2 ventral I (vI1, vI2), 2 in II group (vII1, vII2), and 2 in III (vIII1, vIII2); lateral anterior, 1 in lateral anterior I group (laI1) and 3 in laII group (laII1, laII2, laII3); lateral posterior, 1 in lateral posterior I group (lpI1) and 3 in lpII group (lpII1, lpII2, lpII3). Ambulacrum as illustrated (Figs. 2 and 3). Porotaxy: Dorsal (Fig. 4): Large wax glands – one pair located on the lateral margin of the idiosoma, in segment VIII – WdVIII1 (22/22 = 22 idiosomal sites containing the structure/22 observed idiosomal sites). Lyrifissures – 11 pairs distributed in the following segments: 1 pair in segment III – LdIII5 (22/22); 1 in IV–LdIV2 (19/22); 2 in V – LdV1 (17/22), LdV6 (19/22); 1 in VI – LdVI1 (20/22); 2 in VIII – LdVIII4 (18/22), LdVIII6 (21/22); 2 in IX – LdIX4 (20/22), LdIX6 (21/22); 2 in XII – LdXII4 (20/22), LdXII7 (22/22). Small glands – 40 pairs, 4 on the scutum: 3 pairs in segment III – SdIII1 (21/22), SdIII2 (19/22), and SdIII5 (22/22); 1 in IV – SdIV6 (22/22); and an additional asymmetric small gland on the posterior central margin of the scutum – SdV7 (11/11). On the alloscutum, 36 pairs distributed in the following segments: 1 in IV – SdIV2 (19/22); 4 in V – SdV2 (21/22), SdV3 (21/22), SdV4 (22/22), SdV6 (20/22); 4 in VI – SdVI2 (22/22), SdVI4 (21/22), SdVI5 (22/22), SdVI6 (22/22); 4 in VIII – SdVIII3 (21/22), SdVIII4 (21/22), SdVIII6 (21/22), SdVIII7 (22/22); 3 in IX – SdIX1 (15/22), SdIX2 (22/22), SdIX4 (20/22), SdIX7 (22/22); 5 in X – SdX1 (21/22), SdX3 (15/22), SdX4 (17/22), SdX5
(21/22), SdX6 (22/22), and dorsal fovea (series 7 – 22/22); 3 in XI – SdXI1 (22/22), SdXI2 (22/22), SdXI7 (22/22); 4 in XII – SdXII1 (21/22), SdXII2 (21/22), SdXII5 (21/22), SdXII7 (18/22); 5 in XIII – SdXIII1 (12/22), SdXIII2 (22/22), SdXIII3 (21/22), SdXIII5 (22/22), SdXIII6 (22/22); 2 in XIV – SdXIV1 (22/22), SdXIV2 (22/22). Ventral (Fig. 4): Large wax glands – 4 pairs: 1 in the outer margin of coxa I in segment III – WvIII1 (22/22); 1 posterior to coxa II in segment IV – WvIV4 (22/22); 1 posterior to coxa III in segment V – WvV5 (22/22); 1 on the 5th festoon in segment XIII – WvXIII1 (22/22). Lyrifissures – 13 pairs distributed in the following segments: 1 pair in segment IV – LvIV6 (22/22); 1 in V – LvV6 (22/22); 1 in VIII – LvVIII5 (19/22); 4 in IX – LvIX1 (22/22), LvIX3 (22/22), LvIX5 (19/22), LvIX6 (22/22); 1 in X – LvX2 (21/22); 1 in XI – LvXI2 (22/22); 1 in XII – LvXII3 (22/22); 2 in XIII – LvXIII2 (22/22), LvXIII6 (22/22); 1 in XIV – LvXIV2 (22/22). Small glands – 21 pairs distributed in the following segments: 1 in III – SvIII6 (22/22); 1 in IV–SvIV6, (22/22); 2 in V – SvV1 (20/22), SvV5 (22/22); 1 in VI – SvVI2 (21/22); 1 in VIII – SvVIII1 (22/22); 3 in IX – SvIX2 (22/22), SvIX4 (22/22), SvIX6 (22/22); 3 in X – SvX1 (22/22), SvX3 (21/22), SvX4 (22/22); 2 in XI – SvXI1 (22/22), SvXI3 (19/22); 2 in XII – SvXII2 (22/22), SvXII4 (22/22); 2 in XIII – SvXIII3 (19/22), SvXIII6 (22/22); 3 in XIV – SvXIV1 (21/22), SvXIV3 (19/22), SvXIV5 (22/22). Asymmetric small glands found at low frequency. SdVI3 (3/22), SdX7 (1/22), and SvIX6 (2/22). Discussion The morphological characters and measurements of A. calcaratum were compared with descriptions of other species of the Amblyomma genus from the Neotropical region. The basis capituli of A. calcaratum is triangular, even as in Amblyomma cajennense (Fabricius, 1787) (Famadas et al., 1997), Amblyomma longirostre
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Figs. 5–10. Amblyomma calcaratum, larva: (Fig. 5) Gnathosoma, dorsal view (bar: 30 m); (Fig. 6) Gnathosoma, ventral view (bar: 30 m); (Fig. 7) Larva, dorsal view (bar: 90 m); (Fig. 8) Detail of coxae (bar: 50 m); (Fig. 9) Detail of festoons (bar: 50 m); (Fig. 10) Tarsus I, dorsal view (bar: 15 m).
(Koch, 1844) (Barros-Battesti et al., 2005), Amblyomma ovale Koch, 1844 (Barbieri et al., 2008a), and Amblyomma pacae Aragão, 1911 (Barbieri et al., 2008b). However, in Amblyomma parvum Aragão, 1908, and Amblyomma pseudoparvum Guglielmone, Mangold and Keirans, 1990 (Guglielmone et al., 1990), the basis capituli is slightly subtriangular, and in Amblyomma brasiliense Aragão, 1908 (Sanches et al., 2009) it is rectangular. The idiosoma of A. calcaratum is oval in outline, as well as in A. parvum and A. pseudoparvum (Guglielmone et al., 1990), A. cajennense (Famadas et al., 1997), A. longirostre (Barros-Battesti et al., 2005), A. ovale (Barbieri et al., 2008a), A. pacae (Barbieri et al., 2008b), but is rounded in A. brasiliense. The measurements of the idiosoma of A. calcaratum is similar to those observed in A. brasiliense and A. ovale (Sanches et al., 2009; Barbieri et al., 2008a). On the other hand, larvae of A. parvum, A. pseudoparvum, and A. cajennense (Guglielmone et al., 1990; Famadas et al., 1997) are smaller than A. calcaratum, and A. longirostre and A. pacae (BarrosBattesti et al., 2005; Barbieri et al., 2008b) are larger. The chaetotaxy of the idiosoma, gnathosoma, and tarsus I of the larvae of A. calcaratum was similar to that found in other species of this genus, as reported in other descriptions of larval Amblyomma.
Variation in the numbers of setae was observed for the tibiotarsus of the palpi and for setae in group dII on tarsus I. The palpal tibiotarsus of A. calcaratum carries 10 setae, 6 terminal (Ttt1), 2 paraxial (Ttp), and 2 antiaxial (Tta). The same number of setae was reported for Amblyomma varium Koch, 1844, Amblyomma dubitatum Neumann, 1899, A. parvum, A. pseudoparvum, and Amblyomma oblongoguttatum Koch, 1844 (Amorim and SerraFreire, 1996, 1999; Guglielmone et al., 1990; Barbieri et al., 2012). Larvae of Amblyomma rotundatum Koch, 1844, carry 7 setae on that segment (Amorim and Serra-Freire, 1995), larvae of A. ovale, A. pacae, and Amblyomma romitii Tonelli-Rondelli, 1939, carry 11 (Barbieri et al., 2008a, 2008b; Barros-Battesti et al., 2013), and larvae of A. cajennense, A. longirostre, and A. brasiliense carry 12 (Famadas et al., 1997; Barros-Battesti et al., 2005; Sanches et al., 2009). The dorsal setal arrangement of tarsus I for 9 Amblyomma species considered by Clifford and Anastos (1960) was 2:2:2:2:2. The larvae of A. calcaratum show the same arrangement, except for group dII, which includes 6 setae (Fig. 10). This number was also found in larvae of A. parvum, A. pseudoparvum (Guglielmone et al., 1990), A. dubitatum (Amorim and Serra-Freire 1999), and A. romitii
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(Barros-Battesti et al., 2013), but differs from that of A. rotundatum (Amorim and Serra-Freire, 1995), A. varium (Amorim and SerraFreire, 1996), A. ovale (Barbieri et al., 2008a), A. pacae (Barbieri et al., 2008b), and A. oblongoguttatum (Barbieri et al., 2012), all of which have 5 setae in group dII, and from A. cajennense (Famadas et al., 1997), A. longirostre (Barros-Battesti et al., 2005), and A. brasiliense (Sanches et al., 2009), which have 7 each. The topographical and numerical patterns of integumentary structures on the idiosoma of the larvae of A. calcaratum showed the presence of setae, lyrifissures, small glands, and large wax glands, each of which easily characterized morphologically (Schulze, 1942; Dinnik and Zumpt, 1949; Barbieri et al., 2007). As noted by Klompen et al. (1996), Barbieri et al. (2007, 2008a,b), these structures are found isolated or associated over the entire idiosoma, except on the scutum where large wax glands are absent. The arrangement of the large wax gland on the idiosoma of A. calcaratum larvae was the same as reported by Clifford and Anastos (1960) for Amblyomma species: one pair located dorsally on the lateral margin of the body (segment VIII), and 4 pairs on the ventral surface – one located posterior to each coxa (segments III, IV, and V) and one pair on the 5th festoon (segment XIII). A similar arrangement has been reported for Amblyomma glauerti Keirans, King and Sharrad, 1994, Amblyomma variegatum (Fabricius, 1794), Amblyomma americanum (Linnaeus, 1758) (Klompen et al., 1996); A. cajennense (Famadas et al., 1997); A. parvum, A. rotundatum (Barbieri et al., 2007), A. pacae (Barbieri et al., 2008b), A. brasiliense (Sanches et al., 2009), and A. oblongoguttatum (Barbieri et al., 2012). An additional ventral pair of large wax glands on the 4th festoon (segment XII) has been reported for Amblyomma tuberculatum Marx, 1894, Amblyomma geoemydae (Cantor, 1847), Amblyomma babirussae Schulze, 1933 (Klompen et al., 1996), Amblyomma aureolatum (Pallas, 1772) (Arzua, 2002), A. longirostre (Barros-Battesti et al., 2005; Barbieri et al., 2007), A. ovale (Barbieri et al., 2008a), and A. romitii (Barros-Battesti et al., 2013). Further additional pairs have been reported on segment V for A. geoemydae, segments V and X for A. babirussae, dorsal on segment V (WdV1) for A. longirostre, and on segments IV (WdIV3) and V (WdV1) for A. romitii (Barros-Battesti et al., 2013). The number of lyrifissures present on the idiosoma of A. calcaratum was 24, 11, and 13 on the dorsal and ventral surfaces, respectively. This number and the pattern is similar to that found for other Amblyomma species (Klompen et al., 1996; Barbieri et al., 2007, 2008a,b), but differs from that for larval A. parvum, who carry only 12 lyrifissures on the ventral surface. Klompen et al. (1996) noted that the number and pattern of lyrifissures present in all Parasitiformes is similar, listing total numbers of lyrifissures of 18, 24, 20, 20 (all larvae) to 28 (adult), and 23–24 (adult) for Prostriata, Metastriata, Argas, Carios (Argasidae), Zerconidae, and Amblyseiinae, respectively (the latter 2 are Mesostigmata). Among the integumentary structures, the small glands are the most numerous on the idiosoma, but their frequency and pattern also showed a high degree of variability. This pattern has been observed previously for larvae of other Amblyomma species, such as A. cajennense, A. longirostre, A. parvum, A. rotundatum (Barbieri et al., 2007), A. ovale (Barbieri et al., 2008a), and A. pacae (Barbieri et al., 2008b), as well as for species within the genera Rhipicephalus Koch, 1844 (Nawar and Madbouly, 1985), Amblyomma, Dermacentor Koch, 1844, and Ixodes Latreille, 1795 (Klompen et al., 1996). This variability in the frequency of small glands can be related to the higher number this structure is present on the idiosoma, since the lowest variation has been observed for large wax glands, which had the lowest number on the idiosoma (Barbieri et al., 2007). In general, the integumentary structures on the idiosoma of the larvae of A. calcaratum show patterns similar to those observed in larvae of A. cajennense, A. longirostre, A. parvum, and A. rotundatum (Barbieri et al., 2007), A. ovale (Barbieri et al., 2008a), A. pacae
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(Barbieri et al., 2008b), A. oblongoguttatum (Barbieri et al., 2012), and A. romitii (Barros-Battesti et al., 2013). However, their number, position, and association patterns may help identifying this stage. Thus, larvae of A. calcaratum differ from those of A. longirostre, A. ovale, and A. romitii because these larvae have 2 and one additional pairs of large wax glands, respectively (Barbieri et al., 2007, 2008a; Barros-Battesti et al., 2013); from those of A. parvum because this species has one less pair of lyrifissures (Barbieri et al., 2007). The association between 2 or more structures can also be auxiliary in the identification, for example, the association of lyrifissure and small gland in ventral surface/segment IX/series 6 and lack in A. cajennense and A. oblongoguttatum (Barbieri et al., 2007, 2012), and association of the same structures in ventral surface/segment X, XI, and XII/series 2 in A. pacae and lack in A. calcaratum (Barbieri et al., 2008b). This is the first morphological description of the larva of A. calcaratum. Currently, larval morphological descriptions are available for only 19 of the 29 Amblyomma species known from Brazil (Barbieri et al., 2012). Although many of these descriptions provide general measurements of the body and details on chaetotaxy, complete analyses of integumental structures (lyrifissures, small glands, and large wax glands) have been reported for only 8 species (A. cajennense, A. parvum, A. rotundatum, A. longirostre, A. ovale, A. pacae, A. oblongoguttatum, and A. romitii) (Barbieri et al., 2007, 2008a,b, 2012; Barros-Battesti et al., 2013). Chaetotaxy is generally highly conserved among Amblyomma spp. (Barbieri et al., 2007), but analyses of especially gland distribution patterns in association with other morphological features and bioecological information should eventually yield a taxonomic key for identification of the Amblyomma larvae of the Neotropical Zoogeographic Region.
Acknowledgments The authors are grateful for financial support from CNPqBolsa de Desenvolvimento Científico Regional (no. 350265/2005-4) and Scientific Career Scholarships to MBL and DMBB, CNPq (nos. 479877-2004-1 and 304475/2010-6), FAPESP (no. 07/57749-2) and FAPERJ (no. E-26/170179/2004).
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Original article
Description of the larva of Amblyomma calcaratum Neumann, 1899 (Acari: Ixodidae) by light and scanning electron microscopy Fábio S. Barbieri a,∗ , Luciana G. Brito a , Marcelo B. Labruna b , Darci M. Barros-Battesti c , Luis Marcelo A. Camargo d , Kátia M. Famadas e a
Embrapa Rondônia, Rodovia BR 364, Km 5,5, caixa postal 127, 76815-800, Porto Velho, RO, Brazil Faculdade de Medicina Veterinária e Zootecnia/USP, São Paulo, SP, Brazil c Laboratório Especial de Colec¸ões Zoológicas/Instituto Butantan, São Paulo, SP, Brazil d Instituto de Ciências Biomédicas 5/USP, Monte Negro, RO, Brazil e Departamento de Parasitologia Animal/UFRRJ, Seropédica, RJ, Brazil b
a r t i c l e
i n f o
Article history: Received 19 March 2013 Received in revised form 10 June 2013 Accepted 11 July 2013 Available online 26 October 2013 Keywords: Amblyomma calcaratum Larva Description Morphology Chaetotaxy Integumentary structures
a b s t r a c t The larval stage of Amblyomma calcaratum Neumann is described using optical and scanning electron microscopy. Unfed larvae were obtained from a colony of A. calcaratum originating from engorged females collected on Tamandua tetradactyla in the Jaraguá Mountain (23◦ 40 S, 45◦ 44 W), São Paulo County, Brazil. Eleven larvae were prepared and mounted on slides and observed under a light microscope equipped with a drawing tube. Three specimens were prepared for SEM. Several morphological characters are described, including the chaetotaxy of the idiosoma, palpi, and Haller’s organ, as well as morphological features of the idiosoma, gnathosoma, and legs of A. calcaratum larvae. In addition, topographical and numerical patterns of integumentary structures on the larval idiosoma are described using a recently proposed nomenclature. On the idiosoma, setaes, lyrifissures, small glands, and large wax glands were found. These structures were observed isolated or associated over the entire idiosoma, except on the scutum, which lacks large wax glands. The topographical and numerical patterns of integumentary structures of the A. calcaratum larva showed only minor differences when compared with patterns of other Amblyomma larvae; however, a few key features can be used to differentiate A. calcaratum from other members of this genus. © 2013 Elsevier GmbH. All rights reserved.
Introduction Currently, there are 130 valid species belonging to the genus Amblyomma in the world (Guglielmone et al., 2010). In Brazil, there are 29 established species of Amblyomma (Dantas-Torres et al., 2009), from which the larval stage has been described for only 19 species (Barbieri et al., 2012). Indeed, this low number of descriptions is related to the difficulty of obtaining engorged females (and resultant larvae) of some species, since the majority of Brazilian ticks preferentially feed on wild animals and are hard to breed in the laboratory (Onofrio et al., 2006; Szabó et al., 2009). Amblyomma calcaratum Neumann, 1899, has been reported from Mexico to Argentina, where the adult stage is found feeding almost exclusively on anteaters, while nymphs feed preferably on passerine birds (Jones et al., 1972; Guglielmone et al., 2003; Onofrio et al., 2006; Guzmán-Cornejo et al., 2006; Ogrzewalska et al., 2011). Birds seem also to be preferred hosts for the larva,
∗ Corresponding author. Tel.: +55 69 3901 2520; fax: +55 69 3222 0409. E-mail address: [email protected] (F.S. Barbieri). 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.07.004
although there have been only a very limited number of host records (Ogrzewalska et al., 2010). A. calcaratum is of potential medical importance because a spotted fever group bacterium, identified as a Rickettsia parkeri-like agent, was recently reported infecting nymphs collected from birds in Brazil (Ogrzewalska et al., 2013). In the present study, the larva of A. calcaratum is described and illustrated by optical and scanning electron microscopy (SEM). Materials and methods Unfed larvae were obtained from engorged females of A. calcaratum collected on Tamandua tetradactyla at the Jaraguá Mountain (23◦ 40 S, 45◦ 44 W), São Paulo County, Brazil. Eggs laid by these females under laboratory conditions (27 ◦ C, 90 ± 5% RH) were pooled and placed in modified disposable syringes whose open ends were covered with hydrophilic cotton. Eleven larvae were individually prepared for light microscopy, as previously described (Barbieri et al., 2007) while another 5 specimens were processed for SEM according to Keirans et al. (1976). The A. calcaratum engorged females were determined to species using the keys and redescriptions of Guimarães et al. (2001). The
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Fig. 1. Amblyomma calcaratum, larva. Gnathosoma, dorsal (right) and ventral (left) views. Abbreviations: d, dorsal; v, ventral; a, antiaxial; p, paraxial; t, terminal; F, femur; G, genu; Tt, tibiotarsus.
chaetotaxy terminology of Clifford and Anastos (1960), Hess and Vlimant (1983), and Woolley (1988), and the porotaxy terminology of Barbieri et al. (2007) were used in our larval description. The frequency of integumentary structures was determined by independently analyzing each idiosomal side (left and right), according to Klompen et al. (1996). Thus, a total of 22 idiosomal sides were analyzed. All measurements were made with a Wild M11 light microscope and are reported in millimeters. The mean is followed by the standard error, with the range in parentheses. Four larval specimens were deposited in the Acari Collection of the Instituto Butantan (IBSP 5000), São Paulo, Brazil. Description Amblyomma calcaratum Neumann 1899, Larva (Figs. 1–10) Idiosoma: Dorsal surface (Figs. 4 and 7). Length from apices of scapulae to posterior margin of body 0.602 ± 0.015 (0.579–0.628); greatest width 0.585 ± 0.012 (0.559–0.598); outline oval, with 11 festoons. Setae: 2 central dorsal pairs (Cd1, Cd2); 8 marginal dorsal pairs (Md1–Md8), Md1 and Md2 before a large wax gland present in segment VIII (WdVIII1), and Md3 pair located in the inner side behind this gland, Md4–Md8 pairs posterior to large wax gland, each one in one subsequent festoon. Scutum (Figs. 4 and 7): outline subtriangular; length 0.273 ± 0.012 (0.255–0.296); breadth 0.430 ± 0.015 (0.408–0.455) at level of eyes. Scutum with few punctations. Eyes slightly bulging and shallow; cervical grooves slightly convergent, parallel to the proximities of setae Sc3. Setae: 3 scutal pairs (Sc1, Sc2, Sc3) (Fig. 4). Ventral surface (Figs. 4, 8 and 9): Anal aperture on central portion of opisthosoma. Setae: 3 sternal pairs (St1, St2, St3), 2 preanal pairs (Pa1, Pa2), 4 premarginal pairs (Pm1–Pm4), 5 marginal ventral pairs (Mv1–Mv5), and 1 anal pair (A1) (Figs. 4, 8 and 9). Gnathosoma: Dorsal (Figs. 1 and 5). Basis capituli triangular in outline; length from posterior margin of trochanter to posterior margin of capituli 0.073 ± 0.005 (0.065–0.079), width 0.157 ± 0.004 (0.151–0.163). Posterior margin straight, cornua absent. Palpal grooves on segments well-defined (Fig. 5). Palp length from apices of tibiotarsal segment to posterior margin of trochanter 0.122 ± 0.003 (0.116–0.125), width 0.055 ± 0.004 (0.051–0.061); trochanter length 0.019 ± 0.002 (0.016–0.022),
width 0.044 ± 0.003 (0.039–0.047); femur length 0.051 ± 0.003 (0.046–0.056), width 0.053 ± 0.002 (0.050–0.056); genu length 0.052 ± 0.002 (0.049–0.058), width 0.047 ± 0.003 (0.042–0.053). Femur with sensillum near seta Fa1 (Fig. 5). Ventral: Basis capituli as illustrated (Figs. 1 and 6). Hypostome compact, spatulate, length from apices to posthypostomal seta 0.092 ± 0.002 (0.087–0.096), dental formula 2/2 in file teeth, apical corona usually with 9 small denticles; 1 pair of posthypostomal setae (Ph1) (Fig. 6). Palpal setae (Fig. 1): 10 setae on tibiotarsus, 6 terminal (Ttt1–Ttt6), 2 paraxial (Ttp1, Ttt2), and 2 antiaxial (Tta1, Tta2) (Fig. 6); 6 genual setae, 1 paraxial (Gp1), 1 antiaxial (Ga1), 3 dorsal (Gd1, Gd2, Gd3), and 1 ventral (Gv1); 6 femoral setae, 1 paraxial (Fd1), 2 antiaxial (Fa1, Fa2), 1 dorsal (Fd1), and 2 ventral (Fv1, Fv2); trochanter 0.
Figs. 2 and 3. Amblyomma calcaratum, larva. Tarsus I: (Fig. 2) dorsal view; (Fig. 3) ventral view. Abbreviations: d, dorsal; v, ventral; a, antiaxial; p, paraxial; la, lateral anterior; lp, lateral posterior.
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Fig. 4. Amblyomma calcaratum, larva. Segmentation model of the idiosoma. Segments are indicated by Roman numerals (III–VI and VIII–XIV) and delimitated by dashed lines (- - -); series are indicated by Arabic numbers and delimitated by dotted lines (· · ·). Integumentary structures are illustrated.
Legs: Coxa I with 2 triangular spurs, the external slightly longer than the internal; coxae II and III each with a single, large spur. Setae: 3 on coxa I, 1 anterior (CIa), 1 posterior (CIp), and 1 paraxial (CIpa); coxae II and III each with 2 setae, 1 anterior (CIIa, CIIIa) and 1 posterior (CIIp, CIIIp) (Fig. 8). Trochanter lacking spur. Tarsus I length 0.220 ± 0.006 (0.210–0.227), width 0.085 ± 0.004 (0.076–0.090). Setae (Figs. 2, 3 and 10): dorsal, 2 in dorsal I group (dI1, dI2); 6 dorsal II (dII1–dII6) (Figs. 2 and 10); 2 dorsal III (dIII1, dIII2); 2 dorsal IV (dIV1, dIV2); 0 dorsal V; 2 dorsal VI (dVI1, dVI2); ventral, 2 ventral I (vI1, vI2), 2 in II group (vII1, vII2), and 2 in III (vIII1, vIII2); lateral anterior, 1 in lateral anterior I group (laI1) and 3 in laII group (laII1, laII2, laII3); lateral posterior, 1 in lateral posterior I group (lpI1) and 3 in lpII group (lpII1, lpII2, lpII3). Ambulacrum as illustrated (Figs. 2 and 3). Porotaxy: Dorsal (Fig. 4): Large wax glands – one pair located on the lateral margin of the idiosoma, in segment VIII – WdVIII1 (22/22 = 22 idiosomal sites containing the structure/22 observed idiosomal sites). Lyrifissures – 11 pairs distributed in the following segments: 1 pair in segment III – LdIII5 (22/22); 1 in IV–LdIV2 (19/22); 2 in V – LdV1 (17/22), LdV6 (19/22); 1 in VI – LdVI1 (20/22); 2 in VIII – LdVIII4 (18/22), LdVIII6 (21/22); 2 in IX – LdIX4 (20/22), LdIX6 (21/22); 2 in XII – LdXII4 (20/22), LdXII7 (22/22). Small glands – 40 pairs, 4 on the scutum: 3 pairs in segment III – SdIII1 (21/22), SdIII2 (19/22), and SdIII5 (22/22); 1 in IV – SdIV6 (22/22); and an additional asymmetric small gland on the posterior central margin of the scutum – SdV7 (11/11). On the alloscutum, 36 pairs distributed in the following segments: 1 in IV – SdIV2 (19/22); 4 in V – SdV2 (21/22), SdV3 (21/22), SdV4 (22/22), SdV6 (20/22); 4 in VI – SdVI2 (22/22), SdVI4 (21/22), SdVI5 (22/22), SdVI6 (22/22); 4 in VIII – SdVIII3 (21/22), SdVIII4 (21/22), SdVIII6 (21/22), SdVIII7 (22/22); 3 in IX – SdIX1 (15/22), SdIX2 (22/22), SdIX4 (20/22), SdIX7 (22/22); 5 in X – SdX1 (21/22), SdX3 (15/22), SdX4 (17/22), SdX5
(21/22), SdX6 (22/22), and dorsal fovea (series 7 – 22/22); 3 in XI – SdXI1 (22/22), SdXI2 (22/22), SdXI7 (22/22); 4 in XII – SdXII1 (21/22), SdXII2 (21/22), SdXII5 (21/22), SdXII7 (18/22); 5 in XIII – SdXIII1 (12/22), SdXIII2 (22/22), SdXIII3 (21/22), SdXIII5 (22/22), SdXIII6 (22/22); 2 in XIV – SdXIV1 (22/22), SdXIV2 (22/22). Ventral (Fig. 4): Large wax glands – 4 pairs: 1 in the outer margin of coxa I in segment III – WvIII1 (22/22); 1 posterior to coxa II in segment IV – WvIV4 (22/22); 1 posterior to coxa III in segment V – WvV5 (22/22); 1 on the 5th festoon in segment XIII – WvXIII1 (22/22). Lyrifissures – 13 pairs distributed in the following segments: 1 pair in segment IV – LvIV6 (22/22); 1 in V – LvV6 (22/22); 1 in VIII – LvVIII5 (19/22); 4 in IX – LvIX1 (22/22), LvIX3 (22/22), LvIX5 (19/22), LvIX6 (22/22); 1 in X – LvX2 (21/22); 1 in XI – LvXI2 (22/22); 1 in XII – LvXII3 (22/22); 2 in XIII – LvXIII2 (22/22), LvXIII6 (22/22); 1 in XIV – LvXIV2 (22/22). Small glands – 21 pairs distributed in the following segments: 1 in III – SvIII6 (22/22); 1 in IV–SvIV6, (22/22); 2 in V – SvV1 (20/22), SvV5 (22/22); 1 in VI – SvVI2 (21/22); 1 in VIII – SvVIII1 (22/22); 3 in IX – SvIX2 (22/22), SvIX4 (22/22), SvIX6 (22/22); 3 in X – SvX1 (22/22), SvX3 (21/22), SvX4 (22/22); 2 in XI – SvXI1 (22/22), SvXI3 (19/22); 2 in XII – SvXII2 (22/22), SvXII4 (22/22); 2 in XIII – SvXIII3 (19/22), SvXIII6 (22/22); 3 in XIV – SvXIV1 (21/22), SvXIV3 (19/22), SvXIV5 (22/22). Asymmetric small glands found at low frequency. SdVI3 (3/22), SdX7 (1/22), and SvIX6 (2/22). Discussion The morphological characters and measurements of A. calcaratum were compared with descriptions of other species of the Amblyomma genus from the Neotropical region. The basis capituli of A. calcaratum is triangular, even as in Amblyomma cajennense (Fabricius, 1787) (Famadas et al., 1997), Amblyomma longirostre
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Figs. 5–10. Amblyomma calcaratum, larva: (Fig. 5) Gnathosoma, dorsal view (bar: 30 m); (Fig. 6) Gnathosoma, ventral view (bar: 30 m); (Fig. 7) Larva, dorsal view (bar: 90 m); (Fig. 8) Detail of coxae (bar: 50 m); (Fig. 9) Detail of festoons (bar: 50 m); (Fig. 10) Tarsus I, dorsal view (bar: 15 m).
(Koch, 1844) (Barros-Battesti et al., 2005), Amblyomma ovale Koch, 1844 (Barbieri et al., 2008a), and Amblyomma pacae Aragão, 1911 (Barbieri et al., 2008b). However, in Amblyomma parvum Aragão, 1908, and Amblyomma pseudoparvum Guglielmone, Mangold and Keirans, 1990 (Guglielmone et al., 1990), the basis capituli is slightly subtriangular, and in Amblyomma brasiliense Aragão, 1908 (Sanches et al., 2009) it is rectangular. The idiosoma of A. calcaratum is oval in outline, as well as in A. parvum and A. pseudoparvum (Guglielmone et al., 1990), A. cajennense (Famadas et al., 1997), A. longirostre (Barros-Battesti et al., 2005), A. ovale (Barbieri et al., 2008a), A. pacae (Barbieri et al., 2008b), but is rounded in A. brasiliense. The measurements of the idiosoma of A. calcaratum is similar to those observed in A. brasiliense and A. ovale (Sanches et al., 2009; Barbieri et al., 2008a). On the other hand, larvae of A. parvum, A. pseudoparvum, and A. cajennense (Guglielmone et al., 1990; Famadas et al., 1997) are smaller than A. calcaratum, and A. longirostre and A. pacae (BarrosBattesti et al., 2005; Barbieri et al., 2008b) are larger. The chaetotaxy of the idiosoma, gnathosoma, and tarsus I of the larvae of A. calcaratum was similar to that found in other species of this genus, as reported in other descriptions of larval Amblyomma.
Variation in the numbers of setae was observed for the tibiotarsus of the palpi and for setae in group dII on tarsus I. The palpal tibiotarsus of A. calcaratum carries 10 setae, 6 terminal (Ttt1), 2 paraxial (Ttp), and 2 antiaxial (Tta). The same number of setae was reported for Amblyomma varium Koch, 1844, Amblyomma dubitatum Neumann, 1899, A. parvum, A. pseudoparvum, and Amblyomma oblongoguttatum Koch, 1844 (Amorim and SerraFreire, 1996, 1999; Guglielmone et al., 1990; Barbieri et al., 2012). Larvae of Amblyomma rotundatum Koch, 1844, carry 7 setae on that segment (Amorim and Serra-Freire, 1995), larvae of A. ovale, A. pacae, and Amblyomma romitii Tonelli-Rondelli, 1939, carry 11 (Barbieri et al., 2008a, 2008b; Barros-Battesti et al., 2013), and larvae of A. cajennense, A. longirostre, and A. brasiliense carry 12 (Famadas et al., 1997; Barros-Battesti et al., 2005; Sanches et al., 2009). The dorsal setal arrangement of tarsus I for 9 Amblyomma species considered by Clifford and Anastos (1960) was 2:2:2:2:2. The larvae of A. calcaratum show the same arrangement, except for group dII, which includes 6 setae (Fig. 10). This number was also found in larvae of A. parvum, A. pseudoparvum (Guglielmone et al., 1990), A. dubitatum (Amorim and Serra-Freire 1999), and A. romitii
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(Barros-Battesti et al., 2013), but differs from that of A. rotundatum (Amorim and Serra-Freire, 1995), A. varium (Amorim and SerraFreire, 1996), A. ovale (Barbieri et al., 2008a), A. pacae (Barbieri et al., 2008b), and A. oblongoguttatum (Barbieri et al., 2012), all of which have 5 setae in group dII, and from A. cajennense (Famadas et al., 1997), A. longirostre (Barros-Battesti et al., 2005), and A. brasiliense (Sanches et al., 2009), which have 7 each. The topographical and numerical patterns of integumentary structures on the idiosoma of the larvae of A. calcaratum showed the presence of setae, lyrifissures, small glands, and large wax glands, each of which easily characterized morphologically (Schulze, 1942; Dinnik and Zumpt, 1949; Barbieri et al., 2007). As noted by Klompen et al. (1996), Barbieri et al. (2007, 2008a,b), these structures are found isolated or associated over the entire idiosoma, except on the scutum where large wax glands are absent. The arrangement of the large wax gland on the idiosoma of A. calcaratum larvae was the same as reported by Clifford and Anastos (1960) for Amblyomma species: one pair located dorsally on the lateral margin of the body (segment VIII), and 4 pairs on the ventral surface – one located posterior to each coxa (segments III, IV, and V) and one pair on the 5th festoon (segment XIII). A similar arrangement has been reported for Amblyomma glauerti Keirans, King and Sharrad, 1994, Amblyomma variegatum (Fabricius, 1794), Amblyomma americanum (Linnaeus, 1758) (Klompen et al., 1996); A. cajennense (Famadas et al., 1997); A. parvum, A. rotundatum (Barbieri et al., 2007), A. pacae (Barbieri et al., 2008b), A. brasiliense (Sanches et al., 2009), and A. oblongoguttatum (Barbieri et al., 2012). An additional ventral pair of large wax glands on the 4th festoon (segment XII) has been reported for Amblyomma tuberculatum Marx, 1894, Amblyomma geoemydae (Cantor, 1847), Amblyomma babirussae Schulze, 1933 (Klompen et al., 1996), Amblyomma aureolatum (Pallas, 1772) (Arzua, 2002), A. longirostre (Barros-Battesti et al., 2005; Barbieri et al., 2007), A. ovale (Barbieri et al., 2008a), and A. romitii (Barros-Battesti et al., 2013). Further additional pairs have been reported on segment V for A. geoemydae, segments V and X for A. babirussae, dorsal on segment V (WdV1) for A. longirostre, and on segments IV (WdIV3) and V (WdV1) for A. romitii (Barros-Battesti et al., 2013). The number of lyrifissures present on the idiosoma of A. calcaratum was 24, 11, and 13 on the dorsal and ventral surfaces, respectively. This number and the pattern is similar to that found for other Amblyomma species (Klompen et al., 1996; Barbieri et al., 2007, 2008a,b), but differs from that for larval A. parvum, who carry only 12 lyrifissures on the ventral surface. Klompen et al. (1996) noted that the number and pattern of lyrifissures present in all Parasitiformes is similar, listing total numbers of lyrifissures of 18, 24, 20, 20 (all larvae) to 28 (adult), and 23–24 (adult) for Prostriata, Metastriata, Argas, Carios (Argasidae), Zerconidae, and Amblyseiinae, respectively (the latter 2 are Mesostigmata). Among the integumentary structures, the small glands are the most numerous on the idiosoma, but their frequency and pattern also showed a high degree of variability. This pattern has been observed previously for larvae of other Amblyomma species, such as A. cajennense, A. longirostre, A. parvum, A. rotundatum (Barbieri et al., 2007), A. ovale (Barbieri et al., 2008a), and A. pacae (Barbieri et al., 2008b), as well as for species within the genera Rhipicephalus Koch, 1844 (Nawar and Madbouly, 1985), Amblyomma, Dermacentor Koch, 1844, and Ixodes Latreille, 1795 (Klompen et al., 1996). This variability in the frequency of small glands can be related to the higher number this structure is present on the idiosoma, since the lowest variation has been observed for large wax glands, which had the lowest number on the idiosoma (Barbieri et al., 2007). In general, the integumentary structures on the idiosoma of the larvae of A. calcaratum show patterns similar to those observed in larvae of A. cajennense, A. longirostre, A. parvum, and A. rotundatum (Barbieri et al., 2007), A. ovale (Barbieri et al., 2008a), A. pacae
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(Barbieri et al., 2008b), A. oblongoguttatum (Barbieri et al., 2012), and A. romitii (Barros-Battesti et al., 2013). However, their number, position, and association patterns may help identifying this stage. Thus, larvae of A. calcaratum differ from those of A. longirostre, A. ovale, and A. romitii because these larvae have 2 and one additional pairs of large wax glands, respectively (Barbieri et al., 2007, 2008a; Barros-Battesti et al., 2013); from those of A. parvum because this species has one less pair of lyrifissures (Barbieri et al., 2007). The association between 2 or more structures can also be auxiliary in the identification, for example, the association of lyrifissure and small gland in ventral surface/segment IX/series 6 and lack in A. cajennense and A. oblongoguttatum (Barbieri et al., 2007, 2012), and association of the same structures in ventral surface/segment X, XI, and XII/series 2 in A. pacae and lack in A. calcaratum (Barbieri et al., 2008b). This is the first morphological description of the larva of A. calcaratum. Currently, larval morphological descriptions are available for only 19 of the 29 Amblyomma species known from Brazil (Barbieri et al., 2012). Although many of these descriptions provide general measurements of the body and details on chaetotaxy, complete analyses of integumental structures (lyrifissures, small glands, and large wax glands) have been reported for only 8 species (A. cajennense, A. parvum, A. rotundatum, A. longirostre, A. ovale, A. pacae, A. oblongoguttatum, and A. romitii) (Barbieri et al., 2007, 2008a,b, 2012; Barros-Battesti et al., 2013). Chaetotaxy is generally highly conserved among Amblyomma spp. (Barbieri et al., 2007), but analyses of especially gland distribution patterns in association with other morphological features and bioecological information should eventually yield a taxonomic key for identification of the Amblyomma larvae of the Neotropical Zoogeographic Region.
Acknowledgments The authors are grateful for financial support from CNPqBolsa de Desenvolvimento Científico Regional (no. 350265/2005-4) and Scientific Career Scholarships to MBL and DMBB, CNPq (nos. 479877-2004-1 and 304475/2010-6), FAPESP (no. 07/57749-2) and FAPERJ (no. E-26/170179/2004).
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