Immature argasid ticks: diagnosis and keys for Neotropical region

Many argasid tick species are known only through their larval descriptions, in which the chaetotaxy, together with other external morphological characteristics, has been used to separate genera and species. However, the illustrations of these features are based on optical microscopy alone and many of these features are not clearly defined. Because of the difficulties in determining the larval and nymph stages of some genera, we have prepared illustrated keys for the immature stages of argasids, including an up-to-date list of the known species of the Neotropical region. We have also included an illustrated key for larvae of the Ornithodoros species from Brazil, based on scanning electron microscopy.

The adult and nymphal stages of some argasid species are morphologically very similar, especially within the genus Ornithodoros, which makes it problematic to critically assess distribution and species relationships based on previous contributions.Most descriptions of nymphal stages are poor in details, lacking figures or illustrations of the instars, which hinders morphological differentiation (ESTRADA-PEÑA et al., 2010).In the absence of DNA studies on these species, only larval morphological features have been adequately defined for specific determination (VENZAL et al., 2008).
Because of the difficulties in determining the larvae and nymphs of some genera and species, we have prepared illustrated keys for these immature stages based on optical and scanning electron microcopy, in order to help in identifying the generic taxa of Argasidae in the Neotropics.Here, we also present a current list of all argasid species in the Neotropical region, including a key to the genera of immature stages in this region and also a key to larvae of Ornithodoros species in Brazil.

Materials and Methods
The specimens illustrated in this study were cleaned by means of ultrasound (40 kHz), using distilled water and commercial detergent in the proportions 8:2.The cleaning process was completed in three separate stages: 3 minutes in water + detergent, 2 minutes in distilled water, and an additional 2 minutes in distilled water.Micrographs were made by using a Zeiss LEO 440 digital scanning microscope.The plates were made using CorelDraw X 5, version 2010.The figures were prepared Photoshop CS 6, version 2012.
The larval terminology for Antricola, Argas, and Nothoaspis followed Kohls et al. (1965Kohls et al. ( , 1969Kohls et al. ( , 1970) ) and Nava et al. (2010).Nymphs were determined by means of the original descriptions as well as from specimens obtained from colonies maintained at the parasitology laboratory of the Butantan Institute.The key for larval species of Ornithodoros in Brazil was based on Kohls et al. (1965) with modifications proposed by Venzal et al. (2008) and Labruna and Venzal (2009).

Results and Discussion
The main morphological characteristics of adults and nymphs of the family Argasidae are as follows: tegument granulated, mammillated, coriaceous or tuberculated; dorsal scutum absent.The spiracular plates are small and they are localized lateroposteriorly, between coxae III and IV.There is a pair of coxal glands in a ventral position that open between coxae I and II.All palpal articles are free, and article IV is not inserted in a depression in article III.Eyes, if present, are located laterally, close to the supracoxal folders.Pulvilli absent or rudimentary in nymphs and adults; however, they can be well developed in Antricola larvae.Sexual dimorphism is generally slight, based mainly on the shape of the genital aperture (VENZAL et al., 2006).
Larvae present a vestigial scutum and the capitulum is placed at the terminal position, while in nymphs and adults the capitulum is inserted in the ventral idiosoma; the camerostome is more accentuated in post-larval stages.Few species present eyes.The generic diagnosis for larvae and nymphs of argasid and lists of species in the Neotropical region are shown below.Species present in Brazil are in bold.
Nothoaspis -Larva: Dorsal plate with isosceles triangle shape occupying entire length of the dorsum of unfed specimens; dorsal surface with 12-13 pairs of setae; hypostome with apex pointed, dental formula 2/2 with 20 denticles in each row, corona absent.Nymphs: Idiosoma twice longer than wide, anteriorly more abruptly narrowing than posteriorly; false shield (nothoaspis) covered by cells (irregular in shape and size) occupying the anterocentral area of dorsum, most of them at least with 1 seta; setae short, except for posterior margin of idiosoma, where setae are larger.Ventral surface with integument also covered by cells (irregular in shape and size), except for a narrow area located between coxae I and III; anus subcircular, lateral to coxa IV, valves each with 1 pair of setae; spiracular plate small, similar to that of male.Basis capituli subrectangular in outline, with 1 pair of post-hypostomal setae and at least 7 pairs of sublateral setae, bordered posteriorly by integumental fold; postpalpal setae absent; hood large, broadly rounded, not entirely covering capitulum, cheliceral blades, palpal articles II-IV visible dorsally; ventrally, article I forms elongate flaps protecting the pointed hypostome, dental formula 4/4 apically, 5/5 at base.
List of species (Neotropics, N = 2; Brazil, N = 1): N. reddelli Clifford and Keirans, 1975; and N. amazoniensis Nava, Venzal and Labruna, 2010.Comments: The diagnoses of larvae and nymphs were based on the original descriptions.Two nymphal instars: the first one does not feed and has reduced hypostome (NAVA et al., 2010).
List of species (N = 1): O. megnini (Dugès, 1883).There are two species but only this one occurs in the Neotropical region.
Comments: There have been isolated reports of O. megnini in Brazil (FLECHTMANN, 1985;DINIZ et al., 1987); however, there has been no indication that this species is established in Brazil, even though it is established in several neighboring countries (GUGLIELMONE et al., 2003).

Argasids from Brazil
Genus Antricola.This occurs in hot and humid caves inhabited by bats (Chiroptera), from southern United States to northern Mexico (A. coprophilus), throughout Cuba and the Caribbean areas, to South America (Colombia, Venezuela and northern and northeastern Brazil), mainly on the guano.Many species are known only from the adult stage described in Cuba.Adult ticks have mouthparts incompatible with blood feeding, and there is no evidence of blood feeding in the late nymphal instars (ESTRADA-PEÑA et al., 2008).The larvae, in turn, have a long and terminal hypostome and present well-developed pulvilli that facilitate climbing cave walls.Adults of three species are known in Brazil: A. inexpectata, described from a cave at the locality of Brejinho, municipality of Araripe (13° 47' S, 59° 49' W), state of Ceará; and A. delacruzi and A. guglielmonei, both from a cave in Itabaiana (10° 50' S, 37° 27' W), state of Sergipe (ESTRADA-PEÑA et al., 2004).The latter two species were also collected from a cave in the municipality of Porto Velho (08° 40' S, 63° 51' W), state of Rondônia (LABRUNA et al., 2008).
Genus Argas.Among the 12 species in the Neotropical region, the genus Argas is represented in Brazil only by A. miniatus, for which all stages have been described.The first record of A. miniatus in Brazil was in the state of Rio de Janeiro (MARCHOUX; SALIMBENI, 1903).Rohr (1909) referred to A. miniatus as A. persicus, with occurrence in the municipality of Campinas (state of São Paulo) and in Rio de Janeiro (referred to as the "Federal District").Aragão (1936) considered this tick species to be A. persicus var.dissimile and mentioned its distribution in the states of Paraná, Santa Catarina, São Paulo, Rio de Janeiro, Minas Gerais, Espírito Santo, Mato Grosso, Pernambuco, Paraíba, Maranhão, Ceará, Pará and Bahia.Cançado et al. (2008) included the Pantanal region of Mato Grosso do Sul in its distribution area.Besides Brazil, A. miniatus is distributed in Colombia, Guyana, Panama, Trinidad & Tobago, Cuba, Jamaica, Puerto Rico, Venezuela and the Nearctic region (GUGLIELMONE et al., 2003).This species occurs mainly on chickens but may be found on other birds.It is a vector of Borrelia anserine, the agent of fowl spirochetosis.Although A. persicus has been recorded in many countries of South America, this species is originally from the Palearctic region.However, the Neotropical species A. persicus is probably a sibling species closely related to a true Palearctic species (GUGLIELMONE et al., 2003).
Genus Nothoaspis.Until recently, this genus was represented by a single species, N. redelli, in Mexico.In Brazil, a second species, N. amazoniensis, was recently found in caves in Rondônia (NAVA et al., 2010).The descriptions include larvae, nymphal instars and adults.
The species O. rostratus described in Brazil also occurs in Argentina, Paraguay and Bolivia (ARAGÃO, 1936;NAVA et al., 2007).Adults and larvae were described (GUGLIELMONE et al., 2003).It bites humans and several mammal species (ALMEIDA et al., 2012).The larvae feed for few hours.In Brazil specimens of O. rostratus have been recorded in the states of São Paulo, Mato Grosso do Sul, Mato Grosso, Goiás and Minas Gerais, generally in association with domestic animals (ARAGÃO, 1936;PARDI;ROCHA, 1954;CANÇADO et al., 2008).The species O. brasiliensis is known only from the state of Rio Grande do Sul, where it has been found parasitizing many animals, including humans (MARTINS et al., 2011).Adults and larvae (ARAGÃO, 1923;BARROS-BATTESTI et al., 2012) and all the nymphal instars (LANDULFO et al., 2013) have been described.Some species such as O. jul and O. nattereri have not been reported since their description, and only the adult stage is known (GUGLIELMONE et al., 2003).O. jul was found in a wasp nest used by bats in the municipality of Nova Teutônia, state of Santa Catarina.The type of O. jul was reported to be deposited at the Berlin museum (SCHULZE, 1940); however, it has not been found in this collection.On the other hand, Warburton (1927) mentioned that the type of O. nattereri was deposited at the Vienna Museum (label 86) and that the 12 specimens were from Brazil, but the host is unknown.This species resembles O. rostratus.
Although O. capensis had been considered to be among the Brazilian species of Ornithodoros (DANTAS-TORRES et al., 2012), it may be confused with O. denmarki, O. amblus and O. talaje, among others, which form the "capensis" group.It has wide among marine birds in Neotropical coastal areas and islands, and also in the Ethiopian, Nearctic, Oriental and Palearctic regions (KOHLS et al., 1965;GUGLIELMONE et al., 2003).All stages of O. capensis have been described (GUGLIELMONE et al., 2003).
The species A. hasei (cited as O. dunni) was originally described in Panama (MATHESON, 1935), and Cooley and Kohls (1944) cited a female of this species from Marajó Island, state of Pará, Brazil, collected in 1941, which was found "living in a tree hole with bats".All stages of O. hasei have been described (GUGLIELMONE et al., 2003).Cooley and Kohls (1941) described O. stageri from adult and immature specimens collected from bats in California, Arizona, Oklahoma and Texas.This species was found in Mexico (KOHLS et al., 1965), andJones et al. (1972) enlarged the distribution of O. stageri to include Venezuela, Nicaragua and Brazil.Specimens of A. hasei and O. stageri collected in Brazil are deposited at USNTC (JONES et al., 1972).
The larval morphology of O. rudis resembles O. rostratus and O. brasiliensis, mainly because of the dorsal plate, but it lacks spurs in the dorsal region of palpus I. On the other hand, adults of O. rudis may be confounded with other species of the Alectorobius group (BARROS-BATTESTI et al., 2012).All stages have been described (GUGLIELMONE et al., 2003).
The species O. setosus was described from larvae collected from bats in Piedras Negras, state of Rondônia; the holotype and paratypes were deposited under the number RML 49559, according to Kohls et al. (1969).Larvae have also been collected from bats in Mexico and Venezuela.Only the larval stage is known (GUGLIELMONE et al., 2003).
The species O. talaje forms a species group with wide distribution from the southern United States to Argentina (HOOGSTRAAL, 1985).According to this author, most records before 1950 are questionable because they were based primarily on adult morphology.Venzal et al. (2008) commented that this species may be restricted to Central America, and that the records from South America are probably O. rioplatensis known from Uruguay, or O. puertoricensis, or a yet undescribed closely related species.These authors also commented that the material from Guatemala that they examined had been reared from adults collected close to the type locality of O. talaje.These seem to be the "true" O. talaje larvae, given that the original description of the species by Guérin-Méneville was made from adult specimens collected from a nearby locality.All stages have been described.Two new species were recently described and included in this group: O. guaporensis (larvae and adults, collected from a rocky fissure in the Amazon forest, in Bolivia) and O. microlophi in Chile (larvae collected from lizards of the genus Microlophus) (NAVA et al., 2013;VENZAL et al., 2013a).
The species O. rondoniensis is known from its adult stage collected from a cave in the municipality of Porto Velho, state of Rondônia (LABRUNA et al., 2008) and from caves in the state of Pará (HENRIQUE-SIMÕES et al., 2012).The species O. fonsecai and O. cavernicolous were described from the larvae and adults, and from all stages, respectively (LABRUNA; VENZAL, 2009;DANTAS-TORRES et al., 2012).O. fonsecai is only known from specimens collected from bats on the inner walls of São Miguel cave, located in the rural area of Bonito, state of Mato Grosso do Sul.This species has also been found on bats and on walls of a cave named "Gruta Lagoa Azul", located 80 km from the municipality of Nobres, state of Mato Grosso (BARROS-BATTESTI personal communication).On the other hand, the species O. cavernicolous has wide geographical distribution, with occurrences on bats and in caves in the states of Pará, Ceará, Rio Grande do Norte, Bahia Goiás and Minas Gerais (DANTAS-TORRES et al., 2012).
Larvae of O. mimon were originally collected from bats in Bolivia, with records also from Uruguay and Argentina (VENZAL et al., 2004).In Brazil, adults and nymphs of O. mimon were first collected from a household in the municipality of Araraquara, state of São Paulo.Larvae were reared from females in a laboratory, and were redescribed along with a description of the adults as well as the biology of this species under laboratory conditions (BARROS-BATTESTI et al., 2011;LANDULFO et al., 2012).Nymphal instars of O. mimon were also described (LANDULFO et al., 2013).
The species O. marinkellei is known from Brazil, Colombia, Panama and Venezuela (VENZAL et al., 2006;LABRUNA et al., 2011).In Brazil, adults of this species were found in caves in the municipality of Porto Velho, state of Rondônia (LABRUNA et al., 2011), and in the state of Pará (HENRIQUE-SIMÕES et al., 2012).In the same cave in Porto Velho, larvae were collected from bats; few of these larvae molted to nymphs under the conditions of the cave.Adults and the first nymphal instar were described and the larva was redescribed by Labruna et al. (2011).This species is closely related to O. viguerasi and O. moormops, and all belong to the subgenus "Subparmatus".
Larvae of O. kohlsi were collected from bats of the species Neoplatymops matogrossensis, which were found in a rock crevice in Monte Negro, state of Rondônia, in 2005.This species was previously described as O. boliviensis by Kohls and Clifford (1964), but the name "boliviensis" had been preoccuped.Therefore, Guglielmone and Keirans (2002) proposed the name O. kohlsi.This species occurs on bats of the species Myotis nigricans and Molossus sp. in several localities in Bolivia.According to Kohls et al. (1965), adults and nymphs were found in bat-infested houses, where the tick was found biting humans.
Genus Otobius.This genus is represented by two species in the world, and in the Neotropics only Otobius megnini (Dugès, 1883) has been recorded.Although there have been isolated reports from northern and southeastern Brazil (FLECHTMANN, 1985;DINIZ et al., 1987), the species is not established in this country.All stages have been described.