Morphological Description of Amblyomma Brasiliense Aragão, 1908 (acari: Ixodidae) Larvae and Nymphs

The immature stages of Neotropical ticks are poorly known and in many cases have not been described. This work presents a morphological description of Amblyomma brasiliense larvae (F1) and a redescription of nymphs (F1). A. brasiliense is reported as one of the most aggressive ticks to humans in Brazil. Immature ticks obtained from a laboratory colony initiated from adult specimens collected in the Parque Estadual Intervales (24° 18' S and 48° 24' W), São Paulo, Brazil, were analyzed under scanning electron microscope, and also under light and stereoscopic microscopes. A. brasiliense larvae present basis capituli rectangular; short palpi; rounded idiosoma; coxa I with two spurs, the external one being longer than the internal one; and coxae II and III each with one short spur. Nymphs present basis capituli rectangular with a sharp pointed cornua; oval idiosoma with scutum reaching coxa III; coxa I with two evident spurs, the external one being longer than the internal one; coxae II-III each with one short spur; and coxa IV with a very short spur and chitinous tubercles on internal surface of posterior border of idiosoma. These morphological features, in association with chaetotaxy and porotaxy, should make possible the identification of immature Amblyomma ticks. Resumo Os estágios imaturos dos carrapatos Neotropicais são pouco conhecidos e, em muitos casos, não estão descritos. Este trabalho apresenta uma descrição morfológica das larvas (F1) e redescrição das ninfas (F1) do carrapato Amblyomma brasiliense, relatado como um dos ixodídeos mais agressivos ao homem no Brasil. Para este fim, carrapatos imaturos de uma colônia iniciada com espécimes adultos do Parque Estadual de Intervales, São Paulo, Brasil foram analisados sob da borda posterior do idiosoma. Estas características, associadas à quetotaxia e porotaxia, poderão tornar possível a identificação dos estágios imaturos de carrapatos do gênero Amblyomma.


Introduction
The tick genus Amblyomma Koch 1844 is estimated to include 127 species, 57 of which are found in the Neotropical region, with 33 reported in Brazil (BARROS-BAttEStI et al. 2006).However there are few morphological studies about immature stages of this genus, with larvae and nymphs still undescribed for the majority of the species.Unfortunately, most of the existing descriptions were made only under light microscope, and many structures would be better observed with scanning electron microscopy (BARROS-BAttEStI et al. 2005).
The tick Amblyomma brasiliense Aragão (1908) is endemic to South America with reports in Argentina, Paraguay, and Brazil (GUGlIElMONE et al. 2003), (Rio de Janeiro, São Paulo, Minas Gerais, Pará and Espírito Santo States) (ARAGãO 1936;SzABó et al. 2006;OGRzEWAlSKA et al. 2007).Previous studies reported that larvae and nymphs of A. brasiliense are among the most aggressive ticks to humans in Brazil (ARAGãO, 1936), with local reaction to tick attachment varying from almost imperceptible to intense.Itching especially, but hyperemia and swelling as well, were prominent features of the reaction (SzABó et al. 2006).Nevertheless, morphological descriptions of the larval stage of this tick are unknown and nymphal stage descriptions are restricted to a study by Aragão (1908) using light microscopy.
Therefore, the present study aims to describe immature stages of A. brasiliense by means of light and scanning electron microscopy, thus to enable making fast, accurate identifications of immature stages of this tick.

Origin of ticks
larvae and nymphs were obtained from a laboratory colony derived from unfed adult A. brasiliense ticks collected in the Intervales State Park (24° 18' S and 48° 24' W), São Paulo State, Brazil, from December 2003 to January 2006 and identified to species using the taxonomic keys proposed by Barros-Battesti et al. (2006).The procedures used to obtain larvae and nymphs have been described elsewhere (SANCHES et al. 2008).

Material preparation
A sample of 30 larvae and 30 nymphs originated from different engorged females were kept starving for 30 days, so that the consolidation of exoskeleton took place; afterwards they were preserved in 70% ethanol.Of these, 20 larvae were prepared according to Famadas et al. (1996) to be measured electronically under a MC80DX light microscope coupled with a capture digital camera, using the software Motic Image Plus 2.0 Ml. twenty nymphs were measured under a leica Mz12 stereomicroscope using the same method.
Five specimens of each immature stage were prepared for scanning electron microscopy.The material was dehydrated for 30 minutes in each of the following concentrations of alcohol: 70, 80, 90, and 100% (three times).They were then kept in acetone until undergoing critical point drying.Micrographs were taken in the Electron Microscopy laboratory of the Biology Department at the Bioscience Institute, São Paulo State University (UNESP), Rio Claro, São Paulo, and laboratory of Electron Microscopy, São Paulo State University, Jaboticabal, São Paulo, using a PHIllIPS 505 scanning electron microscope and a JEOl JSM 5410.
The larvae and nymphs used for the description and the slide-mounted specimens were deposited at the Department of Biology, Bioscience Institute, São Paulo State University (UNESP), Rio Claro, São Paulo for follow-up studies focusing on cuticular structures.
*All measurements are given in millimeters, with the mean followed by standard deviation and range in brackets.
Summarizing, the main morphological characteristics of A. brasiliense larvae are rectangular basis capituli, short palpi, rounded idiosoma, large wax glands recorded on the fifth festoon, and spurs on coxae and chaetotaxy as discussed.
the current re-description using electron microscope complements the one made by Aragão (1908) of the nymphal stage of A. brasiliense using light microscopy.
Morphological characteristics and measurements of the idiosoma, gnathosoma, and tarsus I of A. brasiliense nymph were compared with other previously described species such as A. parvum and A. pseudoparvum (GUGlIElMONE et al. 1990) The basis capituli of A. brasiliense nymphs is rectangular with sharp pointed cornua, and short rounded auriculae, similar to A. pseudoparvum.Amblyomma neumanii also presents rectangular basis capituli, but differs in cornua shape (rounded) and in palpi (stronger than in A. brasiliense).The hypostome is spatulate, with a dental formula of 2/2 for all Amblyomma species used for comparison.These characteristics corroborate Aragão (1908).
Idiosoma of A. brasiliense nymphs shows oval dorsal surface, with measurements very close to A. parvum but differing in the length of scutum (shorter in A. brasiliense).Aragão (1908) reported measurements that are relatively bigger, but it is important to consider that (1) in this study the specimens were collected in different geographic regions, and (2) Aragão did not specify the exact site of starting and ending measurements.
Amblyomma brasiliense nymphs have long legs, coxa I with two evident spurs, the external longer than the internal; coxae II-III each with one short spur; and coxae IV with a very short spur, consistent with Aragão (1908).These characteristics are similar to A. parvum, and differ from A. dubitatum in the length of spur on coxa IV (shorter in A. brasiliense).The same distribution of spurs is observed in A. neumanii but the spurs in A. brasiliense are larger.Amblyomma triste with triangular external spur on coxa I, A. tigrinum with narrow external spur and small internal spur on coxa I, and A. maculatum with narrow external spur, all present different numerical and morphological distribution of spurs in comparison with A. brasiliense.
Summarizing, the main morphological characteristics of A. brasiliense nymphs are rectangular basis capituli with a sharp pointed cornua, oval idiosoma with scutum reaching coxa III, and coxa I with two evident spurs, the external one being longer than the internal one; coxae II-III each with one short spur; and coxae IV with a very short spur and chitinous tubercles on internal surface of posterior border of idiosoma.
The morphological characteristics presented here must be used with caution because larvae of many Amblyomma species from the Neotropical region are still unknown.Additional studies of immature stages of other species of this genus are required to allow accurate identification of Amblyomma larvae and nymphs.