A New Collection of Amblyomma parvitarsum (Acari: Ixodidae) in Peru, With Description of a Gynandromorph and Report of Rickettsia Detection

A New Collection of Amblyomma parvitarsum (Acari: Ixodidae) in Peru, With Description of a... Abstract Adult stages of Amblyomma parvitarsum Neumann parasitize wild and domestic camelids of the genera Lama and Vicugna in highlands of Andean Plateau and Patagonia. Within the Peruvian Andes, few reports have documented this tick-host association, and although reported in Chile and Argentina, Rickettsia-infected A. parvitarsum remains undocumented for this country. Here we report a new collection of A. parvitarsum from Peru, the finding of the first gynandromorph for the species and high prevalence of Rickettsia in adult stages. Amblyomma, gynandromorphism, Rickettsia, Peru Amblyomma parvitarsum Neumann is an heteroxenous hard tick (Ixodidae) that inhabits highlands of Argentina, Bolivia, Chile, and Peru (Guglielmone et al. 2014, Muñoz-Leal et al. 2014). Larval and nymphal stages of this tick parasitize lizards of Liolaemus genus (González-Acuña et al. 2004; Muñoz-Leal et al. 2016), and adults exploit South American camelids (Mammalia: Artiodactyla) of Lama and Vicugna genera (Muñoz-Leal et al. 2014, Nava et al. 2017). Few collections of A. parvitarsum have been documented for Peru, and although highlands where South American camelids inhabit include at least seven regions of the country (FAO 2005), this tick has only been reported from Arequipa and Ayacucho. Gynandromorphism is a genetic anomaly that defines the occurrence of female and male characters in the phenotype of an individual of a naturally dimorphic species (Eritja 1996). According to the proportions and distribution of male and female traits, in ticks, this malformation has been classified into five categories: bipartite protogynander, where the proportions of external sexual features are equally represented; deuterogynander, where one sex is restricted to a quadrant (i.e., a fourth part of the idiosoma); metagynander, where one sex is reduced to a small segment (i.e., less than a fourth part of the idiosoma); gynander intriqué, which may present both, protogynander or deuterogynander forms, with ‘islands’ of male or female tegument embedded in areas of the opposite sex; and mosaics, where the separation of the male and female surface is not defined but intimately interlaced without indication of symmetry (Campana-Rouget 1959). Currently, more than 20 cases of gynandromorphism have been reported in the Amblyomma genus worldwide, and eight of them belong to Neotropical species (Brumpt 1934; Fonseca 1935; Sundman 1965, Viñabal et al. 1994; Aguirre et al. 1999; Guglielmone et al. 1999; Labruna et al. 2000, 2002). As in other South American representatives of Amblyomma genus, rickettsial organisms that are phylogenetically related to Rickettsia parkeri have been detected in postlarval stages of A. parvitarsum. Despite the wide distribution of this tick species in the Andean Plateau and Patagonia (Muñoz-Leal et al. 2014), documented reports of infected specimens of A. parvitarsum exist only for Argentina and Chile (Ogrzewalska et al. 2016). Materials and Methods Ticks were collected between July to September 2011, from Vicugna vicugna (Molina) captured during a traditional shear activity, organized by local people within the ‘Salinas y Aguada Blanca’ National Reserve (16º06ʹ07ʹʹS; 71º13ʹ08ʹʹW), located above 4,000 m in the Peruvian Andean Plateau (Arequipa region). Ticks were preserved in 100% ethanol, taken to the laboratory, and identified to species level following Estrada-Peña et al. (2005) and Nava et al. (2017). One gynandromorph was recovered and photographed with a Zeiss AxioCam MRc5 adapted to a SteREO Discovery V12 stereomicroscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). DNA extraction was performed in 30 specimens (13 females and 17 males) using the Guanidine Isothiocyanate technique (Sangioni et al. 2005). A polymerase chain reaction (PCR) using primers 16S + (forward) and 16S − (reverse) targeting a ≈460-bp fragment of the tick mitochondrial 16S rRNA gene (Mangold et al. 1998) was performed to confirm the morphological diagnosis for one male and one female. The presence of Rickettsia was assessed by means of a conventional PCR using primers CS-78 (forward) and CS-323 (reverse), which target a 401-bp conserved fragment of the citrate synthase gene (gltA) of all Rickettsia species (Labruna et al. 2004). Positive tick-extracted DNA was successively submitted to a new PCR using primers Rr190.70 (forward) and 190.701 (reverse), targeting a 632-bp fragment of the 190-kDa outer membrane protein gene (ompA) present only in Rickettsia species belonging to the spotted fever group (Roux et al. 1996). PCR products were purified with ExoSAP-IT (USB Products, Cleveland, OH) and further sequenced using the BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems, Austin, TX) in an ABI-automated sequencer (Applied Biosystems/Thermo Fisher Scientific, model ABI 3500 Genetic Analyser, Foster City, CA) with the same primers used for PCR. Obtained sequences were assembled with Geneious R9 (Kearse et al. 2012) and submitted to a BLAST analysis (www.ncbi.nlm.nih.gov/blast) to infer closest similarities with other known organisms. Results Forty-eight fully and partially engorged females, 44 males, and one gynander were collected from vicugnas and identified as A. parvitarsum. Taxonomical identification was confirmed by molecular analyses for one female and one male, from which a consensus mitochondrial 16S rDNA gene partial sequence was 99% (413/414-bp) identical to A. parvitarsum from Argentina (KX230479) and Chile (KX230482). Examination of the A. parvitarsum gynandromorph revealed an engorged protogynander showing features of a gynander intriqué. In general, the right side demonstrated predominantly male characteristics, while the left side showed female traits. Dorsally, the capitulum was equally divided with a single porose area and more robust cornua on the left side (Fig. 1A). The left palpus was longer than the right one. A dorsal midline separated the scutum in the male side, covering the alloscutum, and showing ornamentation and punctuations typical of males of this species. The female side showed the reduced scutum with ornamentation and typical female traits. The dorsal midline ended at the sixth festoon (Fig. 1B). In the ventral view, the sexual dimorphism was similar to the dorsal view, with the ventral midline extending from the capitulum to the sixth festoon as well, separating the male and female features in the right and left sides, respectively (Fig. 1C). The genital opening was septate with the male component possessing a flap. The anus was surrounded by male cuticle in the right side and female cuticle in the left side; however, there were several traits of female alloscutum (expanded cuticle) in the male side, indicating the intriqué form. In addition, coxae IV of both sides were typically female, with a single short spur. The spiracular plate corresponded to the respective sex in each side of the specimen. Fig. 1. View largeDownload slide Gynandromorph of Amblyomma parvitarsum. (A) Dorsal capitulum. (B) Dorsal view. (C) Ventral view. Fig. 1. View largeDownload slide Gynandromorph of Amblyomma parvitarsum. (A) Dorsal capitulum. (B) Dorsal view. (C) Ventral view. All 13 tested females and 15 of 17 males (overall positivity rate: 93.3%) were positive for Rickettsia. For 10 ticks (five females and five males) submitted to DNA sequencing, a unique haplotype was obtained for each of the gltA and ompA partial sequences. The gltA and ompA haplotypes were 100% identical to corresponding sequences of the gltA (KR296943) and ompA (KR296944) generated from A. parvitarsum ticks collected in Chile and Argentina. This gltA haplotype is 99.7% (349/350-bp) identical to Rickettsia sibirica (MF002541), Rickettsia africae (KX819298), R. parkeri (CP003341), and Rickettsia sp. strain Atlantic rainforest (KJ855083), while the ompA haplotype is 99.3% (586/590-bp) identical to R. parkeri (KY113110) and Rickettsia sp. strain Atlantic rainforest (KX137902) as well, 99.0% (584/590-bp) to R. africae (CP001612), and 98.6% (582/590-bp) to R. sibirica (KY513920). Sequences of tick and rickettsial genes obtained in this study were deposited in GenBank under the following accession numbers: KY705377, KY705378, and KY705379. One male, 10 females, and the gynander were deposited in the tick collection ‘Coleção Nacional de Carrapatos Danilo Gonçalves Saraiva’ (CNC) under accession number CNC-3476. Discussion The current report corresponds to the fourth documented collection of A. parvitarsum from Peru. In conjunct with previous studies (Dale and Venero 1977, Need et al. 1991, Guglielmone et al. 2005), this evidence delimits the occurrence of this tick only within the Arequipa and Ayacucho regions. However, geographical range of highland ecosystems inhabited by domestic and wild camelids also extends into the Apurimac, Cusco, Junin, and Puno regions. For this reason, it is highly possible that known distribution of A. parvitarsum within the Peruvian Andean Plateau might be larger than currently described. The gynandromorph specimen found in the current study was classified as a protogynander intriqué. This type of gynander has been previously reported for several other Amblyomma species, as for example Amblyomma sculptum Berlese (published as Amblyomma cajennense [F.] [Acari: Ixodidae]) in Brazil (Fonseca 1935) and Amblyomma hebraeum Koch in South Africa (Clarke and Rechav 1992). To date, cases of gynandromorphism in the genus Amblyomma include bipartite protogynanders as the most common type (Labruna et al. 2000). Here we describe gynandromorphism for the first time in A. parvitarsum. We detected a spotted fever group rickettsia in A. parvitarsum from Peru for the first time. Our results indicate that this rickettsial agent is the same that was recently reported in A. parvitarsum ticks from Argentina and Chile (Muñoz-Leal et al. 2016, Ogrzewalska et al. 2016). Previous analyses demonstrated that this A. parvitarsum rickettsia is phylogenetically closely related to R. africae, R. parkeri, and R. sibirica, precluding its classification as a new species (Ogrzewalska et al. 2016). High prevalence of this bacterium in A. parvitarsum of the current (93.3%) and a previous study (57.7–64.4%) (Ogrzewalska et al. 2016) might correspond to indirect evidence supporting an endosymbiotic relationship, as observed in other ixodid ticks and their associated Rickettsia (Cheng et al. 2013, Kurtti et al. 2015). On the other hand, pathogenic interactions of Rickettsia sp. of A. parvitarsum with the natural hosts of this tick are still unexplored. Noteworthy to mention, the human pathogen R. africae, phylogenetically closely related to the A. parvitarsum Rickettsia (Ogrzewalska et al. 2016), is also found in high infection rates (>80%) in other Amblyomma species (Fournier et al. 2009, Mediannikov et al. 2010). Therefore, the pathogenic nature of the rickettsial agent from A. parvitarsum cannot be discarded. Finally, in the current study, Rickettsia DNA was detected in fully and partially engorged A. parvitarsum ticks, so the acquisition of these bacteria from the host during feeding cannot be overlooked as well. Acknowledgments This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). SML was funded by CONICYT Programa de Formación de Capital Humano Avanzado, grant #72140079. References Cited Aguirre, D. H., Viñabal A. E., and Guglielmone A. A.. 1999. The life cycle of Amblyomma neumanni Ribaga, 1902 (Acari: Ixodidae) in the laboratory. Exp. Appl. Acarol . 23: 159– 64. Google Scholar CrossRef Search ADS PubMed  Brumpt, E. 1934. Le gynandromorphisme chez les Ixodinés. Un curieux cas obtenu dans un élevage d’Amblyomma dissimile. Annales de Parasitologie Humaine et Comparée . 12: 98– 104. Google Scholar CrossRef Search ADS   Campana-Rouget, Y. 1959. La teratologie des tiques. 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B.. 2005. Rickettsial infection in animals and Brazilian Spotted Fever endemicity. Emerg. Infect. Dis . 11: 265– 270. Google Scholar CrossRef Search ADS PubMed  Sundman, J. A. 1965. A case of gynandromorphism in Amblyomma imitator (Acarina: Ixodidae). Ann. Entomol. Soc. Am . 58: 592– 593. Google Scholar CrossRef Search ADS PubMed  Viñabal, A. E., Mangold A. J., and Guglielmone A. A.. 1994. Ginanadromorfismo en Amblyomma neumanni Ribaga, 1902 (Acari: Ixodidae). Veterinaria Argentina . 11: 257– 259. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Medical Entomology Oxford University Press

A New Collection of Amblyomma parvitarsum (Acari: Ixodidae) in Peru, With Description of a Gynandromorph and Report of Rickettsia Detection

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Abstract

Abstract Adult stages of Amblyomma parvitarsum Neumann parasitize wild and domestic camelids of the genera Lama and Vicugna in highlands of Andean Plateau and Patagonia. Within the Peruvian Andes, few reports have documented this tick-host association, and although reported in Chile and Argentina, Rickettsia-infected A. parvitarsum remains undocumented for this country. Here we report a new collection of A. parvitarsum from Peru, the finding of the first gynandromorph for the species and high prevalence of Rickettsia in adult stages. Amblyomma, gynandromorphism, Rickettsia, Peru Amblyomma parvitarsum Neumann is an heteroxenous hard tick (Ixodidae) that inhabits highlands of Argentina, Bolivia, Chile, and Peru (Guglielmone et al. 2014, Muñoz-Leal et al. 2014). Larval and nymphal stages of this tick parasitize lizards of Liolaemus genus (González-Acuña et al. 2004; Muñoz-Leal et al. 2016), and adults exploit South American camelids (Mammalia: Artiodactyla) of Lama and Vicugna genera (Muñoz-Leal et al. 2014, Nava et al. 2017). Few collections of A. parvitarsum have been documented for Peru, and although highlands where South American camelids inhabit include at least seven regions of the country (FAO 2005), this tick has only been reported from Arequipa and Ayacucho. Gynandromorphism is a genetic anomaly that defines the occurrence of female and male characters in the phenotype of an individual of a naturally dimorphic species (Eritja 1996). According to the proportions and distribution of male and female traits, in ticks, this malformation has been classified into five categories: bipartite protogynander, where the proportions of external sexual features are equally represented; deuterogynander, where one sex is restricted to a quadrant (i.e., a fourth part of the idiosoma); metagynander, where one sex is reduced to a small segment (i.e., less than a fourth part of the idiosoma); gynander intriqué, which may present both, protogynander or deuterogynander forms, with ‘islands’ of male or female tegument embedded in areas of the opposite sex; and mosaics, where the separation of the male and female surface is not defined but intimately interlaced without indication of symmetry (Campana-Rouget 1959). Currently, more than 20 cases of gynandromorphism have been reported in the Amblyomma genus worldwide, and eight of them belong to Neotropical species (Brumpt 1934; Fonseca 1935; Sundman 1965, Viñabal et al. 1994; Aguirre et al. 1999; Guglielmone et al. 1999; Labruna et al. 2000, 2002). As in other South American representatives of Amblyomma genus, rickettsial organisms that are phylogenetically related to Rickettsia parkeri have been detected in postlarval stages of A. parvitarsum. Despite the wide distribution of this tick species in the Andean Plateau and Patagonia (Muñoz-Leal et al. 2014), documented reports of infected specimens of A. parvitarsum exist only for Argentina and Chile (Ogrzewalska et al. 2016). Materials and Methods Ticks were collected between July to September 2011, from Vicugna vicugna (Molina) captured during a traditional shear activity, organized by local people within the ‘Salinas y Aguada Blanca’ National Reserve (16º06ʹ07ʹʹS; 71º13ʹ08ʹʹW), located above 4,000 m in the Peruvian Andean Plateau (Arequipa region). Ticks were preserved in 100% ethanol, taken to the laboratory, and identified to species level following Estrada-Peña et al. (2005) and Nava et al. (2017). One gynandromorph was recovered and photographed with a Zeiss AxioCam MRc5 adapted to a SteREO Discovery V12 stereomicroscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). DNA extraction was performed in 30 specimens (13 females and 17 males) using the Guanidine Isothiocyanate technique (Sangioni et al. 2005). A polymerase chain reaction (PCR) using primers 16S + (forward) and 16S − (reverse) targeting a ≈460-bp fragment of the tick mitochondrial 16S rRNA gene (Mangold et al. 1998) was performed to confirm the morphological diagnosis for one male and one female. The presence of Rickettsia was assessed by means of a conventional PCR using primers CS-78 (forward) and CS-323 (reverse), which target a 401-bp conserved fragment of the citrate synthase gene (gltA) of all Rickettsia species (Labruna et al. 2004). Positive tick-extracted DNA was successively submitted to a new PCR using primers Rr190.70 (forward) and 190.701 (reverse), targeting a 632-bp fragment of the 190-kDa outer membrane protein gene (ompA) present only in Rickettsia species belonging to the spotted fever group (Roux et al. 1996). PCR products were purified with ExoSAP-IT (USB Products, Cleveland, OH) and further sequenced using the BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems, Austin, TX) in an ABI-automated sequencer (Applied Biosystems/Thermo Fisher Scientific, model ABI 3500 Genetic Analyser, Foster City, CA) with the same primers used for PCR. Obtained sequences were assembled with Geneious R9 (Kearse et al. 2012) and submitted to a BLAST analysis (www.ncbi.nlm.nih.gov/blast) to infer closest similarities with other known organisms. Results Forty-eight fully and partially engorged females, 44 males, and one gynander were collected from vicugnas and identified as A. parvitarsum. Taxonomical identification was confirmed by molecular analyses for one female and one male, from which a consensus mitochondrial 16S rDNA gene partial sequence was 99% (413/414-bp) identical to A. parvitarsum from Argentina (KX230479) and Chile (KX230482). Examination of the A. parvitarsum gynandromorph revealed an engorged protogynander showing features of a gynander intriqué. In general, the right side demonstrated predominantly male characteristics, while the left side showed female traits. Dorsally, the capitulum was equally divided with a single porose area and more robust cornua on the left side (Fig. 1A). The left palpus was longer than the right one. A dorsal midline separated the scutum in the male side, covering the alloscutum, and showing ornamentation and punctuations typical of males of this species. The female side showed the reduced scutum with ornamentation and typical female traits. The dorsal midline ended at the sixth festoon (Fig. 1B). In the ventral view, the sexual dimorphism was similar to the dorsal view, with the ventral midline extending from the capitulum to the sixth festoon as well, separating the male and female features in the right and left sides, respectively (Fig. 1C). The genital opening was septate with the male component possessing a flap. The anus was surrounded by male cuticle in the right side and female cuticle in the left side; however, there were several traits of female alloscutum (expanded cuticle) in the male side, indicating the intriqué form. In addition, coxae IV of both sides were typically female, with a single short spur. The spiracular plate corresponded to the respective sex in each side of the specimen. Fig. 1. View largeDownload slide Gynandromorph of Amblyomma parvitarsum. (A) Dorsal capitulum. (B) Dorsal view. (C) Ventral view. Fig. 1. View largeDownload slide Gynandromorph of Amblyomma parvitarsum. (A) Dorsal capitulum. (B) Dorsal view. (C) Ventral view. All 13 tested females and 15 of 17 males (overall positivity rate: 93.3%) were positive for Rickettsia. For 10 ticks (five females and five males) submitted to DNA sequencing, a unique haplotype was obtained for each of the gltA and ompA partial sequences. The gltA and ompA haplotypes were 100% identical to corresponding sequences of the gltA (KR296943) and ompA (KR296944) generated from A. parvitarsum ticks collected in Chile and Argentina. This gltA haplotype is 99.7% (349/350-bp) identical to Rickettsia sibirica (MF002541), Rickettsia africae (KX819298), R. parkeri (CP003341), and Rickettsia sp. strain Atlantic rainforest (KJ855083), while the ompA haplotype is 99.3% (586/590-bp) identical to R. parkeri (KY113110) and Rickettsia sp. strain Atlantic rainforest (KX137902) as well, 99.0% (584/590-bp) to R. africae (CP001612), and 98.6% (582/590-bp) to R. sibirica (KY513920). Sequences of tick and rickettsial genes obtained in this study were deposited in GenBank under the following accession numbers: KY705377, KY705378, and KY705379. One male, 10 females, and the gynander were deposited in the tick collection ‘Coleção Nacional de Carrapatos Danilo Gonçalves Saraiva’ (CNC) under accession number CNC-3476. Discussion The current report corresponds to the fourth documented collection of A. parvitarsum from Peru. In conjunct with previous studies (Dale and Venero 1977, Need et al. 1991, Guglielmone et al. 2005), this evidence delimits the occurrence of this tick only within the Arequipa and Ayacucho regions. However, geographical range of highland ecosystems inhabited by domestic and wild camelids also extends into the Apurimac, Cusco, Junin, and Puno regions. For this reason, it is highly possible that known distribution of A. parvitarsum within the Peruvian Andean Plateau might be larger than currently described. The gynandromorph specimen found in the current study was classified as a protogynander intriqué. This type of gynander has been previously reported for several other Amblyomma species, as for example Amblyomma sculptum Berlese (published as Amblyomma cajennense [F.] [Acari: Ixodidae]) in Brazil (Fonseca 1935) and Amblyomma hebraeum Koch in South Africa (Clarke and Rechav 1992). To date, cases of gynandromorphism in the genus Amblyomma include bipartite protogynanders as the most common type (Labruna et al. 2000). Here we describe gynandromorphism for the first time in A. parvitarsum. We detected a spotted fever group rickettsia in A. parvitarsum from Peru for the first time. Our results indicate that this rickettsial agent is the same that was recently reported in A. parvitarsum ticks from Argentina and Chile (Muñoz-Leal et al. 2016, Ogrzewalska et al. 2016). Previous analyses demonstrated that this A. parvitarsum rickettsia is phylogenetically closely related to R. africae, R. parkeri, and R. sibirica, precluding its classification as a new species (Ogrzewalska et al. 2016). High prevalence of this bacterium in A. parvitarsum of the current (93.3%) and a previous study (57.7–64.4%) (Ogrzewalska et al. 2016) might correspond to indirect evidence supporting an endosymbiotic relationship, as observed in other ixodid ticks and their associated Rickettsia (Cheng et al. 2013, Kurtti et al. 2015). On the other hand, pathogenic interactions of Rickettsia sp. of A. parvitarsum with the natural hosts of this tick are still unexplored. Noteworthy to mention, the human pathogen R. africae, phylogenetically closely related to the A. parvitarsum Rickettsia (Ogrzewalska et al. 2016), is also found in high infection rates (>80%) in other Amblyomma species (Fournier et al. 2009, Mediannikov et al. 2010). Therefore, the pathogenic nature of the rickettsial agent from A. parvitarsum cannot be discarded. 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Journal of Medical EntomologyOxford University Press

Published: Mar 1, 2018

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