Debido al impacto potencial de los vectores sobre la estructura de las comunidades biológicas y al funcionamiento de los ecosistemas, se hace necesario conocer su distribución para implementar acciones de manejo y control. Un enfoque fundamental para la comprensión y gestión de especies invasoras es determinar su distribución potencial. Diversos estudios proporcionan una visión general de la modelación de distribución de especies o una comparación de los métodos de modelación (Anderson et al., 2003; Fielding y Bell, 1997; Guisan y Zimmermann, 2000; Guisan y Thuiller, 2005; Segurado y Araújo, 2004; Zaniewski et al., 2002). Estos modelos de distribución de especies tienen por objeto predecir áreas que describan las condiciones ambientales adecuadas para la supervivencia de las especies; es decir, la distribución potencial o nicho fundamental (Anderson et al., 2003; Guisan y Thuiller, 2005). En general, estos métodos de modelación combinan datos de localidades georeferenciadas de las especies donde ha sido confirmada su presencia con variables ambientales, para crear un modelo de requerimientos de la especie de acuerdo con las variables examinadas (Anderson et
al., 2003). El modelo resultante es proyectado sobre un mapa de la región de estudio que
36 para detectar áreas donde las especies invasoras pueden estar presentes y donde posiblemente estarán en el futuro. El clima es un factor importante que afecta o determina la distribución de los organismos; por tal motivo, los análisis de variables climáticas ayudan a comprender el por qué una especie crece en un determinado sitio y no en otro (Lindenmayer et al., 1991).
37
2 HIPÓTESIS
Amblyomma mixtum es la especie del complejo Amblyomma cajennense (sensu lato) que
3 OBJETIVOS
3.2 Objetivo general
Determinar la distribución espacial y caracterizar morfológicamente las garrapatas del complejo taxonómico Amblyomma cajennense (sensu lato) recolectadas de bovinos, équidos y vegetación en las diez regiones del estado de Veracruz, México.
3.3 Objetivos específicos
• Identificar morfológicamente mediante un microscopio estereoscopio las garrapatas que pertenecen al complejo taxonómico A. cajennense (sensu lato) recolectadas en bovinos, équidos y vegetación en las diez regiones naturales del estado de Veracruz, México.
• Determinar a través de microscopía electrónica de barrido la (s) especie (s) del complejo taxonómico de A. cajennense (sensu lato) recolectadas de bovinos, équidos y de la vegetación en las diez regiones del estado de Veracruz, México.
• Georreferenciar la (s) especie (s) del complejo taxonómico de A. cajennense
(sensu lato) en las diez regiones naturales del estado de Veracruz, México.
• Determinar el área de distribución potencial del complejo taxonómico de A.
cajennense (sensu lato) en las diez regiones naturales del estado de Veracruz,
México.
• Determinar la estructura genética de Amblyomma mixtum en el estado de Veracruz.
39
4 REFERENCIAS
Alekseev, A. N., Dubinina, H. V., Jushkova, O. V. (2004). First report on the coexistence and compatibility of seven tick-born pathogenes in unfed adult Ixodes Persulcatus Schulze (Acari: Ixodidae). International Journal of Medicina and Microbiology, 293 (37): 104-108.
Allsopp, M. T., Lewis, B. D., Penzhorn, B. L. (2007). Molecular evidence for transplacental transmission of Theileria equi from carrier mares to their apparently healthy foals.
Veterinary Parasitology, 148: 130-136.
Álvarez, V. V., Bonilla, R. M. (2007). Adultos y ninfas de la garrapata Amblyomma
cajennense Fabricius (Acari: Ixodidae) en equinos y bovinos. Agronomía Costarrisense, 31(1): 61-69.
Anderson, J.F. y Magnarelli, L.A. (2008). Biology of ticks. Infect Dis Clin North Am; 22:195- 215.
Araya-Anchettaa, A., Buscha, J. D., Scolesb, G. A., Wagne, D. M. (2015) Thirty years of tick population genetics: A comprehensive review. Infection, Genetics and Evolution 29 (2015) 164–179.
Balashov, Yu. S. (1972). Bloodsucking ticks (Ixodoidea)-vectors of diseases of men and animals. Miscelaneous Publications of the Entomological Society of America, 8:163-376.
Balashov, Y. S., Amosova, L. I., Grigor’eva, L. A. (1998). Transovarial and transphasic transmission of Borrelia by the taiga tick Ixodes persulcatus (Ixodidae). Parazitologiya 32: 489–494.
Barker, S. C., Murrell, A. (2004). Systematics and evolution of ticks with a list of valid genus and species names. Parasitology, 129: 15-36.
Beati, L., Nava, S., Burkman, J. E., Barros-Battesti, D. M., Labruna, M. B., Guglielmone, A. A., Cáceres, A. G., Guzmán-Cornejo, C. M., León, R., Durden, L. A., Faccini, J. L. H. (2013). Amblyomma cajenense (Fabricius, 1787) (Acari: Ixodidae), the Cayenne tick: phylogeofraphy and evidence for allopatric speciation. BMC
40 Berdyev, A. (1980). Ecology of Ixodid Ticks of Turkmenistan and Their Role in Natural
Focal Diseases Epizootiology. Ashkhabad, Turkmenistan: Ylym.
Borges, L. M. F., Oliveira, P. R., Lisboa, C. L. M., Ribeiro, M. F. B. (2002). Horse resistance to natural infestation of Anocentor nitens and Amblyomma cajennense (Acari: Ixodidae). Veterinary Parasitology, 104: 265-273.
De la Cruz y Estrada-Peña. (1992). A simple, new improved method for Preparing ticks for examination by Scanning electron microscopy. Acarologia.
De Waal, D. T. (1992). Equine piroplasmosis: a review. The British veterinary journal, 148 (1): 6-14.
Dantas-Torres F.e, Lia R., Otranto D. (2013). Efficiency of flagging and dragging for tick collection. Experimental and applied acarology. (61) 1. 119-127.
Reimer, L. (1998). Escanning Electron Microscopy. Germany. Ed. Board.
Eisen, L. (2008). Climate change and tick-borne diseases: a research field in need of long- term empirical field studies. Int. J. Med. Microbiol. 298, 12–18.
Estrada-Peña, A., Guglielmone, A. A., Mangold, A. J. (2004). The distribution and ecological “preferences”’ of the tick Amblyomma cajennense (Acari: Ixodidae), an ectoparasite of humans and other mammals in the Americas. Annals of Tropical
Medicine and Parasitology, 98 (3): 283–292.
Estrada-Peña A. (2015). Ticks as vectors: taxonomy, biology and ecology. Rev. Sci. Tech. 34(1): 53-65.
Geraci, N. S., Johnston, S. J., Robinson, J. P., Wikel, S. K., Hill, C. A. (2007). Variation in genome size of argasid and ixodid ticks. Insect Biochemestry, 14: 2051-2088. Gordillo-Pérez, G., Vargas, M., Solórzano-Santos, F., Rivera, A., Polaco, O. J., Alvarado,
L., Muñoz, O., Torres, J. (2009). Demonstration of Borrelia burgdorferi sensu stricto infection in ticks from the northeast of Mexico. Clinical Microbiology and Infection, 15: 469-498.
Guglielmone, A. A., Hanadi A., Mangold A. J. (1986). Empleo del dióxido de carbono para la captura de ninfas y adultos de Amblyomma neumanni y Amblyomma
41 Guglielmone, A. A., Mangold, A. J., Oyala, B. C. (1992). Ciclo de vida del Amblyomma
cajennense (Fabricius, 1787) (Acari: Ixodidae) en condiciones de laboratorio. Revista de Medicina Veterinaria, 73 (4): 184-187.
Guglielmone, A. A., Estrada-Peña, A., Keirans, J. E., Robbins, R. G. (2003). Ticks (Acari: xodidae) of the Neotropical Zoogeographic Region. Special Publication,
International Consortium on Ticks and Tick-Borne Diseases, Atalanta, Houten, The Netherlands, pp 174.
Guglielmone, A. A., Robbins, R. G., Apanaskevich, D. A., Petney, T. N., Estrada-Peña, A., Horak, I. G. (2009). Comments on controversial tick (Acari: Ixodida) species names and species described or resurrected from 2003 to 2008. Exp Appl Acarol. 48(4):311-27.
Guzmán-Cornejo, C., Robbins, R., Guglielmone, A. A., Montiel-Parra, G., Pérez, T. M. (2011). The Amblyomma (Acari: Ixodidae) of Mexico: identification keys, distribution and hosts. Zootaxa, 2998: 16-38.
Illoldi-Rangel, P., Chissa-Louise, R., Blake, S., Trout, F., Gordillo-Pérez, G., Rodríguez- Moreno, A., Williamson, P., Montiel-Parra, G., Sánchez-Cordero, V., Sahotra, S. (2012). Species distribution models and ecological suitability analysis for potential tick vectors of Lyme disease in México. Journal of Tropical Medicine, 2012: 10 pp. doi:10.1155/2012/959101.
Jeyaprakash, A., Hoy, M. A. (2009). First divergence time estimate of spiders, scorpions, mites and ticks (subphylum: Chelicerata) inferred from mithocondrial phylogeny.
Experimental and Applied Acarology, 47: 1-18.
Keaney P. (1994). The Effectiveness of Various Trapping Techniques In Collecting Ixodes scapularis and Other Species of Ticks.
Kohls, G.M. (1958) Amblyomma Imitator, a New Species of Ticks from Texas and Mexico and Remarks on the Synonymy of A. Cajennense (Fabricius) (Acarina-Ixodidae). The Journal of Parasitology, 44, 430-433.
Krantz, G. W, Walter, D. E. (2009). A Manual of Acarology. 3erd ed. Lubbock, TX: Texas tech University Press.
42 Labruna, M.B., Kasai, N., Ferreira, F., Faccini, J. L. H., Gennari, S. M. (2002). Seasonal
dynamics of ticks (Acari: Ixodidae) on horses in the state of Sāo Paulo, Brazil.
Veterinary Parasitology, 105: 65-77.
Labruna, M. B., Amaku M., Metzner J. A., Pinter, A., Ferreira, F. (2003). Larval behavioral diapause regulates life cycle of Amblyomma cajennense (Acari: Ixodidae) in Southeast Brazil.J. Med Entomol. 40(2): 170-8.
Labruna, M. B., Soares, J. F., Martins, T. F., Soares, H. S., Cabrera, R. R. (2011). Cross- mating experiments with geographically different populations of Amblyomma
cajennense (Acari: Ixodidae). Experimental and Applied Acarology, 54: 41–49.
Lopes, C. M. L., Leite, R. C., Labruna, M. B., Oliveria, P. R., Borges, L. M. F., Rodrigues, Z. B., Carvalho, H. A., Freitas, C. M. V., Junior, C. R. V. (1998). Host specificity of
Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) with comments on the
drop-off rhythm. Memórias do Instituto Oswaldo Cruz, 93(3): 347-351.
Mans, B. J., Neitz, A. W. (2004). Adaptation of ticks to a blood-feeding environment: evolution from a functional perspective. Insect Biochemestry of Molecular Biology, 34: 1-17.
Mannelli, A., Bertolotti, L., Gern, L., and Gray, J. (2012). Ecology of Borrelia burgdorferi sensu lato in Europe: transmission dynamics in multi-host systems, influence of molecular processes and effects of climate change. FEMS Microbiol. Rev. 36, 837– 861.
Mastropaolo, M., Nava, S., Guglielmone, A. A., Mangold, A. J (2011) Biological differences between two allopatric populations of Amblyomma cajennense (Acari: Ixodidae) in Argentina. Exp Appl Acarol. 53(4):371–375.
Mullen, G. R. and Durden, L. A. San Diego, California: Academic Press; 2002: 517-518. Nava, S., Gugliemone, A. A., Kumthong, R. (2007). The molecular basis of the
Amblyomma americanum tick attachment phase. Experimental and Applied Acarology, 41: 267-287.
Nava, S., Gugliemone, A. A., Mangold, A. J. (2009). An overview of systematic and evolution of ticks. Biological Science, 14: 2857-2877.
Nava, S., Beati, L., Labruna, M. B., Cáceres A. G., Mangold, A. J., y Guglielmone, A. A. (2014). Reassessment of the taxonomic status of Amblyomma cajennense
43 (Fabricius, 1787) with the description of three new species, Amblyomma tonelliae n. sp., Amblyomma interandinum n. sp. and Amblyomma patinoi n. sp., and reinstatement of Amblyomma mixtum Koch, 1844, and Amblyomma sculptum Berlese, 1888 (Ixodida: Ixodidae). Ticks Tick Borne Dis. In press.
Neumann, L. G. (1899). Révision de la famille des Ixodidés (Зème memoire.). Mémoires de la Sociétézoologique de France, 12: 107-294.
Nicholson, W., Sunenshine, D. E., Lane, R. S., Uilenberg, G. (2009). Ticks (Ixoda). En L. A. Durden and G. Mullen (Eds), Medical and Veterinary Entomology. San Diego: Academic Press, pp. 493-542.
Oliveira, P. R., Borges, L. M. F., Leite, R.C., Freitas, C. M. V. (2003). Seasonal dynamics of the Cayenne tick, Amblyomma cajennense on horses in Brazil. Medical and
Veterinary Entomology, 17: 412-416.
Piña, F. T.B., da Silva-Rodrigues, V., de Oliveira-Souza H. L., Garcia, M. V., Barros, J. C., de León, A. A. P., Andreotti, R. (2017). Life cycle of Amblyomma mixtum (Acari: Ixodidae) parasitizing different hosts under laboratory conditions. Exp Appl Acarol. 73(2):257-267.
Ribeiro, J.M. y Francischetti, I.M. (2003). Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu Rev Entomol 48:73-88.
Randolph, S. E., Storey, K. (1999). Impact of microclimate on immature tick-rodent host interactions (Acari: Ixodidae): implications for parasite transmission. J Med Entomol. 36(6):741-8.
Rikihisa, Y. (2015). Molecular Pathogenesis of Ehrlichia chaffeensis Infection Annual Review of Microbiology. 69: 283-304.
Robinson, L. E. (1926). The genus Amblyomma. En Ticks: A monograph of the Ixodoidea. Vol. IV. Cambridge, University Press, pp. 302.
Rodríguez-Vivas, R. I., Rosado A. A., Estrella, B. G., García-Vázquez, Z., Rosario-Cruz, R., Fragoso, S. H. Manual Técnico para el control de garrapatas en el ganado bovino. (2006). Publicación Técnica Número 4. Ed. INIFAP.
Rojas, R., Marini, M. A., Coutinho, M. T. Z. (1999). Wild birds as hosts of Amblyomma
cajennense (Fabricius, 1787) (Acari: Ixodidae). Memórias do Instituto Oswaldo Cruz, 94(3): 315-322.
44 Romero-Castañón, S., Ferguson, B. G., Güiris, D., González, D., López, S., Paredes, A., Weber, M. (2008). Comparative parasitology of wild and domestic ungulates in the Selva Lacandona, Chiapas, Mexico. Comparative Parasitology, 75: 115-126. Rudolph, D., Knulle, W. (1974). Site and mechanism of water vapour uptake from the
atmosphere in ixodid ticks. Nature. 249:84–85
Scoles G. A., Hutcheson., Schlater J. L., Hennager S. G., Pelzel M. A., Knowles, D.P. (2011). Equine Piroplasmosis associated with Amblyomma cajennense Ticks, Texas, USA. Journal of Emerging Infectious Diseases, 17 (10): 1903-1905.
Scoles, G., A, Ueti, M. W. (2013). Amblyomma cajennense is an intrastadial biological vector of Theileria equi. Parasites & Vectors, 6: 306.
Serra Freire, N. M. (1982). Epidemiología de Amblyomma cajennense: Ocurrencia estacional y comportamiento de dos estadios de parásitos en pastos del estado de Río de Janeiro. Arq. Univ. Rur. Rio de J. 5(2): 187-193.
Sonenshine, D. E. (1991). Biology of Ticks I. New York (USA): Oxford University Press, Inc.
Sonenshine, D.E.; Lane, R.S.; Nicholson, W.L. (2002). Ticks (Ixodida). In Medical and Veterinary Entomology. Mullen, G. R. and Durden, L. A. San Diego, California: Academic Press; 2002: 517-518.
Sonenshine, D. E., Roe, R. M. (2014). Biology of Ticks. Oxford University Press. 2nd.
Edition.
Tae Chong, S., Heung C. K., In-Yong, L., Thomas, M. K., Jr., Sancho, A. R., Sames W. J., Klein, T. A. (2013). Comparison of Dragging and Sweeping Methods for Collecting Ticks and Determining Their Seasonal Distributions for Various Habitats, Gyeonggi Province, Republic of Korea. Journal of Medical Entomology. 50(3): 611- 618.
Tonelli-Rondelli, M. (1939). Ixodoidea Parte II-Contributo alla conoscenza della fauna ixodologica sudamericana. Rivista di Parassitologia, 3(1): 39-55.
Topalis, P., Tzavlaki, C., Vestaki, K., Dialynas, E., Sonenshine, D. E., Butler, R., Bruggner, R. V., Stinson, E. O., Collins, F. H., Louis, C. (2008). Anatomical ontologies of mosquitoes and ticks, and their web browsers in VectorBase. Insect Molecular
45 Ueti, M. W., Palmar, G. H., Scoles, G. A., Kappmeyer, L. S., Knowles, D. P. (2008). Persistently infected horses are reservoirs for intrastadial tick-borne transmission of the apicomplexan parasite Babesia equi. Infectious immunology, 76: 3525-3529. Ueti MW, Mealey RH, Kappmeyer LS, White SN, Kumpula-McWhirter N, Pelzel AM, et al. (2012) Re-Emergence of the Apicomplexan Theileria equi in the United States: Elimination of Persistent Infection and Transmission Risk.
Xu, Y., Zhangs, S., Bayin, C., Xuan, X., Igarashi, I., Fujisak, K., Kabeya, H., Maruyamas, M. T. (2003). Seroepidemiologic studies on Babesia equi and Babesia caballi infection in horses in Jilin province of China. Journal of Veterinary Medical Science, 65 (9): 1015-1017.
Zobba, R., Ardu, M., Niccolini, S., Chessa, B., Manna, L., Cocco, R., Parpaglie, M. L. P. (2008). Clinical and laboratory findings in equine piroplasmosis. Journal Equine
46
CAPÍTULO II
Genetic structure analysis of Amblyomma mixtum populations in Veracruz State, Mexico.
Artículo aceptado.
Revista: Ticks & Tick-borne Diseases Factor de impacto: 2.84
Contents lists available atScienceDirect
Ticks and Tick-borne Diseases
journal homepage:www.elsevier.com/locate/ttbdis
Original article
Genetic structure analysis of
Amblyomma mixtum
populations in Veracruz
State, Mexico
Mariel Aguilar-Domíngueza, Sokani Sánchez-Montesb, María Dolores Esteve-Gassentd, Carolina Barrientos-Salcedoe, Adalberto Pérez de Leónc, Dora Romero-Salasa,⁎
aLaboratorio de Parasitología, Posta Zootécnica Torreón del Molino, Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, Veracruz, Mexico bCentro de Medicina Tropical, Unidad de Investigación en Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico cUSDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Kerrville, TX, USA
dDepartament of Veterinary Pathobiology, Texas A&M University, TX, USA eFacultad de Bioanálisis, Universidad Veracruzana, Veracruz, Mexico
A R T I C L E I N F O Keywords: Amblyomma mixtum Population genetics Ectoparasite Cattle Horse Veracruz Mexico A B S T R A C T
Amblyomma mixtumKoch, 1844 parasitizes livestock, humans, and wildlife in Mexico. However, information on population genetics for this tick species in the country is missing. Tick samples were collected from livestock in ten regions across the state of Veracruz (22°28′N, 17°09′S, 93°36′E, 98°39′W) to analyze the genetic structure of A.mixtumpopulations. Ticks were morphologically identified using taxonomic keys. In order to test the intra- specific variability ofA. mixtumfragments of the mitochondrial gene 16S-rRNA and cytochrome oxidase subunit 1 (COI) were amplified. Ninety-six sequences were amplified from the 50 specimens’analyzed (96% amplifi- cation success). Eleven haplotypes were detected in 16S-rRNA gene and 10 more for COI. Neutrality tests showed negative results in most of the locations analyzed, which is indicative of an excess of recently derived haplotypes. However, these results were not statistically significant. Minimal union network analysis revealed that there is no separation of populations by geography, and that there is an overlap of several haplotypes among diverse populations. Significant genetic differentiation was not detected in theA. mixtumpopulations sampled in the state of Veracruz, Mexico, this may be due to the frequent movement of livestock hosts. This is thefirst report on the genetic structure ofA.mixtumpopulations in Mexico.
1. Introduction
Ticks are obligate hematophagous ectoparasites, which can act as vectors of multiple pathogens such as bacteria, viruses, protozoa, and helminths that affect human, domestic animal, and wildlife populations (Baneth, 2014). Particularly in America, several members of theAm- blyomma cajennensespecies complex represent a problem for livestock production and human public health, due to their adaptation to cattle and their anthropophilic habits (Estrada-Peña et al., 2004; Almazan et al., 2018;Labruna, 2018). Among them the species that exhibits the widest distribution is Amblyomma mixtumKoch, 1844, that has been registered from southwestern Texas to Colombia (Beati et al., 2013;
Nava et al., 2014; Rivera-Páez et al., 2016, 2018). This species has adapted to diverse ecological niches, including semi-arid grasslands and subtropical secondary forests (Estrada-Peña et al., 2004).
This tick species parasitizes several domestic and wild mammals, in which it causes direct effects related to blood feeding such as weakness
and weight loss. However, it also serves as a vector transmitting pa- thogens to animals (e.g. Anaplasma marginale) and human (e.g.
Rickettsia rickettsii). In addition, other potentially zoonotic species of
Rickettsiahave been detected (e.g.R. amblyommatis) makingA. mixtum
one of the most important tick species in veterinary medicine and public health in Mexico (Estrada-Peña et al., 2004; Guzmán-Cornejo et al., 2011;Rodríguez-Vivas et al., 2016;Sánchez-Montes et al., 2016;
Álvarez-Hernández et al., 2017;Piña et al., 2017).
However, information related to the genetic diversity ofA. mixtum
in Mexico is lacking. Genetic and demographic studies are essential to determine the magnitude of genetic exchange among tick populations (Araya-Anchetta et al., 2015; Esteve-Gassent et al., 2016). This knowledge has practical implications such as the ability to understand how the movement of livestock across agroecosystems may influence the geneflow between conspecific tick populations, and the possibility to predict the dispersion of genes that confer resistance to ixodicides (Loaiza et al., 2010). Moreover, these studies can yield critical
https://doi.org/10.1016/j.ttbdis.2018.09.004
Received 21 March 2018; Received in revised form 27 August 2018; Accepted 10 September 2018
⁎Corresponding author.
E-mail address:[email protected](D. Romero-Salas).
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
1877-959X/ © 2018 Elsevier GmbH. All rights reserved.
information about the divergence of lineages, discrete populations and the timing of demographic processes that may influence the contribu- tion of vectors in the transmission of several tick-borne pathogens.
Mitochondrial DNA (mtDNA) analysis is a powerful tool for the reconstruction of the phylogeny of species and their demographic his- tory (Mirol et al., 2008). The advantages of mtDNA for studying closely related taxa and populations within species are its low recombination rate, maternal heredity, highly conserved structure, small effective population size and relatively fast mutation rate. In ticks, the 16S-rRNA and cytochrome oxidase subunit 1 (COI) genes have a high mutation rate, rendering them as very useful tools for intra-population studies (Esseghir et al., 1997;Ishikawa et al., 1999;Hodgkinson et al., 2003;de Lima et al., 2017). Therefore, we planned this study to investigate the genetic structure of A. mixtumpopulations infesting livestock in the state of Veracruz, Mexico. Veracruz is the state in Mexico with the largest cattle herd, which includes approximately four million head (Rodriguez-Vivas et al., 2017). TheA. mixtumhost diversity, as well as the variety of ecosystems and cattle movement in the state of Veracruz suggest small differences thus, ticks were sampled across ecological regions in the state to assess genetic divergence by the amplification of the 16S-rRNA and COI genes.
2. Materials and methods
2.1. Tick collection and mapping
A total of 50 (23 female, 27 male)A. mixtumfrom cattle and equine from ten regions were collected and analyzed during August-November 2015 and February-April 2016 (Table 1) in a convenience sampling. The ranch locations sampled across the ten regions in the state of Veracruz include diverse ecosystems that range from tropical coastal plains up to 1,520 m above sea level. Hosts were physically restrained and inspected for the presence of ticks, particularly in the neck, limbs and inguinal region. All ticks were recovered from the hosts using forceps and were fixed and preserved in ethanol. All experimental protocols were approved by the animal care and use committee of the School of Veterinary Medicine, University of Veracruz, Veracruz, Mexico.
2.2. Morphological identification
Morphological characters of each tick were observed under a Motic® stereoscope microscope. These were identified according to the mor- phological keys from Guzmán-Cornejo et al. (2011) andNava et al. (2014).
2.3. DNA extraction
Tick DNA extraction was performed individually. Every specimen was transferred to a 1.5-mL Eppendorf tube, which was placed on the surface of a container with liquid nitrogen. Subsequently the sample was crushed with the help of a sterile pistil. Then, 500μL of 5% Chelex® 100 Chelating Resin (Biorad, USA) solution and 20μL of Proteinase K (SIGMA life sciences, USA) were added per sample, and allowed to incubate at 56 °C for two hours. The samples were then centrifuged at 25,000 ×gfor 15 min and the supernatant was collected in new tubes and stored at−20 °C until further use (Ballados-González et al., 2018).
2.4. Polymerase chain reaction (PCR)
To analyze the intra-specific variability ofA. mixtum, we amplified a fragment of∼400 bp from the 16S-rRNA gene using a pair of oligo- nucleotides and conditions reported by Norris et al. (1996), and a fragment of∼379 bp from the cytochrome oxidase subunit 1 (COI) with the conditions ofHafner et al. (1994). PCR was performed in a volume of 25μL, which contained 300–500 ng of genomic DNA, 1μL of
each primer (2μM), 12.5μL of green GoTaq of Promega, and 8.5μL of free nuclease sterile water. A negative control (reaction mix without DNA), and a positive control (reaction mix andRhipicephalus sanguineus
s. l. DNA) were both included. PCR products were resolved in 2% agarose gels using 1 kb molecular weight marker (nucleic acid markers, Axygen) in 1x TBE buffer. Amplicons of the expected size were sub- mitted in the purification and sequencing to Laboratorio de Biología Molecular y de la Salud, Universidad Nacional Autónoma de México.
2.5. Population genetics analysis
Sequence data were edited, and global alignments were done using Clustal W algorithm in Mega 6.0. Sequences generated in this study were submitted to GenBank using the Bankit tool. In order to ascertain