Characterization of Haemaphysalis flava (Acari: Ixodidae) from Qingling Subspecies of Giant Panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by Morphology and Molecular Markers
Tick is one of important ectoparasites capable of causing direct damage to their hosts and also acts as vectors of relevant infectious agents. In the present study, the taxa of 10 ticks, collected from Qinling giant pandas (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains of China in April 2010, were determined using morphology and molecular markers (nucleotide ITS2 rDNA and mitochondrial 16S). Microscopic observation demonstrated that the morphological features of these ticks were similar to Haemaphysalis flava. Compared with other Haemaphysalis species, genetic variations between Haemaphysalis collected from A. m. qinlingensis and H. flava were the lowest in ITS2 rDNA and mitochondrial 16S, with sequence differences of 2.06%–2.40% and 1.30%–4.70%, respectively. Phylogenetic relationships showed that all the Haemaphysalis collected from A. m. qinlingensis were grouped with H. flava, further confirmed that the Haemaphysalis sp. is H. flava. This is the first report of ticks in giant panda by combining with morphology and molecular markers. This study also provided evidence that combining morphology and molecular tools provide a valuable and efficient tool for tick identification.
References
[1]
O’Brien SJ, Pan W, Lü Z (1994) Pandas, people and policy. Nature 369: 179–180.
[2]
Peng JJ, Jiang ZG, Hu JC (2001) Status and conservation of giant panda (Ailuropoda melanoleuca): a review. Folia Zoologica 50: 81–88.
[3]
Species Survival Commission, IUCN (2008) Ailuropoda melanoleuca. The World Conservation Union, Gland. Available at http://www.redlist.org/search/details.ph？p?species=712 [accessed in 2013].
[4]
Zhang JS, Daszak P, Huang HL, Yang GY, Kilpatrick AM, et al. (2008) Parasite threat to panda conservation. Ecohealth 5: 6–9.
[5]
Zhang ZH, Wei FW (2006) Giant panda ex-situ conservation theory and practice. Beijing: Science Press (In Chinese).
[6]
Wang T, He G, Yang G, Fei Y, Zhang Z, et al. (2008) Cloning, expression and evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag3 antigen in mice. Vaccine 26: 6919–6924.
[7]
Zeng CJ, Pan HJ, Gong SB, Yu JQ, Wan QH, et al. (2007) Giant panda BAC library construction and assembly of a 650-kb contig spanning major histocompatibility complex class II region. BMC Genomics 8: 315.
[8]
Zhang W, Yie S, Yue B, Zhou J, An R, et al. (2012) Determination of Baylisascaris schroederi infection in wild giant pandas by an accurate and sensitive PCR/CE-SSCP method. PLoS ONE 7: e41995.
[9]
He G, Chen S, Wang T, Yan Y, Zhang Z, et al. (2012) Sequence analysis of the Bs-Ag1 gene of Baylisascaris schroederi from the giant panda and an evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag1 antigen in mice. DNA Cell Biol 31: 1174–1181.
[10]
Zhang H, Wang XH, Fan WF, Yuan M (2010) Review of parasitosis of gaint panda. Gansu Anim Vet Sci 212: 40–43 (In Chinese)..
[11]
de la Fuente J, Estrada-Pe？a A, Venzal JM, Tocan KM, Sonenshine DE (2008) Overview: ticks as vectors of pathogens that cause disease in humans and animals. Front Biosci 13: 6938–6946.
[12]
Oliver JH (1989) Biology and systematics of ticks (Acari: Ixodida). Annu Rev Ecol Syst 20: 397–430.
[13]
Wu J, Hu HG (1985) Gaint panda: new host of Haemaphysalis warburtoni. Chin J Vet Med 8: 10 (In Chinese)..
[14]
Chen KL, Shi XQ (1992) Haemaphysalis aponommoides and Haemaphysalis longicornis were found in gaint panda. Shanghai J Anim Husb Vet Med 1: 8 (In Chinese)..
[15]
Qiu MH, Zhu CJ (1987) Parasites in giant panda and their prevention and treatment. Proceedings of giant panda disease treatment conference. Beijing: China Forestry Press 1–9 p (In Chinese).
[16]
Ma GY (1987) Collection of ascarid and ticks in giant panda in Wen county, Gansu province. Sichuan J Zool 6: 34 (In Chinese)..
[17]
Lai CL, Yang ML, Wang Q (1990) Ectoparasites of giant panda. Sichuan J Zool 9: 7 (In Chinese)..
[18]
Yang GY (1998) Progress on parasite and parasitosis of giant panda. Chin J Vet Sci 18: 206–208 (In Chinese)..
[19]
Qin XC, Tian JH, Wang JB, Lu X, Sun QZ, et al. (2011) Identification of Haemaphysalis longicornis and Rhipicephalus microplus. Chin J Epidemiol 32: 608–612 (In Chinese)..
[20]
Padgett KA, Nadler SA, Munson L, Sacks B, Boyce WM (2005) Systematics of Mesocestoides (Cestoda: Mesocestoididae): evaluation of molecular and morphological variation among isolates. J Parasitol 91: 1435–1443.
[21]
Wickstr？m LM, Haukisalmi V, Varis S, Hantula J, Henttonen H (2005) Molecular phylogeny and systematics of anoplocephaline cestodes in rodents and lagomorphs. Syst Parasitol 62: 83–99.
[22]
de Rojas M, Ubeda JM, Cutillas C, Mora MD, Ariza C, et al. (2007) Utility of ITS1-5.8S-ITS2 and 16S mitochondrial DNA sequences for species identification and phylogenetic inference within the Rhinonyssus coniventris species complex (Acari: Rhinonyssidae). Parasitol Res 100: 1041–1046.
[23]
Zhao GH, Li J, Mo XH, Li XY, Lin RQ, et al. (2012) The second transcribed spacer rDNA sequence: an effective genetic marker for inter-species phylogenetic analysis of trematodes in the order Strigeata. Parasitol Res 111: 1467–1472.
[24]
Wang CR, Gao JF, Zhu XQ, Zhao Q (2012) Characterization of Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle by internal transcribed spacers of nuclear ribosomal DNA. Res Vet Sci 92: 99–102.
[25]
Song J, Shi L, Li D, Sun Y, Niu Y, et al. (2012) Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA. PLoS ONE 7: e43971.
[26]
McLain DK, Wesson DM, Collins FH, Oliver JH Jr (1995) Evolution of the rDNA spacer, ITS 2, in the ticks Ixodes scapularis and I. pacificus (Acari: Ixodidae). Heredity 75: 303–319.
[27]
Crampton A, McKay I, Barker SC (1996) Phylogeny of ticks (Ixodida) inferred from nuclear ribosomal DNA. Int J Parasitol 26: 511–5l7.
[28]
Murrell A, Campbell NJ, Barker SC (2001) Recurrent gains and losses of large (84–109 bp) repeats in the rDNA internal transcribed spacer 2 (ITS2) of rhipicephaline ticks. Insect Mol Biol 10: 587–596.
[29]
Hlinka O, Murrell A, Barker SC (2002) Evolution of the secondary structure of the rRNA internal transcribed spacer 2 (ITS2) in hard ticks (Ixodidae, Arthropoda). Heredity 88: 275–279.
[30]
Wesson DM, McLain DK, Oliver JH, Piesman J, Collins FH (1993) Investigation of the validity of species status of Ixodes dammini (Acari: Ixodidae) using rDNA. Proc Natl Acad Sci USA 90: 10221–10225.
[31]
Marrelli MT, Souza LF, Marques RC, Labruna MB, Matioli SR, et al. (2007) Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA. J Med Entomol 44: 222–228.
[32]
Chao LL, Wu WJ, Shih CM (2011) Species identification of Ixodes granulatus (Acari: Ixodidae) based on internal transcribed spacer 2 (ITS2) sequences. Exp App Acarol 54: 51–63.
[33]
Norris DE, Klompen JS, Keirans JE, Black WC 4th (1996) Population genetics of Ixodes scapularis (Acari: Ixodidae) based on mitochondrial 16S and 12S gene. J Med Entomol 33: 78–89.
[34]
Kain DE, Sperling FA, Daly HV, Lane RS (1999) Mitochondrial DNA sequence variation in Ixodes pacificus (Acari: Ixodidae). Heredity 83: 378–386.
[35]
Murrell A, Campbell NJ, Barker SC (2001) A total-evidence phylogeny of ticks provides insight into the evolution of life cycles and biogeography. Mol Phylogenet Evol 21: 244–258.
[36]
Black WC 4th, Piesman J (1994) Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences. Proc Natl Acad Sci USA 91: 10034–10038.
[37]
Beati L, Keirans JE (2001) Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters. J Parasitol 87: 32–48.
[38]
Chitimia L, Lin RQ, Cosoroaba I, Wu XY, Song HQ, et al. (2010) Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes. Exp Appl Acarol 52: 305–311.
[39]
Chen Z, Yang X, Bu F, Yang X, Liu J (2012) Morphological, biological and molecular characteristics of bisexual and parthenogenetic Haemaphysalis longicornis. Vet Parasitol 189: 344–352.
[40]
Levin ML, Studer E, Killmaster L, Zemtsova G, Mumcuoglu KY (2012) Crossbreeding between different geographical populations of the brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae). Exp Appl Acarol 58: 51–68.
[41]
Apanaskevich DA, Horak IG, Matthee CA, Matthee S (2011) A new species of Ixodes (Acari: Ixodidae) from South African mammals. J Parasitol 97: 389–398.
[42]
Dantas-Torres F, Venzal JM, Bernardi LF, Ferreira RL, Onofrio VC, et al. (2012) Description of a new species of bat-associated argasid tick (Acari: Argasidae) from Brazil. J Parasitol 98: 36–45.
[43]
Burger TD, Shao R, Beati L, Miller H, Barker SC (2012) Phylogenetic analysis of ticks (Acari: Ixodida) using mitochondrial genomes and nuclear rRNA genes indicates that the genus Amblyomma is polyphyletic. Mol Phylogenet Evol 64: 45–55.
[44]
Rumer L, Sheshukova O, Dautel H, Donoso Mantke O, Niedrig M (2011) Differentiation of medically important Euro-Asian tick species Ixodes ricinus, Ixodes persulcatus, Ixodes hexagonus, and Dermacentor reticulatus by polymerase chain reaction. Vector Borne Zoonotic Dis 11: 899–905.
[45]
Deng GF (1978) Economic insect fauna of China. Volume 15. Beijing:Science Press 1978: 1–174 p (In Chinese).
[46]
Chitimia L, Lin RQ, Cosoroaba I, Braila P, Song HQ, et al. (2009) Molecular characterization of hard ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA. Parasitol Res 105: 1479–1482.
[47]
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
[48]
Chilton NB, Gasser RB, Beveridge I (1995) Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea). Int J Parasitol 25: 647–651.
[49]
Zhao GH, Mo XH, Zou FC, Li J, Weng YB, et al. (2009) Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes. Vet Parasitol 162: 67–74.
[50]
Swofford DL (2002) PAUP*: Phylogenetic analysis using parsimony (and other methods). Sinauer Associates, Sunderland, MA.
[51]
Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: 357–358.
[52]
Lee WK, Lim JW, Lee SY, Lee IY (1997) Redescription of Haemaphysalis flava and Ixodes tanuki collected from a raccoon dog in Korea. Korean J Parasitol 35: 1–8 (In Korean)..
[53]
Wei F, Hu Y, Zhu L, Bruford WW Zhan X, et al. (2012) Black and white and read all over: the past, present and future of giant panda genetics. Mol Ecol 21: 5660–5674.
[54]
Huo T, Zhang Y, Lin J (2012) Functional annotation from the genome sequence of the giant panda. Protein Cell 3: 602–608.
[55]
Yang GY, Zhang ZH (2013) Parasitic diseases of wildlife. Beijing: Science Press. 41 p (In Chinese).
[56]
Zhu XQ, D’Amelio S, Gasser RB, Yang TB, Paggi L, et al. (2007) Practical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea: Anisakidae) using genetic markers in nuclear ribosomal DNA. Mol Cell Probes 21: 97–102.
[57]
Mangold AJ, Bargues MD, Mas-Coma S (1998) Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae). Parasitol Res 84: 478–484.
[58]
Tian Z, Liu G, Xie J, Yin H, Luo J, et al. (2011) Discrimination between Haemaphysalis longicornis and H. qinghaiensis based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2). Exp Appl Acarol 54: 165–172.
