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A New Species of Pit Mite (Trombidiformes: Harpirhynchidae

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A New Species of Pit Mite (Trombidiformes: Harpirhynchidae & Herpeto gy lo lo gy o : h C Mendoza-Roldan et al., Entomol Ornithol Herpetol it u n r r r e O 2017, 6:3 n , t y Entomology, Ornithology & R g e o l s DOI: 10.4172/2161-0983.1000201 o e a m r o c t h n E Herpetology: Current Research ISSN: 2161-0983 Research Open Access A New Species of Pit Mite (Trombidiformes: Harpirhynchidae) from the South American Rattlesnake (Viperidae): Morphological and Molecular Analysis Mendoza-Roldan JA2,3, Barros-Battesti DM1,2*, Bassini-Silva R2,3, Jacinavicius FC2,3, Nieri-Bastos FA2, Franco FL3 and Marcili A4 1Departamento de Patologia Veterinária, Unesp-Jaboticabal, Jaboticabal-SP, Brazil 2Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Brazil 3Laboratório Especial de Coleções Zoológicas, Instituto Butantan, São Paulo-SP, Brazil 4Departamento de Medicina e Bem-Estar Animal, Universidade de Santo Amaro, UNISA, São Paulo-SP, Brazil *Corresponding author: Barros-Battesti DM, Departamento de Patologia Veterinária, Unesp-Jaboticabal, Jaboticabal-SP, Paulo Donato Castellane s/n, Zona rural, CEP 14884-900, Brazil, Tel: +55 16 997301801; E-mail: [email protected] Received date: August 10, 2017; Accepted date: September 07, 2017; Publish date: September 14, 2017 Copyright: © 2017 Mendoza-Roldan JA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Background: Mites of the genus Ophioptes, parasitize a wide range of snakes’ species worldwide. Pit mites develop in capsules inside the connective tissue or scales of their hosts and all stages have a genital-anal opening with no connection to the midgut. To this date, there are 15 known species, of which five occur in the Neotropical region. In South America four species have been described from Colubrid snakes. Methods: Mites were collected from the chin shields and infralabial area of the head, and the anterior third portion of the snake. Comparisons of South American species of pit mites are provided for identification purposes. SEM imaging and illustration were made to provide morphological details of the new species. DNA extraction, sequencing, and phylogeny inference were performed of the new mite species and other species of Trombidiformes mites found on reptiles and amphibians. Results: Ophioptes ekans n. sp. is described from the pits made by the mite on the scales and skin of a South American rattlesnake (Crotalus durissus terrificus) in Campo Limpo Paulista, São Paulo state, Brazil, captured on January 2014. The Genbank accession numbers of the new species are KU891263, KU891264 and KU891265. DNA sequences were used for molecular phylogenetic inference. Three nymhpal stages were observed for this species. Conclusion: This is the first record of a viper snake from the sub-family Crotaline parasitized by Ophioptes mites. Molecular analyses showed that molecular systematic of Trombidiformes mites is still unclear and more sequences and other genes are needed do better elucidate the relationships within the group. These are the first DNA sequences (18rRNA V4 region) of mites from the Ophioptinae subfamily. Keywords: Ophioptes; New species; Pit mite; Rattlesnake; Brazil Ohioptinae probably migrated from passerine birds onto the colubroid snakes [12]. Introduction To this date, the genus Ophioptes includes 15 species [7-10]. Of Mites of the genus Ophioptes, are also called pit mites due to the pit- these, five species occur in the New World, four of them described like lesions produced over the scales and connective tissue of their parasitizing South American snakes (O. parkeri Sambon, 1928 in hosts (reptiles), which are solely snakes (Colubridae, Dipsadidae and Bolivia, Brazil, and Argentine; O. tropicalis Ewing, 1933 in Guiana; O. Elapidae) [1-10]. These mites develop under the skin or scales of their longipilis and O. brevipilis Lizaso, 1981 in Brazil). The species O. hosts and adults emerge and reproduce over the body of the snake. Life dromicus Allerd, 1958 was described from Cuba. cycle of these mites is divided in 4 phases egg, larva, nymph (both In the present study, we described a new species of Ophioptes legless stages), and adults [7]. The development of immature stages parasitizing the South American rattlesnake Crotalus durissus occurs in the soft tissues and at the base of the scales. Larvae and terrificus Laurenti, 1768 (Crotalinae: Viperidae), from the State of São nymphs are legless [1,7,11]. Paulo, Brazil. Molecular data was produced and those DNA sequences Ophioptes mites belong to the family Harpirhynchidae (permanent from GenBank of some species of Trombidiformes mites found on and highly specialized parasites of birds and snakes). The subfamily reptiles and amphibians were used to infer phylogenetic relationships Ophioptinae parasitizes snakes of the families: Colubridae, Dipsadidae, of these mite groups and for barcoding purposes. Elapidae, and Lamprophiidae (Ophidia: Colubroidea). All development of these mites proceeds within the scales. The ancestor of Entomol Ornithol Herpetol, an open access journal Volume 6 • Issue 3 • 1000201 ISSN: 2161-0983 Citation: Mendoza-Roldan JA, Barros-Battesti DM, Mendoza-Roldan JA, Bassini-Silva R, Jacinavicius FC, et al. (2017) A New Species of Pit Mite (Trombidiformes: Harpirhynchidae) from the South American Rattlesnake (Viperidae): Morphological and Molecular Analysis. Entomol Ornithol Herpetol 6: 201. doi:10.4172/2161-0983.1000201 Page 2 of 8 Materials and Methods The DNA samples were subjected to polymerase chain reaction (PCR) amplification of mite 18S region V4 rRNA gene (forward primer- Mite collection and preparation ATATTGGAGGGCAAGTC TGG; reverse primer- TGGCATCGTTTATGGTTAG) [21]. PCR products of the desired size During triage procedure at the herpetology section of the (~480 bp) were purified and sequenced in an automated sequencer Laboratório Especial de Coleções Zoológicas, Instituto Butantan, a (ABI Prism 310). The nucleotide sequences were deposited in the female adult South American rattlesnake was found infested with mites GenBank database, and the accession numbers of the new species are that were either moving slowly over the animal or embedded in the KU891263, KU891264 and KU891265 (Table 1). skin or scales of the anterior portion (Figure 1A). Embedded mites were collected via delicate scarification technique [10]. Mites were Phylogenetic inference (Barcoding) preserved in 100% ethanol before mounting in Hoyer’s medium over slides. The specimens were clarified using hydroxyl potassium 5%, The newly generated sequences were aligned with sequences acetic acid 10% and lacto-phenol, to eliminate the guanine mass previously deposited of other Trombidiformes mites available in (common in this genus) that clouds the posterior portion (idiosoma) GenBank (Table 1) using ClustalW [22] and were adjusted manually of the mites in all the stages of development [7]. Another group of with GeneDoc program [23]. The alignment was used to construct mites was prepared and used for scanning electron microscopy (SEM) phylogenetic trees using maximum parsimony and maximum like- under QUANTA 250 SEM microscopes in the Laboratório de Biologia hood with MEGA 6.06 [24] applying 500 bootstrap replicates, and Celular, Instituto Butantan. Drawings of the mites were made using a Bayesian analysis was performed with MrBayes v.3.1.2 [25] with LEICA DM 4000 B microscope and measurement with a LEICA DM 2,000,000 generations, using a HKY+R model. The first 25% of the 2500 microscope and Leica Application Suite Version 3.3.0 software. trees represented ‘burn-in’, and the remaining trees were used to The leg and idiosomal chaetotaxy of the species description follows calculate Bayesian posterior probabilities. Grandjean [13,14] adapted by Kethley [15]. Palpal setation terminology follows Fain [16], and Grandjean [17] with adaptations Ethical approval proposed by Bochkov [18]. All measurements are given in micrometres The animals were caught and manipulated accordingly to the (µm) and were taken according to the standard method [19,20]. recommendations of the Ethical Principles in Animal Experimentation adopted by the Brazilian College of Animal Experimentation (COBEA), and it was approved by the Ethics Committee on Animal Experimentation (CEUA) of the Universidade de São Paulo (FMVZ- USP) - no 3069/2013. Results Family HARPIRHYNCHIDAE Dubinin, 1957 Subfamily Ophioptinae Southcott, 1956 Genus Ophioptes Sambon, 1928 Ophioptes ekans n. sp. Mendoza-Roldan & Barros-Battesti Description Female: (Holotype Figure 1B) Body, including gnathosoma, 370 long (range 360-380 in 2 paratypes) and 439 wide (435-480). Gnathosoma 123 long (100-125), about 100 wide. Palps 55 long (50-60) and 122 wide (110-130). Palpal setae dF, dG (Puffed setae), and I”G pectinate, subequal in length, 17-22 long; IT setae 16-17 long (Figures 2A-D, and 3B). Subcapitulum ventrally with setae n 39-40 long, setae m bulked and 13-14 long. Idiosoma saccate, and smooth, 355 long (320-340) (Figures 3A, 4 and 5A-B). Genital-anal opening situated ventrally near the apex of the idiosoma, with three pairs anal- genital setae (spicules), g1-g3, and four pairs of genital setae,h1,h2,f1,f2 Figure 1: Ophioptes sp. n. A. Image in stereomicroscope of mites (22-23 long) around the genital-anal opening (Figures 2E, 3A and 4). embedded beneath the scales of the infralabial
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