Introduction
Parasitism is one of the most common interactions among species within ecosystems (Gardner & Campbell, Reference Gardner and Campbell1992; Thomas et al., Reference Thomas, Renaud and Guégan2005), and it affects individual health, population growth, community structure and even ecosystem functioning (Grenfell, Reference Grenfell1992; Morand et al., Reference Morand, Ivanova and Vaucher1996; Morand & Arias-Gonzalez, Reference Morand and Arias-Gonzalez1997; Poulin, Reference Poulin1998). Small mammal hosts, including rodents, marsupials and bats, are infected by a variety of parasitic species, and may act as reservoirs of zoonosis, such as leishmaniasis, Chagas disease and schistosomiasis (Gentile et al., Reference Gentile, Costa Neto and D'Andrea2010; Orozco et al., Reference Orozco, Piccinali, Mora, Enriquez, Cardinal and Gürtler2014). Despite the importance of studies concerning mammal–parasite interactions for addressing the occurrence of diseases in natural and human-disturbed environments (Simões et al., Reference Simões, Gentile, Rademaker, D'Andrea, Herrera, Freitas, Lanfredi and Maldonado2010; Moreira et al., Reference Moreira, Giese, Melo, Simões, Thiengo, Maldonado and Santos2013; Cardoso et al., Reference Cardoso, Simões, Luque, Maldonado and Gentile2016), little is known about the helminth fauna, their geographic distribution, host specificity and prevalence in Neotropical small mammals. Moreover, studies containing detailed taxonomical and structural descriptions of these helminths are still scarce.
Necromys lasiurus (Lund, 1840) is a small (35 g), terrestrial rodent species which is known to be a reservoir of leishmaniasis (Brandão-Filho et al., Reference Brandão-Filho, Brito, Carvalho, Ishikawa, Cupolillo, Floeter-Winter and Shaw2003) and hantavirus pulmonary syndrome (Limongi et al., Reference Limongi, Moreira, Peres, Suzuki, Ferreira, Souza, Pinto and Pereira2013). Its distribution extends through central Brazil to south of the Amazon River, including north-eastern Argentina, extreme south-eastern Peru, Paraguay and Bolivia (Redford & Eisenberg, Reference Redford and Eisenberg1999), thus inhabiting the grasslands of Cerrado, Caatinga, as well as open areas in the Atlantic Forest biome in Brazil (Bonvicino et al., Reference Bonvicino, Oliveira and D'Andrea2008). It is considered to be a generalist species, being favoured by anthropogenic disturbances due to its tolerance of habitat modification and broad diet spectrum, including leaves, seeds, fruits and insects (Vieira & Baumgarten, Reference Vieira and Baumgarten1995; Redford & Eisenberg, Reference Redford and Eisenberg1999). Moreover, due to its high density and short life cycle (Francisco et al., Reference Francisco, Magnusson and Sanaiotti1995), N. lasiurus may contribute to persistence of parasite populations and their transmission among hosts (Oliveira et al., Reference Oliveira, Guterres, Fernandes, D'Andrea, Bonvicino and de Lemos2014; Sabino-Santos et al., Reference Sabino-Santos, Maia, Jonsson, Goodin, Salazar-Bravo and Figueiredo2016).
Nematodes of the family Rictulariidae Froelich, 1802, infect mammals worldwide, including bats, marsupials, rodents, carnivores and primates. The morphological characteristics of the genus Pterygodermatites are based on the oral opening position and the total number cuticular projections, the number of prevulvar cuticular projections in females, and the positions of the papillae at the posterior end and the size of the spicules in males (Quentin, Reference Quentin1967). Pterygodermatites (Paucipectines) zygodontomis was first described by Quentin (Reference Quentin1967) from the small intestine of the rodent N. lasiurus collected in Exú, in the state of Pernambuco, Brazil. However, some morphological aspects were not described in detail, and this species has not been reported by scanning electron microscopy and molecular analysis.
Within the phylum Nematoda, phylogenetic studies based on molecular data have been carried out for the order Spirurida by Blaxter et al. (Reference Blaxter, De Ley, Garey, Liu, Scheldeman, Vierstraete and Vida1998), Wijová et al. (Reference Wijová, Moravec, Horak, Modry and Lukes2005) and Nadler et al. (Reference Nadler, Carreno, Mejía-Madrid, Ullberg, Pagan, Houston and Hugot2007). Nevertheless, so far, no molecular phylogeny has included the family Rictulariidae. In fact, just one small sequence from a member of this family is available in public databases.
In the present study, P. (P.) zygodontomis was analysed by light and scanning electron microscopy, adding new details to taxonomic characteristics. Additionally, phylogenies including representatives of this nematode genus were inferred, based on partial sequences of the mitochondrially encoded cytochrome c oxidase I gene, to determine relationships of the superfamily Rictularioidea within the order Spirurida.
Materials and methods
Study sites and habitat description
Rodents were captured in Uberlândia (18°55′07″S, 48°17′19″W), in the state of Minas Gerais, Brazil, which is located in the Cerrado biome. Cerrado is the largest South American savannah, characterized by a tropical climate with a dry winter from April to September and a wet season from October to March. Collection was carried out in rural areas, including grassland and cornfields, and in the borders of Cerrado vegetation preserved areas, called Cerrado sensu stricto.
Collection and examination of rodents
In collaboration with the staff of the municipal government, 102 specimens of N. lasiurus were collected and analysed for helminth parasites during an investigation of hantavirus cases. Animals were captured with Tomahawk® (16 × 5 × 5 inches) and Sherman® traps (3 × 3.75 × 12 inches) baited with a mixture of peanut butter, banana, oats and bacon. Trapping occurred between December 2011 and November 2012. Biosafety techniques were used during all procedures involving biological samples (Lemos & D'Andrea, Reference Lemos and D'Andrea2014).
Helminth recovery and morphological analysis
The abdominal and thoracic cavities of the rodents were examined for the presence of helminths. Organs were placed separately in Petri dishes, washed twice in physiological saline solution, and dissected under a stereomicroscope. Worms were washed twice in saline solution to remove tissue debris and fixed in AFA (2% glacial acetic acid, 3% formaldehyde, 95% ethanol), or alternatively preserved in 70% ethanol for DNA isolation. For study of morphological characters, nine male and ten female specimens were cleared in 80% phenol (70% ethanol and phenolic acid), mounted on temporary slides and examined using a Zeiss Scope Z1 light microscope (Zeiss, Göttingen, Germany). The structures were measured via digital images captured by a Zeiss Axio Cam HRC using the accessory software Axio Vision Rel. 4.7.
For scanning electron microscopy (SEM), six specimens (three males and three females) were fixed in 2.5% glutaraldehyde and 4% freshly prepared formaldehyde in 0.1 m cacodylate buffer, pH 7.2, washed in 0.1 m cacodylate buffer, post fixed for 2 h in 1% osmium tetroxide and 0.8% potassium ferricyanide, pH 7.2, dehydrated in a graded ethanol series (20–100° GL) for 20 min each step and dried to critical point (Mafra & Lanfredi Reference Mafra and Lanfredi1998). Specimens were examined using a JEOL JSM-6390 microscope (JEOL, Tokyo, Japan) at the Rudolf Barth Electron Microscopy Platform of the Oswaldo Cruz Institute.
Voucher specimens of the helminths were deposited in the Coleção Helmintológica do Instituto Oswaldo Cruz (P. zygodontomis numbers: CHIOC 38398, female only, from Exú, Pernambuco state and CHIOC 38399, male and female, from Uberlândia, Minas Gerais state; Pterygodermatites jaegerskioldi numbers: CHIOC 38400 and 38501 from Corumbá, Mato Grosso do Sul state).
Molecular and phylogenetic analysis
Genomic DNA samples were isolated from mid-section fragments of P. zygodontomis, P. jaegerskioldi, Physocephalus lassancei and Protospirura numidica adult worms (table 1). DNA isolation used the QIAGEN QIAamp® DNA Mini Kit according to the manufacturer's protocol (QIAGEN, Hilden, Germany). Each reaction was performed using only one specimen. Before DNA extraction, each specimen was clarified in alcohol/glycerol, identified morphologically and subsequently washed in 70% ethanol and distilled water.
Table 1. Species, geographic locality, host and GenBank accession number of species used for this study.
n/a, Not available.
DNA amplification by polymerase chain reaction (PCR) methodology was conducted using the primer cocktail: NemF1_t1 5′-TGT AAA ACG ACG GCC AGT CRA CWG TWA ATC AYA ARA ATA TTG G-3′, NemF2_t1 5′-TGT AAA ACG ACG GCC AGT ARA GAT CTA ATC ATA AAG ATA TYG G-3′, NemF3_t1 5′-TGT AAA ACG ACG GCC AGT ARA GTT CTA ATC ATA ARG ATA TTG G-3′, NemR1_t1 5′-CAG GAA ACA GCT ATG ACT AAA CTT CWG GRT GAC CAA AAA ATC A-3′, NemR2_t1 5′-CAG GAA ACA GCT ATG ACT AWA CYT CWG GRT GMC CAA AAA AYC A-3′ and NemR3_t1 5′-CAG GAA ACA GCT ATG ACT AAA CCT CWG GAT GAC CAA AAA ATC A-3′, as described by Prosser et al. (Reference Prosser, Velarde-Aguilar, León-Règagnon and Hebert2013), for the barcode region of the mitochondrial cytochrome c oxidase subunit I gene (MT-CO1). Each PCR contained 2.5 μl of 10× PCR buffer, 2 μl of 50 mm MgCl2, 0.5 μl of each primer cocktail (10 μm of a three-forward-primers mix, and 10 μm of a three-reverse-primers mix), 0.5 μl of 10 mm deoxynucleotide triphosphate solution (dNTPs), 0.2 μl of Invitrogen™ Platinum™ Taq DNA polymerase (500 U/μl) (Invitrogen, São Paulo, Brazil), 2.0 μl of genomic DNA and ultrapure water, in a total reaction volume of 25 μl. Thermal cycling conditions were 94°C for 1 min; five cycles at 94°C for 40 s, 45°C for 40 s, 72°C for 1 min; followed by 35 cycles at 94°C for 40 s, 51°C for 40 s, 72°C for 1 min; and a final extension at 72°C for 5 min (Prosser et al., Reference Prosser, Velarde-Aguilar, León-Règagnon and Hebert2013). The resulting amplicons were visualized on 2% agarose gels using GelRed™ nucleic acid gel stains (Biotium, Hayward, California, USA).
Successfully amplified amplicons were purified using the GE Healthcare illustra™ GFX™ PCR DNA and Gel Band Purification Kit following the manufacturer's protocol (GE Healthcare Little Chalfont, Bucks, UK) and then cycle sequenced using the Applied Biosystems™ BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, California, USA), individually for each primer mentioned above for better accuracy. Sequencing was performed using the Applied Biosystems™ ABI 3730 DNA Analyzer. Both procedures and cycle-sequenced product precipitation were conducted at the DNA sequencing platform of the Oswaldo Cruz Institute, PDTIS/FIOCRUZ. Fragments were assembled into contigs and edited for ambiguities using the Geneious 9.1.8 software (http://www.geneious.com; Kearse et al., Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Mentjies and Drummond2012), resulting in a consensus sequence for each individual worm.
The MT-CO1 dataset included sequences from representatives of the superfamilies Rictularioidea (our two Pterygodermatites species), Acuarioidea, Camallanoidea, Dracunculoidea, Filarioidea, Gnathostomatoidea, Habronematoidea, Physalopteroidea, Spiruroidea and Thelazioidea (table 1). Plectoidea was included as the outgroup. Substitution saturation in the dataset was assessed using the test by Xia et al. (Reference Xia, Xie, Salemi, Chen and Wang2003) and Xia & Lemey (Reference Xia, Lemey, Lemey, Salemi and Vandamme2009) with the software DAMBE version 6.4.79 (Xia, Reference Xia2017).
Phylogenetic reconstructions were carried out using the software Treefinder version of March 2011 (Jobb, Reference Jobb2011) for maximum likelihood (ML) as optimality criteria and MrBayes version 3.2.6 (Ronquist et al., Reference Ronquist, Teslenko, van derMark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012) for Bayesian inference (BI). Both ML and BI analyses were performed using distinct models per codon position, to account for different evolutionary processes at each of the three codon positions.
In the ML analyses, evolutionary models were chosen by the Bayesian information criterion (BIC) (Schwarz, Reference Schwarz1978). ML-pairwise distances were computed using the same codon-based partitioned models using Treefinder. Robustness of nodes in ML was assessed by non-parametric bootstrap percentages (BP) after 1000 pseudoreplicates and by the expected-likelihood weights applied to local rearrangements of tree topology (LR-ELW) after 1000 replicates.
In the BI analyses, distinct GTR + I + G models were used for each codon position, with unlinking of base frequencies and parameters. Markov chain Monte Carlo (MCMC) sampling was performed for 10,000,000 generations with four simultaneous chains, in two runs. Robustness of nodes in BI was assessed by Bayesian posterior probabilities (BPP) calculated from trees that were sampled every 100 generations, after removing the first 25% generations as a ‘burn-in’ stage. Adequacy of BI analyses sampling was assessed through the effective sample size (ESS) of each parameter, calculated using the software Tracer version 1.6 (Rambaut, Reference Rambaut2012). Values above 100 effectively independent samples were considered sufficient.
Results
Morphology by light and scanning electron microscopy
Description
Adult helminths exhibited sexual dimorphism. The female body was larger and more robust than the male, and both showed two columns of spine-like cuticular projections organized in pairs located ventrally, beginning below the buccal capsule and extending until near the end of the body. The oral cavity was triangulate, surrounded by six labial papillae (2 ventral, 2 lateroventral and 2 dorsal) and four external cephalic papillae (1 pair ventral and 1 pair dorsal) (figs 1A and 2B–D). There was a pair of amphids between the lateral and the dorsal labial papillae (fig. 2B). The buccal capsule with thick walls was sclerotized, trapezoidal in shape, with three oesophageal teeth (fig. 1A). The oral opening was surrounded by a toothed strip of 21 denticles in females (9 dorsal and 6 on each ventrolateral side) and 17 in males (7 dorsal and 5 on each ventrolateral side) (figs 1A and 2E). The female presented 81 ventrolateral spine-like cuticular projections arranged in pairs, being 38 prevulvar and 43 postvulvar (fig. 1B). The vulvar opening was posterior to the oesophageal–intestinal junction (fig. 1C). The tail was conical and fig. 1D indicates the position of the anus.
Fig. 1. Light microscopy of Pterygodermatites (Paucipectines) zygodontomis. (A) Female buccal capsule with three oesophageal teeth (te) showing oral opening (asterisk). Details of apical view showing three developed lips and six labial papillae (lp); (B) vulvar (vu) region, lateral view, and spine-like posvulvar cuticular projections (sl); (C) region of transition of oesophagus (es) and intestine and vulva (vu); (D) female posterior region, lateral view, anus (a); (E) male posterior region, ventral view, details of four fans (fa) and spicules (sp); (F) right spicule (spr), left spicule (spl) and gubernaculum (g), lateral view.
Fig. 2. Scanning electron microscopy of P. (P.) zygodontomis. (A) Male, spine-like cuticular projections; (B) female, anterior end showing a pair of well-defined longitudinal cuticular elements, ventrolaterally located (v); (C) female, anterior end, apical view and external cephalic papillae (cp); (D) apical view, amphid (a), ventral region (v), dorsal region (d) and labial papillae (asterisk); (E) anterior end showing oral opening surrounded by a crown of teeth.
Males presented 41 spine-like ventrolateral cuticular projections (fig. 2A) with three or four fans ventral and anterior to the cloaca in juvenile and adult worms, respectively, in finfold shape (figs 1E, 2A). The posterior end of the male had nine pairs of papillae: 2 pairs precloacal, 1 pair ad-cloacal and 6 pairs postcloacal, organized in two groups. The first group was made up of two pairs of papillae and the second with three pairs of papillae near the tail tip. The ninth pair of papillae was located at the tip of the male tail, ending bluntly (figs 3B–D). A pair of phasmids was also observed between the eighth and the ninth pair of papillae (fig. 3C). The spicules were unequal in size with similar shape, at a ratio of 1:2 (right:left) (fig. 1F). The presence of a rectangular gubernaculum was observed (fig. 1F). These traits and the morphometric characteristics allowed us to identify the specimens to the species level by comparing them with other specimens belonging to P. (P.) zygodontomis recovered from N. lasiurus (table 2).
Fig. 3. Scanning electron microscopy of P. (P.) zygodontomis. (A) Male, posterior portion, ventral view, details of four fans (fa) and spicules (sp); (B) pairs of papillae (asterisk); (C) the tail, a pair of phasmids (ph) and papillae (asterisk); (D) tip of male with papillae (asterisk).
Table 2. Comparison of morphometric characteristics (in μm) of Pterygodermatites (Paucepectines) zygodontomis among specimens of Necromys lasiurus of Brazilian rodents.
dfae, Distance from anterior end.
Molecular and phylogenetic analysis
Alignment of sequences resulted in a matrix comprising 46 taxa and 876 characters, of which 309 were constant and 507 were variable characters, informative for parsimony. The test by Xia & Lemey (Reference Xia, Lemey, Lemey, Salemi and Vandamme2009) for substitution saturation provided evidence for saturation only at the third codon positions, whereas overall there was little saturation in the matrix (data not shown).
Our phylogenies, based on the MT-CO1, inferred using two different optimality criteria (ML and BI), resulted in similar topologies with little variation in nodes and support values (fig. 4). The ML method resulted in a tree with score lnL = –12339.81. The evolutionary models selected through BIC were: TN+G in the first codon positions, TVM+G in the second codon positions, and J2+G in the third codon positions, all models with optimized substitution rates, frequencies of bases and gamma distributions. The BI Markov chains provided highly significant estimated sample sizes (ESS) for all parameters.
Fig. 4. Bayesian phylogenetic reconstruction, based on the MT-CO1 gene. Values at the nodes are the Bayesian posterior probabilities greater than 90%.
MT-CO1 sequences formed two well-supported reciprocally monophyletic groups with P. jaergerskioldi (LR-ELW = 94%, ML-BP = 98%, BPP = 100%) and P. zygodontomis (LR-ELW = 89%, ML-BP = 98%, BPP = 100%). These two clades formed a strongly supported monophyletic group (LR-ELW = 100%, ML-BP = 100%, BPP = 100%), representing the superfamily Rictularioidea.
Sequences representing the superfamily Spiruroidea were not recovered as monophyletic in any topology. Within Spiruroidea, families Spiruridae, represented by Protospirura species, and Spirocercidae, represented by Cylicospirura, Physocephalus and Spirocerca species, were not monophyletic, although Cylicospirura and Spirocerca species formed a well-supported monophyletic group (LR-ELW = 99%, ML-BP = 97%, BPP = 100%).
Although relationships between superfamilies Rictularioidea, Filarioidea, Habronematoidea Physalopteroidea, Spiruroidea and Thelazioidea were generally poorly supported, their sequences formed a strongly supported monophyletic group (LR-ELW = 100%, ML-BP = 92%, BPP = 100%) (see supplementary table S1).
ML-pairwise genetic distances of the MT-CO1 gene between Pterygodermatites species ranged from 3 to 4%, between P. (P.) jaergerskioldi and P. (P.) zygodontomis. The intraspecific distances between P. (P.) zygodontomis specimens ranged from 0.04%, between the specimens from N. lasiurus and Rhipidomys mastacalis from Uberlândia, state of Minas Gerais, to 0.05% between the specimens from N. lasiurus from Uberlândia, state of Minas Gerais, and from Exú, state of Pernambuco. The intraspecific distance between P. (P.) jaegerskioldi specimens was 0.04% (table 3).
Table 3. Interspecific and intraspecific MT-CO1 pairwise ML genetic distances, minimum and maximum percentages within spirurid genera.
*, Not applicable.
Discussion
The genus Pterygodermatites is characterized by the oral opening shape, presence of three oesophageal teeth, and number of spine-like prevulvar cuticular projections. Moreover, the subgenus Paucipectines includes the arrangement of the caudal papillae in males (Quentin, Reference Quentin1969; Anderson et al., Reference Anderson, Chabaud and Willmott2009). In the Americas, 17 species belonging to the subgenus Paucipectines have been reported parasitizing rodents of the families Cricetidae and Sciuridae, marsupials of the families Caenolestidae and Didelphidae, and bats of the family Molossidae as definitive hosts (Lent & Freitas, Reference Lent and Freitas1935; Quentin, Reference Quentin1967; Sutton, Reference Sutton1979; Chabaud & Bain, Reference Chabaud and Bain1981; Sutton, Reference Sutton1984; Navone, Reference Navone1989; Navone & Suriano, Reference Navone and Suriano1992; Ramallo & Claps, Reference Ramallo and Claps2007; Torres et al., Reference Torres, Maldonado and Lanfredi2007; Jiménez & Patterson, Reference Jiménez and Patterson2012; Miño et al., Reference Miño, Rojas Herrera, Notarnicola, Robles and Navone2012; Lynggaard et al., Reference Lynggaard, García-Prieto, Guzmán-Cornejo and Osorio-Sarabia2014).
Pterygodermatites (Paucipectines) zygodontomis was described as Rictularia zygodontomis by Quentin (Reference Quentin1967) and later transferred to Pterygodermatites by Quentin (Reference Quentin1969). Within the subgenus Paucipectines, this species can be distinguished from other species (P. coloradensis, P. peromysci, P. parkeri, P. ondatrae, P. azarai, P. microti, P. onychomis and P. hymanae) by the greater number of spine-like prevulvar cuticular projections (Lichtenfels, Reference Lichtenfels1970; Lynggaard et al., Reference Lynggaard, García-Prieto, Guzmán-Cornejo and Osorio-Sarabia2014). Furthermore, P. kozeki, P. chaetophracti, P. spinicaudatis, P. microti and P. dipodomis differ from P. zygodontomis due to the lower number of total spine-like cuticular projections in females.
In addition, P. zygodontomis differs from P. baiomydis, P. azarai and P. elegans by the smaller distance between the vulva and the oesophageal–intestinal junction. The nematodes P. jaegerskioldi and P. massoiai, both described only on females, are distinct from P. zygodontomis by the smaller distance from the spine-like cuticular projection to the tip of the tail (Sutton, Reference Sutton1979; Torres et al., Reference Torres, Maldonado and Lanfredi2007).
In the original description, P. zygodontomis was reported to present three unpaired fans in males in the posterior ventral region (Quentin, Reference Quentin1967). We observed four fans at the posterior ventral end using scanning electron microscopy. Probably, the first structure is not totally developed and cannot be visualized under light microscopy.
Although Quentin (Reference Quentin1967) described males with ten pairs of papillae, we clearly observed a pair of phasmids between the eighth and ninth pairs of papillae at the posterior end. Thus, P. (P.) zygodontomis presented nine pairs of papillae and not ten as reported previously. To date, P. (P.) zygodontomis has been described as a parasite of N. lasiurus, sharing morphological characteristics such as the number of spine-like prevulvar cuticular projections and fans, in males, and unequal spicules. However, female specimens examined in this study and by Grossmann (Reference Grossmann2015) were smaller in body length than those found by Quentin (Reference Quentin1967). Among the characteristics used to identify the species, we noted that the distance from the last spine-like cuticular projection to the tip of the tail in females was smaller than the distance observed by Quentin (Reference Quentin1967), and the distance from the vulva to the oesophago-intestinal region was also smaller in the present study. In addition, we found differences in the distance from the cloaca to the tip of the tail in males. These differences may be due to different development stages of the specimens analysed by Quentin and in the present study.
The original morphological description of P. (P.) zygodontomis by Quentin (Reference Quentin1967) was confirmed in the present study using light microscopy and SEM. We also added new characteristics using SEM, such as details of the oral opening surrounded by a toothed strip of 21 denticles, four fans and a pair of phasmids.
Additionally, our findings increase the host spectrum and the geographic distribution of P. (P.) zygodontomis, through the description of this species infecting N. lasiurus and R. mastacalis in the Cerrado biome, while the original description of the species was restricted to N. lasiurus individuals in the Caatinga biome. Cerrado and Caatinga are neighbouring biomes forming the so-called South American Dry Diagonal, a large belt of land characterized by high seasonality and low rainfall. Their mammal faunas are largely shared (120 species, including 22 rodents) (Carmignotto et al., Reference Carmignotto, de Vivo, Langguth, Patterson and Costa2012), which contributes to similarities in the helminth fauna of mammalian hosts. The presence of this parasite in both biomes may be linked to the extended distribution of N. lasiurus. Moreover, infection of R. mastacalis by P. (P.) zygodontomis may be accidental, although indicating the low host-specificity of this parasite. Distribution and feeding habits in these rodents may explain their infection by P. (P.) zygodontomis. Rodents of both species (N. lasiurus and R. mastacalis) inhabit the Cerrado biome (Carmignotto et al., Reference Carmignotto, de Vivo, Langguth, Patterson and Costa2012) and have an insectivorous–omnivorous diet (Talamoni et al., Reference Talamoni, Couto, Cordeiro and Diniz2008; Pinotti et al., Reference Pinotti, Naxara and Pardini2011), which may favour infection through the ingestion of insects that act as intermediate hosts. However, while Necromys is a genus endemic to open formations (D'Elia, Reference D'Elia2003), R. mastacalis is an arboreal species, commonly found in forest physiognomies (Atlantic Forest, woodlands and gallery or semi-deciduous forests in the Cerrado biome), although also occurring in the Caatinga. Hence, conversion of natural ecosystems to open formations, following anthropogenic disturbances occurring in the Cerrado during the past few decades, may have contributed to the transmission dynamics of P. (P.) zygodontomis between rodent species.
In our molecular phylogenies, based on the MT-CO1 gene, no phylogenetic affinities could be unambiguously established between Pterygodermatites species and other Spirurid families or superfamilies. In addition, a close relationship between families Rictulariidae and Physalopteridae, as suggested by Vicente et al. (Reference Vicente, Rodrigues, Gomes and Pinto1997), was not supported in our phylogenetic analysis. Nevertheless, we have found strong support for a group including the superfamilies Filarioidea, Habronematoidea Physalopteroidea, Rictularioidea, Spiruroidea and Thelazioidea. This group is consistent with the classifications proposed by Ley & Blaxter (Reference Ley, Blaxter and Lee2002), for an infraorder Spiruromorpha, or by Hodda (Reference Hodda and Zhang2011), for a suborder Spirurina, once excluding Camallanoidea; or is consistent with the classification proposed by Anderson et al. (Reference Anderson, Chabaud and Willmott2009), for a suborder Spirurina, once excluding Gnathostomatoidea. Conversely, Gnathostomatoidea, Dracunculoidea and Camallanoidea were more distantly related to other spirurids; with a Dracunculoidea–Camallanoidea clade supporting the suborder Camallanina proposed by Anderson et al. (Reference Anderson, Chabaud and Willmott2009).
Additionally, in topologies, our sequences of P. numidica did not form a clade with the sequence of Protospirura muricola from GenBank. That would not be expected if these specimens belonged to the same genus. Presumably, the sample from GenBank may have been erroneously assigned to the genus Prostospirura, while in fact belonging to the genus Mastophorus. Because of the inappropriate choice of characters, Protospirura has had a long and difficult history of confusion with the similar Mastophorus, as stated by Smales et al. (Reference Smales, Harris and Behnke2009). A complete morphological examination of the GenBank specimen of P. muricola could clear this matter. Moreover, genetic distances between Protospirura species were approximately 5%, which were larger than the distances between species of Brugia, Cylicospirura, Gnathostoma, Habronema, Onchocerca and Pterygodermatites, and smaller than the distances between Thelazia species.
In conclusion, the geographic distribution of P. (P) zygodontomis was expanded with this study and new morphological characters were added. In addition, this is the first report of molecular phylogenetic analyses of the Rictulariidae family, enhancing its molecular dataset and contributing to future research that may compare ancestry among the Pterygodermatites subgenera, as proposed by Quentin (Reference Quentin1969). Further studies are needed for a better understanding of the relationships between the spirurid superfamilies, including representatives of the Aproctoidea and Diplotriaenoidea superfamilies.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0022149X17000736
Acknowledgements
We thank Dr Paulo D'Andrea for providing us with his ICMBio licence; Dr Helene Santos Barbosa for the use of the Rudolf Barth Electron Microscopy Platform (PDTIS/FIOCRUZ); and Socrates Costa-Neto from the Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios for assistance with the collection of rodents and helminths. We also thank the staff of the Plataforma de Sequenciamento de DNA do Instituto Oswaldo Cruz (PDTIS/FIOCRUZ) for assistance with sequencing samples in this study. Finally, we are grateful to Dr Marcelo Knoff for loaning specimens from the Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC).
Financial support
This study received financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES, Instituto Oswaldo Cruz (FIOCRUZ), CNPq and FAPEMIG.
Conflict of interest
None.
Ethical standards
Licences for animal capture were provided by Chico Mendes Institute for Biodiversity Conservation (ICMBio authorization number 13373). All protocols followed the guidelines for capture, handling and care of the Ethics Committee on Animal Use of the Oswaldo Cruz Institute (according to licences L-049/08 and 066/08).
