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Ancylostoma ailuropodae n. sp. (Nematoda: Ancylostomatidae), a new hookworm parasite isolated from wild giant pandas in Southwest China

Ancylostoma ailuropodae n. sp. (Nematoda: Ancylostomatidae), a new hookworm parasite isolated... Background: Hookworms belonging to the genus Ancylostoma (Dubini, 1843) cause ancylostomiasis, a disease of considerable concern in humans and domestic and wild animals. Molecular and epidemiological data support evidence for the zoonotic potential among species of Ancylostoma where transmission to humans is facilitated by rapid urbanization and increased human-wildlife interactions. It is important to assess and describe these potential zoonotic parasite species in wildlife, especially in hosts that have physiological similarities to humans and share their habitat. Moreover, defining species diversity within parasite groups that can circulate among free-ranging host species and humans also provides a pathway to understanding the distribution of infection and disease. In this study, we describe a previously unrecognized species of hookworm in the genus Ancylostoma in the giant panda, including criteria for morphological and molecular characterization. Methods: The hookworm specimens were obtained from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China in November 2013. They were microscopically examined and then genetically analyzed by sequencing the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in two representative specimens (one female and one male, FTZ1 and FTZ2, respectively). Results: Ancylostoma ailuropodae n. sp. is proposed for these hookworms. Morphologically the hookworm specimens differ from other congeneric species primarily based on the structure of the buccal capsule in males and females, characterized by 2 pairs of ventrolateral and 2 pairs of dorsolateral teeth; males differ in the structure and shape of the copulatory bursa, where the dorsal ray possesses 2 digitations. Pairwise nuclear and mitochondrial DNA comparisons, genetic distance analysis, and phylogenetic data strongly indicate that A. ailuropodae from giant pandas is a separate species which shared a most recent common ancestor with A. ceylanicum Looss, 1911 in the genus Ancylostoma (family Ancylostomatidae). (Continued on next page) * Correspondence: guangyou1963@aliyun.com Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xie et al. Parasites & Vectors (2017) 10:277 Page 2 of 18 (Continued from previous page) Conclusion: Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms. Keywords: Ancylostoma ailuropodae n. sp, Ailuropoda melanoleuca, Morphology, Phylogeny, Ancylostomatidae Background Cuvier; A. pluridentatum in Puma concolor coryi Hookworms (Nematoda: Ancylostomatidae) are one of (Bangs); A. kusimaense in Nyctereutes procyonoides the most common soil-transmitted helminths, causing viverrinus Temminck; A. taxideae in Taxidea taxus serious iron-deficiency anemia and protein malnutrition taxus (Schreber); A. genettae in Genetta genetta (Lin- in humans and domestic and wild mammals [1–3]. Both naeus); A. protelesis in Proteles cristata (Sparrman); and major genera Ancylostoma (Dubini, 1843) and Necator A. somaliense in C. mesomelas [5–12]. Although a di- Stiles, 1903, relegated to two distinct subfamilies, are re- verse assemblage of carnivorans is recognized as hosts sponsible for morbidity and socioeconomic burdens [4]. for Ancylostoma, only one species had been documented Unlike species in the genus Necator, most Ancylostoma or described previously among the Ursidae [7]; species hookworms are considered to be of greater medical and of the distantly related Uncinaria Frölich, 1789, are con- veterinary importance because of distribution, preva- sidered typical in ursine hosts [13]. lence, and multiple zoonotic species [2]. Currently there Recent molecular-based genetic and epidemiological are fourteen valid species identified in the genus Ancy- investigations have shown that among certain wild or lostoma that are often considered in the context of the domestic animal-derived species of Ancylostoma, A. cey- range of hosts that are typically infected. For example, lanicum is becoming the second most common hook- the ‘anthrophilic’ form is limited to Ancylostoma duode- worm found to infect and complete its life-cycle in nale (Dubini, 1843) which principally infects humans. humans [12, 14–18]. Similar transmission and cross- ‘Anthropozoonotic’ forms, capable of circulating among infection cases have been reported for other congeneric free-ranging wild hosts, some domestic hosts and species, notably A. caninum [12, 19, 20] and A. brazi- humans include Ancylostoma caninum (Ercolani, 1859), liense [12]. Such situations highlight the public health Ancylostoma braziliense Gomes de Faria, 1910 and Ancy- significance of hookworm infection and the necessity to lostoma ceylanicum Looss, 1911. Other species, includ- assess their prevalence and distribution, and to identify ing most of the recognized diversity in the genus are their wildlife hosts. This has become especially important considered to be primarily of veterinary importance, in- for wildlife hosts that may have recently adapted to the cluding Ancylostoma tubaeforme (Zeder, 1800), Ancylos- human environment due to rapid urbanization [14, 21] toma malayanum (Alessandrini, 1905), Ancylostoma leading to increased interactions with people in conserva- pluridentatum (Alessandrini, 1905), Ancylostoma para- tion centers and zoological gardens constructed for en- duodenale Biocca, 1951, Ancylostoma kusimaense dangered and valuable animals [22]. Regrettably, little Nagayosi, 1955, Ancylostoma buckleyi Le Roux & Biocca, attention has been broadly paid to the species of Ancylos- 1957, Ancylostoma taxideae Kalkan & Hansen, 1966, toma because of a limited understanding of their diversity, Ancylostoma genettae Macchioni, 1995, Ancylostoma abundance and distribution and the difficulty in etiological protelesis Macchioni, 1995, and Ancylostoma somaliense and epidemiological sampling in the wild [12, 14]. Macchioni, 1995 [5, 6]. It is noteworthy that nearly all of The giant panda, Ailuropoda melanoleuca (David), these species can also be found in wildlife, such as A. one of the most endangered and rare species of China, is duodenale in Crocuta crocuta (Erxleben); A. caninum regarded as one of the preeminent species for wildlife and A. braziliense in Acinonyx jubatus (Schreber) and conservation in the world. Higher taxonomic status for Canis mesomelas Schreber; A. ceylanicum in Canis lupus these enigmatic carnivorans had been unresolved, until dingo Meyer; A. paraduodenale in Leptailurus serval relatively recent decisions that unequivocally placed (Schreber); A. malayanum in Ursus thibetanus G. giant pandas among the Ursidae (e.g. [23–26]). Wild Xie et al. Parasites & Vectors (2017) 10:277 Page 3 of 18 giant pandas currently inhabit six small mountain ranges fresh Ancylostoma specimens and provided an oppor- of China i.e. Qinling, Minshan, Qionglai, Daxiangling, tunity to fill some of these gaps in our knowledge. We Xiaoxiangling and Liangshan (Fig. 1), with an estimated have used DNA sequence and morphological analysis, population size of ~1,864 [27–30]. Since the 1950s, nu- applying clear species criteria established in a phylogen- merous natural reserves, conservation centers, research etic context [42], to recognize and describe a previously bases and zoological gardens were specifically estab- unknown hookworm species from the giant panda. A lished by the Chinese government to protect this threat- putative sister-species relationship with the ‘anthropo- ened species [31]. Some of these wild giant pandas have zoonotic’ A. ceylanicum suggests a possible zoonotic risk become closely associated with humans as they are for transmission and infection to humans. housed for artificial breeding and conservation and bio- logical investigations. Also, some pandas have been dis- Methods played publically as the ‘messenger of peace and Parasite collection and microscopic examination friendship’ around the world [32]. Although ecological, In November 2013, a wild female giant panda was found genetic and etiological studies have shown that the dead in the Fengtongzai Natural Nature Reserve, panda faces the threat of extinction due to habitat loss, Sichuan Provence of China (Fig. 1). After a routine nec- poor reproduction and low resistance to infectious dis- ropsy, seventeen hookworm specimens (seven males and eases [33, 34], recent surveys strongly indicate that para- ten females) were collected from the small intestine sitic infections represent the leading health threat to under the Scientific Procedures Premises License for the giant pandas of China [35–40]. College of Veterinary Medicine, Sichuan Agricultural Hookworm parasites have been frequently observed in University (Sichuan, China). In addition, parasite eggs the intestines of wild dead giant pandas since 1995 [28] were isolated from the intestinal content by the and the first record, attributed to a species of Ancylos- centrifuge-flotation method using saturated MgSO [43]. toma, was reported by Zhang et al. in 2005 [41]. How- After washing in physiological saline, the hookworm ever, detailed morphological descriptions, determination specimens were either directly fixed in Berland’s fluid of taxonomic status and indicators of pathogenicity of (95% glacial acetic acid and 5% formaldehyde) for mor- the Ancylostoma sp. derived from giant panda are lack- phological analysis or stored in 70% ethanol for subse- ing. The recent collection of parasites from a wild giant quent molecular profiling. For morphology, the panda that died in the Fengtongzai Natural Reserve in hookworms were identified to the genus level on the Sichuan Province of China resulted in the recovery of basis of the existing taxonomic keys and descriptions of Fig. 1 Sampling site in China (red circle) for Ancylostoma ailuropodae n. sp. in the giant panda. The distribution of the giant panda populations in Shaanxi, Gansu and Sichuan provinces of China is indicated in black with the names of mountain ranges Xie et al. Parasites & Vectors (2017) 10:277 Page 4 of 18 Ancylostoma spp. (e.g. [44]). In brief, the worms (n = 15; examined on agarose (1%) gels to verify that they repre- 6 males and 9 females) were prepared as temporary sented the target bands. The corrected gel-isolated whole mounts in glycerin after clearing in lactophenol amplicons were column-purified and sequenced in both and examined under both dissecting and light micros- directions using terminator-based cycle sequencing with copy at magnifications of 10–40× and 40–200×, respect- BigDye chemistry (Applied Biosystems, Foster City, CA, ively; male and female specimens were characterized USA) on an ABI 3730 DNA sequencer (Applied Biosys- morphologically including photo-micrographic imaging tems) in TaKaRa Biotechnology Co. Ltd. (Dalian, China). and morphometrics. Measurements are given in micro- To ensure maximum accuracy, each amplicon was se- metres (μm) unless specified otherwise and presented quenced three times independently. The consensus se- with the range followed by the mean within parentheses. quences were utilized for the following bioinformatic In addition, some key characteristics of the adults were analyses and added to GenBank under the accession drawn with the aid of serial photographs for morpho- numbers KP842923 (FTZ1) and KP842924 (FTZ2) for logical comparison and differentiation from other related ITS1-5.8S-ITS2 and KP842921 (FTZ1) and KP842922 species. These specimens including the type-series and (FTZ2) for cox1. vouchers for molecular analyses have been deposited in Sequences of ITS1-5.8S-ITS2 and cox1of Ancylostoma the Department of Parasitology, Sichuan Agricultural sp. in the present study were separately aligned with ref- University (accession numbers code GYY-XY). erence sequences from closely related species (Table 1), including the congeneric species A. ceylanicum, A. cani- Molecular profiles and phylogeny num, A. duodenale, A. braziliense and A. tubaeforme as For molecular analysis, two adult specimens of Ancylos- well as other hookworm species Necator americanus toma sp. (one female and one male; sample codes FTZ1 (Stiles, 1902), Uncinaria hamiltoni Baylis, 1933 [45], U. and FTZ2, respectively) preserved in 70% ethanol were lucasi Stiles & Hassall, 1901, U. stenocephala (Railliet, air-dried and their mid-body regions (~1 cm) were ex- 1884), U. sanguinis Marcus, Higgins, Slapeta & Gray, cised individually for extraction of genomic DNA using 2014 [46], Uncinaria sp., and Bunostomum phleboto- the Universal Genomic DNA Extraction Kit (TaKaRa, mum (Railliet, 1900), using the Clustal X 1.83 program Dalian, China) according to the manufacturer’s protocol. [47]. During the procedure, the nucleotide alignment of The cephalic and caudal extremities of each specimen cox1 was further adjusted by a codon-guided protein were retained as archived vouchers. The DNA extract alignment. Given the presence of the ambiguous regions was used as template for PCR amplifications at the nu- within these alignments, an online version of GBlocks clear internal transcribed spacer ITS1-5.8S-ITS2 region (http://molevol.cmima.csic.es/castresana/Gblocks_server. (734 bp) and mitochondrial cytochrome c oxidase sub- html) was also introduced here. After refining the align- unit 1 (cox1) locus (393 bp) using primer pairs designed ments using Gblocks, the sequence datasets were used based on the alignments of the relatively conserved re- for phylogenetic analyses using both maximum parsi- gions of the congeneric species A. ceylanicum, A. cani- mony (MP) (PAUP* 4.10b [48]) and Bayesian inference num, and A. duodenale in GenBank. Two PCR primer (BI) methods (MrBayes 3.2 [49]). In the MP analysis, sets were as follows: ITS1-5.8S-ITS2, forward: 5′-GTC heuristic searches were executed by branch-swapping GAA GCC TTA TGG TTC CT-3′ and reverse: 5′-TAA utilizing tree-bisection-reconnection (TBR) algorithm CAG AAA CAC CGT TGT CAT ACT A-3′; cox1, for- and 1,000 random-addition sequence replicates with 10 ward: 5′-ATT TTA ATT TTG CCT GCT TTT G-3′ trees held at each step, and finally the optimal topology and reverse: 5′-ACT AAC AAC ATA ATA GGT ATC with bootstrapping frequencies (BF) was obtained using ATG TAA-3′. The PCR reactions contained ~20 ng of Kishino-Hasegawa, as described previously [50]. For the genomic DNA were performed in 50-μl reaction vol- BI analysis, the nucleotide substitution model GTR + I + umes containing 25 μl 2× Phusion High-Fidelity PCR G was determined using the Bayesian Information Cri- Master Mix (Finnzymes OY, Espoo, Finland), 3 μl teria (BIC) test in jModeltest v. 2.1.6 [51], and the trees gDNA, 3 μL of each primer and 16 μl of ddH O. PCR were constructed employing the Markov chain Monte cycling conditions carried out in a Mastercycler Gradi- Carlo (MCMC) method (chains = 4) over 100,000 (cox1) ent 5331 thermocycler (Eppendorf, Germany) were an or 1,000,000 (ITS1-5.8S-ITS2) generations with every initial denaturation at 95 °C for 5 min; then for ITS1- 100th (cox1) or 1000th (ITS1-5.8S-ITS2) tree being 5.8S-ITS2, 35 cycles of 95 °C for 30 s, 39.8 °C for 30 s, saved; when the average standard deviation of the split and 72 °C for 45 s; but for cox1, 35 cycles at 95 °C for frequencies reduced to less than 0.01, 25% of the first 30 s, 44.1 °C for 30 s, and 72 °C for 30 s; followed by a saved trees were discarded as “burn-in” and the consen- final step at 72 °C for 10 min. For each amplification, sus (50% majority rule) trees were inferred from all samples without parasite gDNA and host DNA as nega- remaining trees and further plotted in TreeviewX tive controls were also included. All PCR products were (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html), Xie et al. Parasites & Vectors (2017) 10:277 Page 5 of 18 Table 1 Information of Ancylostoma species used for molecular identification in the present study Species Gender Host Geographical origin GenBank accession Reference species number ITS1-5.8S-ITS2 cox1 ITS1-5.8S-ITS2 cox1 Ancylostoma ailuropodae Female Giant pandas China (Sichuan) China (Sichuan) KP842923 KP842921 This study n. sp. A. ailuropodae n. sp. Male Giant pandas China (Sichuan) China (Sichuan) KP842924 KP842922 This study Ancylostoma braziliense – Dogs Brazil (Belo Horizonte) – DQ438055 – e Silva et al. [64] A. braziliense – Dogs Brazil (Belo Horizonte) – DQ438056 – e Silva et al. [64] A. braziliense – Dogs Brazil (Belo Horizonte) – DQ438050 – e Silva et al. [64] A. braziliense – Dogs Brazil (Campo Grande) – DQ438060 – e Silva et al. [64] A. braziliense – Dogs Brazil (Belo Horizonte) – DQ438052 – e Silva et al. [64] Ancylostoma caninum Male Humans – Japan (Shiga) – AB751617 Unpublished A. caninum Male Dogs – Australia (Townsville) – NC_012309 Jex et al. [65] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438074 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438071 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438075 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438077 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438072 – e Silva et al. [64] Ancylostoma ceylanicum Male Dogs UK (Nottingham) – DQ381541 – Traub et al. [66] A. ceylanicum – Dogs India (Assam) – DQ780009 – Traub et al. [66] A. ceylanicum – Humans – Cambodia (Preah Vihear) – KF896599 Inpankaew et al. [16] A. ceylanicum – Dogs – Cambodia (Preah Vihear) – KF896602 Inpankaew et al. [16] A. ceylanicum – Humans – Cambodia (Preah Vihear) – KF896604 Inpankaew et al. [16] A. ceylanicum – Humans – Cambodia (Preah Vihear) – KF896601 Inpankaew et al. [16] Ancylostoma duodenale – Humans – China (Zhejiang) – AJ407968 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – AJ407959 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – AJ407942 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – AJ407953 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – NC_003415 Hu et al. [68] A. duodenale –– – China (Xiamen) EU344797 – Unpublished Ancylostoma tubaeforme – Cats – Australia (Townsville) – AJ407940 Hu et al. [67] Xie et al. Parasites & Vectors (2017) 10:277 Page 6 of 18 Table 1 Information of Ancylostoma species used for molecular identification in the present study (Continued) Species Gender Host Geographical origin GenBank accession Reference species number ITS1-5.8S-ITS2 cox1 ITS1-5.8S-ITS2 cox1 A. tubaeforme – Cats – USA (Michigan) JQ812691 – Lucio-Forster et al. [69] Uncinaria hamiltoni Female Sea lions Argentina (Punta Leon) – HQ262116 – Nadler et al. [70] U.hamiltoni Female Fur seals Uruguay (Lobos Island) – HQ262109 – Nadler et al. [70] U.hamiltoni Female Fur seals Uruguay (Cabo Polonio) – HQ262100 – Nadler et al. [70] U.hamiltoni Female Sea lions Uruguay (Cabo Polonio) – HQ262119 – Nadler et al. [70] Uncinaria lucasi Male Sea lions USA (Hazy Island) – HQ262131 – Nadler et al. [70] U. lucasi Female Sea lions Russia (Iony Island) – HQ262149 – Nadler et al. [70] U. lucasi Female Sea lions USA (Hazy Island) – HQ262140 – Nadler et al. [70] U. lucasi Female Sea lions USA (Hazy Island) – HQ262138 – Nadler et al. [70] U. lucasi Female Sea lions USA (Lowry Island) – HQ262142 – Nadler et al. [70] U. lucasi Female Fur seals USA (Reef Rookery) – HQ262078 – Nadler et al. [70] U. lucasi Male Fur seals USA (Adams Cove) – HQ262088 – Nadler et al. [70] U. lucasi Male Sea lions Russia (Iony Island) – HQ262154 – Nadler et al. [70] U. lucasi Female Fur seals Russia (Commander – HQ262067 – Nadler Islands) et al. [70] Uncinaria sanguinis – Sea lions – Australia (Kangaroo Island) – NC_025267 Haynes et al. [71] U. sanguinis – Sea lions – Australia (Kangaroo Island) – KF924756 Haynes et al. [71] Uncinaria stenocephala Female Foxes USA (San Miguel Island) – HQ262052 – Nadler et al. [70] U. stenocephala Female Foxes USA (San Miguel Island) – HQ262053 – Nadler et al. [70] U. stenocephala Male Foxes USA (San Miguel Island) – HQ262054 – Nadler et al. [70] U. stenocephala Male Foxes USA (San Miguel Island) – HQ262055 – Nadler et al. [70] Uncinaria sp. Female Elephant Australia (Macquarie – HQ262127 – Nadler seals Island) et al. [70] Uncinaria sp. Female Elephant Australia (Macquarie – HQ262130 – Nadler seals Island) et al. [70] Uncinaria sp. Female Elephant Australia (Macquarie – HQ262124 – Nadler seals Island) et al. [70] Necator americanus – Humans – China (Zhejiang) – AJ417719 Hu et al. [68] N. americanus – Humans – China (Zhejiang) – NC_003416 Hu et al. [68] N. americanus –– – Togo (−) – AJ556134 Hu et al. [72] Xie et al. Parasites & Vectors (2017) 10:277 Page 7 of 18 Table 1 Information of Ancylostoma species used for molecular identification in the present study (Continued) Species Gender Host Geographical origin GenBank accession Reference species number ITS1-5.8S-ITS2 cox1 ITS1-5.8S-ITS2 cox1 N. americanus – Humans – Central African Republic AB793527 – Hasegawa (−) et al. [73] N. americanus Male Humans – Guatemala (−) AF217891 – Nadler et al. [74] N. americanus –– – China (−) KM891738 – Unpublished N. americanus – Humans – Laos (Thakhek) LC036565 – Unpublished Bunostomum – Sheep China (Heilongjiang) – GQ859497 – Wang phlebotomum et al. [75]; (Outgroup) Male Calf – South Africa (Pretoria) – NC_012308 Jex et al. [65] Sample localities in parentheses with nodal supports expressed as posterior probabil- ZooBank registration: To comply with the regulations ities (PP). The livestock hookworm B. phlebotomum set out in article 8.5 of the amended 2012 version of the was used as outgroup reference and included in each International Code of Zoological Nomenclature (ICZN) phylogenetic analysis. Paralleled to the phylogenies, [53], details of the new species have been submitted to among the genus Ancylostoma the new hookworm ZooBank. The Life Science Identifier (LSID) of the art- species coupled with A. ceylanicum, A. caninum, A. icle is urn:lsid:zoobank.org:pub:A2492E99-AA70-4A58- duodenale and A. tubaeforme was also subjected to detec- AB70-7FED78E726A3. The LSID for the new name tion of synonymous and non-synonymous mutations in Ancylostoma ailuropodae n. sp. is urn:lsid:zoobank.or- the mitochondrial cox1 gene using their corresponding g:act:2C6B6C1E-5F70-49B7-A303-B4D5AE9C7847. protein sequences, followed by determination of genetic Etymology: The new species is named for the type-host. distances between them using a distance matrix based on the maximum composite likelihood model in MEGA [52]. Description General. Slender, relatively small nematodes of white col- Results oration in life (Fig. 2a). Body cylindrical, tapering toward Family Ancylostomatidae Looss, 1905 cephalic and caudal extremities with fine transversely stri- Genus Ancylostoma (Dubini, 1843) ated cuticle; head oriented dorsally in males and females. Buccal capsule widening posteriorly to prominent oral Ancylostoma ailuropodae Yang, Hoberg & Xie n. sp. aperture, possessing two pairs of ventrolateral teeth and Type-host: Giant panda Ailuropoda melanoleuca (David) two pairs of triangular dorsolateral teeth (Fig. 2b-f). (Mammalia: Carnivora: Ursidae). Ventrolateral teeth vary in size and shape, with small, sub- Type-locality: Fengtongzai Natural Reserve (30°42′12″ aduncate inner and large triangular outer teeth extending N, 102°56′14″E), Baoxing, Sichuan Province, China. dorsally. Dorsal gland well developed, associated with rod- Type-material: Holotype, adult male (GYY-XY 1301); like oesophagus, slightly swollen posteriorly, terminating allotype, adult female (GYY-XY 1308); paratypes, three in a lobed valve at junction with intestine (Fig. 4a, b). adult males (GYY-XY 1302-4) and three females (GYY-XY Nerve-ring at midlevel of oesophagus. Cervical papillae 1309-11). All materials, together with nine vouchers (three well developed, conical, situated posterior to level of males, GYY-XY1305-7; six females, GYY-XY13012-17) con- nerve-ring. Excretory pore opens at level between cervical taining one male and one female represented by cephalic papillae and nerve-ring (Fig. 5a1, 2). and caudal extremities, with the mid-body sub-sampled for DNA sequence analysis, are deposited at the Department of Male. [Based on the holotype and three males.] Body Parasitology in Sichuan Agricultural University, Sichuan, length 8.60–12.00 (10.30) mm, maximum width at mid- China. Collectors: GY Yang, TF Zhang and Y Xie. body 500–520 (510). Buccal capsule 180–220 (200) long, Site in host: Small intestine (most in the duodenum). 120–160 (140) wide in dorsoventral view; oesophagus Representative DNA sequences: Representative nuclear 960–1,500 (1,230) long, 150–190 (170) wide; ribosomal and mitochondrial DNA sequences were de- oesophageal length 12% of total body. Cervical papillae posited in the GenBank database under the accession 600–750 (680), excretory pore 500–580 (530), nerve-ring numbers KP842923–KP842924 (ITS1-5.8S-ITS2) and 390–520 (425) posterior to cephalic extremity. Copula- KP842921–KP842922 (cox1). tory bursa well developed, broader than long; dorsal lobe Xie et al. Parasites & Vectors (2017) 10:277 Page 8 of 18 ab c de f Fig. 2 Photomicrographs of adults of Ancylostoma ailuropodae n. sp. a Total view of males (top) and females (down); b-f Cephalic extremity: lateral view of mouth (b and c), showing dorsolateral and ventrolateral teeth; dorsoventral view of mouth (d-f), showing dorsolateral (d) and ventrolateral (e and f) teeth with their positions, shapes and sizes. The arrangements of dorsolateral (2 pairs; b-d) and ventrolateral (2 pairs; b, c, e and f) teeth are indicated by red arrows small with lateral lobes projecting in direction of lateral extremity. Vulva opens ventrally in posterior third of trunks (Figs. 3a-f, 5a5). Dorsal ray thick, 280–390 (350) body, at 2,450–4,686 (3,480) from caudal extremity; vagina in length, 40–60 (52) in maximum width; bifurcating at relatively short. Female reproductive system amphidelphic, 270–295 (280) from anterior into 2 branches; each with poorly differentiated vestibule, paired sphincters and branch further dividing into 2 sub-branches; externodor- infundibula confluent with uterine and ovarian stems sal rays arcuate, arising from dorsal ray at same level (Fig. 5a3). Tail 90–370 (230) long, terminating in acute, (Figs. 3e-f, 5a5). Lateral rays slender, tapering, and arcu- spine-like point 9–25 (17) in length (Figs. 4g, 5a4). Eggs ate with a common stem. Anterolateral ray bending oval, 54–71 × 28–38 (62 × 33) (n = 20) (Fig. 5a6). anteriad, with medio- and posterolateral rays projecting in parallel, extending to edge of bursa (Figs. 3a-b, 5a5). Remarks Antero- and posteroventral rays merge at base and then Ancylostoma ailuropodae n. sp. is established based on divide, continuing parallel deep into cleft (Figs. 3c-d, comparisons to available descriptions among congeners 5a5). Spicules tawny colored, paired, equal, filiform, in the global fauna [6, 7, 9, 10, 54–62]. Ancylostoma 2,000–2,900 (2,450) long (Figs. 4e-f, 5a5). Gubernaculum ailuropodae is unequivocally differentiated from conge- fusiform, 80–120 (90) long, 12–20 (16) wide (Figs. 4c, ners by structural characteristics of male and female 5a5). Cloacal papillae (n = 7) (Figs. 4d, 5a5): 1 pair dis- specimens including body size, arrangement, number posed dorsally, 1 pair laterally, 3 single papillae ventrally. and dimensions of buccal teeth and shape of the buccal capsule, and in males by the configuration of the dorsal Female. [Based on the allotype and three females.] Body ray and bursa and lengths of spicules and gubernaculum, 9.80–16.00 (12.90) mm long, with maximum width at respectively (see Table 2 and Fig. 5b). Of note, tooth- mid-body 560–740 (650); width at anus 270–340 (285). number appears to represent one of the key morpho- Buccal capsule 170–250 (210) long, 130–190 (160) wide logical characters separating A. ailuropodae from other in dorsoventral view; oesophagus 1,280–1,320 (1,300) species of Ancylostoma. Specifically, (i) A. ailuropodae long, 170–250 (200) in maximum width near base. Cer- differs from A. caninum, A. tubaeforme and A. taxideae vical papillae 800–1,230 (900), excretory pore 760–950 by the number (2 vs 3 pairs) of ventrolateral teeth; and (ii) (820), nerve-ring 600–650 (620) posterior to cephalic from A. ceylanicum, A. braziliense, A. duodenale, A. Xie et al. Parasites & Vectors (2017) 10:277 Page 9 of 18 50 m µ 50 m µ 50 m µ a ce 50 m µ 50 m µ 50 m µ bd f Fig. 3 Photomicrographs of Ancylostoma ailuropodae n. sp. male, caudal extremity. a, b Lateral view of bursa showing position of lateral rays and genital cone. c, d Ventral view of bursa showing configuration of antero- and postero-ventral rays. e, f Dorsal view of bursa showing relationships of the dorsal and externodorsal rays; note configuration of the bifurcations of the dorsal ray. Arrows in a, c and e denote the rays which are magnified in panels b, d and f, respectively kusimaense, A. paraduodenale and A. malayanum by the Sequence characterization number (2 vs 0/1 pairs) of triangular dorsolateral teeth. For ITS1-5.8S-ITS2, the 734 bp sequences from FTZ1 Furthermore, the shape of the dorsal rays appears to be and FTZ2 were identical and had 52.2% A + T content. another potential species-specific morphological indicator BLAST analysis revealed that A. ailuropodae shared the (Fig. 5b). Specimens of A. ailuropodae n. sp. vary from A. highest identity with A. ceylanicum (99.6%), followed by tubaeforme by differences in cleft length of two digitations 98.8% identity with A. duodenale, 97.2% with A. tubae- in each branch (Fig. 5b9 and 10) and further from A. taxi- forme, 95.8% with A. caninum, and 92.6% with A. brazi- deae, A. duodenale, A. paraduodenale, A. caninum, A. liense. Based on the identities, there were a total of 59 malayanum, A. kusimaense, A. ceylanicum and A. brazi- variable positions found in the pairwise alignment of liense by the absence of a third digitation in each branch ITS1-5.8S-ITS2, including 17 parsimony-informative (Fig. 5b1–8 and 10). Verified specimens of A. genettae, A. and 42 singleton sites (data not shown). Within cox1 se- protelesis and A. somaliense have not yet been described quences, same base composition (A = 23.4%; C = 9.7%; and these three species were not included in the compari- G = 22.6%; T = 44.3%) and sequence length (393 bp) son above. Notably, the adults of both A. pluridentatum were also observed in these two representative indi- and A. buckleyi can be distinguished from the new species viduals of A. ailuropodae,withanA+T contentof by the number of ventrolateral teeth, given that A. pluri- 67.7%, a typical mitochondrial nucleotide feature in dentatum has only one pair while A. buckleyi has three nematodes (towards AT). BLAST search against Gen- pairs according to the original descriptions (e.g. [60, 63]). Bank/DDBJ/EMBL databases once again showed the Based on these morphological attributes, A. ailuropodae is highest nucleotide identity existing between the new considered to be a previously unrecognized species within species and A. ceylanicum (92.6%), followed by 89.2% the genus Ancylostoma. identity between A. ailuropodae and A. tubaeforme, 88.6% between A. ailuropodae and A. duodenale,and Molecular characterization 86.0% between A. ailuropodae and A. caninum,to- To further probe the taxonomic position of A. ailuropodae, gether corresponding to 99.2–100% identities at the both nuclear ITS1-5.8S-ITS2 and mitochondrial cox1se- amino-acid level. In terms of identity comparisons, quences from two representative specimens (codes FTZ1 there were a total of 78 variable positions in the and FTZ2, respectively) were obtained and subjected to se- 378 bp pairwise alignment, including 28 parsimony- quence characterization and phylogenetic analyses. informative and 50 singleton sites. Xie et al. Parasites & Vectors (2017) 10:277 Page 10 of 18 a cd 50 m µ 50 m µ b f 50 m µ 100 m µ 100 m µ 50 m µ Fig. 4 Photomicrographs of adults of Ancylostoma ailuropodae n. sp. a Dorsoventral view of anterior region of female, showing buccal capsule and entire oesophagus. b Lobed valves between oesophagus and intestine. c Ventrolateral view of male tail, showing gubernaculum. d Ventral view of male tail, showing cloacal papillae. e, f Ventral and ventrolateral views of male tail, showing spicules from both proximal (e) and distal (f) extremities. g Lateral view of female tail with spine-like point. Arrows indicate some small structures, including cervical papillae (a), lobed valve (b), gubernaculum (c), cloacal papillae (d), spicules (e, f) and spine-like point of female tail (g) Further, we located these sites and determined if there tubaeforme, 0.127 to A. duodenale, and 0.151 to A. cani- were non-synonymous substitutions apparent via com- num (not shown). parison of their protein sequences, and the results are shown in Fig. 6. Out of 78 variable base sites, 13 were Phylogenetic characterization unique for A. ailuropodae (in red); 16 were identical be- Phylogenetic relationships between A. ailuropodae and tween A. ailuropodae and one of A. ceylanicum, A. duo- other species were inferred from the respective se- denale, A. caninum and A. tubaeforme (in orange); and quences of ITS1-5.8S-ITS2 and cox1 using both MP and 49 were shared between A. ailuropodae and any two or BI algorithms and their corresponding tree topologies three of these four congeneric species (in yellow). are shown in Fig. 7. Although the two consistent struc- Among the 49 variable sites, however, the non- tures (MP/BI) topologically varied from each other synonymous substitutions A/G in A. ceylanicum and due to the different reference species included, both T/A in A. caninum led to their amino acid changes: I trees provided an identical, robust phylogenetic reso- (Ilu)→ V (Val) in the former and I (Ilu)→ N (Asn) in lution for A. ailuropodae within the genus Ancylos- the latter (see Fig. 6). In addition, analysis of genetic dis- toma and for the genus Ancylostoma within the tances using maximum composite likelihood estimates family Ancylostomatidae. Specifically, (i) the two A. ailur- placed A. ailuropodae close to A. ceylanicum with the opodae specimens clustered together as a monophyletic minimum interspecific evolutionary divergence (0.084), group that was separated from the other Ancylostoma spe- compared with 0.121 evolutionary divergence to A. cies. (ii) When the congeneric species A. ceylanicum, A. 100 µm Xie et al. Parasites & Vectors (2017) 10:277 Page 11 of 18 200 µm 1 2 3 4 5 6 7 8 9 10 Fig. 5 Line drawings of Ancylostoma ailuropodae n. sp. and comparison of dorsal rays among Ancylostoma spp. a Morphological structures of A. ailuropodae n. sp.: 1, dorsoventral view of anterior region; 2, lateral view of anterior region; 3, lateral view of female vulval region; 4, lateral view of female caudal region; 5, dorsal view of male caudal region; 6, egg. b Ten Ancylostoma species for comparison of dorsal rays: 1, A. taxideae [10]; 2, A. duodenale [9]; 3, A. paraduodenale [6]; 4, A. caninum [58]; 5, A. malayanum [7]; 6, A. kusimaense [9]; 7, A. ceylanicum [9, 11]; 8, A. braziliense [9, 11]; 9, A. tubaeforme [58]; 10, A. ailuropodae n. sp caninum A. duodenale and A. tubaeforme were considered re-construct this phylogenetic relationship using the in our cox1-based analysis (Fig. 7a), A. ailuropodae and A. ITS1-5.8S-ITS2 data (Fig. 7b), A. ailuropodae remained as ceylanicum were more closely related to each other than the putative sister of A. ceylanicum,regardless of isolate to A. caninum, A. tubaeforme and A. duodenale,withro- origins (one from the UK and another from India; see bust support for tree topology (BP = 95 and PP = 0.99). Table 1), with high statistical support (BP = 89 and PP = (iii) When another species, A. braziliense, was added to 0.91), which was in agreement with the inferences from 200 µm 200 µm 200 µm 200 µm 20 µm Xie et al. Parasites & Vectors (2017) 10:277 Page 12 of 18 Table 2 Key comparisons between A. ailuropodae n. sp. and other congeneric Ancylostoma spp Species Body size (mm) Ventrolateral teeth Spicules (μm) Gubernaculum (μm) Hosts References A. caninum M: 11.0–13.0 × 3 pairs 730–960 nr Dogs; cats; humans; Burrows [58] 0.34–0.39 (860) wild canids and felids (11.7 × 0.37); F: 14.0–20.5 × 0.50–0.56 (17.0 × 0.52) A. ceylanicum M: 7.91 ± 0.04 × 2 pairs (outer large; 740 ± 20 77 ± 1.64 × 10 Dogs; cats; humans; Yoshida [9] 0.35 ± 0.02; inner very small) wild canids and felids F: 9.48 ± 0.81 × 0.42 ± 0.04 A. braziliense M: 6.84 ± 0.50 × 2 pairs(outer large; 800 ± 70 73 ± 1.94 × Humans Yoshida [9]; 0.24 ± 0.02; inner minute) 10 ± 0.44 Norris[59] F: 8.67 ± 0.68 × 0.34 ± 0.01 A. duodenale M: 10.67 ± 0.17 × 2 pairs (similar in 1,800 ± 90 131 ± 1.49 × Humans Yoshida [9] 0.47 ± 0.03; both size and shape) 13 ± 0.42 F: 12.67 ± 1.12 × 0.64 ± 0.03 A. kusimaense M: 7.82 ± 0.20 × 2 pairs (outer large; 840 ± 4 84 ± 0.71 × Raccoon dogs Yoshida [9] 0.28 ± 0.01; inner small) 10 ± 0.44 F: 9.12 ± 0.55 × 0.33 ± 0.02 A. tubaeforme M: 6.84 ± 0.50 × 3 pairs 1,100–1,470 nr Cats Burrows [58] 0.24 ± 0.02; (1,290) F: 8.67 ± 0.68 × 0.34 ± 0.01 A. paraduodenale M: 5.0–8.0 × 2 pairs (outer stouter 1,100–1,500 80 × 20 Servals Biocca [6] 0.21–0.24 than inner) (1,250) (6.8 × 0.23): F: 6.5–8.5 × 0.26–0.32 (7.7 × 0.29) A. malayanum M: 11.02– 2 pairs (outer large, 2,490–2,620 112 Bears Wu et al. [7] 13.80 × vertical; inner small, 0.46–0.51; subaduncate; one F: 20.40 × 0.54 pair of triangular dorsolateral teeth) A. taxideae M: 8.37 ± 1.92 × 3 pairs (one pair 1,470 ± 87 138 ± 2 × 15 Badgers Kalkan & 0.32 ± 0.02; of triangular Hansen [10] F: 16.05 ± 1.30 × dorsolateral teeth) 0.46 ± 0.05 A. ailuropodae n. sp. M: 10.30 ± 1.70 × 2 pairs (similar in 2,000–2,900 80–120 × Giant panda This study 0.51 ± 0.01; both size and shape; (2,450) 12–20 F: 12.90 ± 3.10 × two pairs of triangular (90 × 16) 0.63 ± 0.09 dorsolateral teeth) Abbreviation: nr not reported; the source paper presented no data on the species under consideration Only the length of gubernaculum was found in the original description [7] the cox1 gene analysis (see Fig. 7a). (iv) The inter- Discussion relationships of A. ailuropodae, A. ceylanicum, A. cani- Hookworms in the genus Ancylostoma cause significant num, A. duodenale, A. braziliense and A. tubaeforme in medical and veterinary disease (ancylostomiasis) in vari- the genus Ancylostoma; U. sanguinis, U. hamiltoni, U. ous hosts including humans and domestic and wild lucasi, U. stenocephala and Uncinaria sp. in the genus mammals [2, 71]. Recent epidemiological surveys Uncinaria;and N. americanus in the genus Necator,dem- revealed that some wild animal-derived species of Ancy- onstrated phylogenetic stability of these monophyletic lostoma are emerging as important helminthic zoonotic groups, with the current analyses being consistent with agents because of rapid urbanization and increased previously proposed molecular phylogenies of the hook- human-wildlife interactions [11, 13–21]. The giant worms based on the nuclear ribosomal and mitochondrial panda, for example, is an endangered and rare wild spe- DNA data [64–75]. cies in China that has been artificially protected and Xie et al. Parasites & Vectors (2017) 10:277 Page 13 of 18 Fig. 6 Simultaneous alignments of nucleotide and amino-acid sequences of mitochondrial cox1 genes from Ancylostoma ailuropodae n. sp. and its congeneric species. For the alignments, the nucleotide sequences of cox1 genes were retrieved from the GenBank database (species and accession numbers are indicated in parentheses): Aai (A. ailuropodae n. sp.; KP842921), Ace (A. ceylanicum; KF896601), Aca (A. caninum; AB751617), Adu (A. duodenale; NC_003415), and Atu (A. tubaeforme; AJ407940). The corresponding protein sequences were deduced based on the Invertebrate Mitochondrial Code. Both nucleotide and amino-acid sequences were aligned with Clustal X 1.83 program. Regions of identity in either nucleotide (*) or amino-acid (#) are indicated. Variable base loci in Aai unique for A. ailuropodae n. sp. are highlighted in red; those shared between A. ailuropodae n. sp. and one of A. ceylanicum, A. duodenale, A. caninum and A. tubaeforme are highlighted in orange; and those shared between A. ailuropodae n. sp. and any two or three of these four congeneric species are highlighted in yellow.The 250 251 non-synonymous substitutions A/G in A. ceylanicum and T/A in A. caninum as well as their amino-acid changes: I (Ilu)/V (Val) and I (Ilu)/N (Asn) are noted in red with a red star. Percentages of nucleotide and amino-acid identities with respect to Aai are shown at the end of each sequence even partially housed for decades due to habitat loss Ancylostoma spp. [55, 56]. Among this assemblage, it is [33]. Clinically unidentified specimens of Ancylostoma in important to note that A. ailuropodae is clearly structur- giant pandas had been confirmed by veterinarians and ally distinct from A. malayanum, the only other species wildlife biologists since the last century, but their poten- of Ancylostoma known in ursid hosts (e.g. Ursus thibeta- tial zoonotic importance remains to be defined [41]. In nus) (Table 2), with the implication that each of these the present study, A. ailuropodae n. sp. was isolated species endemic to China may be more closely related to from the giant panda, morphologically characterized and other congeners within the genus. Specimens upon demonstrated to be closely related to the anthropozoo- which the description and differentiation of A. ailuropo- notic A. ceylanicum by molecular analysis. dae n. sp. was based were restricted to fully developed In general, morphological identification is a conven- adults and eggs. Further work, using a combined tional and authoritative approach to define a new laboratory-egg cultivation and Baermann technique, to nematode parasite species. Concerning the genus Ancy- describe the morphology of developmentally advanced lostoma, several common species can be morphologically larval stages is needed to complement morphological differentiated by key characters such as body size, teeth characteristics of the new species, and to provide valu- of the buccal capsule and shape of bursal rays (see able information assisting in species identification and Table 2 and Fig. 5b; cf. [9]). Similarly, specimens of A. differentiation in this genus [55, 76]. ailuropodae from giant pandas are separated from other Following our morphological evidence, A. ailuropodae hookworms on the basis of either ventrolateral and from giant pandas was further confirmed as an inde- dorsolateral teeth or dorsal rays, supporting the previous pendent species by molecular analysis. For example, the conclusions that teeth and rays were reliable morpho- internal transcribed spacer region (ITS1-5.8S-ITS2) logical indicators in the differential diagnosis of of the nuclear ribosomal DNA is regarded as an Xie et al. Parasites & Vectors (2017) 10:277 Page 14 of 18 ab Fig. 7 Phylogenetic relationships of hookworms isolated from the giant panda with the related hookworms in the family Ancylostomatidae. Phylogeny was inferred on the basis of mitochondrial cox1(a) and nuclear ITS1-5.8S-ITS2 (b) sequences using both maximum parsimony (MP) and Bayesian inference (BI) methods. The livestock hookworm Bunostomum phlebotomum represented the outgroup species. Taxa belonging to the three major genera including Ancylostoma, Uncinaria and Necator in the family Ancylostomatidae are indicated by differently colored rectangles and shown in both phylogenetic topologies. The numbers along the branches indicate bootstrap values resulting from different analyses in the order MP/BI; values less than 50% are shown as “-” appropriate genetic marker to resolve nematode rela- Based on the results from integrated molecular and tionships at the species level [77]. Pairwise comparisons morphological comparisons, we propose that A. ailuro- of ITS1-5.8S-ITS2 in A. ailuropodae with congeneric podae of giant pandas is a previously unrecognized and species available in the GenBank database revealed a separate species that is closely related to the anthropo- species-specific sequence feature (containing 59 variable zoonotic A. ceylanicum within the genus Ancylostoma. informative sites) and overall identity of 92.6–99.6% Additional information regarding the ultrastructure and among A. ceylanicum, A. tubaeforme, A. caninum and A. genomics of this species and other related hookworms is braziliense. Furthermore, high bootstrap support was still required. Broader taxonomic comparisons can pro- evident, based on phylogenetic analysis of ITS1-5.8S- vide an increasingly precise morphological and molecu- ITS2 that demonstrated monophyly of A. ailuropodae as lar basis for species recognition among hookworms. In the putative sister of A. ceylanicum (see Fig. 7b). addition, there were two non-synonymous base substitu- Critically, similar conclusions were reinforced by ana- tions detected in cox1 genes of A. ceylanicum (A/G ) lysis of the mitochondrial cox1 gene. It should also be and A. caninum (T/A ) (Fig. 6) that were confirmed to noted that cox1 analysis was included because recent be fixed and species-specific after homologous compari- studies of the substitution patterns for nematode mito- sons with other A. ceylanicum or A. caninum isolates chondrial genes (e.g. cox1 and nad4) revealed that they from two sites in the same geographic area. have utility in identifying and differentiating novel or Ancylostoma ailuropodae identified here is the fourth cryptic species among closely related taxa due to as- hookworm to be described from the Ursidae. Previously, sumed faster evolutionary rates than nuclear genes, fea- the hookworm Uncinaria yukonensis (Wolfgang, 1956) tures of maternal inheritance and absence of was characterized in black bears and Uncinaria rauschi recombination [78–80]. Compared to the nuclear ITS, (Olsen, 1968) in grizzly and black bears [81, 82]. On the the cox1of A. ailuropodae appeared to have more vari- basis of comparisons of morphometric and distribution able informative sites (n = 78, including 13 unique loci). data of ursine hookworms as well as the historical bio- Nevertheless, results based on cox1 were consistent with geography of bears, Catalano et al. [13] proposed that inference from ITS, in revealing a sister-species relation- there was a relatively recent host-switching event of U. ship with A. ceylanicum among a broader assemblage of rauschi from black bears to grizzly bears. congeners in the genus. Phylogenetic analysis of cox1 The occurrence of A. ailuropodae appears consistent data (Fig. 7a) also supported the contention that A. with speciation following a host colonization event to ailuropodae n. sp. is an independent species which is giant pandas apparently from a carnivoran source in clearly differentiated from A. ceylanicum, A. caninum, sympatry, and further indicates a history of independent A. tubaeforme and A. duodenale. association with ursine hosts for the broader Xie et al. Parasites & Vectors (2017) 10:277 Page 15 of 18 ancylostomatid hookworm assemblage. The timing and phylogenies [23, 24]. These relationships alone would geographic source for these hookworms cannot be eluci- serve to refute a coevolutionary hypothesis for Ancylos- dated based on the currently available data and the re- toma hookworms among bears, conversely supporting a duced and relictual distribution for pandas, but a history history of independent events of host colonization that of host colonization is compatible with the current tree have structured this fauna. topology (for parasites and hosts) and distribution of Unlike U. yukonensis and U. rauschi in bears, the carnivore hosts for other species of Ancylostoma (e.g. hookworm from giant panda is genetically similar to [24]). We suggest that acquisition of Ancylostoma by other Ancylostoma species (Fig. 7). These respective gen- giant pandas likely occurred prior to 7 million years era are referred to two independent subfamilies within ago (MYA) when a shift from an omnivorous diet to the Ancylostomatide, namely Ancylostomatinae Looss, one dominated strictly by bamboo (by 2.4 MYA) was 1905 for Ancylostoma and Bunostominae Looss, 1911 underway [25]. for Uncinaria, consistent with extended evolutionary Divergence of A. ailuropodae appears to have occurred trajectories for these taxa among the hookworms. This prior to acquisition of A. ceylanicum by humans in suggests the independent origin of A. ailuropodae, sup- Southeast Asia, and prior to the intense bottlenecking of porting monophyly of A. ailuropodae and congeneric giant panda populations that has characterized the past species A. ceylanicum, A. duodenale, A. tubaeforme, A. century (cf. [33, 83] for details about the history of giant caninum and A. braziliense, and strengthens the close pandas). This interpretation is significant, as it would re- relationship between the giant panda hookworm and A. late to the historical independence of A. ailuropodae and ceylanicum within the clade. Concurrently it suggests A. ceylanicum before the current intensified conserva- that Uncinaria spp. from pinnipeds and ursids are a dis- tion campaign for maintaining giant pandas, and the po- tinct monophyletic group in the family Ancylostomati- tential for cross-transmission of both hookworm species dae [70]. The apparent genetic differences of A. when infected humans are in contact. The unique niche ailuropodae n. sp. in pandas and U. rauschi and U. yuko- and specialized bamboo-feeding habits of giant pandas nensis in bears, coupled with their divergent biogeo- suggest that colonization in ecological time, related to graphic and ecological histories suggest this system as a the source or origin of A. ailuropodae, was unlikely good model for exploring the complexities of diversifica- given relative isolation with respect to a sympatric as- tion and faunal assembly in the evolution of host range semblage of carnivorans or other mammals that may and associations among hookworms (e.g. [85–88]). serve as hosts for species of hookworms [25, 26, 33]. The potential for genetic partitioning among possible Parasitological inventory among potential carnivoran disjunct populations of hookworms in giant pandas hosts in Sichuan and nearby regions remains necessary should be considered, as it will reflect information about to demonstrate that A. ailuropodae has a narrow host the timing of colonization to giant pandas and the dur- range and may now be limited to the giant panda [84]; ation of the history of association. Further, the history of apparent narrow host range, however, does not preclude fragmentation and isolation for giant pandas across now the potential or capacity for contemporary host switches isolated mountain systems in southwestern China sug- to humans as a zoonotic parasite given opportunity due gests a complex relationship among hosts and hook- to permissive ecological circumstances [85–88]. worms in this region. Such history could be explored Phylogenetic and historical isolation of giant pandas through fecal-based approaches in conjunction with mo- from the broader assemblage of ursids and ursine bears lecular diagnostics to examine occurrence and the extent (e.g. [23, 24]) in conjunction with apparent structural di- of genetic diversity and distribution for hookworm para- vergence (e.g. teeth and configuration of the dorsal ray; sites among populations and subspecies of giant pandas. Table 2 and Fig. 5b5 and 10) of A. ailuropodae and A. malayanum suggests that independent events of host Conclusions colonization, separated in space and time, were essential This study is the first to describe and define a new mem- in the process of speciation for these hookworms; mo- ber of the genus Ancylostoma, A. ailuropodae, in the lecular data, particularly from A. malayanum, is still wild giant panda using morphological and molecular cri- needed to explore this hypothesis. Moreover, phylogen- teria. Morphological characters (e.g. ventrolateral (two etic hypotheses for the Ursidae have placed giant pandas pairs) and dorsolateral (two pairs) teeth and dorsal rays) distantly from species of Ursus (and other ursines) near distinctly separate A. ailuropodae n. sp. from other con- the base of an extensive radiation for bears that unfolded generic species in the genus Ancylostoma. Further, nu- across the late Miocene and Pliocene [24]. Among ursine clear ITS1-5.8S-ITS2 and mitochondrial cox1-based hosts for Ancylostoma, U. thibetanus (Asiatic black bear) genetic distance analysis and phylogenies supported the is regarded as the sister of U. americanus (American black assertion that A. ailuropodae is independent and shares bear) and placed among crown species in ursid a sister-species relationship with the anthropozoonotic Xie et al. Parasites & Vectors (2017) 10:277 Page 16 of 18 A. ceylanicum. Although additional molecular evidence of Agriculture, Agricultural Research Service, Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, Beltsville, is warranted, this finding should enhance public aware- Maryland 20705, USA. United States Department of Agriculture, Agricultural ness of parasitic hookworms in giant pandas, especially Research Service, Beltsville Agricultural Research Center, Animal Parasitic in captive populations that have frequent contact with Disease Laboratory, Beltsville, Maryland 20705, USA. Department of Civil and Environmental Engineering, University of Maryland, College Park, Maryland breeders, veterinarians and even tourists. Moreover, the 20740, USA. morphological and molecular data presented here en- hances the information on species within the genera Received: 7 April 2017 Accepted: 18 May 2017 Ancylostoma, Uncinaria, and Necator and contributes to a more complete understanding of the taxonomy, diag- References nostics and evolutionary biology of hookworms. 1. Hotez PJ, Brooker S, Bethony JM, Bottazzi ME, Loukas A, Xiao S. Hookworm infection. N Engl J Med. 2004;351:799–807. Abbreviations 2. Brooker S, Bethony J, Hotez PJ. Human hookworm infection in the 21st BF: Bootstrapping frequencies; BI: Bayesian inference; BIC: Bayesian century. Adv Parasitol. 2004;58:197–288. information criteria; cox1: Cytochrome c oxidase subunit 1; ITS: Internal 3. Schad GA, Warren KS. Hookworm disease: current status and new transcribed spacer (ITS1-5.8S-ITS2); MCMC: Markov chain Monte Carlo; directions. London: Taylor & Francis; 1990. MP: Maximum parsimony; MYA: Million years ago; PP: Posterior probabilities; 4. 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Ancylostoma ailuropodae n. sp. (Nematoda: Ancylostomatidae), a new hookworm parasite isolated from wild giant pandas in Southwest China

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References (92)

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Springer Journals
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Copyright © 2017 by The Author(s).
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Biomedicine; Parasitology; Entomology; Tropical Medicine; Infectious Diseases; Veterinary Medicine/Veterinary Science; Virology
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1756-3305
DOI
10.1186/s13071-017-2209-2
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28576124
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Abstract

Background: Hookworms belonging to the genus Ancylostoma (Dubini, 1843) cause ancylostomiasis, a disease of considerable concern in humans and domestic and wild animals. Molecular and epidemiological data support evidence for the zoonotic potential among species of Ancylostoma where transmission to humans is facilitated by rapid urbanization and increased human-wildlife interactions. It is important to assess and describe these potential zoonotic parasite species in wildlife, especially in hosts that have physiological similarities to humans and share their habitat. Moreover, defining species diversity within parasite groups that can circulate among free-ranging host species and humans also provides a pathway to understanding the distribution of infection and disease. In this study, we describe a previously unrecognized species of hookworm in the genus Ancylostoma in the giant panda, including criteria for morphological and molecular characterization. Methods: The hookworm specimens were obtained from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China in November 2013. They were microscopically examined and then genetically analyzed by sequencing the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in two representative specimens (one female and one male, FTZ1 and FTZ2, respectively). Results: Ancylostoma ailuropodae n. sp. is proposed for these hookworms. Morphologically the hookworm specimens differ from other congeneric species primarily based on the structure of the buccal capsule in males and females, characterized by 2 pairs of ventrolateral and 2 pairs of dorsolateral teeth; males differ in the structure and shape of the copulatory bursa, where the dorsal ray possesses 2 digitations. Pairwise nuclear and mitochondrial DNA comparisons, genetic distance analysis, and phylogenetic data strongly indicate that A. ailuropodae from giant pandas is a separate species which shared a most recent common ancestor with A. ceylanicum Looss, 1911 in the genus Ancylostoma (family Ancylostomatidae). (Continued on next page) * Correspondence: guangyou1963@aliyun.com Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xie et al. Parasites & Vectors (2017) 10:277 Page 2 of 18 (Continued from previous page) Conclusion: Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms. Keywords: Ancylostoma ailuropodae n. sp, Ailuropoda melanoleuca, Morphology, Phylogeny, Ancylostomatidae Background Cuvier; A. pluridentatum in Puma concolor coryi Hookworms (Nematoda: Ancylostomatidae) are one of (Bangs); A. kusimaense in Nyctereutes procyonoides the most common soil-transmitted helminths, causing viverrinus Temminck; A. taxideae in Taxidea taxus serious iron-deficiency anemia and protein malnutrition taxus (Schreber); A. genettae in Genetta genetta (Lin- in humans and domestic and wild mammals [1–3]. Both naeus); A. protelesis in Proteles cristata (Sparrman); and major genera Ancylostoma (Dubini, 1843) and Necator A. somaliense in C. mesomelas [5–12]. Although a di- Stiles, 1903, relegated to two distinct subfamilies, are re- verse assemblage of carnivorans is recognized as hosts sponsible for morbidity and socioeconomic burdens [4]. for Ancylostoma, only one species had been documented Unlike species in the genus Necator, most Ancylostoma or described previously among the Ursidae [7]; species hookworms are considered to be of greater medical and of the distantly related Uncinaria Frölich, 1789, are con- veterinary importance because of distribution, preva- sidered typical in ursine hosts [13]. lence, and multiple zoonotic species [2]. Currently there Recent molecular-based genetic and epidemiological are fourteen valid species identified in the genus Ancy- investigations have shown that among certain wild or lostoma that are often considered in the context of the domestic animal-derived species of Ancylostoma, A. cey- range of hosts that are typically infected. For example, lanicum is becoming the second most common hook- the ‘anthrophilic’ form is limited to Ancylostoma duode- worm found to infect and complete its life-cycle in nale (Dubini, 1843) which principally infects humans. humans [12, 14–18]. Similar transmission and cross- ‘Anthropozoonotic’ forms, capable of circulating among infection cases have been reported for other congeneric free-ranging wild hosts, some domestic hosts and species, notably A. caninum [12, 19, 20] and A. brazi- humans include Ancylostoma caninum (Ercolani, 1859), liense [12]. Such situations highlight the public health Ancylostoma braziliense Gomes de Faria, 1910 and Ancy- significance of hookworm infection and the necessity to lostoma ceylanicum Looss, 1911. Other species, includ- assess their prevalence and distribution, and to identify ing most of the recognized diversity in the genus are their wildlife hosts. This has become especially important considered to be primarily of veterinary importance, in- for wildlife hosts that may have recently adapted to the cluding Ancylostoma tubaeforme (Zeder, 1800), Ancylos- human environment due to rapid urbanization [14, 21] toma malayanum (Alessandrini, 1905), Ancylostoma leading to increased interactions with people in conserva- pluridentatum (Alessandrini, 1905), Ancylostoma para- tion centers and zoological gardens constructed for en- duodenale Biocca, 1951, Ancylostoma kusimaense dangered and valuable animals [22]. Regrettably, little Nagayosi, 1955, Ancylostoma buckleyi Le Roux & Biocca, attention has been broadly paid to the species of Ancylos- 1957, Ancylostoma taxideae Kalkan & Hansen, 1966, toma because of a limited understanding of their diversity, Ancylostoma genettae Macchioni, 1995, Ancylostoma abundance and distribution and the difficulty in etiological protelesis Macchioni, 1995, and Ancylostoma somaliense and epidemiological sampling in the wild [12, 14]. Macchioni, 1995 [5, 6]. It is noteworthy that nearly all of The giant panda, Ailuropoda melanoleuca (David), these species can also be found in wildlife, such as A. one of the most endangered and rare species of China, is duodenale in Crocuta crocuta (Erxleben); A. caninum regarded as one of the preeminent species for wildlife and A. braziliense in Acinonyx jubatus (Schreber) and conservation in the world. Higher taxonomic status for Canis mesomelas Schreber; A. ceylanicum in Canis lupus these enigmatic carnivorans had been unresolved, until dingo Meyer; A. paraduodenale in Leptailurus serval relatively recent decisions that unequivocally placed (Schreber); A. malayanum in Ursus thibetanus G. giant pandas among the Ursidae (e.g. [23–26]). Wild Xie et al. Parasites & Vectors (2017) 10:277 Page 3 of 18 giant pandas currently inhabit six small mountain ranges fresh Ancylostoma specimens and provided an oppor- of China i.e. Qinling, Minshan, Qionglai, Daxiangling, tunity to fill some of these gaps in our knowledge. We Xiaoxiangling and Liangshan (Fig. 1), with an estimated have used DNA sequence and morphological analysis, population size of ~1,864 [27–30]. Since the 1950s, nu- applying clear species criteria established in a phylogen- merous natural reserves, conservation centers, research etic context [42], to recognize and describe a previously bases and zoological gardens were specifically estab- unknown hookworm species from the giant panda. A lished by the Chinese government to protect this threat- putative sister-species relationship with the ‘anthropo- ened species [31]. Some of these wild giant pandas have zoonotic’ A. ceylanicum suggests a possible zoonotic risk become closely associated with humans as they are for transmission and infection to humans. housed for artificial breeding and conservation and bio- logical investigations. Also, some pandas have been dis- Methods played publically as the ‘messenger of peace and Parasite collection and microscopic examination friendship’ around the world [32]. Although ecological, In November 2013, a wild female giant panda was found genetic and etiological studies have shown that the dead in the Fengtongzai Natural Nature Reserve, panda faces the threat of extinction due to habitat loss, Sichuan Provence of China (Fig. 1). After a routine nec- poor reproduction and low resistance to infectious dis- ropsy, seventeen hookworm specimens (seven males and eases [33, 34], recent surveys strongly indicate that para- ten females) were collected from the small intestine sitic infections represent the leading health threat to under the Scientific Procedures Premises License for the giant pandas of China [35–40]. College of Veterinary Medicine, Sichuan Agricultural Hookworm parasites have been frequently observed in University (Sichuan, China). In addition, parasite eggs the intestines of wild dead giant pandas since 1995 [28] were isolated from the intestinal content by the and the first record, attributed to a species of Ancylos- centrifuge-flotation method using saturated MgSO [43]. toma, was reported by Zhang et al. in 2005 [41]. How- After washing in physiological saline, the hookworm ever, detailed morphological descriptions, determination specimens were either directly fixed in Berland’s fluid of taxonomic status and indicators of pathogenicity of (95% glacial acetic acid and 5% formaldehyde) for mor- the Ancylostoma sp. derived from giant panda are lack- phological analysis or stored in 70% ethanol for subse- ing. The recent collection of parasites from a wild giant quent molecular profiling. For morphology, the panda that died in the Fengtongzai Natural Reserve in hookworms were identified to the genus level on the Sichuan Province of China resulted in the recovery of basis of the existing taxonomic keys and descriptions of Fig. 1 Sampling site in China (red circle) for Ancylostoma ailuropodae n. sp. in the giant panda. The distribution of the giant panda populations in Shaanxi, Gansu and Sichuan provinces of China is indicated in black with the names of mountain ranges Xie et al. Parasites & Vectors (2017) 10:277 Page 4 of 18 Ancylostoma spp. (e.g. [44]). In brief, the worms (n = 15; examined on agarose (1%) gels to verify that they repre- 6 males and 9 females) were prepared as temporary sented the target bands. The corrected gel-isolated whole mounts in glycerin after clearing in lactophenol amplicons were column-purified and sequenced in both and examined under both dissecting and light micros- directions using terminator-based cycle sequencing with copy at magnifications of 10–40× and 40–200×, respect- BigDye chemistry (Applied Biosystems, Foster City, CA, ively; male and female specimens were characterized USA) on an ABI 3730 DNA sequencer (Applied Biosys- morphologically including photo-micrographic imaging tems) in TaKaRa Biotechnology Co. Ltd. (Dalian, China). and morphometrics. Measurements are given in micro- To ensure maximum accuracy, each amplicon was se- metres (μm) unless specified otherwise and presented quenced three times independently. The consensus se- with the range followed by the mean within parentheses. quences were utilized for the following bioinformatic In addition, some key characteristics of the adults were analyses and added to GenBank under the accession drawn with the aid of serial photographs for morpho- numbers KP842923 (FTZ1) and KP842924 (FTZ2) for logical comparison and differentiation from other related ITS1-5.8S-ITS2 and KP842921 (FTZ1) and KP842922 species. These specimens including the type-series and (FTZ2) for cox1. vouchers for molecular analyses have been deposited in Sequences of ITS1-5.8S-ITS2 and cox1of Ancylostoma the Department of Parasitology, Sichuan Agricultural sp. in the present study were separately aligned with ref- University (accession numbers code GYY-XY). erence sequences from closely related species (Table 1), including the congeneric species A. ceylanicum, A. cani- Molecular profiles and phylogeny num, A. duodenale, A. braziliense and A. tubaeforme as For molecular analysis, two adult specimens of Ancylos- well as other hookworm species Necator americanus toma sp. (one female and one male; sample codes FTZ1 (Stiles, 1902), Uncinaria hamiltoni Baylis, 1933 [45], U. and FTZ2, respectively) preserved in 70% ethanol were lucasi Stiles & Hassall, 1901, U. stenocephala (Railliet, air-dried and their mid-body regions (~1 cm) were ex- 1884), U. sanguinis Marcus, Higgins, Slapeta & Gray, cised individually for extraction of genomic DNA using 2014 [46], Uncinaria sp., and Bunostomum phleboto- the Universal Genomic DNA Extraction Kit (TaKaRa, mum (Railliet, 1900), using the Clustal X 1.83 program Dalian, China) according to the manufacturer’s protocol. [47]. During the procedure, the nucleotide alignment of The cephalic and caudal extremities of each specimen cox1 was further adjusted by a codon-guided protein were retained as archived vouchers. The DNA extract alignment. Given the presence of the ambiguous regions was used as template for PCR amplifications at the nu- within these alignments, an online version of GBlocks clear internal transcribed spacer ITS1-5.8S-ITS2 region (http://molevol.cmima.csic.es/castresana/Gblocks_server. (734 bp) and mitochondrial cytochrome c oxidase sub- html) was also introduced here. After refining the align- unit 1 (cox1) locus (393 bp) using primer pairs designed ments using Gblocks, the sequence datasets were used based on the alignments of the relatively conserved re- for phylogenetic analyses using both maximum parsi- gions of the congeneric species A. ceylanicum, A. cani- mony (MP) (PAUP* 4.10b [48]) and Bayesian inference num, and A. duodenale in GenBank. Two PCR primer (BI) methods (MrBayes 3.2 [49]). In the MP analysis, sets were as follows: ITS1-5.8S-ITS2, forward: 5′-GTC heuristic searches were executed by branch-swapping GAA GCC TTA TGG TTC CT-3′ and reverse: 5′-TAA utilizing tree-bisection-reconnection (TBR) algorithm CAG AAA CAC CGT TGT CAT ACT A-3′; cox1, for- and 1,000 random-addition sequence replicates with 10 ward: 5′-ATT TTA ATT TTG CCT GCT TTT G-3′ trees held at each step, and finally the optimal topology and reverse: 5′-ACT AAC AAC ATA ATA GGT ATC with bootstrapping frequencies (BF) was obtained using ATG TAA-3′. The PCR reactions contained ~20 ng of Kishino-Hasegawa, as described previously [50]. For the genomic DNA were performed in 50-μl reaction vol- BI analysis, the nucleotide substitution model GTR + I + umes containing 25 μl 2× Phusion High-Fidelity PCR G was determined using the Bayesian Information Cri- Master Mix (Finnzymes OY, Espoo, Finland), 3 μl teria (BIC) test in jModeltest v. 2.1.6 [51], and the trees gDNA, 3 μL of each primer and 16 μl of ddH O. PCR were constructed employing the Markov chain Monte cycling conditions carried out in a Mastercycler Gradi- Carlo (MCMC) method (chains = 4) over 100,000 (cox1) ent 5331 thermocycler (Eppendorf, Germany) were an or 1,000,000 (ITS1-5.8S-ITS2) generations with every initial denaturation at 95 °C for 5 min; then for ITS1- 100th (cox1) or 1000th (ITS1-5.8S-ITS2) tree being 5.8S-ITS2, 35 cycles of 95 °C for 30 s, 39.8 °C for 30 s, saved; when the average standard deviation of the split and 72 °C for 45 s; but for cox1, 35 cycles at 95 °C for frequencies reduced to less than 0.01, 25% of the first 30 s, 44.1 °C for 30 s, and 72 °C for 30 s; followed by a saved trees were discarded as “burn-in” and the consen- final step at 72 °C for 10 min. For each amplification, sus (50% majority rule) trees were inferred from all samples without parasite gDNA and host DNA as nega- remaining trees and further plotted in TreeviewX tive controls were also included. All PCR products were (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html), Xie et al. Parasites & Vectors (2017) 10:277 Page 5 of 18 Table 1 Information of Ancylostoma species used for molecular identification in the present study Species Gender Host Geographical origin GenBank accession Reference species number ITS1-5.8S-ITS2 cox1 ITS1-5.8S-ITS2 cox1 Ancylostoma ailuropodae Female Giant pandas China (Sichuan) China (Sichuan) KP842923 KP842921 This study n. sp. A. ailuropodae n. sp. Male Giant pandas China (Sichuan) China (Sichuan) KP842924 KP842922 This study Ancylostoma braziliense – Dogs Brazil (Belo Horizonte) – DQ438055 – e Silva et al. [64] A. braziliense – Dogs Brazil (Belo Horizonte) – DQ438056 – e Silva et al. [64] A. braziliense – Dogs Brazil (Belo Horizonte) – DQ438050 – e Silva et al. [64] A. braziliense – Dogs Brazil (Campo Grande) – DQ438060 – e Silva et al. [64] A. braziliense – Dogs Brazil (Belo Horizonte) – DQ438052 – e Silva et al. [64] Ancylostoma caninum Male Humans – Japan (Shiga) – AB751617 Unpublished A. caninum Male Dogs – Australia (Townsville) – NC_012309 Jex et al. [65] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438074 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438071 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438075 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438077 – e Silva et al. [64] A. caninum – Dogs Brazil (Belo Horizonte) – DQ438072 – e Silva et al. [64] Ancylostoma ceylanicum Male Dogs UK (Nottingham) – DQ381541 – Traub et al. [66] A. ceylanicum – Dogs India (Assam) – DQ780009 – Traub et al. [66] A. ceylanicum – Humans – Cambodia (Preah Vihear) – KF896599 Inpankaew et al. [16] A. ceylanicum – Dogs – Cambodia (Preah Vihear) – KF896602 Inpankaew et al. [16] A. ceylanicum – Humans – Cambodia (Preah Vihear) – KF896604 Inpankaew et al. [16] A. ceylanicum – Humans – Cambodia (Preah Vihear) – KF896601 Inpankaew et al. [16] Ancylostoma duodenale – Humans – China (Zhejiang) – AJ407968 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – AJ407959 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – AJ407942 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – AJ407953 Hu et al. [67] A. duodenale – Humans – China (Zhejiang) – NC_003415 Hu et al. [68] A. duodenale –– – China (Xiamen) EU344797 – Unpublished Ancylostoma tubaeforme – Cats – Australia (Townsville) – AJ407940 Hu et al. [67] Xie et al. Parasites & Vectors (2017) 10:277 Page 6 of 18 Table 1 Information of Ancylostoma species used for molecular identification in the present study (Continued) Species Gender Host Geographical origin GenBank accession Reference species number ITS1-5.8S-ITS2 cox1 ITS1-5.8S-ITS2 cox1 A. tubaeforme – Cats – USA (Michigan) JQ812691 – Lucio-Forster et al. [69] Uncinaria hamiltoni Female Sea lions Argentina (Punta Leon) – HQ262116 – Nadler et al. [70] U.hamiltoni Female Fur seals Uruguay (Lobos Island) – HQ262109 – Nadler et al. [70] U.hamiltoni Female Fur seals Uruguay (Cabo Polonio) – HQ262100 – Nadler et al. [70] U.hamiltoni Female Sea lions Uruguay (Cabo Polonio) – HQ262119 – Nadler et al. [70] Uncinaria lucasi Male Sea lions USA (Hazy Island) – HQ262131 – Nadler et al. [70] U. lucasi Female Sea lions Russia (Iony Island) – HQ262149 – Nadler et al. [70] U. lucasi Female Sea lions USA (Hazy Island) – HQ262140 – Nadler et al. [70] U. lucasi Female Sea lions USA (Hazy Island) – HQ262138 – Nadler et al. [70] U. lucasi Female Sea lions USA (Lowry Island) – HQ262142 – Nadler et al. [70] U. lucasi Female Fur seals USA (Reef Rookery) – HQ262078 – Nadler et al. [70] U. lucasi Male Fur seals USA (Adams Cove) – HQ262088 – Nadler et al. [70] U. lucasi Male Sea lions Russia (Iony Island) – HQ262154 – Nadler et al. [70] U. lucasi Female Fur seals Russia (Commander – HQ262067 – Nadler Islands) et al. [70] Uncinaria sanguinis – Sea lions – Australia (Kangaroo Island) – NC_025267 Haynes et al. [71] U. sanguinis – Sea lions – Australia (Kangaroo Island) – KF924756 Haynes et al. [71] Uncinaria stenocephala Female Foxes USA (San Miguel Island) – HQ262052 – Nadler et al. [70] U. stenocephala Female Foxes USA (San Miguel Island) – HQ262053 – Nadler et al. [70] U. stenocephala Male Foxes USA (San Miguel Island) – HQ262054 – Nadler et al. [70] U. stenocephala Male Foxes USA (San Miguel Island) – HQ262055 – Nadler et al. [70] Uncinaria sp. Female Elephant Australia (Macquarie – HQ262127 – Nadler seals Island) et al. [70] Uncinaria sp. Female Elephant Australia (Macquarie – HQ262130 – Nadler seals Island) et al. [70] Uncinaria sp. Female Elephant Australia (Macquarie – HQ262124 – Nadler seals Island) et al. [70] Necator americanus – Humans – China (Zhejiang) – AJ417719 Hu et al. [68] N. americanus – Humans – China (Zhejiang) – NC_003416 Hu et al. [68] N. americanus –– – Togo (−) – AJ556134 Hu et al. [72] Xie et al. Parasites & Vectors (2017) 10:277 Page 7 of 18 Table 1 Information of Ancylostoma species used for molecular identification in the present study (Continued) Species Gender Host Geographical origin GenBank accession Reference species number ITS1-5.8S-ITS2 cox1 ITS1-5.8S-ITS2 cox1 N. americanus – Humans – Central African Republic AB793527 – Hasegawa (−) et al. [73] N. americanus Male Humans – Guatemala (−) AF217891 – Nadler et al. [74] N. americanus –– – China (−) KM891738 – Unpublished N. americanus – Humans – Laos (Thakhek) LC036565 – Unpublished Bunostomum – Sheep China (Heilongjiang) – GQ859497 – Wang phlebotomum et al. [75]; (Outgroup) Male Calf – South Africa (Pretoria) – NC_012308 Jex et al. [65] Sample localities in parentheses with nodal supports expressed as posterior probabil- ZooBank registration: To comply with the regulations ities (PP). The livestock hookworm B. phlebotomum set out in article 8.5 of the amended 2012 version of the was used as outgroup reference and included in each International Code of Zoological Nomenclature (ICZN) phylogenetic analysis. Paralleled to the phylogenies, [53], details of the new species have been submitted to among the genus Ancylostoma the new hookworm ZooBank. The Life Science Identifier (LSID) of the art- species coupled with A. ceylanicum, A. caninum, A. icle is urn:lsid:zoobank.org:pub:A2492E99-AA70-4A58- duodenale and A. tubaeforme was also subjected to detec- AB70-7FED78E726A3. The LSID for the new name tion of synonymous and non-synonymous mutations in Ancylostoma ailuropodae n. sp. is urn:lsid:zoobank.or- the mitochondrial cox1 gene using their corresponding g:act:2C6B6C1E-5F70-49B7-A303-B4D5AE9C7847. protein sequences, followed by determination of genetic Etymology: The new species is named for the type-host. distances between them using a distance matrix based on the maximum composite likelihood model in MEGA [52]. Description General. Slender, relatively small nematodes of white col- Results oration in life (Fig. 2a). Body cylindrical, tapering toward Family Ancylostomatidae Looss, 1905 cephalic and caudal extremities with fine transversely stri- Genus Ancylostoma (Dubini, 1843) ated cuticle; head oriented dorsally in males and females. Buccal capsule widening posteriorly to prominent oral Ancylostoma ailuropodae Yang, Hoberg & Xie n. sp. aperture, possessing two pairs of ventrolateral teeth and Type-host: Giant panda Ailuropoda melanoleuca (David) two pairs of triangular dorsolateral teeth (Fig. 2b-f). (Mammalia: Carnivora: Ursidae). Ventrolateral teeth vary in size and shape, with small, sub- Type-locality: Fengtongzai Natural Reserve (30°42′12″ aduncate inner and large triangular outer teeth extending N, 102°56′14″E), Baoxing, Sichuan Province, China. dorsally. Dorsal gland well developed, associated with rod- Type-material: Holotype, adult male (GYY-XY 1301); like oesophagus, slightly swollen posteriorly, terminating allotype, adult female (GYY-XY 1308); paratypes, three in a lobed valve at junction with intestine (Fig. 4a, b). adult males (GYY-XY 1302-4) and three females (GYY-XY Nerve-ring at midlevel of oesophagus. Cervical papillae 1309-11). All materials, together with nine vouchers (three well developed, conical, situated posterior to level of males, GYY-XY1305-7; six females, GYY-XY13012-17) con- nerve-ring. Excretory pore opens at level between cervical taining one male and one female represented by cephalic papillae and nerve-ring (Fig. 5a1, 2). and caudal extremities, with the mid-body sub-sampled for DNA sequence analysis, are deposited at the Department of Male. [Based on the holotype and three males.] Body Parasitology in Sichuan Agricultural University, Sichuan, length 8.60–12.00 (10.30) mm, maximum width at mid- China. Collectors: GY Yang, TF Zhang and Y Xie. body 500–520 (510). Buccal capsule 180–220 (200) long, Site in host: Small intestine (most in the duodenum). 120–160 (140) wide in dorsoventral view; oesophagus Representative DNA sequences: Representative nuclear 960–1,500 (1,230) long, 150–190 (170) wide; ribosomal and mitochondrial DNA sequences were de- oesophageal length 12% of total body. Cervical papillae posited in the GenBank database under the accession 600–750 (680), excretory pore 500–580 (530), nerve-ring numbers KP842923–KP842924 (ITS1-5.8S-ITS2) and 390–520 (425) posterior to cephalic extremity. Copula- KP842921–KP842922 (cox1). tory bursa well developed, broader than long; dorsal lobe Xie et al. Parasites & Vectors (2017) 10:277 Page 8 of 18 ab c de f Fig. 2 Photomicrographs of adults of Ancylostoma ailuropodae n. sp. a Total view of males (top) and females (down); b-f Cephalic extremity: lateral view of mouth (b and c), showing dorsolateral and ventrolateral teeth; dorsoventral view of mouth (d-f), showing dorsolateral (d) and ventrolateral (e and f) teeth with their positions, shapes and sizes. The arrangements of dorsolateral (2 pairs; b-d) and ventrolateral (2 pairs; b, c, e and f) teeth are indicated by red arrows small with lateral lobes projecting in direction of lateral extremity. Vulva opens ventrally in posterior third of trunks (Figs. 3a-f, 5a5). Dorsal ray thick, 280–390 (350) body, at 2,450–4,686 (3,480) from caudal extremity; vagina in length, 40–60 (52) in maximum width; bifurcating at relatively short. Female reproductive system amphidelphic, 270–295 (280) from anterior into 2 branches; each with poorly differentiated vestibule, paired sphincters and branch further dividing into 2 sub-branches; externodor- infundibula confluent with uterine and ovarian stems sal rays arcuate, arising from dorsal ray at same level (Fig. 5a3). Tail 90–370 (230) long, terminating in acute, (Figs. 3e-f, 5a5). Lateral rays slender, tapering, and arcu- spine-like point 9–25 (17) in length (Figs. 4g, 5a4). Eggs ate with a common stem. Anterolateral ray bending oval, 54–71 × 28–38 (62 × 33) (n = 20) (Fig. 5a6). anteriad, with medio- and posterolateral rays projecting in parallel, extending to edge of bursa (Figs. 3a-b, 5a5). Remarks Antero- and posteroventral rays merge at base and then Ancylostoma ailuropodae n. sp. is established based on divide, continuing parallel deep into cleft (Figs. 3c-d, comparisons to available descriptions among congeners 5a5). Spicules tawny colored, paired, equal, filiform, in the global fauna [6, 7, 9, 10, 54–62]. Ancylostoma 2,000–2,900 (2,450) long (Figs. 4e-f, 5a5). Gubernaculum ailuropodae is unequivocally differentiated from conge- fusiform, 80–120 (90) long, 12–20 (16) wide (Figs. 4c, ners by structural characteristics of male and female 5a5). Cloacal papillae (n = 7) (Figs. 4d, 5a5): 1 pair dis- specimens including body size, arrangement, number posed dorsally, 1 pair laterally, 3 single papillae ventrally. and dimensions of buccal teeth and shape of the buccal capsule, and in males by the configuration of the dorsal Female. [Based on the allotype and three females.] Body ray and bursa and lengths of spicules and gubernaculum, 9.80–16.00 (12.90) mm long, with maximum width at respectively (see Table 2 and Fig. 5b). Of note, tooth- mid-body 560–740 (650); width at anus 270–340 (285). number appears to represent one of the key morpho- Buccal capsule 170–250 (210) long, 130–190 (160) wide logical characters separating A. ailuropodae from other in dorsoventral view; oesophagus 1,280–1,320 (1,300) species of Ancylostoma. Specifically, (i) A. ailuropodae long, 170–250 (200) in maximum width near base. Cer- differs from A. caninum, A. tubaeforme and A. taxideae vical papillae 800–1,230 (900), excretory pore 760–950 by the number (2 vs 3 pairs) of ventrolateral teeth; and (ii) (820), nerve-ring 600–650 (620) posterior to cephalic from A. ceylanicum, A. braziliense, A. duodenale, A. Xie et al. Parasites & Vectors (2017) 10:277 Page 9 of 18 50 m µ 50 m µ 50 m µ a ce 50 m µ 50 m µ 50 m µ bd f Fig. 3 Photomicrographs of Ancylostoma ailuropodae n. sp. male, caudal extremity. a, b Lateral view of bursa showing position of lateral rays and genital cone. c, d Ventral view of bursa showing configuration of antero- and postero-ventral rays. e, f Dorsal view of bursa showing relationships of the dorsal and externodorsal rays; note configuration of the bifurcations of the dorsal ray. Arrows in a, c and e denote the rays which are magnified in panels b, d and f, respectively kusimaense, A. paraduodenale and A. malayanum by the Sequence characterization number (2 vs 0/1 pairs) of triangular dorsolateral teeth. For ITS1-5.8S-ITS2, the 734 bp sequences from FTZ1 Furthermore, the shape of the dorsal rays appears to be and FTZ2 were identical and had 52.2% A + T content. another potential species-specific morphological indicator BLAST analysis revealed that A. ailuropodae shared the (Fig. 5b). Specimens of A. ailuropodae n. sp. vary from A. highest identity with A. ceylanicum (99.6%), followed by tubaeforme by differences in cleft length of two digitations 98.8% identity with A. duodenale, 97.2% with A. tubae- in each branch (Fig. 5b9 and 10) and further from A. taxi- forme, 95.8% with A. caninum, and 92.6% with A. brazi- deae, A. duodenale, A. paraduodenale, A. caninum, A. liense. Based on the identities, there were a total of 59 malayanum, A. kusimaense, A. ceylanicum and A. brazi- variable positions found in the pairwise alignment of liense by the absence of a third digitation in each branch ITS1-5.8S-ITS2, including 17 parsimony-informative (Fig. 5b1–8 and 10). Verified specimens of A. genettae, A. and 42 singleton sites (data not shown). Within cox1 se- protelesis and A. somaliense have not yet been described quences, same base composition (A = 23.4%; C = 9.7%; and these three species were not included in the compari- G = 22.6%; T = 44.3%) and sequence length (393 bp) son above. Notably, the adults of both A. pluridentatum were also observed in these two representative indi- and A. buckleyi can be distinguished from the new species viduals of A. ailuropodae,withanA+T contentof by the number of ventrolateral teeth, given that A. pluri- 67.7%, a typical mitochondrial nucleotide feature in dentatum has only one pair while A. buckleyi has three nematodes (towards AT). BLAST search against Gen- pairs according to the original descriptions (e.g. [60, 63]). Bank/DDBJ/EMBL databases once again showed the Based on these morphological attributes, A. ailuropodae is highest nucleotide identity existing between the new considered to be a previously unrecognized species within species and A. ceylanicum (92.6%), followed by 89.2% the genus Ancylostoma. identity between A. ailuropodae and A. tubaeforme, 88.6% between A. ailuropodae and A. duodenale,and Molecular characterization 86.0% between A. ailuropodae and A. caninum,to- To further probe the taxonomic position of A. ailuropodae, gether corresponding to 99.2–100% identities at the both nuclear ITS1-5.8S-ITS2 and mitochondrial cox1se- amino-acid level. In terms of identity comparisons, quences from two representative specimens (codes FTZ1 there were a total of 78 variable positions in the and FTZ2, respectively) were obtained and subjected to se- 378 bp pairwise alignment, including 28 parsimony- quence characterization and phylogenetic analyses. informative and 50 singleton sites. Xie et al. Parasites & Vectors (2017) 10:277 Page 10 of 18 a cd 50 m µ 50 m µ b f 50 m µ 100 m µ 100 m µ 50 m µ Fig. 4 Photomicrographs of adults of Ancylostoma ailuropodae n. sp. a Dorsoventral view of anterior region of female, showing buccal capsule and entire oesophagus. b Lobed valves between oesophagus and intestine. c Ventrolateral view of male tail, showing gubernaculum. d Ventral view of male tail, showing cloacal papillae. e, f Ventral and ventrolateral views of male tail, showing spicules from both proximal (e) and distal (f) extremities. g Lateral view of female tail with spine-like point. Arrows indicate some small structures, including cervical papillae (a), lobed valve (b), gubernaculum (c), cloacal papillae (d), spicules (e, f) and spine-like point of female tail (g) Further, we located these sites and determined if there tubaeforme, 0.127 to A. duodenale, and 0.151 to A. cani- were non-synonymous substitutions apparent via com- num (not shown). parison of their protein sequences, and the results are shown in Fig. 6. Out of 78 variable base sites, 13 were Phylogenetic characterization unique for A. ailuropodae (in red); 16 were identical be- Phylogenetic relationships between A. ailuropodae and tween A. ailuropodae and one of A. ceylanicum, A. duo- other species were inferred from the respective se- denale, A. caninum and A. tubaeforme (in orange); and quences of ITS1-5.8S-ITS2 and cox1 using both MP and 49 were shared between A. ailuropodae and any two or BI algorithms and their corresponding tree topologies three of these four congeneric species (in yellow). are shown in Fig. 7. Although the two consistent struc- Among the 49 variable sites, however, the non- tures (MP/BI) topologically varied from each other synonymous substitutions A/G in A. ceylanicum and due to the different reference species included, both T/A in A. caninum led to their amino acid changes: I trees provided an identical, robust phylogenetic reso- (Ilu)→ V (Val) in the former and I (Ilu)→ N (Asn) in lution for A. ailuropodae within the genus Ancylos- the latter (see Fig. 6). In addition, analysis of genetic dis- toma and for the genus Ancylostoma within the tances using maximum composite likelihood estimates family Ancylostomatidae. Specifically, (i) the two A. ailur- placed A. ailuropodae close to A. ceylanicum with the opodae specimens clustered together as a monophyletic minimum interspecific evolutionary divergence (0.084), group that was separated from the other Ancylostoma spe- compared with 0.121 evolutionary divergence to A. cies. (ii) When the congeneric species A. ceylanicum, A. 100 µm Xie et al. Parasites & Vectors (2017) 10:277 Page 11 of 18 200 µm 1 2 3 4 5 6 7 8 9 10 Fig. 5 Line drawings of Ancylostoma ailuropodae n. sp. and comparison of dorsal rays among Ancylostoma spp. a Morphological structures of A. ailuropodae n. sp.: 1, dorsoventral view of anterior region; 2, lateral view of anterior region; 3, lateral view of female vulval region; 4, lateral view of female caudal region; 5, dorsal view of male caudal region; 6, egg. b Ten Ancylostoma species for comparison of dorsal rays: 1, A. taxideae [10]; 2, A. duodenale [9]; 3, A. paraduodenale [6]; 4, A. caninum [58]; 5, A. malayanum [7]; 6, A. kusimaense [9]; 7, A. ceylanicum [9, 11]; 8, A. braziliense [9, 11]; 9, A. tubaeforme [58]; 10, A. ailuropodae n. sp caninum A. duodenale and A. tubaeforme were considered re-construct this phylogenetic relationship using the in our cox1-based analysis (Fig. 7a), A. ailuropodae and A. ITS1-5.8S-ITS2 data (Fig. 7b), A. ailuropodae remained as ceylanicum were more closely related to each other than the putative sister of A. ceylanicum,regardless of isolate to A. caninum, A. tubaeforme and A. duodenale,withro- origins (one from the UK and another from India; see bust support for tree topology (BP = 95 and PP = 0.99). Table 1), with high statistical support (BP = 89 and PP = (iii) When another species, A. braziliense, was added to 0.91), which was in agreement with the inferences from 200 µm 200 µm 200 µm 200 µm 20 µm Xie et al. Parasites & Vectors (2017) 10:277 Page 12 of 18 Table 2 Key comparisons between A. ailuropodae n. sp. and other congeneric Ancylostoma spp Species Body size (mm) Ventrolateral teeth Spicules (μm) Gubernaculum (μm) Hosts References A. caninum M: 11.0–13.0 × 3 pairs 730–960 nr Dogs; cats; humans; Burrows [58] 0.34–0.39 (860) wild canids and felids (11.7 × 0.37); F: 14.0–20.5 × 0.50–0.56 (17.0 × 0.52) A. ceylanicum M: 7.91 ± 0.04 × 2 pairs (outer large; 740 ± 20 77 ± 1.64 × 10 Dogs; cats; humans; Yoshida [9] 0.35 ± 0.02; inner very small) wild canids and felids F: 9.48 ± 0.81 × 0.42 ± 0.04 A. braziliense M: 6.84 ± 0.50 × 2 pairs(outer large; 800 ± 70 73 ± 1.94 × Humans Yoshida [9]; 0.24 ± 0.02; inner minute) 10 ± 0.44 Norris[59] F: 8.67 ± 0.68 × 0.34 ± 0.01 A. duodenale M: 10.67 ± 0.17 × 2 pairs (similar in 1,800 ± 90 131 ± 1.49 × Humans Yoshida [9] 0.47 ± 0.03; both size and shape) 13 ± 0.42 F: 12.67 ± 1.12 × 0.64 ± 0.03 A. kusimaense M: 7.82 ± 0.20 × 2 pairs (outer large; 840 ± 4 84 ± 0.71 × Raccoon dogs Yoshida [9] 0.28 ± 0.01; inner small) 10 ± 0.44 F: 9.12 ± 0.55 × 0.33 ± 0.02 A. tubaeforme M: 6.84 ± 0.50 × 3 pairs 1,100–1,470 nr Cats Burrows [58] 0.24 ± 0.02; (1,290) F: 8.67 ± 0.68 × 0.34 ± 0.01 A. paraduodenale M: 5.0–8.0 × 2 pairs (outer stouter 1,100–1,500 80 × 20 Servals Biocca [6] 0.21–0.24 than inner) (1,250) (6.8 × 0.23): F: 6.5–8.5 × 0.26–0.32 (7.7 × 0.29) A. malayanum M: 11.02– 2 pairs (outer large, 2,490–2,620 112 Bears Wu et al. [7] 13.80 × vertical; inner small, 0.46–0.51; subaduncate; one F: 20.40 × 0.54 pair of triangular dorsolateral teeth) A. taxideae M: 8.37 ± 1.92 × 3 pairs (one pair 1,470 ± 87 138 ± 2 × 15 Badgers Kalkan & 0.32 ± 0.02; of triangular Hansen [10] F: 16.05 ± 1.30 × dorsolateral teeth) 0.46 ± 0.05 A. ailuropodae n. sp. M: 10.30 ± 1.70 × 2 pairs (similar in 2,000–2,900 80–120 × Giant panda This study 0.51 ± 0.01; both size and shape; (2,450) 12–20 F: 12.90 ± 3.10 × two pairs of triangular (90 × 16) 0.63 ± 0.09 dorsolateral teeth) Abbreviation: nr not reported; the source paper presented no data on the species under consideration Only the length of gubernaculum was found in the original description [7] the cox1 gene analysis (see Fig. 7a). (iv) The inter- Discussion relationships of A. ailuropodae, A. ceylanicum, A. cani- Hookworms in the genus Ancylostoma cause significant num, A. duodenale, A. braziliense and A. tubaeforme in medical and veterinary disease (ancylostomiasis) in vari- the genus Ancylostoma; U. sanguinis, U. hamiltoni, U. ous hosts including humans and domestic and wild lucasi, U. stenocephala and Uncinaria sp. in the genus mammals [2, 71]. Recent epidemiological surveys Uncinaria;and N. americanus in the genus Necator,dem- revealed that some wild animal-derived species of Ancy- onstrated phylogenetic stability of these monophyletic lostoma are emerging as important helminthic zoonotic groups, with the current analyses being consistent with agents because of rapid urbanization and increased previously proposed molecular phylogenies of the hook- human-wildlife interactions [11, 13–21]. The giant worms based on the nuclear ribosomal and mitochondrial panda, for example, is an endangered and rare wild spe- DNA data [64–75]. cies in China that has been artificially protected and Xie et al. Parasites & Vectors (2017) 10:277 Page 13 of 18 Fig. 6 Simultaneous alignments of nucleotide and amino-acid sequences of mitochondrial cox1 genes from Ancylostoma ailuropodae n. sp. and its congeneric species. For the alignments, the nucleotide sequences of cox1 genes were retrieved from the GenBank database (species and accession numbers are indicated in parentheses): Aai (A. ailuropodae n. sp.; KP842921), Ace (A. ceylanicum; KF896601), Aca (A. caninum; AB751617), Adu (A. duodenale; NC_003415), and Atu (A. tubaeforme; AJ407940). The corresponding protein sequences were deduced based on the Invertebrate Mitochondrial Code. Both nucleotide and amino-acid sequences were aligned with Clustal X 1.83 program. Regions of identity in either nucleotide (*) or amino-acid (#) are indicated. Variable base loci in Aai unique for A. ailuropodae n. sp. are highlighted in red; those shared between A. ailuropodae n. sp. and one of A. ceylanicum, A. duodenale, A. caninum and A. tubaeforme are highlighted in orange; and those shared between A. ailuropodae n. sp. and any two or three of these four congeneric species are highlighted in yellow.The 250 251 non-synonymous substitutions A/G in A. ceylanicum and T/A in A. caninum as well as their amino-acid changes: I (Ilu)/V (Val) and I (Ilu)/N (Asn) are noted in red with a red star. Percentages of nucleotide and amino-acid identities with respect to Aai are shown at the end of each sequence even partially housed for decades due to habitat loss Ancylostoma spp. [55, 56]. Among this assemblage, it is [33]. Clinically unidentified specimens of Ancylostoma in important to note that A. ailuropodae is clearly structur- giant pandas had been confirmed by veterinarians and ally distinct from A. malayanum, the only other species wildlife biologists since the last century, but their poten- of Ancylostoma known in ursid hosts (e.g. Ursus thibeta- tial zoonotic importance remains to be defined [41]. In nus) (Table 2), with the implication that each of these the present study, A. ailuropodae n. sp. was isolated species endemic to China may be more closely related to from the giant panda, morphologically characterized and other congeners within the genus. Specimens upon demonstrated to be closely related to the anthropozoo- which the description and differentiation of A. ailuropo- notic A. ceylanicum by molecular analysis. dae n. sp. was based were restricted to fully developed In general, morphological identification is a conven- adults and eggs. Further work, using a combined tional and authoritative approach to define a new laboratory-egg cultivation and Baermann technique, to nematode parasite species. Concerning the genus Ancy- describe the morphology of developmentally advanced lostoma, several common species can be morphologically larval stages is needed to complement morphological differentiated by key characters such as body size, teeth characteristics of the new species, and to provide valu- of the buccal capsule and shape of bursal rays (see able information assisting in species identification and Table 2 and Fig. 5b; cf. [9]). Similarly, specimens of A. differentiation in this genus [55, 76]. ailuropodae from giant pandas are separated from other Following our morphological evidence, A. ailuropodae hookworms on the basis of either ventrolateral and from giant pandas was further confirmed as an inde- dorsolateral teeth or dorsal rays, supporting the previous pendent species by molecular analysis. For example, the conclusions that teeth and rays were reliable morpho- internal transcribed spacer region (ITS1-5.8S-ITS2) logical indicators in the differential diagnosis of of the nuclear ribosomal DNA is regarded as an Xie et al. Parasites & Vectors (2017) 10:277 Page 14 of 18 ab Fig. 7 Phylogenetic relationships of hookworms isolated from the giant panda with the related hookworms in the family Ancylostomatidae. Phylogeny was inferred on the basis of mitochondrial cox1(a) and nuclear ITS1-5.8S-ITS2 (b) sequences using both maximum parsimony (MP) and Bayesian inference (BI) methods. The livestock hookworm Bunostomum phlebotomum represented the outgroup species. Taxa belonging to the three major genera including Ancylostoma, Uncinaria and Necator in the family Ancylostomatidae are indicated by differently colored rectangles and shown in both phylogenetic topologies. The numbers along the branches indicate bootstrap values resulting from different analyses in the order MP/BI; values less than 50% are shown as “-” appropriate genetic marker to resolve nematode rela- Based on the results from integrated molecular and tionships at the species level [77]. Pairwise comparisons morphological comparisons, we propose that A. ailuro- of ITS1-5.8S-ITS2 in A. ailuropodae with congeneric podae of giant pandas is a previously unrecognized and species available in the GenBank database revealed a separate species that is closely related to the anthropo- species-specific sequence feature (containing 59 variable zoonotic A. ceylanicum within the genus Ancylostoma. informative sites) and overall identity of 92.6–99.6% Additional information regarding the ultrastructure and among A. ceylanicum, A. tubaeforme, A. caninum and A. genomics of this species and other related hookworms is braziliense. Furthermore, high bootstrap support was still required. Broader taxonomic comparisons can pro- evident, based on phylogenetic analysis of ITS1-5.8S- vide an increasingly precise morphological and molecu- ITS2 that demonstrated monophyly of A. ailuropodae as lar basis for species recognition among hookworms. In the putative sister of A. ceylanicum (see Fig. 7b). addition, there were two non-synonymous base substitu- Critically, similar conclusions were reinforced by ana- tions detected in cox1 genes of A. ceylanicum (A/G ) lysis of the mitochondrial cox1 gene. It should also be and A. caninum (T/A ) (Fig. 6) that were confirmed to noted that cox1 analysis was included because recent be fixed and species-specific after homologous compari- studies of the substitution patterns for nematode mito- sons with other A. ceylanicum or A. caninum isolates chondrial genes (e.g. cox1 and nad4) revealed that they from two sites in the same geographic area. have utility in identifying and differentiating novel or Ancylostoma ailuropodae identified here is the fourth cryptic species among closely related taxa due to as- hookworm to be described from the Ursidae. Previously, sumed faster evolutionary rates than nuclear genes, fea- the hookworm Uncinaria yukonensis (Wolfgang, 1956) tures of maternal inheritance and absence of was characterized in black bears and Uncinaria rauschi recombination [78–80]. Compared to the nuclear ITS, (Olsen, 1968) in grizzly and black bears [81, 82]. On the the cox1of A. ailuropodae appeared to have more vari- basis of comparisons of morphometric and distribution able informative sites (n = 78, including 13 unique loci). data of ursine hookworms as well as the historical bio- Nevertheless, results based on cox1 were consistent with geography of bears, Catalano et al. [13] proposed that inference from ITS, in revealing a sister-species relation- there was a relatively recent host-switching event of U. ship with A. ceylanicum among a broader assemblage of rauschi from black bears to grizzly bears. congeners in the genus. Phylogenetic analysis of cox1 The occurrence of A. ailuropodae appears consistent data (Fig. 7a) also supported the contention that A. with speciation following a host colonization event to ailuropodae n. sp. is an independent species which is giant pandas apparently from a carnivoran source in clearly differentiated from A. ceylanicum, A. caninum, sympatry, and further indicates a history of independent A. tubaeforme and A. duodenale. association with ursine hosts for the broader Xie et al. Parasites & Vectors (2017) 10:277 Page 15 of 18 ancylostomatid hookworm assemblage. The timing and phylogenies [23, 24]. These relationships alone would geographic source for these hookworms cannot be eluci- serve to refute a coevolutionary hypothesis for Ancylos- dated based on the currently available data and the re- toma hookworms among bears, conversely supporting a duced and relictual distribution for pandas, but a history history of independent events of host colonization that of host colonization is compatible with the current tree have structured this fauna. topology (for parasites and hosts) and distribution of Unlike U. yukonensis and U. rauschi in bears, the carnivore hosts for other species of Ancylostoma (e.g. hookworm from giant panda is genetically similar to [24]). We suggest that acquisition of Ancylostoma by other Ancylostoma species (Fig. 7). These respective gen- giant pandas likely occurred prior to 7 million years era are referred to two independent subfamilies within ago (MYA) when a shift from an omnivorous diet to the Ancylostomatide, namely Ancylostomatinae Looss, one dominated strictly by bamboo (by 2.4 MYA) was 1905 for Ancylostoma and Bunostominae Looss, 1911 underway [25]. for Uncinaria, consistent with extended evolutionary Divergence of A. ailuropodae appears to have occurred trajectories for these taxa among the hookworms. This prior to acquisition of A. ceylanicum by humans in suggests the independent origin of A. ailuropodae, sup- Southeast Asia, and prior to the intense bottlenecking of porting monophyly of A. ailuropodae and congeneric giant panda populations that has characterized the past species A. ceylanicum, A. duodenale, A. tubaeforme, A. century (cf. [33, 83] for details about the history of giant caninum and A. braziliense, and strengthens the close pandas). This interpretation is significant, as it would re- relationship between the giant panda hookworm and A. late to the historical independence of A. ailuropodae and ceylanicum within the clade. Concurrently it suggests A. ceylanicum before the current intensified conserva- that Uncinaria spp. from pinnipeds and ursids are a dis- tion campaign for maintaining giant pandas, and the po- tinct monophyletic group in the family Ancylostomati- tential for cross-transmission of both hookworm species dae [70]. The apparent genetic differences of A. when infected humans are in contact. The unique niche ailuropodae n. sp. in pandas and U. rauschi and U. yuko- and specialized bamboo-feeding habits of giant pandas nensis in bears, coupled with their divergent biogeo- suggest that colonization in ecological time, related to graphic and ecological histories suggest this system as a the source or origin of A. ailuropodae, was unlikely good model for exploring the complexities of diversifica- given relative isolation with respect to a sympatric as- tion and faunal assembly in the evolution of host range semblage of carnivorans or other mammals that may and associations among hookworms (e.g. [85–88]). serve as hosts for species of hookworms [25, 26, 33]. The potential for genetic partitioning among possible Parasitological inventory among potential carnivoran disjunct populations of hookworms in giant pandas hosts in Sichuan and nearby regions remains necessary should be considered, as it will reflect information about to demonstrate that A. ailuropodae has a narrow host the timing of colonization to giant pandas and the dur- range and may now be limited to the giant panda [84]; ation of the history of association. Further, the history of apparent narrow host range, however, does not preclude fragmentation and isolation for giant pandas across now the potential or capacity for contemporary host switches isolated mountain systems in southwestern China sug- to humans as a zoonotic parasite given opportunity due gests a complex relationship among hosts and hook- to permissive ecological circumstances [85–88]. worms in this region. Such history could be explored Phylogenetic and historical isolation of giant pandas through fecal-based approaches in conjunction with mo- from the broader assemblage of ursids and ursine bears lecular diagnostics to examine occurrence and the extent (e.g. [23, 24]) in conjunction with apparent structural di- of genetic diversity and distribution for hookworm para- vergence (e.g. teeth and configuration of the dorsal ray; sites among populations and subspecies of giant pandas. Table 2 and Fig. 5b5 and 10) of A. ailuropodae and A. malayanum suggests that independent events of host Conclusions colonization, separated in space and time, were essential This study is the first to describe and define a new mem- in the process of speciation for these hookworms; mo- ber of the genus Ancylostoma, A. ailuropodae, in the lecular data, particularly from A. malayanum, is still wild giant panda using morphological and molecular cri- needed to explore this hypothesis. Moreover, phylogen- teria. Morphological characters (e.g. ventrolateral (two etic hypotheses for the Ursidae have placed giant pandas pairs) and dorsolateral (two pairs) teeth and dorsal rays) distantly from species of Ursus (and other ursines) near distinctly separate A. ailuropodae n. sp. from other con- the base of an extensive radiation for bears that unfolded generic species in the genus Ancylostoma. Further, nu- across the late Miocene and Pliocene [24]. Among ursine clear ITS1-5.8S-ITS2 and mitochondrial cox1-based hosts for Ancylostoma, U. thibetanus (Asiatic black bear) genetic distance analysis and phylogenies supported the is regarded as the sister of U. americanus (American black assertion that A. ailuropodae is independent and shares bear) and placed among crown species in ursid a sister-species relationship with the anthropozoonotic Xie et al. Parasites & Vectors (2017) 10:277 Page 16 of 18 A. ceylanicum. Although additional molecular evidence of Agriculture, Agricultural Research Service, Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, Beltsville, is warranted, this finding should enhance public aware- Maryland 20705, USA. United States Department of Agriculture, Agricultural ness of parasitic hookworms in giant pandas, especially Research Service, Beltsville Agricultural Research Center, Animal Parasitic in captive populations that have frequent contact with Disease Laboratory, Beltsville, Maryland 20705, USA. Department of Civil and Environmental Engineering, University of Maryland, College Park, Maryland breeders, veterinarians and even tourists. 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