(Petrochenko, 1949) (Acanthocephala ...

First report on cystacanths of Sphaerirostris lanceoides (Petrochenko, 1949) (Acanthocephala: Centrorhynchidae) from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in China Jing Kang & Liang Li

Systematic Parasitology An International Journal ISSN 0165-5752 Volume 95 Number 5 Syst Parasitol (2018) 95:447-454 DOI 10.1007/s11230-018-9794-0

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Author's personal copy Syst Parasitol (2018) 95:447–454 https://doi.org/10.1007/s11230-018-9794-0

First report on cystacanths of Sphaerirostris lanceoides (Petrochenko, 1949) (Acanthocephala: Centrorhynchidae) from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in China Jing Kang . Liang Li

Received: 4 January 2018 / Accepted: 25 March 2018 / Published online: 10 April 2018 Ó Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Everted cystacanths of Sphaerirostris lanceoides (Petrochenko, 1949) Golvan 1956 are reported from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) for the first time. The prevalence was 1.96% and the intensity ranged between 1.0–3.0 acanthocephalans. SEM observations revealed the morphology of the gonopore and the presence of a flat, bare region on the apical part of the proboscis. Moreover, S. lanceoides was characterised using molecular approaches by sequencing the ribosomal ITS1-5.8S-ITS2 region and the mitochondrial cox1 gene. The resulting ITS sequences were identical and the cox1 sequences showed a divergence of 0–0.75%. Sphaerirostris lanceoides is the first species of the genus for which the ITS1-5.8S-ITS2 and cox1 loci have been sequenced to aid species identification.

commonly parasitic in the digestive tract of various birds worldwide (Petrochenko, 1949, 1956; Yamaguti, 1963; Dimitrova et al., 1997; Amin, 2013). During our recent parasitological survey of the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura), a total of 11 everted cystacanths were collected from the intestine of this host. The detailed morphology of the cystacanths was studied using light and scanning electron microscopy. They were identified morphologically as S. lanceoides (Petrochenko, 1949). In addition, the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the ribosomal internal transcribed spacer (ITS1-5.8S-ITS2) of S. lanceoides were sequenced for the first time to aid the molecular identification of this poorly known species.

Materials and methods Introduction The genus Sphaerirostris Golvan, 1956 currently includes 27 nominal species, adults of which are

This article is part of the Topical Collection Acanthocephala. J. Kang L. Li (&) Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, Hebei Province, P. R. China e-mail: [email protected]

Light and scanning electron microscopy A total of 562 B. gargarizans were examined for parasites. Acanthocephalans isolated from the digestive tract of toads were kept in tap water for a few hours until the proboscis everted, and then fixed and stored in 80% ethanol until studied. For light microscopical examination, the parasites were cleared in lactophenol. Drawings were made with the aid of a Nikon microscope drawing attachment. For scanning electron microscopy (SEM), the specimens were post-fixed in 1% osmium tetroxide, dehydrated in an ethanol series and acetone, and then critical point dried. The

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specimens were coated with gold and examined using a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 20 kV. Measurements (the range, followed by the mean in parentheses) are given in micrometres unless otherwise stated. Voucher specimens are deposited in the College of Life Sciences, Hebei Normal University, Hebei Province, P. R. China. Molecular sequencing Five specimens were randomly selected for molecular analysis of the cox1 and ITS1-5.8S-ITS2 regions. Genomic DNA from each individual was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer’s instructions. DNA was eluted in elution buffer and kept at 20°C until use. The partial cox1 gene was amplified by PCR using the forward primer (50 -GGT CAA CAA ATC ATA AAG ATA TTG G-30 ) and the reverse primer (50 -TAA ACT TCA GGG TGA CCA AAA AAT CA30 ) (Gome´z et al., 2002). The ITS1-5.8S-ITS2 region was amplified by PCR using the forward primer (50 GTC GTA ACA AGG TTT CCG TA-30 ) and the reverse primer (50 -TAT GCT TAA ATT CAG CGG GT-30 ) (Kra´l’ova´-Hromadova´ et al., 2003). The cycling conditions were as described previously: 94°C for 5 min (initial denaturation), followed by 30 cycles at 94°C for 30 s (denaturation), 55°C for 30 s (annealing), 72°C for 70 s (extension), and a final extension step at 72°C for 7 min (Li et al., 2017a, b). PCR amplicons were checked on GoldView-stained 1.5% agarose gels and purified with Column PCR Product Purification Kit (Shanghai Sangon, China). Sequencing was carried out using a DyeDeoxyTerminator Cycle Sequencing Kit (v.2, Applied Biosystems, California, USA) and an automated sequencer (ABI-PRISM 377). Sequencing for each sample was carried out for both strands using the PCR primers. Sequences were aligned using ClustalW2 and adjusted manually. The DNA sequences obtained herein were compared (using the algorithm BLASTn) with those available in the GenBank NCBI database. Family Centrorhynchidae Van Cleave, 1916 Genus Sphaerirostris Golvan, 1956 Sphaerirostris lanceoides (Petrochenko, 1949) Host: Bufo gargarizans Cantor (Amphibia: Anura), Asiatic toad.

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Locality: Yuyao County (120°590 2200 E, 30°030 3800 N), Zhejiang Province, P. R. China. Voucher material: Eleven specimens (4 males and 7 females) were submitted to College of Life Sciences, Hebei Normal University, Shijiazhuang, P. R. China under the accession number HBNU-A-2017009L. Site in host: Intestine. Prevalence and intensity: Eleven of 562 (1.96%) B. gargarizans were infected with an intensity of 1.0–3.0 (mean 1.4) acanthocephalans. Representative DNA sequences: The cox1 and ITS15.8S-ITS2 sequences of S. lanceoides are deposited in the GenBank database under the accession numbers MG931939-MG931943 (cox1) and MG931944MG931946 (ITS1-5.8S-ITS2). Description (Figs. 1–2) General. Polymorphida, Centrorhynchidae, with characters of Sphaerirostris. Worms small, yellowishbrown when alive. Trunk spindle-shaped, gradually tapering toward both extremities, rounded posteriorly (Fig. 1A, B, F). Proboscis divided into two parts separated by constriction, bare and flat apically (Figs. 1C, 2A, C). Anterior proboscis nearly spherical, with 36–38 longitudinal rows of 11–13 hooks each; anterior 8–9 hooks of each row with simple posteriorly directed roots; roots of posterior 3–4 (usually 3) hooks not observed clearly (Figs. 1C, D, 2A, C). Posterior proboscis cylindrical, with more widely-spaced armature, 32–36 (usually 34) longitudinal rows of 3–4 hooks each; roots of posterior proboscis hooks not observed (Fig. 1C, D). All hooks emerged from elevated round rims on proboscis surface (Fig. 2B, D). Proboscis receptacle double-walled. Neck short. Lemnisci subequal, relatively longer than proboscis receptacle (Fig 1. A, B). Male [Based on 4 juvenile male specimens.] Trunk 2.56–3.16 (2.88) mm long, with maximum width 683–707 (699). Proboscis 600–710 (653) long in two parts; anterior proboscis 440–480 9 300–420 (458 9 383); posterior proboscis 140–250 9 350–390 (195 9 365). Length of anterior proboscis hooks from anterior edge: 20–35 (27), 28–45 (38), 38–48 (41), 35–48 (43), 40–53 (46), 33–48 (40), 28–40 (30), 25–35 (30), 25–28 (27); length of posterior proboscis hooks from anterior edge: 28–33 (30), 23–33 (29), 25–33 (27), 23–28 (26). Proboscis receptacle 830–950 9 200–250

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Fig. 1 Sphaerirostris lanceoides collected from Bufo gargarizans in China. A, Juvenile male; B, Juvenile female; C, Proboscis of juvenile female; D, Hooks per row of juvenile female (roots of posterior proboscis hooks not observed in the present material); E, Testes and cement glands; F, Posterior extremity of juvenile female. Scale bars: A–C, E, 200 lm; D, F, 100 lm

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Fig. 2 Scanning electron micrographs of Sphaerirostris lanceoides collected from Bufo gargarizans in China. A, Proboscis of juvenile female, lateral view; B, Magnified image of anterior proboscis hooks; C, Proboscis of juvenile female, apical view; D, Magnified image of posterior proboscis hook; E, Magnified image of gonopore. Scale bars: A, 60 lm; B, 6 lm; C, 40 lm; D, E, 3 lm

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(875 9 225). Lemnisci almost equal in size; left lemniscus 552–740 9 50–75 (646 9 64); right lemniscus 584–690 9 50–63 (651 9 58). Testes oval, pre-equatorial (Fig. 1A). Anterior testis 155–174 9 111–174 (163 9 132); posterior testis 155–173 9 87–140 (166 9 113). Cement-glands 4, tubular, closely arranged together, 377–725 (571) long, 19–48 (38) wide (Fig. 1A, E). Saefftigen’s pouch just posterior to cement glands. Copulatory bursa 487–602 9 144–213 (576 9 179), not extended out of the body (Fig. 1A). Female [Based on 7 juvenile female specimens.] Trunk 2.86–3.64 (3.22) mm long, with maximum width 659–860 (745). Proboscis 630–800 (721) long in two parts; anterior proboscis 440–550 9 410–500 (497 9 453); posterior proboscis 170–260 9 370–500 (221 9 413). Length of anterior proboscis hooks from anterior edge: 19–43 (34), 29–53 (41), 36–53 (48), 39–53 (48), 40–53 (49), 27–50 (40), 24–45 (35), 24–38 (33), 27–43 (29), 27–33 (28); length of posterior proboscis hooks from anterior edge: 19–35 (27), 24–40 (28), 24–31 (26), 19–28 (25). Proboscis receptacle 830–1020 9 250–420 (924 9 301). Lemnisci almost equal, 584–780 9 48–72 (678 9 58). Uterine bell funnel-shaped, 295–343 9 77–128 (331 9 93). Uterus 33–126 9 43–72 (62 9 61); vagina 136–163 9 68–95 (149 9 83) (Fig. 1B, F). Eggs absent. Gonopore terminal (Fig. 2F).

Molecular characterisation Five cox1 sequences for S. lanceoides generated in this study were all 669 bp in length and differed by 0–0.75%. Currently, no cox1 data are registered on GenBank for species of Sphaerirostris. However, we found sequences for two centrorhynchid species, Centrorhynchus aluconis (Mu¨ller, 1780) (GenBank: DQ089716) and Centrorhynchus sp. (GenBank: NC_029765). Pairwise comparison between S. lanceoides and these two species revealed 25.4–26.0% (vs Centrorhynchus sp.) and 19.4–20.0% (vs C. aluconis) divergence in the cox1 gene. Three ITS1-5.8S-ITS2 sequences for S. lanceoides generated herein were all identical (711 bp in length). There is no species of Sphaerirostris or a member of the Centrorhynchidae with ITS data registered on GenBank.

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Discussion The adults of S. lanceoides parasitise Turdus merula intermedius (Richmond) (Passeriformes: Turdidae) and Pluvialis squatarola (Linnaeus) (Charadriiformes: Charadriidae) (see Petrochenko, 1949, 1956; Yamaguti, 1963). The cystacanths of S. lanceoides have been reported from Nyctereutes procyonoides viverrinus (Gray) (Carnivora: Canidae), Procyon lotor (Linnaeus) (Carnivora: Procyonidae) and Meles meles (Linnaeus) (Carnivora: Mustelidae) in Japan and Italy, respectively (Sato et al., 2005, 2006; Torracca et al., 2010). This is the first report of cystacanths of S. lanceoides in the Asiatic toad B. gargarizans. Because our specimens are all juvenile males and females (no eggs found in the uterus), we assume that the Asiatic toad possibly acts as a paratenic host of S. lanceoides. A similar situation can also occur in some species of Centrorhynchus Lu¨he, 1911, which could utilise reptiles (snakes and lizards) and amphibians (frogs and toads) as paratenic hosts to connect the intermediate hosts (usually arthropods) and the definitive hosts (predatory birds) (Kennedy, 2006; Krasnoshchekov & Lisitsyna, 2009). Most aspects of the morphology and measurements of our specimens agree with the descriptions of the cystacanths of S. lanceoides by Sato et al. (2005, 2006) and Torracca et al. (2010), including the size and morphology of the trunk, proboscis and hooks, the length of the proboscis receptacle, the number of the longitudinal rows of proboscis hooks and the hooks per longitudinal row (see Table 1 for details). However, the body size of our specimens is distinctly smaller than the descriptions of S. lanceoides by Petrochenko (1949, 1956) (see Table 1 for details), It is easy to understand the morphometric difference considering that Petrochenko’s (1949, 1956) morphometric data were based on mature adult specimens collected from the definitive hosts (T. merula intermedius and P. squatarola) (vs our specimens being all juvenile). Although our specimens are still juvenile, the size and morphology of the anterior and posterior proboscis, the length of the proboscis receptacle, the number of the longitudinal rows of proboscis hooks and the hooks per longitudinal row are identical to Petrochenko’s (1949, 1956) mature adult specimens. Consequently, we considered our specimens to be conspecific with S. lanceoides. However, we did not observe the roots of posterior proboscis hooks, which

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Table 1 Morphometric data for cystacanths of Sphaerirostris lanceoides collected from Bufo gargarizans Cantor (Amphibia: Anura) in China with previous descriptions Feature

Present studya (n = 11)

Sato et al. (2006)a (n = ?)

Torracca et al. (2010)a (n = 1) Meles meles (Linnaeus)

Sato et al. (2005)a (n = 9) Procyon lotor (Linnaeus)

Petrochenko (1949, 1956)b (n = ?)

Host

Bufo gargarizans Cantor Zhejiang Province, China

Nyctereutes procyonoides viverrinus (Gray) Yakushima Island, Japan

Tuscany and Liguria, Italy

Honshu, Japan

Kirghiz SSR, Turkmen SSR, USSR

ST (mm)

2.56–3.64 9 0.66–0.86

2.76 9 0.76

3.85 9 –

3.60–5.20 9 0.70–1.20

7.47 9 1.52

SP

600–800 9 300–500

790 9 550

700 9 –

600–850 9 220–360

630–690 9 410–420

ASP

440–550 9 300–500

550 9 420

540 9 470

–

410–450 9 –

PSP

140–260 9 350–500

240 9 370

250 9 –

–

200–250 9 –

NHR

36–38

36

36

32–36

36–38

NHPR

11–13

12

12

12–13

11–12

LL

550–780

1180

–

–

–

SPR

830–1,020 9 200–420

860 9 200

–

–

1,030 9 400

Locality

Turdus merula intermedius (Richmond), Pluvialis squatarola (Linnaeus)

a

Cystacanths. bMature adults. Abbreviations: ST, size of trunk; SP, size of proboscis; ASP, size of anterior proboscis; PSP, size of posterior proboscis; NHR, number of longitudinal rows of proboscis hooks; NHPR, number of hooks per longitudinal row; LL, lemnisci length; SPR, size of proboscis receptacle

have been reported in the previous studies (Petrochenko, 1949, 1956; Sato et al., 2006). In addition, the length of lemnisci in our material is smaller than that described by Sato et al. (2006) and Petrochenko (1949, 1956) (see Table 1 for details). This morphometric difference is likely due to intraspecific variation, possibly resulting from different hosts, stage of development and/or geographical locations. Petrochenko (1949, 1956) stated the presence of three cement glands in his specimens. However, there may be some variability of the number of cement glands in different specimens of S. lanceoides. Yamaguti (1963), Belopolskaya (1983), Florescu & Ienistea (1984) all considered that S. lanceoides has four cement glands. It seems that this character has a restricted taxonomic value for distinguishing species of Sphaerirostris. For example, Sphaerirostris turdi (Yamaguti, 1939) also possesses three or four cement glands (Yamaguti, 1939; Dimitrova et al., 1997; Lisitsyna et al., 2012). Torracca et al. (2010) reported the anterior proboscis of their material as being only 0.29 mm wide. However, according to the photo of

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proboscis provided by Torracca et al. (2010), we considered that the original value of the maximal width of anterior proboscis in Torracca et al. (2010) is evidently wrong. Our SEM observations revealed for the first time the morphology of the gonopore and the presence of a flat, bare region on the apical part of proboscis. The morphometrics of the testes, cementglands, copulatory bursa, uterus, uterine bell and vagina of this juvenile form of S. lanceoides are also reported for the first time. Sphaerirostris picae (Rudolphi, 1819) is a common acanthocephalan species parasitic in Pica pica Linnaeus (Corvidae) worldwide (Dimitrova et al., 1995, 1997; Amin et al., 2010). Morphologically this species is very similar to S. lanceoides. However, we can easily distinguish S. picae from S. lanceoides by the presence of a receptacle process in the anterior proboscis in the former species, which was first described by Amin et al. (2010). Amin et al. (2010) considered that this unique structure (receptacle process in the anterior proboscis) in S. picae can be used for diagnosis of this species. In addition, S. picae


has only 8–10 hooks in each longitudinal row on the anterior proboscis, that is slightly less than S. lanceoides (with 11–13 hooks in each longitudinal row on the anterior proboscis). Sphaerirostris turdi has been reported from some birds of the Turdidae, Emberizidae, Sturnidae and Cinclidae in Japan, the Philippines, Hungary and Bulgaria, respectively (Yamaguti, 1939; Dimitrova et al., 1995, 1997; Lisitsyna et al., 2012). According to Dimitrova et al. (1997), this species also has three or four cement glands in some specimens, but differs from S. lanceoides by having fewer longitudinal rows on the anterior proboscis in the male (26–29 longitudinal rows in S. turdi vs 36–38 longitudinal rows in S. lanceoides) and a much larger body size in the female (trunk 4.80–19.0 mm long in the mature females vs trunk only 2.86–3.64 mm long in our juvenile females). Sphaerirostris lanceoides is the first species of the family Centrorhynchidae for which the ITS1-5.8SITS2 region has been sequenced to aid species identification in future studies. The levels of the cox1 interspecific nucleotide divergence is distinctly higher than the level of intraspecific nucleotide differences in S. lanceoides (19.4–26.0% vs 0–0.75%). Acknowledgements The authors wish to thank Dr David I. Gibson (Natural History Museum, London, UK) for providing us some important literature. This study was supported by the Youth Top Talent Support Program of Hebei Province to Dr Liang Li. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval All applicable institutional, national and international guidelines for the care and use of animals were followed.

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