Zhijun Hou · Lei Han · Ying Sun · Dongdong Shen · Zhiwei Peng · Lixin Wang ·Qian Zhai · Yanqiang Zhou · Yaxian Lu · Liwei Teng · Hongliang Chai

Abstract During a helminthological study of waterfowl in China, a new species ( Cloacotaenia cygnimorbus sp.nov.) of hymenolepidid cestodes (tapeworm) was found in the small intestine of whooper swan ( Cygnus cygnus, Linnaeus, 1758). The rudimentary rostellum and four unarmed muscular suckers, proglottids with distinct craspedote and three spherical testes were coincident with the characters of Cloacotaenia or Hymenolepis, but phylogenetic analysis of 28S rRNA and cox1 gene revealed that the new species is Cloacotaenia rather than Hymenolepis. Its morphology was also clearly differentiated from C. megalops in the arrangement of its testes in a triangle instead of in line and the cirrus unarmed rather than spined. Compared with C.megalops, the new species has more elongated neck, much larger mature proglottids and much smaller testes, cirrus sac, ovary, vitellarium and uterine proglottid. In addition, it infected the host intestine not the cloacae. Phylogenetic analysis of cox1 gene of the new species shows that it had a level of sequence variation (10.52—23.06%) with the sequences of C. megalops. The considerable morphological and molecular differences between those two parasites support C. cygnimorbus sp. nov. as a new species.

Keywords Cloacotaenia · Hymenolepididae ·Hymenolepis · Whooper s wan · 28S rR NA · cox1

Introduction

Cestodes (tapeworms) of the family Hymenolepididae Ariola, 1899 have a cosmopolitan distribution and parasitize a broad range of mammalian and avian hosts, such as rodents,bats, and waterfowl (Czaplinski and Vaucher 1994). The latest comprehensive systematic revision of Hymenolepididae was undertaken by Czaplinski and Vaucher ( 1994); however,they did not systematically revise every genus in the family.The genus ofHymenolepisin bird is characterized by the presence of three testes, unarmed suckers, proglottids with visibly craspedote, and absent or rudimentary rostellum,without hooks (Czaplinski and Vaucher 1994); however,those characters are also shared byCloacotaenia(Muniz-Pereira and Amato 1998). The genus ofCloacotaeniaWolff-hügel, 1938 was recognized as a synonym ofHymenolpisin bird hosts by Czaplinski and Vaucher ( 1994), while others consideredHymenolepis megalopsas a synonym ofCloacotaenia megalops(Haukos and Neaville 2003; Green et al.2011), while even more researchers consideredCloacotaeniaandHymenolpisto be independent genera (Nowak et al.2011; Caira and Jensen 2017).

Identification of closely related hymenolepidid cestode species can be very challenging in part due to the phenotypic plasticity of the organisms, variation of the host and paucity of informative morphological features (Casanova et al. 2001). However, isoenzymatic patterns and molecular studies using ribosomal DNA and 28S regions have been used to differentiate and identify hymenolepidid cestodes species (Blaxter et al. 1998; Mariaux 1998). For instance, two new species of hymenolepidid cestodes belonging to the genusNomadolepiswere described based on molecular phylogenetic analysis and morphological characters (Makarikov et al. 2015). Mitochondrial DNA(mtDNA) is also a useful for phylogenetic analysis of the Hymenolepididae species (Guo 2016). Further improvement in the classification of Hymenolepididae requires the combined use of morphological and molecular criteria(Haukisalmi et al. 2010; Greiman and Tkach 2012).

During a helminthological study of waterfowl, several specimens of hymenolepidid cestode from a whooper swan (Cygnus cygnus, Linnaeus 1758) in China were found. The purpose of this study was to explore the taxonomic level and phylogenetic position of these hymenolepidid specimens isolated from the whooper swan on the basis of combined nucleotide sequences and morphological features.

Materials a nd methods

Sampling

Nine specimens of the cestode species were collected from the small intestine of a weak, wild whooper swan,which then died after its rescue in Swan Lake City Wetland Park, Sanmenxia, Henan Province, China in October 2015. Collecting the cestode species from the wild whooper swan was approved by Swan Lake City Wetland Park. Specimens were rinsed in saline, relaxed in water and fixed in 70% ethanol for both morphological and molecular study. They were stained with Ehrlich’s hematoxylin, dehydrated in an ethanol series, cleared in clove oil and mounted in Canada balsam. One of the scoleces and some fragments of the specimens were mounted in Berlese’s medium to facilitate examination of the suckers and cirrus armature. Eggs from gravid proglottids of one specimen were released in water to study their morphology. The type specimens have been deposited in the Parasitology Research Centre of Northeast Forestry University. All experimental designs and animal handling was approved by the Institutional Animal Care and Use Committee of Northeast Forestry University.

PCR am plif ication

Genomic DNA was extracted with the QIAamp DNA Mini kit (QIAamp, Hilden, Germany) and the manufacturer’s instructions from a fragment of single adult worms after preliminary morphological identif ication. Scolex and the rest of the strobilae were mounted on slides as vouchers. Part of the 28S rRNA was PCR amplif ied using primers 28s1 (5′-TAC CCG CTG AAC TTA AGC ATAT-3′) and 28s2 (5′-CTC CTT GG-TCC GTG TTT CAA GAC-3′) (Zehnder and Mariaux 1999), and part of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplif ied using the degenerate forward primer MplatCOX1dF (5′-TGT AAA ACG ACG GCC AGT TTWCITTRGA TCA TAA G-3′) and degenerate reverse primer MplatCOX1dR (5′-CAG GAA AC -AGC TAT GAC TGA AAY AAY AIIGGATCICCACC-3′) (Moszczynska et al. 2009).

PCR reactions (25 μL volumes) were performed in 12 μL of ddH2Owater, 2.5 μL PCR buffer, 2.5 μL dNTP, 1 μL of each primer, 2 μL MgCl 2 , 3 μL DNA and 1 μL Taq DNA Polymerase (Thermo Scientif ic) under the following conditions: (28S) 2 min initial denaturation at 94 °C; 30 cycles of 30 s at 94 °C, 15 s at 58 °C, 1.5 min at 72 °C; and 7 min f inal extension at 72 °C; (cox1) 2 min initial denaturation at 94 °C; 30 cycles of 30 s at 94 °C, 15 s at 50 °C, 45 s at 72 °C; and 7 min f inal extension at 72 °C. PCR reaction products (5 μL) were separated and visualized in 1.0% agarose gel using Goldview I Nuclear staining dye (10,000×,Solarbio, Beijing, China). PCR products were sent to Boshi Company (Harbin, China) for sequencing using a primerwalking strategy, checked against the NCBI database using the Basic Local Alignment Search Tool (BLAST; www.ncbi.nib.gov/BLAST/) and deposited in GenBank (28S rRNA:KX129948 andcox1: KU980902).

Molecular ph ylogenetic anal ysis

Seventeen partial 28S rRNA sequences of 11 hymenolepidid genera isolated from rodents, birds and waterfowl were selected to infer the phylogeny of the new species among family Hymenolepididae. In addition, the partialcox1gene was used to identify the taxonomic level of these newly obtained unknown specimens (C. cygnimorbussp.nov.). Sequences ofSchistosoma japonicumwere used as outgroups. Sequences were aligned using E-INS-i of the program MAFFT (Katoh and Standley 2013) and ambiguously aligned regions were excluded using Gblocks-0.91 (Talavera and Castresana 2007) with the default parameters using the options for a less stringent selection. The best-f itting nucleotide substitution model for the 28S rRNA sequences andcox1gene phylogenetic analyses was GTR + I + G,selected using Akaike’s information criterion in MODELTEST 3.7 (Posada and Crandall 1998). Phylogenetic trees based on the 28S rRNA andcox1sequences were constructing using the Bayesian approach and MrBayes v.3.2(Ronquist and Huelsenbeck 2003) with parameters nst = 6,rates = invgamma. Four Markov chains were run for 2 runs from random starting trees for 5 million generations, and trees were sampled every 100 generations. The 25% generations were discarded as burn-in, and the remaining samples were used to calculate Bayesian posterior probabilities(BPP). Phylograms were drawn using FigTree v1.4.2 ( https://tree.bio.ed.ac.uk/softw are/f igtr ee).

Results

Description

Cloacotaenia cygnimorbussp. nov. (Fig. 1).

Diagnosis (based on 5 stained mounted specimens and 1 scolex cleared in Berlese’s medium). Measurements of the holotype are followed by the range, mean values and number of measured specimens in parentheses.

Fig. 1 Cloacotaenia cygnimorbus sp. nov. a Dorsoventral view of scolex, b neck proglottids, c premature proglottids, d mature proglottids, e gravid proglottids, f posterior end. Scale bars: a 500 μm, b— f 300 μm

Worm of medium size. Proglottids visibly craspedote,broader than long. Scolex almost square when mounted,1441 μm wide (n= 1). Four muscular suckers, round nearly, unarmed, directed forward, 523—583 × 408—521 μm(559 × 476 μm,n= 4). Rostellum rudimentary; rostellar sac vestigial, circular, with no other apical structures present(Fig. 1 a). Neck 738—877 × 120—168 μm (786 × 138 μm,n= 15), clearly differentiated from scolex. Dorsal osmoregulatory canals thin, 23—38 μm wide (n= 9), ventral to genital ducts (Fig. 1 b).

Maturation relatively is slow. Male and female gonads attain maturity almost simultaneously via 9—12 premature proglottids (Fig. 1 c). Mature proglottids 716—850 × 157—213 μm (768 × 186 μm,n= 11), transversely elongate, trapezoid. Three elliptical testes, relatively small,formed an elongated triangle with an obtuse angle. Poral testes separated from 2 antiporal testes by female gonads.Poral testis 89—105 × 59—78 μm (94 × 66 μm,n= 14), median testis 85—104 × 52—72 μm (95 × 60 μm,n= 11), aporal testis 72—87 × 45—68 μm (80 × 57 μm,n= 12) (Fig. 1 d). External seminal vesicle elongate-oval, in upper part of proglottid and close to female gonads. Internal seminal vesicle elongate, occupying nearly half of cirrus sac. Cirrus sac tubular,relatively short, 134—168 × 28—36 μm (144 × 32 μm,n= 1).Antiporal end of cirrus sac usually reaching the osmoregulatory canals. Genital atrium is simple, infundibular, deep,opens laterally approximately in the middle of right proglottid margin. Cirrus unarmed. Genital pores unilateral, dextral.

Ovary 55—71 × 24—56 μm (63 × 39 μm,n= 16) f lattened,slightly lobate, irregularly shaped, situated in the midline of proglottids. Vitellarium 11—19 × 16—24 μm (14 × 21,n= 9)oval to irregular in shape, positioned between ovary and median testis. Vitellarium ventral close to ovary. Vagina inconspicuous, thin walled (Fig. 1 d).

Uterus f irst appears as perforated transversely elongate sac occupying median f ield of proglottids, situated ventrally to genital ducts and testes. With proglottid development, fully developed uterus 410—679 × 243—295 μm(515 × 262 μm,n= 9) rectangular, occupying almost all space in gravid proglottids. Testis and vitellarium disappear,with only cirrus sac remaining (Fig. 1 e). At the end of the worm, cirrus sac also disappears and proglottids are f illed with numerous eggs (Fig. 1 f). Developed uterus contains numerous (up to 260—320) small eggs. Eggs oval, 34 μm in diameter (30—39 μm) (n= 16), with embryophore.

Taxonomic s ummary

Type host:Cygnus cygnus(Linnaeus 1758).

Site in host: small intestine.

Type locality: Swan Lake City Wetland Park, Sanmenxia,Henan Province, China (111.13743 E, 34.77941 N).

Type specimens deposited: Holotype NEFU-HY012(deposited at the Parasitology Research Centre of Northeast Forestry University), single slide, from type host species and type locality, collected by Hongliang Chai, 2 October 2015.Paratype NEFU-HY012P (deposited at the Parasitology Research Centre of Northeast Forestry University), f ive slides, from type host species and type locality, collected by Hongliang Chai, 2 October 2015.

Etymology: Latinmorbus= disease,cygnus= Latin name of host, referring to the species causing disease in swans.

Molecular ph ylogenetic anal ysis

A sequence of 1277 nucleotides for the 28S rRNA ofC.cygnimorbussp. nov. was obtained by PCR amplif ication.The phylogenetic analysis (Fig. 2) of 28S rRNA sequences of Hymenolepididae species from rodents, birds, and waterfowl showed two main clades. The typicalHymenolepis,H.nanaandH. diminutaeach grouped in one cluster (Clades 1 and 2), andC. cygnimorbusformed a parallel cluster (Clade 3) with them.

The phylogenetic analysis of the 685-nt sequence obtained for thecox15′ barcode sequence showed thatC.cygnimorbusclustered withC. megalopsas monotypical clade (Clade B; Fig. 3), and the typeHymenolepisof mammals is in Clade A;H. nanaandH. diminutaare in Clade C.

Discussion

Based on typical morphological criteria, such as four muscular suckers unarmed, rostellum rudimentary, three testis,the new species would be identif ied asHymenolepissp.(Czaplinski and Vaucher 1994). However, these morphological characters also coincide with those ofCloacotaenia(Muniz-Pereira and Amato 1998).CloacotaeniaandHymenolepisare typical members of the family Hymenolepididae, have similar morphological traits and differ from other Hymenolepididae genera. Thus, species ofHymenolepisandCloacotaeniain birds share the typical morphological characters if they are not synonymy for each others.

Fig. 2 Phylogenetic relationships among hymenolepidid species based on Bayesian analysis (5,000,000 generations) of partial 28S rRNA sequences. Schistosoma japonicum was used as the outgroup.Posterior probabilities greater than 50% are shown. Branch length scale bar indicates number of substitutions per site

Fig. 3 Phylogenetic relationships among hymenolepidid species based on Bayesian analysis (5,000,000 generations) of partial cox1 gene. Schistosoma japonicum was used as the outgroup. Posterior probabilities greater than 50% are shown. Branch length scale bar indicates number of substitutions per site

In thecox1phylogenetic tree, theHymenolepisspecies were broadly distributed in each clade with other genera.The new species clustered withCloacotaenia megalops, paralleling the other two clades withH. nanaandH. dimituna,respectively, indicating that the new species isCloacotaenia,and that the current circumscription ofHymenolepiscomprises multiple genera. A similar situation was described by Sharma et al. ( 2016), who found that the typicalHymenolepis,H. nana, was more closed related toStaphylocystisspecies and thatH. diminutawas more closely related toArostrilepisspecies andPseudanoplocephalaspecies in two separate clades.

Cloacotaenia megalopsis one of the most common globally distributed hymenolepidid tapeworms parasitizing waterfowl, but regrettably, only mitochondrial genes forC. megalopshave been deposited in GenBank. Based on the 28S rDNA phylogenetic tree, the new species described here clustered withRetinometra guberianaisolated from swans (waterfowl). Besides those two species,Rodentolepis evaginataisolated fromOndatra zibethicus, which lives in swamps and marshes, grouped into a monophyletic group with high support (BPP = 84) rather than with any species ofHymenolepis. Although the new species and theHynenolepishave a rudimentary rostellum and unarmed suckers, there was a rather distant phylogenetic relationship between them.Otherwise, the new species had the closest relationship with the two species that have an armed rostellum (Retinometra guberianaandRodentolepis evaginata). Thus, the presence of rostellum rudimentary and unarmed muscular suckers in hymenolepidid species is not always indicative of the closest evolutionary relationship.

Although the morphological characters of the new species were not enough to diagnose it asCloacotaeniaorHymenolepis, thecox1and 28S rDNA phylogenic results were sufficient for separating the new species as a member ofCloacotaeniaand not a member ofHymenolepis. So, theCloacotaeniaandHymenolepisare different genera and not in synonymy as previously supposed. This result is coincident with the latest cestode classif ication by Caira and Jensen ( 2017). Actually, based on molecular evidence of the 28S rRNA, others have recommended splitting the hymenolepidid cestodes into multiple genera as a more stable and practical classif ication (Haukisalmi et al. 2010; Greiman and Tkach 2012).

zhikov Ry References B ez ub ik (N o w a k e t a l. 2 0 1 1)e t al. 2 0 1 1)ak ow(N o va (N o w a k e t a l. 2 0 1 1)Maksim n iz-P er ei ra (N o w a k e t a l. 2 0 1 1)Mu N ow ak et al. ( 2 0 1 1)n iz-P er ei ra a n d A m a t o ( 1 9 9 8)Mu Present study g di am-e t e r (μ m)0)—6 6)9)2—4 6 5—3 Eg—5 40—5 46 60 42—4 33 (20 44 (42 th U te ru s w i d t h ×leng m)2 (2 43—2 9 5) × 5(μ////1 5 (4 1 0—6 7 9) r = 0.5 1 3 4 (3 0 9 (6 30—1 5 8 4) ×e p r o g l o t t i d w i d t h × l e n g th 8 (1 4 4—5 4 0) r = 3.4 0/28 8 (3 6 6—5 4 9) r = 2.7 6/5 (4 10—6 7 9) × 2—1 9 2 0) × 4 6(860—29 5) r = 1.9 7 2 6 62 Uterin m)90 43(2(μ////) 97 05—1 00.5 00 24—1—1 0—9 17—1 0 0) × —1 9) × 2 1 6—(1(μm)V it el la ri um (μ m)80 80 60 44—1 7 3 (5 1—1 1 7) r = 1.1 1 1 2) r = 1.5 0 5 1 th g th O va ry w i d t h ×leng 0—300 20 0—180 0—180 7 (95—2 0 0) × 6 2(50—9 0) r = 2.2 1 6 5 (3 2 5—2 4 1) × 1 1 4 (17 60—520) 12(55—7 1) ×3 (9 5—1 4 6) r = 1.8 0 6 6 (4 4(40 5—6 3 0)1 5 3 9(24—5 6) r = 1.6 2 1 4 (1 1) ×/(32 0—6 0 0)0 4 1 (2 5—7 5) × 3 C ir ru s sa c w i d t h × l e n 00(60—700) ×59) r = 0.1 1 1 3 60)3 2 (2 8—3 6) × 1(250—5 7 (1 1 7—1 8 3)7 20 (5 90—1 02 0) 2 0) r = 0.2 2 6 3 44 68 70(55—(4(134—1 nimorbus 60 ×1020 ×)66 Cloacotaenia cyg Testis (μm 0 16 0 12 0 14 2—1 7 5) × 1 3 110 —(59—78 100 —102 100 —94 (75 —155)egalops and m)th (μ M at ur e p r o g l o t t i d s w i d t h × l e n g/////—1 6 3 0) r = 3.5 3 1 49 (1 0 0 (2 75—3 6 6) × 1 1 3 0 (8 6 0 32 8 6 (1 5 7—2 1 3) r = 4.1 3 9 9 (8 9—1 0 5) ×8 (7 16—8 5 0) × 1 76 6 (4 0 8—5 2 1)7 86 (7 38—1 3 8) × 1 68)ia m(μm)—262 38 43—1 20 taen/Neck(2////(1 f Cloaco 3—5 8 3) × 4 7 cker diameter (μ m)res o 5 15 (4 10—6 7 9) × 2 95)490—600 Su Morphological featu 500—600 500—600 531—659 414—693 1—659)559 (52 i am et er (μ m)8 0) r = 1.1 3 6 04 (5 3 9010 23 0—41(134 Scolex d 14///1590 (11/1441 ×—1 9 2 0) × 1 8 egalop/s s egalop s s s s Table 1 Character C. m C. m egalop C. m egalop C. m egalop C. m egalop C. m C. cy gn im or- b u s idth/length r w

Morphologically, the new species can be differentiated morphologically from the only other species in the genus(C. megalops). Comparing withC. megalops, the new species has testes arranged in a triangle instead of a line,unarmed cirrus instead of spined, a more elongated, much larger mature proglottids and much smaller testes, cirrus sac,ovary, vitellarium and uterine proglottid (Table 1). In addition, in the phylogenetic analysis ofcox1sequences, showed a variation of 10.52—23.06% with sequences ofC. megalops,whereas variation ofcox1within a helminth species is usually less than 5% (Blouin 2002; Hu et al. 2002).

The new species infected the intestine of its swan rather than the cloaca (Muniz-Pereira and Amato 1998). This site of infection, combined with the morphological and sequence differences incox1between the two parasite species, supports the delimitation ofC. cygnimorbussp. nov. as a new species of Hymenolepididae.

Conclusion

A potential novel species,C. cygnimorbussp. nov., from a whooper swan was identif ied. It is closely related toC.megalops, but clearly distinct based on differences in morphological and molecular characteristics.

AcknowledgementsThanks are especially due to Professor David Blair of the School of Marine and Tropical Biology, James Cook University, Professor Jean Mariaux, Natural History Museum in Geneva,Switzerland, and reviewers for assistance with the morphological description and language editing.