Phylogenetic Location of the Spirometra Sparganum Isolates from China, Based on Sequences of 28S rDNA D1.

BACKGROUND: The phylogenetic location of Chinese Spirometra sparganum isolates remains unclear. The aim of this study was to explore the phylogenetic location of the Spirometra sparganum isolates from China.
METHODS: The 28S ribosomal DNA (rDNA) D1 sequences of 14 Spirometra sparganum isolates collected from thirteen locations in China were analyzed by using Neighbor-Joining (NJ), maximum parsimony (MP) and Bayesian inference (BI), respectively. To investigate the deep variance of 28S rDNA D1 region among included species, the secondary structure of 28S rDNA D1 region was also calculated using the program RNA structure.
RESULTS: The genus Spirometra as a monophyletic group was evidenced by two inference methods (MP and BI). All sequences within the genus Spirometra had a bulge of a cytosine residue (Bulge C) in the stem 13 of the secondary structure model of 28S rRNA D1 region. Varietal sites in sequences from all thirteen Chinese isolates were appeared in loops. In loops, adenine was the most abundant base (averagely 41.9%) followed by guanine (averagely 30.0%), and cytosine (averagely 15.1%). In stems, the average percentage of G + C (58.3%) was higher than the percentage of A + T (41.7%).
CONCLUSION: The 'Bulge C' in the stem 13 of the 28S rDNA D1 secondary structure could be as a suitable mark to identify the Spirometra species.

Introduction

Tapeworms of the genus Spirometra belong to the class Cestoda, order Pseudophyllidea, family Diphyllobothriidae. The plerocercoid larvae (spargana) of Spirometra can lodge in the subcutaneous tissues and sometimes invades the abdominal cavity, eye, and central nervous system of human causing a serious parasitic zoonosis: sparganosis; human infection results mainly from ingesting raw fleshes of frogs and snakes infected with plerocercoids, drinking raw water contaminated with cyclops harboring procercoids, or placing frog or snake flesh on open wound for treatment of skin ulcers or eye inflammations (1-6). Sparganosis poses a serious threat to human health and also cause significant economic losses (7). Phylogenetic studies to such an important parasite group is therefore of great significance to the prevention and control of human sparganosis as well as to understand the genetic structure of parasites. However, the knowledge about the phylogenetic position of the genus Spirometra, and its affiliation to other Diphyllobothriid species (such as the genera Diphyllobothrium, Digramma, Duthiersia and Schistocephalus) are still fragmentary. Therefore, there is a pressing requirement to investigate the phylogenetic relationships of Spirometra, Diphyllobothrium and other important species within the family Diphyllobothriidae.

The nuclear rDNA gene repeat unit harbors different regions that evolve at varying rates, thus adds useful and often significant resolution to molecular systematic estimates of phylogeny at a number of different taxonomic levels (8, 9). The large subunit RNA gene (lsrDNA or 28S rDNA) has been extensively utilized in estimation of the relationships existing within and among the Cestoda (9-12). In the phylogentic study, the secondary structures of the transcribed rRNA are more conserved than the primary sequences due to the compensatory or semi-compensatory mutations, and some changes of a certain helix could be specific to a taxon to help a lot in species identification (13-15).

So, the secondary structures have drawn lots of attention from phylogenetic scientists (15-17). However, until now, few researchers have been concentrated their studies on the phylogeny of Spirometra with the 28S rDNA sequences, even more considered the secondary structures.

The main aim of this study was to explore the phylogenetic location of the Spirometra sparganum isolates from China based on the primary and corresponding secondary structures of partial 28S rDNA D1 sequences. In addition, the relationships of species among Spirometra, Diphyllobothrium and other important genera within the family Diphyllobothriidae were established using the molecular data obtained.

Materials and Methods

Taxon selection and sampling

The plerocercoids (spargana) of Spirometra were collected from subcutaneous tissue and muscles of the naturally infected wild frogs (Rana nigromaculata, R. rugulosa, R. temporaria, R. limmochari) and snakes (Enhydris chinensis) at thirteen locations of China (Table 1). Spargana dissected from frogs and snakes were wrinkled, whitish, and ribbon-shaped worms, which continuously crept in normal saline.

Table 1

Geographical origins (different locations in China) of Spirometra sparganum isolates and related taxa of the family Diphyllobothriidae used in this study, as well as their GenBank accession numbers for sequences of 28S rDNA D1 region.

Genus	Species	Sample codes	Location	Host	GenBank No.
Spirometra	S. erinaceieuropaei	GX-NN1	Nanning, Guangxi	Rana rugulosa	KF874629*
	S. erinaceieuropaei	GX-NN2	Nanning, Guangxi	R. rugulosa	KF874630*
	S. erinaceieuropaei	GX-NN-Yn1	Yongning, Guangxi	Enhydris chinensis	KF874631*
	S. erinaceieuropaei	GX-NN-Yn2	Yongning, Guangxi	E. chinensis	KF874632*
	S. erinaceieuropaei	GX-NN-Xxt1	Xixiangtang, Guangxi	E. chinensis	KF874633*
	S. erinaceieuropaei	GX-NN-Xxt2	Xixiangtang, Guangxi	E. chinensis	KF874634*
	S. erinaceieuropaei	GX-NN-Xxt3	Xixiangtang, Guangxi	R. temporaria	KF874635*
	S. erinaceieuropaei	GX-NN-Xxt4	Xixiangtang, Guangxi	R. temporaria	KF874636*
	S. erinaceieuropaei	GX-YL-Lc1	Luchuan, Guangxi	R. rugulosa	KF874637*
	S. erinaceieuropaei	GX-YL-Lc2	Luchuan, Guangxi	R. rugulosa	KF874638*
	S. erinaceieuropaei	GX-GL1	Guilin, Guangxi	R. rugulosa	KF874639*
	S. erinaceieuropaei	GX-GL2	Guilin, Guangxi	R. rugulosa	KF874640*
	S. erinaceieuropaei	GX-LG-Lt1	Lingui, Guangxi	R. rugulosa	KF874641*
	S. erinaceieuropaei	GX-LG-Lt2	Lingui, Guangxi	R. rugulosa	KF874642*
	S. erinaceieuropaei	GX-LG-Wt1	Lingui, Guangxi	R. temporaria	KF874643*
	S. erinaceieuropaei	GX-LG-Wt2	Lingui, Guangxi	R. temporaria	KF874644*
	S. erinaceieuropaei	HeN-KF1	Kaifeng, Henan	R. temporaria	KF874645*
	S. erinaceieuropaei	HeN-KF2	Kaifeng, Henan	R. temporaria	KF874646*
	S. erinaceieuropaei	HeN-KF-Tx1	Tongxu, Henan	R. limmochari	KF874647*
	S. erinaceieuropaei	HeN-KF-Tx2	Tongxu, Henan	R. limmochari	KF874648*
	S. erinaceieuropaei	HeN-LH1	Luohe, Henan	R. limmochari	KF874649*
	S. erinaceieuropaei	HeN-LH2	Luohe, Henan	R. limmochari	KF874650*
	S. erinaceieuropaei	HeN-XX1	Xinxiang, Henan	R. limmochari	KF874651*
	S. erinaceieuropaei	HeN-XX2	Xinxiang, Henan	R. limmochari	KF874652*
	S. erinaceieuropaei	HeN-ZK-Fg1	Fugou, Henan	R. limmochari	KF874653*
	S. erinaceieuropaei	HeN-ZK-Fg2	Fugou, Henan	R. limmochari	KF874654*
	S. erinaceieuropaei	HeN-ZZ1	Zhengzhou, Henan	R. nigromaculata	KF874655*
	S. erinaceieuropaei	HeN-ZZ2	Zhengzhou, Henan	R. nigromaculata	KF874656*
	S. erinaceieuropaei	N/a	N/a	N/a	AF004717
	S. mansonoides	N/a	N/a	N/a	AF004718
Diphyllobothrium	D. latum	N/a	N/a	N/a	AF004719
	D. latum	N/a	Russia	Gymnocephalus cernuus	DQ925326
	D. latum	N/a	Finland	N/a	AB302387
	D. nihonkaiense	N/a	Japan	N/a	AB302388
	D. pacificum	N/a	Peru	Homo sapiens	DQ925327
	D. stemmacephalum	N/a	USA	Lagenorhynchus acutus	AF286943
Digramma	D. interrupta	N/a	Russia	Hemiculter lucidus	DQ925325
Duthiersia	D. fimbriata	N/a	Ghana	Varanus exanthematicus	DQ925328
Schistocephalus	S. solidus	N/a	USA	Gasterosteus aculatus	AF286944

Asterisks indicate sequences newly reported in this study (N/a = Not available)

These spargana were 1-13 cm long and 1-2.5 mm wide. To study the phylogenetic relationships among diphyllobothroid cestodes, other members of the genera Spirometra, Digramma, Diphyllobothrium, Duthiersia and Schistocephalus within the family Diphyllobothriidae were considered in the present study (Table 1), with two species of the family Taeniidae (Taenia saginata AF096224 and T. taeniaeformis AF004721) as out-group to root the resulting trees.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from individual plerocercoid sample using the Tiangen DNeasy Blood and Tissue Kit (Tiangen, China) following the manufacturer's protocol. The 28S rDNA D1 region was amplified by PCR using the primer combination of Lee et al. 2007 (9): forward primer (JB10, 5′-GATTACCCGCTGAACTTAAGCATA-3′) and reverse primer (JB9, 5′-GCTGCATTCAC-AAACACCCCGACTC-3′).

Polymerase chain reactions (PCRs) (25μl) were performed in 2mM MgCl2, 2.5μM of each primer, 2.5μl 10 × rTaq buffer, 0.5mM of each deoxyribonucleoside triphosphate (dNTP), 1.25U of rTaq DNA polymerase (Takara, China), and 1μl of DNA (5-10 ng) sample. The amplification profile for 28S rDNA D1 region consisted of an initial denaturation for 2 min at 94 °C, 40 cycles of: 20 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C; followed by a 6 min final extension at 72 °C. PCR products were purified using the High Pure PCR Product Purification Kit (Takara, China) and sequenced in both directions using an automated sequencer (ABI Prism 3730 XL DNA Analyzer; ABI Prism, Foster City, CA) at the Genwiz Company (Beijing, China).

Sequence analysis

The 28S rDNA D1 sequences were aligned using the computer program Clustal X 2.0 (18). Molecular Evolutionary Genetics Analysis (MEGA) software version 5.0 (19) was employed to analyze the nucleotide composition, conserved sites, variable sites, parsim-info sites and singleton sites, as well as to compute uncorrected pairwise divergence. The program DAMBE 5.0 (20) was used to measure the nucleotide substitution saturation using the method of Xia et al., 2003 (21) as the substitution saturation masked the phylogenetic signal.

Phylogenetic analysis

The aligned 28S rDNA D1 sequences were analyzed by using three methods of Neighbor-Joining (NJ), maximum parsimony (MP) and Bayesian inference (BI), respectively. NJ analysis was performed in PAUP*4b10 (22) using the Kimura two-parameter distance selected by Modeltest 3.7 (23) under the Akaike information criterion and the “heuristics” search option with the “simple” addition sequence and TBR (tree bisection-reconnection) swapping. MP analysis was also performed in PAUP*4b10 (22) using heuristic searches with TBR branch swapping and 2,000 random addition sequences. Confidence in each node was assessed by boot-strapping (2000 pseudo-replicates, heuristic search of 20 random addition replicates with TBR option). BI analyses were performed in MrBayes v3.1 (24) with 5,000,000 generations, sampling trees every 100 generations. Stationarity was assessed using a convergence diagnostic. An average standard deviation of the split frequencies (ASDSF) < 0.03 were used as criteria of convergence between both runs.

Predication of secondary structure

The thermodynamic secondary structure of 28S rDNA D1 region was calculated using the program RNA structure 5.6 (25). We recognized standard Watson-Crick base pairs and noncanonical G: U interactions in all putative stem positions, because G: U can replace a Watson-Crick pair to maintain a helical region and may even have a special function in some instances (26). Information on compensatory and/or semi-compensatory base substitutions was also used to confirm local secondary structures. The RNAviz 2.0 (27) was used to produce the figures of the structures.

Results

The 28S rDNA D1 fragments of the Spirometra sparganum isolates from thirteen locations of China were successfully amplified, and sequenced to be 298 bp in eleven isolates (isolates from Luchuan, Yongning, Xixiangtang, Nanning, Guilin, Liutang and Wutong of Guangxi Zhuang Autonomous Region; and Kaifeng, Tongxu, Xinxiang and Zhengzhou of Henan province), 297 bp in two Henan isolates from Luohe and Fugou. The alignment of the fourteen 28S rDNA D1sequences obtained in the present study and the eleven sequences of the family Diphyllobothriidae obtained from the GenBank resulted in a total of 302 characters including gaps. The MEGA analysis presented 97.32% (290/298) sequence identity, 8 variable sites, and 2 singletons in the fourteen sparganum isolates from China. The average contents of A, C, G and T in these sparganum isolates were 24.8, 22.8, 31.6, and 20.8%, respectively. The nucleotide frequencies were a little biased toward G + C, averaging 54.4 %. The genetic divergence of 28S rDNA D1 sequences of species within the family Diphyllobothriidae ranged from 0 to 6.1%. However, when only took account of Chinese isolates, the upper limits would decline to 0.4%.

The test of substitution saturation showed that the observed index of substitution saturation (Iss) for 28S rDNA D1-alignments was 0.0461, and the corresponding critical index substitution saturation (Iss.c) was 0.6149 for a symmetrical tree and 0.3805 for an extreme asymmetrical tree. The index of Iss was significantly lower than the corresponding Iss.c (p = 0.0000), indicating that there was little saturation in our sequences. The most suitable substitution model was K2 for 28S rDNA D1 region following model selection by Modeltest.

In order to explore the phylogenetic location of the Spirometra sparganum isolates from China and the relationships of important species within the family Diphyllobothriidae, phylogenetic trees were reconstructed based on partial 28S rDNA D1 sequences under NJ, MP, BI three inference methods, respectively, (Fig. 1). As shown in Fig. 1, the monophyly of the family Diphyllobothriidae was supported by all three methods with high support values (100/100/1). Within Diphyllobothriidae, the genus Duthiersia was in the basal of the family, the genera Schistocephalus, Digramma, Diphyllobothrium and Spirometra made up a monophyletic group (60/69/0.72). The clade including all isolates from China and two species (Spirometra erinaceieuropaei AF004717 and Spirometra mansonoides AF004718) obtained from the GenBank was supported by MP and BI methods (69/0.99). The genera Schistocephalus, Digramma and Diphyllobothrium were recovered as a single clade but with very weak support.

Fig 1

Phylogenetic relationship among the examined Spirometra erinaceieuropaei sparganum isolates from China and other Diphyllobothriid species inferred by Neighbor-Joining (NJ), maximum parsimony (MP) and Bayesian inference (BI) analyses based on 28S rRNA D1 sequences. The numbers along branches indicate bootstrap values and posterior probabilities resulting from different analyses in the order: NJ/MP/BI. The bootstrap values lower than 60 and the posterior probabilities lower than 0.6 are given as ‘Weak’

Our core secondary structure model of 28S rRNA D1 region based on the Spirometra isolate from Naning of China is shown in Fig. 2. We recognized totally fifteen stems, which were numbered 1-15. Positions within stems were indicated by numbers after dashes: 1-1 indicates the first [5’-side] base in stem 1, paired with its complement. Two of the fifteen stems were supported by positional covariance among the 25 Diphyllobothriid sequences included in this analysis. One was position 9-3 in stem 9 of Diphyllobothrium nihonkaiense, D. pacificum, D. stemmacephalum and Digramma interrupta, respectively; the other was position 10-4 in stem 10 of Duthiersia fimbriata and Spirometra mansonoides (Fig. 3). All sequences within the genus Spirometra had a bulge of a cytosine residue (Bulge C in Fig. 2) in the stem 13, but the bulge structure was absent in the genera Diphyllobothrium, Digramma, Duthiersia and Schistocephalus (Fig. 3). Total and varietal sites, and nucleotide percentages for Diphyllobothriid 28S rRNA D1 stems and loops are given in Table 2. Varietal sites in sequences from all Chinese isolates were appeared in loops. However, these sites were more likely to reveal in stems of Diphyllobothrium, Digramma, Duthiersia and Schistocephalus. In loops, adenine is the most abundant base (averagely 41.9%) followed by guanine (averagely 30.0%), and cytosine (averagely 15.1%). In stems, the average percentage of G + C (58.3%) was higher than the percentage of A + T (41.7%).

Fig 2

A Core secondary structure model for the Diphyllobothriid 28S rRNA D1 region illustrated using a Spirometra erinaceieuropaei sparganum isolate from Nanning of China (GenBank Accession No. KF874629). Base pairing is indicated as follows: standard canonical pairs by lines (G-C, A-U), wobble G:U pairs by dots (G•U)

Fig 3

Secondary structure models for the 28S rRNA D1 region of species within genera Diphyllobothrium, Digramma, Duthiersia, Schistocephalus and Spiromtra mansonoides. Base pairing is indicated as follows: standard canonical pairs by lines (G-C, A-U), wobble G:U pairs by dots (G•U)

Table 2

Molecular properties of Diphyllobothriid 28S rRNA D1 stems and loops

Sequences	Length (bp)	Total sites		Varietal sites		Loops				Stems
		Loop	Stem	Loop	Stem	%A	%C	%G	%T	%A	%C	%G	%T
Spirometra
GX-NN1	298	91	190	0	0	42.9	15.4	29.7	12.0	16.8	24.7	33.2	25.3
GX-NN-Yn1	298	91	190	1	0	41.8	15.4	30.8	12.0	16.8	24.7	33.2	25.3
GX-NN-Xxt1	298	91	190	1	0	41.8	15.4	30.8	12.0	16.8	24.7	33.2	25.3
GX-NN-Xxt3	298	91	190	1	0	41.8	16.5	29.7	12.0	16.8	24.7	33.2	25.3
GX-YL-Lc1	298	91	190	2	0	42.9	15.4	30.8	10.9	16.8	24.7	33.2	25.3
GX-GL1	298	91	190	2	0	41.8	14.3	30.8	13.1	16.8	24.7	33.2	25.3
GX-LG-Lt1	298	91	190	3	0	40.7	14.3	30.8	14.2	16.8	24.7	33.2	25.3
GX-LG-Wt1	298	91	190	1	0	42.9	14.3	29.7	13.1	16.8	24.7	33.2	25.3
HeN-KF1	298	90	190	1	0	42.2	15.6	30.0	12.2	16.8	24.7	33.2	25.3
HeN-KF-Tx1	298	91	190	2	0	40.7	15.4	31.9	12.0	16.8	24.7	33.2	25.3
HeN-LH1	297	90	190	2	0	41.1	15.6	31.1	12.2	16.8	24.7	33.2	25.3
HeN-XX1	298	91	190	2	0	40.7	15.4	31.9	12.0	16.8	24.7	33.2	25.3
HeN-ZK-Fg1	297	90	190	1	0	42.2	15.6	30.0	12.2	16.8	24.7	33.2	25.3
HeN-ZZ1	298	91	190	3	0	40.7	15.4	30.8	13.1	16.8	24.7	33.2	25.3
S. erinaceieuropaei	302	92	192	1	2	42.4	15.2	29.3	13.1	17.2	24.5	32.8	25.5
S. mansonoides	251	84	152	3	4	39.3	14.3	34.5	11.9	17.1	25.7	32.9	24.3
Diphyllobothrium
D. latum (AF004719)	299	93	188	3	0	40.9	15.1	30.1	13.9	17.0	25.0	33.0	25.0
D. latum (DQ925326)	297	89	192	2	3	42.7	14.6	28.1	14.6	16.1	25.0	33.9	25.0
D. latum (AB302387)	249	69	146	1	1	37.7	17.4	31.9	13.0	16.4	26.0	33.6	24.0
D. nihonkaiense	297	89	192	2	4	43.8	14.6	28.1	13.5	16.1	25.0	33.9	25.0
D. pacificum	297	89	192	2	4	43.8	14.6	27.0	14.6	16.1	25.5	33.9	24.5
D. stemmacephalum	297	89	192	2	4	42.7	14.6	28.1	14.6	16.1	25.5	33.9	24.5
Digramma interrupta	297	89	192	2	4	42.7	14.6	28.1	14.6	16.1	25.0	33.9	25.0
Duthiersia fimbriata	297	93	188	4	8	45.2	15.1	25.8	13.9	14.4	24.5	35.6	25.5
Schistocephalus solidus	296	89	192	1	3	42.7	14.6	29.2	13.5	16.1	25.0	33.4	25.5
Average	294.0	89.4	187.1	1.8	1.5	41.9	15.1	30.0	13.0	16.6	24.9	33.4	25.1

Discussion

In this study, the phylogenetic position of the Spirometra sparganum isolates from China was explored based on the 28S rDNA D1 sequence, and the secondary structure of 28S rDNA D1 region was inferred. The phylogeny reveals that all thirteen Chinese sparganum isolates combined with Spirometra erinaceieuropaei and S. mansonoides species make up a monophyletic group. The secondary structure of 28S rDNA D1 sequences within the genus Spirometra (S. erinaceieuropaei and S. mansonoides) has a bulge of a cytosine residue (Bulge C) in the stem 13.

Unlike the nucleotide frequencies of Diphyllobothriid mitochondrial sequences which are more biased toward A + T (28, 29), our analysis of the 28S rDNA D1 region shows that the G + C nucleotide frequencies are higher than A + T, which is consistent with the study of Lee et al. 2007 (9). This may indicates that the Diphyllobothriid nuclear ribosomal DNA sequence is generally GC rich. In the phylogenetic trees, a high posterior probability (0.99) supports the monophyly of the genus Spirometra. Inside the clade of Spirometra, the species Spirometra mansonoides was in the basal, all isolates from China and the species Spirometra erinaceieuropaei made up a sub-clade. As the species Spirometra erinaceieuropaei is mainly distributed in Asia, and S. mansonoides is the most common in North America (30, 31), the sparganum isolates in China should be represent S. erinaceieuropaei. The genera Schistocephalus, Digramma and Diphyllobothrium were revealed as a monophyletic group, which is consistent with the conclusion of Brabec et al. 2006 (11) based on both small and large subunit nuclear ribosomal RNA genes. And the close relationship between Schistocephalus and Diphyllobothrium is also supported by the analysis of Olson et al. 2001 (10). In our analysis, the sister-group relationship between Spirometra and Schistocephalus + Digramma + Diphyllobothrium was moderate supported by NJ, MP and BI inference methods, this tree topology indicated that the two important species groups (Spirometra and Diphyllobothrium) of the family Diphyllobothriidae might be sibling relationship. Certainly, the confirmed relationship between Spirometra and Diphyllobothrium should wait for an even more comprehensive phylogenetic study with broader sampling of the more terminal representative taxa and more other effective molecular markers as well as morphological data.

In the secondary structure of 28S rDNA D1 region, a higher percentage of adenine in unpaired regions and higher nucleotide frequencies of G + C are consistent with results of previous studies (32, 33). As insertion or deletion, the multiple sequence alignments sometimes seem less reliable during the phylogenetic research, but the secondary structures could help to make a more reliable assignment of nucleotide homology in this situation (14). What's more, some changes, such as expansions and deletions, of a certain helix could be specific to a taxon to help a lot in species identification (15).

Conclusion

According to the results of phylogenetic analyses, the thirteen sparganum isolates from China represent S. erinaceieuropaei. The ‘Bulge C’ in the stem 13 of the 28S rDNA D1 secondary structure could be used as a suitable mark to identify the Spirometra species.