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Accepted Manuscript
Molecular analyses reveal two geographic and genetic lineages for tapeworms,Taenia solium and Taenia saginata, from Ecuador using mitochondrial DNA
Danilo Solano, Juan Carlos Navarro, Antonio León-Reyes, Washington Benítez-Ortiz,Richar Rodríguez-Hidalgo
PII: S0014-4894(16)30251-X
DOI: 10.1016/j.exppara.2016.10.015
Reference: YEXPR 7325
To appear in: Experimental Parasitology
Received Date: 20 May 2016
Revised Date: 30 September 2016
Accepted Date: 14 October 2016
Please cite this article as: Solano, D., Navarro, J.C., León-Reyes, A., Benítez-Ortiz, W., Rodríguez-Hidalgo, R., Molecular analyses reveal two geographic and genetic lineages for tapeworms, Taeniasolium and Taenia saginata, from Ecuador using mitochondrial DNA, Experimental Parasitology (2016),doi: 10.1016/j.exppara.2016.10.015.
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Molecular analyses reveal two geographic and genetic lineages for tapeworms, Taenia solium and 1
Taenia saginata, from Ecuador using mitochondrial DNA. 2
Danilo Solanoa,e, Juan Carlos Navarroa,c Antonio León-Reyesd, Washington Benítez-Ortiza,b, Richar 3
Rodríguez-Hidalgoa,b* 4
a Centro Internacional de Zoonosis (CIZ), Universidad Central del Ecuador, Quito, Ecuador. 5
b Facultad de Medicina Veterinaria y Zootecnia, Universidad Central del Ecuador, Quito, Ecuador. 6
c Universidad Internacional SEK, Facultad de Ciencias Naturales y Ambientales. Quito, Ecuador 7
d Laboratorio de Biotecnología Agrícola y de Alimentos, Universidad San Francisco de Quito, Quito, 8
Ecuador. 9
e Carrera Biotecnología, Universidad de las Fuerzas Armadas (ESPE), Quito, Ecuador. 10
*Corresponding Author: Tel.: +593 98 5028 169. 11
E-mail: [email protected] (R. Rodríguez-Hidalgo) 12
13
Abstract 14
Tapeworms Taenia solium and Taenia saginata are the causative agents of taeniasis / cysticercosis. 15
These are diseases with high medical and veterinary importance due to their impact on public health 16
and rural economy in tropical countries. The re-emergence of T. solium as a result of human migration, 17
the economic burden affecting livestock industry, and the large variability of symptoms in several 18
human cysticercosis, encourage studies on genetic diversity, and the identification of these parasites 19
with molecular phylogenetic tools. Samples collected from the Ecuadorian provinces: Loja, Guayas, 20
Manabí, Tungurahua (South), and Imbabura, Pichincha (North) from 2000 to 2012 were performed 21
under Maximum Parsimony analyses and haplotype networks using partial sequences of mitochondrial 22
DNA, cytochrome oxidase subunit I (COI) and NADH subunit I (NDI), from Genbank and own 23
sequences of Taenia solium and Taenia saginata from Ecuador. Both species have shown reciprocal 24
monophyly, which confirms its molecular taxonomic identity. The COI and NDI genes results suggest 25
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phylogenetic structure for both parasite species from south and north of Ecuador. In T. solium, both 26
genes gene revealed greater geographic structure, whereas in T. saginata, the variability for both genes 27
was low. In conclusion, COI haplotype networks of T. solium suggest two geographical events in the 28
introduction of this species in Ecuador (African and Asian lineages) and occurring sympatric, probably 29
through the most common routes of maritime trade between the XV-XIX centuries. Moreover, the 30
evidence of two NDI geographical lineages in T. solium from the north (province of Imbabura) and the 31
south (province of Loja) of Ecuador derivate from a common Indian ancestor open new approaches for 32
studies on genetic populations and eco-epidemiology. 33
Keywords: mtDNA lineages, cysticercosis, taeniasis, COI, NADH-I, Taenia solium, Taenia saginata, 34
Ecuador. 35
1. Introduction 36
Infestations by Taenia spp., and Echinococcus spp., are common, not only in developing countries but 37
also in industrialized countries, where apart from being a threat to health, they represent a socio-38
economic impact. Cysticercosis causes a debilitating disease in humans as well as losses in the meat 39
industry due to the condemnation of meat from infected animals (Jiménez et al., 2002; Raether and 40
Hänel, 2003; Rodriguez Hidalgo, 2007; Schantz, 1999; Tsai et al., 2013) 41
The Andean region of Ecuador was described as hyperendemic for taeniasis / cysticercosis, with 42
prevalences in rural communities up to 1.60% for taeniasis and 14.4% for T. solium-cysticercosis by. 43
The prevalence of T. saginata in Ecuador is not well established, however, a moderate prevalence of 44
bovine cysticercosis with 0.37% on veterinary inspection and 4.03% by serological techniques has been 45
reported (Cayo-Rojas et al., 2011; Cruz et al., 1989; Rodriguez-Hidalgo et al., 2006; Rodríguez-46
Hidalgo et al., 2010, 2003; Rodriguez Hidalgo, 2007). 47
As a result of their great importance in Ecuador, these two cestodes species have been extensively 48
studied. However, the strategies aiming to control these parasitoses have major limitations i.a. it has not 49
been possible to accurately identify the prevalence of Taenia spp. and the determination of strains by 50
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means of ecological, biological or morphological criteria is difficult (Ito et al., 2003). The efficiency of 51
control depends on detailed epidemiological information including identification and precise 52
characterization of the causative agent in each endemic area (Gasser et al., 1999; Jia et al., 2010; 53
McManus, 1990). 54
The genus Taenia has been successfully identified using, enzyme electrophoresis and mitochondrial 55
molecular markers to differentiate between species and also to infer phylogenies (Hoberg et al., 2000; 56
Nakao et al., 2010; Queiroz and Alkire, 1998). However, little is known about the genetic intra-specific 57
variation in cestodes (Pawlowsky Zbigniew, 2002) and considerable research indicates a great 58
heterogeneity in pathology caused by Taenia solium but there is a misunderstanding about the role of 59
genetic diversity and adaptability of species (Del Brutto, 2013; Finsterer and Auer, 2012; Marquez and 60
Arauz, 2012; Román, 2014; Sotelo, 2011). Based on this issue, mitochondrial DNA analysis provides 61
complementary tools for characterization of a population. Gene fragments or complete genome of 62
mitochondrial DNA have been successfully used in population genetics, ecology and identification of 63
tapeworms (Jia et al., 2010). Genetic variation associated with different hosts is a well-known fact in 64
several cestodes species e.g. Echinococcus granulosus and Ito et al., (Ito et al., 2003) suspected that for 65
Taenia saginata and Taenia solium it may well be equally the case. 66
Nakao et al., (Nakao et al., 2002), using the complete genes Cytochrome Oxidase subunit I and 67
Cytochrome b of the mitochondrial DNA, showed the existence of two lineages of Taenia solium 68
worldwide: an Asian group and an African and Latin-American group of strains. A sequence from 69
Ecuador was included in these studies, showing minimal variation with the rest of the sequences in the 70
analysis, albeit this sequence represents only a small portion of the Ecuadorian gene pool. Yanagida et 71
al., (Yanagida et al., 2014) using different mitochondrial genes, report two genetic sympatric lineages 72
of T. solium in Madagascar close related with Asia and Africa/Latin America as consequence of 73
historical human migration. These authors shown the Africa/Latin America lineage connected by 74
haplotypes network with the Ecuadorian haplotype AB066491 here also used. 75
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It is important to complete more detailed variability studies for this species, even more so when genetic 76
variation of tapeworms from various geographic locations might be linked to clinical and pathological 77
differences found in human cysticercosis (Maravilla et al., 2003; Vega et al., 2003). 78
The aim of this research is to determine the genetic diversity within populations of Taenia solium and 79
Taenia saginata collected in six locations, from the north and the south of Ecuador. We also 80
hypothesized that different geographical origins or introductions, can be assessed by means of presence 81
of geographic phylogenetic structuration within sequences-populations and/or haplotypes networks 82
analysis. The results in this study can underpin epidemiological research and the control of these 83
parasites. Furthermore, it provides a great source of information and support for new diagnostic 84
methods and reinforces vaccines developing in the fight against these important diseases (Assana et al., 85
2010; Fernadez et al., 2006; Maravilla et al., 2008; Sciutto et al., 2013; Tsai et al., 2013). 86
2. Materials and methods 87
2.1. Source of Taenia spp. specimens. 88
Specimens used in this research belong to the biological bank of the Centro Internacional de Zoonosis 89
(CIZ) at Universidad Central del Ecuador from localities of a former study and control program. 90
Samples have been conserved in ethanol solution 70%, and kept frozen at -20°C. Samples were 91
collected from the Ecuadorian provinces of: Loja, Guayas, Manabí, Tungurahua (South), and 92
Imbabura, Pichincha (North) from 2000 to 2012 (Supplementary data 1). Specimens were isolated form 93
human faecal material after anthelmintic treatment. Patients became from both urban and rural areas. 94
The vouchers of remains specimens are deposited in the bank of CIZ. 95
2.2. DNA extraction, amplification and sequencing of COI and NDI genes. 96
For DNA extraction of Taenia’s proglottids, the Wizard Genomic DNA Purification of Promega® 97
commercial kit was used (Promega, 2010) following the manufacturer's protocol. The DNA samples 98
obtained from proglottids of Taenia spp. were analysed in agarose gel 0.8% to ascertain presence, 99
quality and size of the extracted material. The quantification of genomic DNA extracted from Taenia 100
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spp. was performed using the Invitrogen fluorometer QUBIT®. We used the Quant-iT™ Broad-Range 101
DNA Assay Kit according to the manufacturer's instructions (Data not shown). 102
The reactions were performed in 25 µL, using a thermo cycler TECHNE TC-412, using the primers for 103
NDI sequence (Bowles and McManus, 1993) and JB3-JB4.5 for COI (Bowles et al., 1992). For each 104
reaction buffer PCR 1X, 3mM of MgCl2, 0.5 µM of oligonucleotide, 0.15 mM of dNTP´s and 0,5 U of 105
Taq Polymerase (Invitrogen) was added. The procedure was as follows: 92 °C for 5 minutes for initial 106
denaturation; 35 cycles of 94 °C, 30 s (denaturation); 55°C (COI)/ 57 °C (NDI), 30 s (annealing); 72 °C, 107
30 s/ 1 min (extension), followed by a final extension at 72 °C for 5 minutes. For each batch of 108
reactions, a negative control (molecular biology grade water) and a positive control (DNA from T. 109
saginata or T. solium) were included. 110
Amplified fragments of COI and NDI were purified using PureLink® PCR Purification Kit (Invitrogen) 111
according to the manufacturer's instructions, subsequently the fragments were sent to Macrogen Inc. 112
(Korea) for sequencing in duplicate (forward and reverse). 113
2.3. Phylogenetic analysis and haplotype networks 114
Own sequences were contig assembly performed using the Sequencer 4.2.2 (Gene Codes, Ann Arbor, 115
MI) software and identity confirmed by BLAST in NCBI resources. The consensus sequences of gene 116
cytochrome oxidase subunit 1 (COI) and NAD dehydrogenase subunit 1 (NDI) from the two groups 117
(Taenia solium and Taenia saginata) were edited in MacClade software (Maddison and Wayne, 2011), 118
resulting sequences of 404 bp (COI) and 459 bp (NDI). Sequences deposited in GenBank NCBI from 119
other species of Taenia were included as reference/external groups and outgroups (Nixon and 120
Carpenter, 1993) (Supplementary data 2) in order to get a wide geographic diversity covering distinct 121
continents and to test the problem species monophyly. DNA sequences were aligned using MacVector 122
7.2 (Accelrys, Madison, WI) by ClustalW algorithm with gap creation and extension penalties by 123
default (Supplementary data 3 and 4). Parsimony analyses were implemented in PAUP 4.0b10 124
(Swofford, 2001) using the heuristic search option with a Tree Bisection Reconnection branch-125
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swapping algorithm with at random stepwise addition of 10 replicates for each search and 100-1,000 126
replications per analysis. Gaps were treated both as missing data and as a fifth character state. The 127
characters were treated as unordered, and equally weighted, after that the characters were weighted by 128
consistence index. The robustness of the trees was estimated using parsimony bootstrap with 500 129
pseudoreplicates after excluding uninformative characters (Carpenter, 1996). We also performed a 130
Maximum Likelihood (ML, substitution model estimated by ModelTest, Posada and Crandall, (1998) 131
on PAUP) and a distance analysis (Neighbour-joining, NJ) using PAUP 4.0b10 (Swofford, 2001). 132
Later, a Nexus Matrix of sequences for each species was used to construct haplotype networks for TCS 133
2.1.1 with 95% of connection limit (Clement et al., 2000). We use the monophyly (based on % of 134
bootstrapping, location in clades on the tree, and a genetic divergence intra species bigger than inter 135
species) and phylogenetic species concept to delimit the different taxa. Then, the paraphyletic position 136
in the tree topology of sequences from GenBank under with the same label means a different species 137
and as consequence a misidentified specimen. 138
3. Results and Discussion 139
3.1. PCR amplification of genes COI and NDI 140
A total of 53/68 specimens of T. solium and T. saginata were successfully amplified. Most samples 141
were complete adult specimens (without scolex), with one third of the samples were proglottids only. 142
Tapeworms were previously characterized using morphology and RFLP analysis of 12S gene 143
(Rodriguez-Hidalgo et al., 2002). 144
3.2 Phylogenetic Analysis 145
3.2.1 Cytochrome oxidase subunit I gene (COI) 146
The maximum parsimony analysis of the 67 sequences used, characters re-weighted, yielded a single 147
parsimonious tree whose length values, consistency index and retention index values of L= 364.82, CI= 148
0.84 and RI= 0.91 (Fig. 1). The ML and NJ yielded the same topology of MP. The substitution model 149
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that fit wit the matrix alignment calculated by ModelTest with Bayesian Information Criteria (BIC) for 150
ML was TrN+G (Supplemtary data 5). 151
The topologies of phylogenetic trees obtained using COI gene (weight and reweighted) reveal the 152
internal grouping of clades, corresponding to sequences of a monophyletic derivated clade Taenia 153
solium, Taenia saginata in a monophyletic clade plus T. asiatica and the outgroups sequences in a 154
basal location. The Taenia saginata group does not show a phylogenetic structure, therefore samples 155
from distant locations like Japan, Belgium, Kenya and Ecuador present values of divergence between 156
0% and 0.26%, evidenced in the tree as a polytomy. A more detailed analysis of the alignment showed 157
two changes: E42.Tsag. Tungurahua, G by A in position 303, and Tsag. FRANCE T by C in position 158
243. 159
In a similar fashion, the internal distribution of the Taenia solium clade shows a phylogenetic structure. 160
Within the internal or derivated clade of T. solium, the sequences showed two subclades with 161
geographical correspondence: an ancestral Asian subclade + Peruvian sequence (Fig. 1, in yellow and 162
green colours) and a derivated African + America subclade (Fig. 1, in blue colour) with Ecuadorian 163
sequences included in a politomy (not-resolved branches). These findings are consistent with the results 164
found by Nakao et al. (Nakao et al., 2002). The distance (p-uncorrected) matrix (Table 1, 165
Supplementary data 6) shows that genetic divergence of the COI gene in samples from Imbabura 166
(North Ecuador) and Loja (South Ecuador), varies between 0 % and 1.06%, therefore, no significant 167
difference was observed among Ecuadorian samples from different geographic locations. 168
The difficulty of taxonomic identification of metacestodes of Taenia spp. is evidenced when some 169
particular sequences found in gene databases do not correspond to the species labelled based on their 170
location in our trees. In our study, two COI sequences: T. solium-Mexico (EU747650) and T. solium-171
Peru (AF360866), appear as different species from those originally published. In the first case 172
(EU747650), the sequence shows a clear homology and sister relationship with T. hydatigena, which 173
has pigs as a common intermediate host (Conlan et al., 2009) suggesting a misidentify. The sequence 174
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(AF360866) is presented as a particular case, the topology analysis locates it as an ancestor of T. 175
solium, however the divergence with this species is considerably high: 11.94%. The divergence 176
between T. solium and the closest sequence T. hydatigena is 12.40%, therefore, this specimen 177
(AF360866) could be a genetic variation of T. solium from Peru (conservative point of view), or an 178
intermediate species between T. solium and T. hydatigena, this however, requires further research. 179
In order to resolve the polytomies observed in the tree and to detect mutational ancestor-descendant 180
changes between geographical sequences, we have performed the haplotype network (TCS) shown in 181
Fig. 2. We found eleven haplotypes of worldwide distribution grouped into two different (not 182
connected) networks with Ecuadorian haplotypes located in both linked with African-American and 183
Asian haplotypes. 184
3.2.2. NAD dehydrogenase subunit I gene (NDI) 185
The maximum parsimony analysis of the 31 sequences and re-weighted yielded a single tree with 186
values of L= 344.89, CI= 0.76 e RI= 0.83 (Fig. 3). The ML and NJ yielded the same topology of MP. 187
The substitution model that fit wit the matrix alignment calculated by ModelTest with Bayesian 188
Information Criteria (BIC) for ML was TVM+G (Supplementary data 7). 189
The topology of the tree shows three consecutive basal clades corresponding to the outgroups taxa. 190
Then, internally two sister and reciprocal monophyletic clades: T. solium clade + T. asiatica (T. 191
saginata clade plus T. krabbei and T. multiceps). In the derivate group of T. saginata we found a 192
polytomy with no geographic structure. The sequences of T. asiatica, T. krabei and T. multiceps are 193
located as ancestral taxa to T. saginata. 194
The T. solium clade shows a geographical structure, forming two groups: one group with all sequences 195
of America (Fig. 3, in red colour), Ecuador included; and another group with Asian sequences (Fig. 3, 196
in yellow colour). The strict consensus from three topologies shows that the distribution of the clades 197
has the same geographical pattern. The bootstrap analysis with 1,000 repetitions shows clades with 198
strong support by statistical re-sampling of the alignment matrix (Fig. 3). 199
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In addition, topologies of phylogenetic trees obtained using NDI gene show a similar distribution to the 200
one found in COI gene trees, however, within the clade of Taenia solium two subclades were found 201
from the north (Imbabura) and from the south (Loja) of Ecuador. The distance (p-uncorrected) matrix 202
(Table 1, Supplementary data 8) shows that genetic divergence of the NDI gene in samples from 203
Imbabura and Loja is around 1,2% which is much less than in case of an interspecies divergence (e.g. 204
12%, between T. asiatica and T. saginata it is 12%). These findings demonstrate higher variability in 205
NDI than COI in the mtDNA of Taenia spp., in accordance with Jia et al. (Jia et al., 2010). The clade 206
of Taenia saginata as well as the results shown in the COI gene did not show evidence of significant 207
geographic differentiation. 208
In order to resolve the polytomies observed in the reweighed tree and to detect ancestor-descendant 209
mutational changes between geographical sequences (Fig. 4) we used the TCS software. The haplotype 210
network constructed is shown in Fig. 4. Five haplotypes worldwide distributed were grouped into a 211
single network with H4 and H5 from South Ecuador and North Ecuador respectively with the India 212
haplotype H1 as close related based on mutational changes. 213
3.3. Haplotype Networks 214
Searching for deeper lineage structure than in phylogenetic analysis, a network of haplotypes was 215
performed, in order to discriminate patterns of genetic divergence through specific mutational changes. 216
The network analysis using COI molecular marker resulted in two not connected networks of eleven 217
haplotypes. The first network consists of haplotypes from Brazil, Cape Verde (South Africa), Colombia 218
and Southern Ecuador (Loja) showing the detailed connection of mutational changes that the 219
politomies in the phylogenetic tree does not, for instance, the ancestor-descendent mutations 220
relationship among Colombia and Cape Verde haplotypes with Loja-Ecuador. The second network is 221
formed by haplotypes from France, Mexico, Haiti, South Korea, China, India and our North-South 222
Ecuador (Imbabura + Loja) including the Ecuadorian sequence in GenBank. Those results showing a 223
clear correlation of the Ecuadorian samples with African and Europe-Asian sequences both occurring 224
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sympatric in the Country similar to Yanagida et al., (2014) results. The two networks of haplotypes 225
analysed are correlated with one of the most common routes of maritime trade between the XV-XIX 226
centuries, supporting the hypothesis of Nakao et al, (Nakao et al., 2002) on the introduction of Taenia 227
solium from Europe to Africa and Latin America. 228
The haplotype network obtained from NDI shows a most probable introduction of Taenia solium to 229
Ecuador from a haplotype from India and subsequently the sequences from Ecuador (north and south) 230
show two separate lineages of mutational changes, with 2 changes to North Ecuador lineage and four 231
mutations to South Ecuador lineage. This network is consistent with the results reported by Martinez-232
Hernández et al. (Martinez-Hernandez et al., 2009) using COI and Cytb, who found a strong correlation 233
among sequences from Latin America (Peru and Mexico) and India. 234
In conclusion, both networks COI and NDI, suggest that the introduction of Taenia solium to Ecuador 235
occurred probably in two different events and geographical origins. Those differences might have been 236
maintained in spite of anthropogenic pressure such as intensive human migrations, and trading of 237
animals and meat products. This meta-population seems to have undergone two colonization processes, 238
with differentiation between northern and southern Ecuador. The Southern colonization influenced 239
from Asia and Africa haplotypes introductions and the Northern Ecuador mainly influenced by Asian 240
introductions. In summary, this research reports the pattern of the genetic variability and gene flow 241
among Ecuadorian localities of both Taenia species. This study provides important genetic baseline 242
data and new approaches for studies on genetic populations, eco-epidemiology and control. 243
Acknowledgements 244
We thank Jef Brandt who provided important comments and suggestion to our manuscript. 245
References 246
Assana, E., Kyngdon, C.T., Gauci, C.G., Geerts, S., Dorny, P., De Deken, R., Anderson, G.A., Zoli, A.P., 247
Lightowlers, M.W., 2010. Elimination of Taenia solium transmission to pigs in a field trial of the TSOL18 248
vaccine in Cameroon. Int. J. Parasitol. 40, 515–9. doi:10.1016/j.ijpara.2010.01.006 249
Bowles, J., Blair, D., McManus, D.P., 1992. Genetic variants within the genus Echinococcus identified by 250
Page 12
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
11
mitochondrial DNA sequencing. Mol. Biochem. Parasitol. 54, 165–73. 251
Bowles, J., McManus, D.P., 1993. NADH dehydrogenase 1 gene sequences compared for species and strains of 252
the genus Echinococcus. Int. J. Parasitol. 23, 969–72. 253
Carpenter, J.M., 1996. Uninformative bootstrapping. Cladistics. doi:10.1006/clad.1996.0013 254
Cayo-Rojas, F., Mamani-Linares, W., Gallo, C., Valenzuela, G., 2011. Revisión de Cisticercosis Bovina 255
(Cysticercus bovis) en ganado faenado: Prevalencia, Distribución y viabilidad del cisticerco. J. Selva 256
Andin. Res. Soc. 2, 53–70. 257
Clement, M., Posada, D., Crandall, K.A., 2000. TCS: a computer program to estimate gene genealogies. Mol. 258
Ecol. 9, 1657–1659. doi:10.1046/j.1365-294x.2000.01020.x 259
Conlan, J. V, Vongxay, K., Fenwick, S., Blacksell, S.D., Thompson, R.C.A., 2009. Does interspecific 260
competition have a moderating effect on Taenia solium transmission dynamics in Southeast Asia? Trends 261
Parasitol. 25, 398–403. doi:10.1016/j.pt.2009.06.005 262
Cruz, M., Davis, A., Dixon, H., Pawlowski, Z.S., Proano, J., 1989. Operational studies on the control of Taenia 263
solium taeniasis/cysticercosis in Ecuador. Bull. World Health Organ. 67, 401–7. 264
Del Brutto, O.H., 2013. Neurocysticercosis. Curr. Opin. Neurol. 26, 289–294. 265
doi:10.1097/WCO.0b013e32836027fa 266
Fernadez, M., Muñoz, A., Corredor, M., 2006. Determinación por medio de marcadores moleculares SSCP y 267
RAPD de la diversidad genética en la especie Taenia solium en Colombia. Parasitol. Latinoam. 61, 101–268
110. doi:10.4067/S0717-77122006000200001 269
Finsterer, J., Auer, H., 2012. Parasitoses of the human central nervous system. J. Helminthol. 270
doi:10.1017/S0022149X12000600 271
Gasser, R.B., Zhu, X., Woods, W., 1999. Genotyping Taenia tapeworms by single-strand conformation 272
polymorphism of mitochondrial DNA. Electrophoresis 20, 2834–2837. doi:10.1002/(SICI)1522-273
2683(19991001)20:14<2834::AID-ELPS2834>3.0.CO;2-F 274
Hoberg, E.P., Jones, A., Rausch, R.L., Eom, K.S., Gardner, S.L., 2000. A phylogenetic hypothesis for species of 275
the genus Taenia (Eucestoda : Taeniidae). J. Parasitol. 86, 89–98. doi:10.1645/0022-276
3395(2000)086[0089:APHFSO]2.0.CO;2 277
Ito, A., Yamasaki, H., Nakao, M., Sako, Y., Okamoto, M., Sato, M.O., Nakaya, K., Margono, S.S., Ikejima, T., 278
Kassuku, A.A., Afonso, S.M.S., Ortiz, W.B., Plancarte, A., Zoli, A., Geerts, S., Craig, P.S., 2003. Multiple 279
genotypes of Taenia solium—ramifications for diagnosis, treatment and control. Acta Trop. 87, 95–101. 280
doi:10.1016/S0001-706X(03)00024-X 281
Jia, W.-Z., Yan, H.-B., Guo, A.-J., Zhu, X.-Q., Wang, Y.-C., Shi, W.-G., Chen, H.-T., Zhan, F., Zhang, S.-H., 282
Fu, B.-Q., Littlewood, D.T.J., Cai, X.-P., 2010. Complete mitochondrial genomes of Taenia multiceps, T. 283
hydatigena and T. pisiformis: additional molecular markers for a tapeworm genus of human and animal 284
health significance. BMC Genomics 11, 447. doi:10.1186/1471-2164-11-447 285
Jiménez, S., Pérez, A., Gil, H., Schantz, P.M., Ramalle, E., Juste, R.A., 2002. Progress in control of cystic 286
echinococcosis in La Rioja, Spain: decline in infection prevalences in human and animal hosts and 287
economic costs and benefits. Acta Trop. 83, 213–221. doi:10.1016/S0001-706X(02)00091-8 288
Maddison, D.M., Wayne, 2011. MacClade. 289
Maravilla, P., Gonzalez-Guzman, R., Zuñiga, G., Peniche, A., Dominguez-Alpizar, J.L., Reyes-Montes, R., 290
Page 13
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
12
Flisser, A., 2008. Genetic polymorphism in Taenia solium cysticerci recovered from experimental 291
infections in pigs. Infect. Genet. Evol. 8, 213–6. doi:10.1016/j.meegid.2007.11.006 292
Maravilla, P., Souza, V., Valera, A., Romero-Valdovinos, M., Lopez-Vidal, Y., Dominguez-Alpizar, J.L., 293
Ambrosio, J., Kawa, S., Flisser, A., 2003. Detection of genetic variation in Taenia solium. J. Parasitol. 89, 294
1250–4. doi:10.1645/GE-2786RN 295
Marquez, J.M., Arauz, A., 2012. Cerebrovascular Complications of Neurocysticercosis. Neurologist. 296
doi:10.1097/NRL.0b013e31823d7a80 297
Martinez-Hernandez, F., Jimenez-Gonzalez, D.E., Chenillo, P., Alonso-Fernandez, C., Maravilla, P., Flisser, A., 298
2009. Geographical widespread of two lineages of Taenia solium due to human migrations: can population 299
genetic analysis strengthen this hypothesis? Infect. Genet. Evol. 9, 1108–14. 300
doi:10.1016/j.meegid.2009.09.005 301
McManus, D., 1990. Charactereization of taeniid cestodes by DNA analysis. Rev. Sci. Tech. 9, 489–510. 302
Nakao, M., Okamoto, M., Sako, Y., Yamasaki, H., Nakaya, K., Ito, a, 2002. A phylogenetic hypothesis for the 303
distribution of two genotypes of the pig tapeworm Taenia solium worldwide. Parasitology 124, 657–662. 304
doi:10.1017/S0031182002001725 305
Nakao, M., Yanagida, T., Okamoto, M., Knapp, J., Nkouawa, A., Sako, Y., Ito, A., 2010. State-of-the-art 306
Echinococcus and Taenia: phylogenetic taxonomy of human-pathogenic tapeworms and its application to 307
molecular diagnosis. Infect. Genet. Evol. 10, 444–52. doi:10.1016/j.meegid.2010.01.011 308
Nixon, K.C., Carpenter, J.M., 1993. On outgroups. Cladistics. 309
Pawlowsky Zbigniew, 2002. Taenia solium: Basic Biology and transmission, in: Singh, G Prabhakar, S. (Ed.), 310
Taenia Solium Cysticercosis: From Basic to Clinical Science. CABI Publishing, pp. 1–14. 311
Posada, D., Crandall, K., 1998. Modeltest: testing the model of DNA substitution. Bioinformatics. 312
Queiroz, A. De, Alkire, N., 1998. The phylogenetic placement of Taenia cestodes that parasitize humans. J. 313
Parasitol. 314
Raether, W., Hänel, H., 2003. Epidemiology, clinical manifestations and diagnosis of zoonotic cestode 315
infections: an update. Parasitol. Res. 316
Rodríguez-Hidalgo, R., Benítez-Ortiz, W., Brandt, J., Geerts, S., Dorny, P., 2010. Observaciones sobre la 317
cisticercosis bovina en el Ecuador , su importancia zoonosica en la salud publica humana - Observations on 318
bovine cisticercosis in ecuador , its zoonotic importance in human public health. Redvet 11. 319
Rodríguez-Hidalgo, R., Benítez-Ortiz, W., Dorny, P., Geerts, S., Geysen, D., Ron-Román, J., Proaño-Pérez, F., 320
Chávez-Larrea, M.A., Barrionuevo-Samaniego, M., Celi-Erazo, M., Vizcaíno-Ordóñez, L., Brandt, J., 321
2003. Taeniosis-cysticercosis in man and animals in the Sierra of Northern Ecuador. Vet. Parasitol. 118, 322
51–60. doi:10.1016/j.vetpar.2003.09.019 323
Rodriguez-Hidalgo, R., Benitez-Ortiz, W., Praet, N., Saa, L.R., Vercruysse, J., Brandt, J., Dorny, P., 2006. 324
Taeniasis-cysticercosis in Southern Ecuador: Assessment of infection status using multiple laboratory 325
diagnostic tools. Mem. Inst. Oswaldo Cruz 101, 779–782. doi:10.1590/S0074-02762006000700012 326
Rodriguez-Hidalgo, R., Geysen, D., Benítez-Ortiz, W., Geerts, S., Brandt, J., 2002. Comparison of conventional 327
techniques to differentiate between Taenia solium and Taenia saginata and an improved polymerase chain 328
reaction-restriction fragment length polymorphism assay using a mitochondrial 12S rDNA fragment. J. 329
Parasitol. 88, 1007–1011. doi:10.1645/0022-3395(2002)088[1007:COCTTD]2.0.CO;2 330
Page 14
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
13
Rodriguez Hidalgo, R., 2007. The epidemiology of Taenia spp. and cysticercosis in Ecuador. 331
Román, G.C., 2014. Tropical myelopathies. Handb. Clin. Neurol. 121, 1521–1548. doi:10.1016/B978-0-7020-332
4088-7.00102-4 333
Schantz, P.M., 1999. Taenia solium Cysticercosis: Taeniasis is a potentially eradicable disease, in: García, 334
Héctor Hugo; Martinez, S. (Ed.), Taenia Solium, Taeniasis/Cysticercosis. Universo, Lima. 335
Sciutto, E., Fragoso, G., Hernández, M., Rosas, G., Martínez, J.J., Fleury, A., Cervantes, J., Aluja, A., Larralde, 336
C., 2013. Development of the S3Pvac vaccine against porcine Taenia solium cysticercosis: a historical 337
review. J. Parasitol. 99, 686–92. doi:10.1645/GE-3102.1 338
Sotelo, J., 2011. Clinical manifestations, diagnosis, and treatment of neurocysticercosis. Curr. Neurol. Neurosci. 339
Rep. 11, 529–535. doi:10.1007/s11910-011-0226-7 340
Swofford, D., 2001. Paup*: Phylogenetic analysis using parsimony (and other methods) 4.0. B5. 341
Tsai, I.J., Zarowiecki, M., Holroyd, N., Garciarrubio, A., Sanchez-Flores, A., Brooks, K.L., Tracey, A., Bobes, 342
R.J., Fragoso, G., Sciutto, E., Aslett, M., Beasley, H., Bennett, H.M., Cai, J., Camicia, F., Clark, R., 343
Cucher, M., De Silva, N., Day, T.A., Deplazes, P., Estrada, K., Fernández, C., Holland, P.W.H., Hou, J., 344
Hu, S., Huckvale, T., Hung, S.S., Kamenetzky, L., Keane, J.A., Kiss, F., Koziol, U., Lambert, O., Liu, K., 345
Luo, X., Luo, Y., Macchiaroli, N., Nichol, S., Paps, J., Parkinson, J., Pouchkina-Stantcheva, N., Riddiford, 346
N., Rosenzvit, M., Salinas, G., Wasmuth, J.D., Zamanian, M., Zheng, Y., Cai, X., Soberón, X., Olson, 347
P.D., Laclette, J.P., Brehm, K., Berriman, M., 2013. The genomes of four tapeworm species reveal 348
adaptations to parasitism. Nature 496, 57–63. doi:10.1038/nature12031 349
Vega, R., Piñero, D., Ramanankandrasana, B., Dumas, M., Bouteille, B., Fleury, A., Sciutto, E., Larralde, C., 350
Fragoso, G., 2003. Population genetic structure of Taenia solium from Madagascar and Mexico: 351
implications for clinical profile diversity and immunological technology. Int. J. Parasitol. 33, 1479–1485. 352
doi:10.1016/S0020-7519(03)00206-6 353
Yanagida, T., Carod, J.F., Sako, Y., Nakao, M., Hoberg, E.P., Ito, A., 2014. Genetics of the pig tapeworm in 354
Madagascar reveal a history of human dispersal and colonization. PLoS One 9. 355
doi:10.1371/journal.pone.0109002 356
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Figure legends 359
Fig. 1. Phylogenetic tree with characters reweighed from the COI gene showing the phylogenetic 360
relationships of Taenia spp. Right side brackets show the species outgroups, and the taxonomic 361
nomination of sequences. Numbers on branches are the bootstrap values. Lines-branches in colors 362
shown the geographical association into each subclades. 363
Fig. 2. Global Network haplotypes of Taenia solium for COI gene. Two not connected networks 364
shows the two geographical lineages. Ecuador haplotypes are located in both networks (Africa-Latin 365
America and European-Asia networks). 366
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Fig. 3. Phylogenetic tree with NDI gene and characters reweighted showing the phylogenetic 367
relationships of Taenia spp. Right side brackets show the species outgroups, and the taxonomic 368
nomination of sequences. Numbers on branches are the bootstrap values. Lines-branches in colours 369
shown the geographical association into each subclades. 370
Fig. 4. Global Network haplotypes of Taenia solium using data from NDI gene. Ecuador 371
haplotypes are located in two separate lines (Southern and Northern Ecuador) after mutational steps 372
from India haplotype. 373
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Gene Specie Collection Place Specie Collection Place Divergence Percentage
(%)
COI
E. granulosus Turkey (JN810792)
T.solium (E44) Southern Ecuador (Loja) 19,003
T.solium (E11) Northern Ecuador (Imbabura) 19,365
T. saginata (E40) Southern Ecuador (Guayas) 19,155
T. saginata (E55) Northern Ecuador (Pichincha) 19,155
T. parva Spain (EU544580)
T.solium (E44) Southern Ecuador (Loja) 16,259
T.solium (E11) Northern Ecuador (Imbabura) 16,686
T. saginata (E40) Southern Ecuador (Guayas) 14,663
T. saginata (E55) Northern Ecuador (Pichincha) 14,663
T. solium
India (AF360869) T.solium (E44) Southern Ecuador (Loja) 1,558
T.solium (E11) Northern Ecuador (Imbabura) 2,023
Cameroon (FN995666) T.solium (E44) Southern Ecuador (Loja) 1,061
T.solium (E11) Northern Ecuador (Imbabura) 0
T. saginata
Belgium (AB107242) T. saginata (E40) Southern Ecuador (Guayas) 0
T. saginata (E55) Northern Ecuador (Pichincha) 0
China (AB533172) T. saginata (E40) Southern Ecuador (Guayas) 0
T. saginata (E55) Northern Ecuador (Pichincha) 0
Kenya (AM503327) T. saginata (E40) Southern Ecuador (Guayas) 0
T. saginata (E55) Northern Ecuador (Pichincha) 0
NDI
E. granulosus Turkey (HM563034)
T.solium (E45) Southern Ecuador (Loja) 26,051
T.solium (E12) Northern Ecuador (Imbabura) 26,651
T. saginata (E40) Southern Ecuador (Guayas) 26,932
T. saginata (E55) Ecuador North (Pichincha) 26,932
T. parva Spain (EU544633)
T.solium (E45) Southern Ecuador (Loja) 28,3
T.solium (E12) Northern Ecuador (Imbabura) 28,926
T. saginata (E40) Southern Ecuador (Guayas) 28,719
T. saginata (E55) Northern Ecuador (Pichincha) 28,507
T. solium
India (EF076753) T.solium (E45) Southern Ecuador (Loja) 0,664
T.solium (E12) Northern Ecuador (Imbabura) 1,081
Australia (AJ239107) T.solium (E44) Southern Ecuador (Loja) 13,387
T.solium (E11) Northern Ecuador (Imbabura) 13,388
T. saginata
Australia (AJ239106) T. saginata (E40) Southern Ecuador (Guayas) 0,85
T. saginata (E55) Northern Ecuador (Pichincha) 0,639
Kenya (AM503345) T. saginata (E40) Southern Ecuador (Guayas) 0,207
T. saginata (E55) Northern Ecuador (Pichincha) 0
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Figure 1. Phylogenetic tree with characters reweighed from the COI gene showing
the phylogenetic relationships of Taenia spp.
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Figure 2. Global Network haplotypes of Taenia solium for COI gene.
?
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Figure 3. Phylogenetic tree with NDI gene and characters reweighted showing the
phylogenetic relationships of Taenia spp.
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Figure 4. Global Network haplotypes of Taenia solium using data from NDI gene.
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• Taenia solium and Taenia saginata cause taeniasis/cysticercosis, a NTD in
Ecuador.
• Maximum Parsimony analyses in Taenia solium revealed greater geographic
structure.
• COI haplotype networks suggest two geographical events in the introduction of
T. solium in Ecuador.
• Two NDI geographical lineages in T. solium derivate from a common Indian
ancestor