research article genetic variation and population genetics...

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2013 Article ID 310605 9 pageshttpdxdoiorg1011552013310605

Research ArticleGenetic Variation and Population Genetics of Taenia saginatain North and Northeast Thailand in relation to Taenia asiatica

Malinee Anantaphruti Urusa Thaenkham Teera Kusolsuk Wanna MaipanichSurapol Saguankiat Somjit Pubampen and Orawan Phuphisut

Department of Helminthology Faculty of Tropical Medicine Mahidol University 4206 Ratchawithi Road Bangkok 10400 Thailand

Correspondence should be addressed to Malinee Anantaphruti tmmtrmahidolacth

Received 24 April 2013 Accepted 2 June 2013

Academic Editor Wej Choochote

Copyright copy 2013 Malinee Anantaphruti et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Taenia saginata is the most common human Taenia in Thailand By cox1 sequences 73 isolates from four localities in north andnortheast were differentiated into 14 haplotypes 11 variation sites and haplotype diversity of 0683 Among 14 haplotypes haplotypeA was the major (521) followed by haplotype B (219) Clustering diagram of Thai and GenBank sequences indicated mixedphylogeny among localities By MJ analysis haplotype clustering relationships showed paired-stars-like network having two maincores surrounded by minor haplotypes Tajimarsquos D values were significantly negative in T saginata world population suggestingpopulation expansion Significant Fursquos 119865

119904values in Thai as well as world population also indicate that population is expanding

and may be hitchhiking as part of selective sweep Haplotype B and its dispersion were only found in populations fromThailandHaplotype B may evolve and ultimately become an ancestor of future populations inThailand Haplotype A seems to be dispersionhaplotype not just in Thailand but worldwide High genetic T saginata intraspecies divergence was found in contrast to its sisterspeciesT asiatica among 30 samples from seven countries its haplotype diversity was 0067 while only 2 haplotypes were revealedThis extremely low intraspecific variation suggests that T asiatica could be an endangered species

1 Introduction

Human taeniasis occurs worldwide It is caused by Taeniasaginata and T solium A third species T asiatica is anadditional source of intestinal infection in a number of Asiancountries Cattle are the most common source of T saginatainfection while themost common cause of bothT solium andT asiatica infection is swine T solium metacestodes calledCysticercus cellulosae reside in the animalrsquos muscle whereasT asiatica metacestodes called C viscerotropica parasitizethe liver In its evolution speciation of Taenia appearsto be linked primarily to host switching among carnivoredefinitive hosts [1] This association between Taenia andhumans is thought to have developed about 10000 yearsago coincidental with the development of agriculture andthe domestication of food animals like cattle and pigs [2]Contraction of Taenia tapeworms by humans happenedindependently twice both times by host switching fromcarnivore definitive hosts to primate definitive hosts [3] One

is an ancestor of T saginata + T asiatica and the otheris T solium Geographical distribution has been extensivelymodified by European exploration and colonization since the1500s and by ongoing globalization of agriculture and thechanging patterns of human migration [4]

Understanding the genetic population structure of par-asites helps to elucidate parasite transmission patterns anddevelop control measures [5] The population structureand genetic variation of T solium revealed two separategroups Asian and AfricanLatin American genotypes [6]Intraspecies strain variation among T solium has been foundto beminimal [7 8] Livestock farming of intermediate swinehosts may be reducing the possibility of genetic variation inT solium On the other hand cattle an intermediate host ofT saginata are found in herds in a wide range of pasturesWith different farmingmethods of this intermediate host thepopulation structure of T saginata requires investigation

Cytochrome c oxidase subunit I (cox1) genes of mito-chondrial DNA have been commonly used for studying

2 Journal of Parasitology Research

N

U

K

BangkokΘ

Figure 1 Map of Thailand showing study areas N Nan U UbonRatchathani K Khon Kaen

phylogenetic relationships among taeniid cestodes Distinctintraspecific variations have been detected among variousspecies for example Echinococcus granulosus [9] E multiloc-ularis [10] and T taeniaeformis [11] However little is knownabout cox1 genetic variation within a human Taenia speciesA minor variation was observed in one isolate of T saginatafrom Kenya and Poland [12] which compared mitochondrialcox1 and nuclear rDNA 28S sequences In this study wefocused on the genetic variation ofT saginata among samplescollected from various geographical localities in north andnortheast Thailand where this parasite is highly prevalentamong local inhabitants [13 14] In-depth studies of thegenetic divergence of T saginata specimens inThailand havenever been conducted Indeed the genetic structure amongpopulations of this species and its evolution in Thailandand throughout the world remains limited Our aim usingpartial sequencing of the mitochondrial cox1 gene was toexamine intraspecific variations and the population geneticsof T saginata inThailandThe genetic divergence of the sisterspecies T asiatica fromThailand was also considered

2 Materials and Methods

21 Studied Host Population and Parasites Parasites werecollected during the years 2009-2010 from four sites in northand northeast Thailand The two sites in the northmdashone inthe lowlands the other in the uplandhill tribe communitiesmdashare both in the Thung Chang district Nan province an areaon the northern border with Lao PDR The two sites in thenortheastern region are in two different provinces UbonRatchathani and Khon Kaen Ubon Ratchathani shares bor-ders with both southern Lao PDR and northern CambodiaKhon Kaen lies closer to the center in the upper half ofThailand (Figure 1)

T saginata was collected from both Taenia egg-positivepersons and from persons who spontaneously discharged

gravid proglottids The worms were identified morphologi-cally as T saginata by scolex andor gravid proglottids Maleinfection rateswere almost double females at a ratio of 43 23The ages of the infected individuals ranged between 12 and83 years The worms were fixed in 70 ethanol for molecularanalysis This study was approved by the Ethics Committeesof the Faculty of Tropical Medicine Mahidol University andthe Ministry of Public Health Thailand Informed consentwas obtained prior to subject participation

Twelve T asiatica previously collected from Kan-chanaburi Province [15] were coprocessed with T saginataThe cox1 sequences of 33 T saginata and 18 T asiatica fromvarious different countries in the world deposited in theGenBank database (Table 1) were also analyzed

22 Molecular Studies

221 DNA Analysis Partial proglottid fragments of individ-ual strobila were separated and washed with distilled waterto remove any ethanol remaining from the fixation processThe genomic DNA of each worm was extracted using aGenomic DNA Mini Kit (Geneaid Sijhih City Taiwan) perthe manufacturerrsquos instructions DNA was resuspended in50120583L elution buffer (provided as part of the kit) The PCRamplicons were amplified using two oligonucleotide primerscox1 (forward) 51015840-CATGGAATAATAATGATTTTC-31015840 andcox1 (reverse) 51015840-ACAGTACACACAATTTTAAC-31015840 Theseprimers were designed from the alignment of T saginataand T asiatica mitochondrial cox1 genes (AB533171 andAB533175 resp) PCR amplicons were produced in 50120583L ofreaction mixture containing 10 ng genomic DNA 05120583Mof each primer and 1x TopTaq Master Mix Kit (comprisingTopTaq DNA Polymerase PCR Buffer with 15mM MgCl

2

and 200120583M each dNTP) (QIAGEN Germany) Amplifi-cation conditions were as follows initial heating at 94∘Cfor 3min followed by 30 amplification cycles consisting ofdenaturation at 95∘C for 30 sec annealing at 53∘C for 30 secand elongation at 72∘C for 50 sec PCR products were runinto 12 agarose gel and visualized with a UV illumina-tor The PCR amplicons were purified and sequenced bydideoxyterminationmethod using an ABI3730XL sequencerand BigDye v 31 (Applied Biosystems Foster City CA USA)atMacrogen Inc (Geumcheon-gu Seoul Republic of Korea)DNA sequences were aligned using the BioEdit programversion 70 [16] There is no conflict of interests with thecommercial identities in this paper

222 Population Genetic Analysis The 924 bp cox1 genepopulation genetics ofT saginata samples from four differentlocalities was analyzed Genetic diversity values includingpolymorphic sites between populations (S) haplotype num-bers (h) haplotype diversity (Hd) nucleotide diversity (120587)theta-w (120579119908) and theta-120587 (120579120587) estimators to measure DNApolymorphism [17ndash19] were calculated using DnaSP version40 [20] and the Arlequin computer program version 31[21 22]These programswere also used to evaluate the geneticstructure of the parasites under the population expansioneffect via Tajimarsquos D test and Fursquos 119865

119904test [23]

Journal of Parasitology Research 3

Table 1 Accession numbers of 73 T saginata cox1 sequences in this study 33 from 11 different geographical countries and 30 T asiaticaisolates from 7 different countries deposited in GenBank

Species Number of samples Locality (country) Accession numbers

T saginata 5 China AB107239 AB107247 AB533168 AB533169 andAB533171

1 Korea AB4652461 Japan AB4652442 Indonesia AB107240 AB4652401 Cambodia AB4652411 Nepal AB1072432 Ecuador AB107238 AB4652433 Brazil AB107237 AB107246 and AB4652383 Ethiopia AB107241 AB465237 and AB4652451 Belgium AB107242

13 ThailandAB107244 AB107245 AB465231 AB465232 AB465233

AB465234 AB465235 AB465236 AB465239AB465242 AB465247 AB465248 and AB533173

73 Thailand this study JN986646ndashJN986718T asiatica 4 China AB465211 AB465212 AB465213 and AB465227

1 Taiwan AB4652302 Korea AB465224 AB4652253 Japan AB608736 AB608739 and AB6087421 Philippines AB4652293 Indonesia AB465215 AB465216 and AB4652284 Thailand AB533174 AB533175 AB465222 and AB46522312 Thailand this study JQ517298ndashJQ517309

223 Clustering Diagram and Haplotype Network AnalysisThe cox1 sequences were aligned by ClustalX version 20 [24]and the haplotypes then distinguished A neighbor-joining(NJ) phylogram was constructed under p-distance model byMEGA version 50 [25] Bootstrap analyses were conductedusing 1000 replicates A median-joining (MJ) network ofcox1 haplotypes was illustrated by Network 4516 Software(Fluxus Technology Ltd (httpwwwfluxus-engineeringcom))TheT saginataworld populations (Table 1) were alsotested for genetic differentiation without regional separationby global AMOVA

3 Results

31 Parasites and Infections A total of 73 Taenia saginataisolates were collected from 66 cases across the four studysites in north (Nan lowland NL Nan highland NH) andnortheast (Ubon Ratchathani UB Khon Kaen KK)Thailand(Figure 1) The samples studied were 16 and 32 isolates fromlowland and highland Nan Province in the north and 9 and16 isolates fromUbonRatchathani andKhonKaen provincesin the northeast

32 Mitochondrial cox1 DNA Sequence Analyses Total DNAwas extracted from 73 Taenia samples from four differentgeographical localities and then processed for sequencingThe partial cytochrome c oxidase subunit 1 (cox1) sequences

confirmed that they were all T saginata (GenBank accessionnos JN986646 to JN986718) The 924 bp cox1 sequences ofthese samples were divided into 14 discrete groups rep-resented as haplotypes AndashN and revealed 11 segregation(polymorphic) sites (S) Percentage intraspecific variationwas 12 with 1ndash5 nucleotide substitutions (Tables 2 and3) Among these diverse haplotypes two main ones had thehighest ratio Haplotype A was the most dominant haplotype(3873 521 of samples) in total and across all four localitiesHaplotype B was the second most dominant (1673 isolates219) and was also detected in all localities Between thetwo main haplotypes A and B there were two nucleotidesubstitutions (02)The other haplotypes (haplotypes CndashN)were detected in only 1ndash3 isolates (Table 2)

33 Population Genetics The genetic diversity value of the 73samples taken from the four localities determined by haplo-type diversity (Hd) was 0683 plusmn 005 the nucleotide diversity(120587) was 000146 plusmn 000017 120579119908 was greater than 120579120587 TajimarsquosD test of neutrality showed no significant value (minus1102 119875 =0112) in these samples A significant Fursquos 119865

119904value was

however revealed (minus7565 119875 = 0001) (Table 3) The cox1gene sequences of 33 T saginata in different geographicalareas from the GenBank database (Table 1) were includedin the analysis From 106 samples 23 haplotypes (h) and23 polymorphic sites (S) were revealed Entire intraspecificvariation was 25 Mean haplotype diversity (Hd) was


Table2Haplotypenucleotidev

ariatio

nsites

ofpartialcox1g

ene(924b

pleng

th)andfre

quency

of73

Taeniasaginatasamples

inthisstu

dy(THAlowast

haplotype

AndashN)and

33fro

m11different

geograph

icalcoun

trieslowastlowast

(Haplotype

OndashW

)

Num

berHaplotype

Positionof

nucle

otidec

hange

Num

bero

find

ividualsperp

opulation

00

11

22

23

34

56

66

77

77

88

99

9NLNH

UB

KKSubtotal

THAlowast

THA

CHN

KOR

JPN

IND

CAM

NEP

ECU

BRA

ETH

BELTo

tal

36

78

13

30

89

72

38

12

69

09

01

29

34

69

17

01

20

19

71

39

86

01

24

1A

CG

GA

CA

CT

AA

AG

AC

TT

AT

CA

CG

T10

175

638

72

11

2mdash

11

21

157

2B

sdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotTsdotsdot

16

36

163

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

193

CsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdot

12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

34

DsdotsdotsdotGsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdot

12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

35

EsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotGsdotsdotsdotsdotsdotsdotsdotsdotsdotsdot

11

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

26

FsdotsdotsdotsdotsdotsdotsdotsdotGsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdot

mdash2

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

27

GsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotTsdotsdot

mdash1

mdash1

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

28

HsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotCsdotsdotsdotsdotsdotsdotsdotsdot

mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

19

IsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdot

mdashmdash

1mdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

JsdotsdotsdotsdotsdotsdotsdotsdotGsdotsdotsdotsdotsdotsdotsdotsdotCsdotsdotsdotsdotsdot

1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

111

KsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotCsdotsdotsdot

1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

112

LsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotsdotsdotsdot

mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

113

MsdotsdotsdotsdotsdotsdotsdotsdotGsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotC

Tsdotsdot

mdash1

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

114

NsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotTsdotsdotsdotAsdotC

Tsdotsdot

mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

115

OsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotTsdotC

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1

16PsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotTsdotA

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1

17QsdotsdotsdotsdotsdotsdotsdotsdotsdotGsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdot

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1

18RsdotAsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdot

mdash1

mdashmdash

mdash1

mdashmdash

mdashmdash

mdash2

19SsdotsdotAsdotsdotGsdotsdotsdotsdotsdotAsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdot

mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1

20TsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdot

mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1

21UsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotTsdotsdotsdotsdot

mdashmdash

mdashmdash

mdashmdash

mdash1

mdashmdash

mdash1

22V

TsdotsdotsdotsdotsdotTsdotsdotsdotsdotsdotsdotsdotsdotCsdotsdotsdotsdotTsdotsdot

mdashmdash

mdashmdash

mdashmdash

mdashmdash

1mdash

mdash1

23W

sdotsdotsdotsdotsdotsdotsdotCsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotsdotAsdot

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash2

mdash2

Dotsreprepresenth

omolog

ywith

haplotypeA

sequ

ence

NLNan

lowland

NHN

anhigh

land

UB

Ubo

nRa

tchathaniKK

Kho

nKa

en

lowastlowast

11coun

triesincluding

THATh

ailand

CHNC

hinaK

OR

KoreaJPNJapanINDInd

onesiaC

AMC

ambo

diaNEP

NepalE

CUE

cuadorB

RAB

razilET

HE

thiopiaandBE

LBe

lgium

Then

umberin

gof

nucleotid

eposition

of1ndash924referred

topo

sition40

0ndash1324

ofthec

ompletem

tDNAsequ

ence

(1620b

p)of

Tsaginata

(GenBa

nkacc

noA

B066

495)


0686 andmean intrapopulation nucleotide diversity (120587) was000155 120579119908 was greater than 120579120587 (Table 3) The nucleotidesequence of Haplotype A in our study (38) was identical to Tsaginata inGenBank data forThailand(7) and the other coun-tries analyzed (China(2) Korea(1) Japan(1) Indonesia(2)Nepal(1) Ecuador(1) Brazil(2) Ethiopia(1) and Belgium(1))The nucleotide sequence of Haplotype B was unique amongthe Thailand isolates that is 16 samples in this study and3 from GenBank The frequency of Haplotype A was 538(57106 samples) the frequency of Haplotype B was 179 (19from 106 samples) Our sample sequences of Haplotype CndashNand the GenBank sequences of Haplotype OndashQ were uniqueamong the Thailand isolates (Table 2) Some samples fromChina Cambodia Ecuador Brazil and Ethiopia showednonidentical sequences (Haplotype RndashW) (Table 2) In theGenBank data samples analyzed noteworthy TajimarsquosD valueand Fursquos 119865

119904value (minus1878 minus18798) were observed (Table 3)

In looking at the sister species T asiatica significant dif-ferences between T asiatica and T saginata were discoveredThe 924 bp cox1 gene sequences of the 12 isolates from Kan-chanaburi Province Thailand were all identical Among 30samples from seven different countriesmdashChina (4) Taiwan(1) Korea (2) Japan (3) the Philippines (1) Indonesia (3)andThailand (16)mdashonly two haplotypes of the cox1 gene werefound where the major haplotype comprised 29 samplesOnly one sample from China had other haplotypes and onlyone polymorphic site was found Haplotype diversityHd was0067 However the value of nucleotide diversity 120587 of each ofthe two species was very low (Table 3)

34 Clustering Diagram and Haplotype Network The clus-tering diagram of T saginata cox1 gene indicated no sig-nificant genetic differentiation among the populations fromthe four different localities studied T saginata could notbe separated by geographical locality that is upper northeast of northeast and central northeast Thailand (Figure 2)SimilarlyT saginata from the different countries (see Table 1)was not geographically discriminated The median-joining(MJ) clustering relationship was constructed as a haplotypenetwork (Figure 3) All haplotypes appeared to be separatedinto two groupswhich each cored at the twomain haplotypesA and B Connectivity between themwas seen with a numberof shared divergent haplotypes In addition unique sequencesof samples from different geographical countries Haplo-types QndashW showed connectivity to Haplotype A Amongthese haplotypes 1ndash4 nucleotide substitutions were revealed(Table 2) Additionally of samples fromThailand only in theGenBank database three had Haplotype B sequences twounique sequence haplotypes (O and P) were connected toHaplotype B (Figure 3)

4 Discussion

In this study we investigated the population variation ofTaenia saginata inThailandThe results showed 11 haplotypeswhere distribution was not related to geographical localitySamples in our study from the north (119899 = 48) northeast (119899 =25) and west (119899 = 8) (AB465231-3 AB465242 AB465247-8

24BP 01DT 65SS 42JO 19LT 59NP 51SS 64MK 08SS 03IP 21PS 04IP 16LP 18PT 26PP 46PK

07CI 44PS 41WS 73SS35JS 67PS 57PM 17ST 56SM 31SP

53RH 13BK

66

10TK29LP66PS02DT49PS58NW45PT

33TP71NM50PM14SI09PJ

23

12

37

12BK30PP34JW

30

64

37

5544

61BT

39SK28SP

15KP

36JS

37PP 68SM

32AS27CP06BS38LH48KT54TP69YN55SK52SM63MK74TS43NP62TS25MP70CN60TC

05WI

75SW

D

EI

H

JF

KL

C

B

GN

M

A

A

A

A

15

00002

72SS

6320TT11TT22PK

15

19

47SS

Nan (lowland)Nan (highland)

Ubon RatchathaniKhon Kaen

Figure 2 A neighbor-joining phylogram of mitochondrialcox1 gene of 73 T saginata from 4 localities of Thailand A to Nindicates haplotype

AB465236 and AB465239) of Thailand were mixed togetherIntraspecific variations in cox1 genes have been reportedamong T saginata samples from a number of different local-ities including China Ethiopia France Indonesia KoreaLao PDR the Philippines TaiwanThailand and SwitzerlandGlobal genetic divergence was 12ndash18 in the nucleotidevariant positions of the total 1620 bp length [26 27] In


Table 3Genetic diversity and test ofT saginata population 73 from4different geographical regions ofThailand 33 from 11 different countriesand of T asiatica 12 from Kanchanaburi province and 18 from 7 different countries

Species Population Number ofsamples ℎ 119878 () Genetic diversity Neutrality tests

Hd 120587 Theta-119908 Theta-120587 Tajimarsquos119863(119875 value)

Fursquos 119865119904(119875

value)

T saginata Thai population 73 14 11 (12) 0683 plusmn 0050 000146 plusmn 0000 2263 plusmn 0875 1350 plusmn 0093 minus1102(0112)minus7565lowast(0001)

World population 106 23 23 (25) 0686 plusmn 0045 00155 plusmn 00001 4194 plusmn 1319 1487 plusmn 1002 minus1878lowast

(0004)minus18798lowast(0000)

T asiatica Thai population 12 0 0 0 0 ND ND ND NDAsian population 30 2 1 0067 0000 ND ND minus115 ND

ℎ haplotype numbers 119878 number of segregation sitesHd haplotype diversity 120587 nucleotide diversityTheta-119908 Wattersonrsquos theta based on 119878 Theta-120587 the thetabased on 120587 lowastSignificance (119875 lt 005)

THAlowast

THACHNKORJPNIND

NEPECUBRAETHBELCAM

1

A57

CB19 3

R2 E2D3

H1

I1S1

Q1

W2

U1

J1

F2K1

M1N1

O1

G2

P1L1

V1T1

Figure 3 A median-joining network of T saginata from Thailand (THAlowast 119899 = 73) and other 11 countries (119899 = 33) Haplotype codes (A-Ware shown insideadjacent to the circlesThe size of circle denotes that a haplotype is proportional to the number of isolates of each haplotypeshown insideadjacent to the circle Small circles indicate the number of nucleotide substitutions THAThailand CHN China KOR KoreaJPN Japan IND Indonesia NEP Nepal ECU Ecuador BRA Brazil ETH Ethiopia BEL Belgium and CAM Cambodia

terms of nucleotide divergence of large-scale samples fromThailand 11S 12 displayed the results of isolates fromglobal infection However among the worldwide T saginatapopulations (119899 = 106) the Thai isolates (119899 = 73) andGenBank isolates (119899 = 33) used in this analysis a high level ofgenetic variation (23S 25) was foundThe genetic diversityvalues haplotype diversity (Hd) and nucleotide diversity (120587)found in populations in this study and in other combinedpopulations were similar

By MJ network Haplotype B showed connectivity toHaplotype A in the T saginata world population In theT saginata sequences 19 of 33 (576) from GenBankwere identical to Haplotype A Consequently the star-likeexpansion in theMJ network of themajor haplotype confirms

Haplotype A as an ancestor among the T saginata worldpopulation It also suggests that the subpopulation of minorhaplotypes recently experienced a significant increase fromits ancestors Haplotype B and its star-like expansion networkwere unique to theThailand isolates Indeed it is possible forHaplotype B diverged genetically to be the recent commonancestor of T saginata in Thailand However the majorHaplotype A contained half (119899 = 57) of the total populationwe analyzed This means that T saginata Haplotype A mayshare its genetic ancestry with populations from a varietyof different geographical areas in Asia as well as thosein other continents 120579120587 less than 120579119908 indicates purifyingselection which results in selective removal of the deleteriousallele in the population [18] Tajimarsquos D value is significantly


negative in the world population but not in the Thailandpopulation The significant Fursquos 119865

119904value revealed in isolates

from both Thailand and the world population howeversuggests that the population is growing and is hitchhikingdue to population expansion and selective sweep [23] Thesamples analyzed from each country were too small to beable to estimate this expansion and the genetic structure ofT saginata populations worldwide

T asiatica is the third Taenia tapeworm of humans and isreported only in Asian countries It is distributed in specificareas across several countries including Taiwan [28] Korea[29] China [30] Vietnam [31] Indonesia [32] and Thailand[33] It is estimated that T saginata and T asiatica divergedfrom other human tapeworms about one million years ago(Mya) 0414ndash1616Mya T saginata lineages emerged at anearlier period thanT asiatica Lineages ofT saginata emergedat 238000 years while that of T asiatica were at 41000years [34] Very low intraspecies diversity of T asiaticahas been observed Identical partial cox1 gene nucleotidesequences have been found in 5 isolates (366 bp) fromunspecified areas of Taiwan [12] 17 isolates (337 bp) havebeen found across different localities in Korea [27] and 12isolates (924 bp) have been found in Kanchanaburi Provinceof Thailand Of the total 1620 bp sequence length only twovariant nucleotide positions (01) were detected in 5 isolatesfrom China (2) the Philippines (1) and Korea (2) [26]The low genetic variation of T asiatica suggests populationsof T asiatica tapeworm to be small The prevalence of Tasiatica is low when compared to T saginata infections inmost countries [35ndash37] Also Tajimarsquos D value revealed nogene flow in T asiatica a result which indicates obviousdifferences in the population structures of T saginata andT asiatica To date since the emergence of T saginata andT asiatica cattle have been known to be the intermediatehosts for T saginata and swine for T asiatica The livestockmanagement of these two intermediate host species has beendifferent and this may be an impact factor for these parasitesPigs are raised feeding in restricted shed areas gene flowamong parasite populations in pigs therefore is diminishedIn cattle farming especially in Thailand the animals areherded by grazing on naturally grown pastures particularlypastures of postharvest rice which often cover wide distancesFurthermore such cattle are frequently untransported whiledaily herds are moved to a main city slaughterhouse over arelatively long period of time The chances of cattle cominginto contact with contaminated Taenia eggs whether fromhuman carriers during grazing or whilst drinking streamwater from place to place remain This supports no specificlocality in T saginata populations for each genotype It mayeven suggest that T saginata tapeworm populationsmigratedduring host cattle farming Gene flow among T saginatamay have been influenced by host population migrationThe difference in population genetics found in T saginatasuggests that intraspecies populations are growing On theother hand its sister species T asiatica reveals very lowgenetic diversity Such low divergence may indicate a lossof potential adaptive alleles for surviving in a changingenvironment which could lead to the overall reduction of Tasiatica populations

Cattle also act as definitive hosts for the liver flukeFasciola spp Ichikawa et al [38] investigated the 535 bppartial nucleotide sequences of the nad1 gene in 88 adultFasciola flukes from three localities in Myanmar and found27 substitution sites that yielded 20 haplotypes A majorhaplotype revealed 546 (4888 flukes) frequency and wasseen in all three areas regardless of locality The intraspeciesgenetic variation in Fasciola spp is thought to have beenintroduced to Myanmar through ancient anthropogeneticmovements of domestic ruminants This seems to be themain factor determining mixing of the parasite populationLikewise cattle host movements suggest intraspecific geneticvariations of T saginata populations in our study Despitethe fact that cattle serve as the definitive host for Fasciolaspp but the intermediate hosts for T saginata this statusof intermediate host and definitive host does not influencedifferent genetic variation of parasite species

Our work shows thatT saginata adult worm isolates fromhumans from two locations in the north and two provinces innortheastThailand exploited intraspecific genetic variabilitywithout correlation with the geographical region of originThe phylogenetic network of cox1 sequences revealed 14haplotypes from 73 samples Thirty-three sequences fromGenBank were added and 23 haplotypes were exploitedamong the 106 samples The genetic divergence of world Tsaginata populations was 25 Two main haplotypes A andB showed connectivity between them Haplotype A seemsto be an ancestor of T saginata in the world populationHaplotype B and its dispersion are unique to the Thailandpopulation Intensive studies and a greater number of samplesfrom different geographical areas are required to clarify thepopulation genetics of T saginata both in Thailand andworldwide

Acknowledgments

Special thanks are extended to staff of the various healthcenters at each study site and particularly to Mr Somjet Yos-alai at Thung Chang Hospital in Nan Province The authorswould also like to thank the Director of the NortheasternRegion Hydro Power Plant of the Electricity GeneratingAuthority ofThailand and its staff for aiding their cooperationwith local health centers and communities and for providingaccommodation at Sirindhorn Dam and Ubolratana Damduring their stay in Ubon Ratchathani and Khon KaenThe authors sincerely thank Dr Yukifumi Nawa for hiscomments and suggestions for improving this Paper Thisstudy was supported by an internal research grant fromMahidol University

References

[1] E P Hoberg A Jones R L Rausch K S Eom and S LGardner ldquoA phylogenetic hypothesis for species of the genusTaenia (Eucestoda Taeniidae)rdquo Journal of Parasitology vol 86no 1 pp 89ndash98 2000

[2] E P Hoberg N L Alkire A De Queiroz and A Jones ldquoOut ofAfrica origins of theTaenia tapeworms in humansrdquoProceedings


of the Royal Society B Biological Sciences vol 268 no 1469 pp781ndash787 2001

[3] A DeQueiroz andN L Alkire ldquoThe phylogenetic placement ofTaenia cestodes that parasitize humansrdquo Journal of Parasitologyvol 84 no 2 pp 379ndash383 1998

[4] E P Hoberg ldquoTaenia tapeworms their biology evolution andsocioeconomic significancerdquo Microbes and Infection vol 4 no8 pp 859ndash866 2002

[5] G Campbell H H Garcia M Nakao A Ito and P S CraigldquoGenetic variation in Taenia soliumrdquo Parasitology Internationalvol 55 supplement pp S121ndashS126 2006

[6] M Nakao M Okamoto Y Sako H Yamasaki K Nakayaand A Ito ldquoA phylogenetic hypothesis for the distribution oftwo genotypes of the pig tapeworm Taenia solium worldwiderdquoParasitology vol 124 no 6 pp 657ndash662 2002

[7] K Hancock D E Broughel I N S Moura et al ldquoSequencevariation in the cytochrome oxidase I internal transcribedspacer 1 and Ts14 diagnostic antigen sequences ofTaenia soliumisolates from South and Central America India and AsiardquoInternational Journal for Parasitology vol 31 no 14 pp 1601ndash1607 2001

[8] M Okamoto M Nakao Y Sako and A Ito ldquoMolecularvariation ofTaenia solium in the worldrdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 32 no 2 pp 90ndash932001

[9] R B Gasser and N B Chilton ldquoCharacterisation of taeniidcestode species by PCR-RFLP of ITS2 ribosomal DNArdquo ActaTropica vol 59 no 1 pp 31ndash40 1995

[10] M Okamoto Y Bessho M Kamiya T Kurosawa and THorii ldquoPhylogenetic relationships within Taenia taeniaeformisvariants and other taeniid cestodes inferred from the nucleotidesequence of the cytochrome c oxidase subunit I generdquo Parasitol-ogy Research vol 81 no 6 pp 451ndash458 1995

[11] H Azuma M Okamoto Y Oku andM Kamiya ldquoIntraspecificvariation of Taenia taeniaeformis as determined by variouscriteriardquo Parasitology Research vol 81 no 2 pp 103ndash108 1995

[12] J Bowles and D P McManus ldquoGenetic characterization of theAsian Taenia a newly described taeniid cestode of humansrdquoAmerican Journal of Tropical Medicine and Hygiene vol 50 no1 pp 33ndash44 1994

[13] P Radomyos B Radomyos and A Tungtrongchitr ldquoMulti-infection with helminths in adults from northeast Thailandas determined by post-treatment fecal examination of adultwormsrdquo Tropical Medicine and Parasitology vol 45 no 2 pp133ndash135 1994

[14] B Radomyos T Wongsaroj P Wilairatana et al ldquoOpisthorchi-asis and intestinal fluke infections in Northern ThailandrdquoSoutheast Asian Journal of Tropical Medicine and Public Healthvol 29 no 1 pp 123ndash127 1998

[15] M T Anantaphruti U Thaenkham D Watthanakulpanichet al ldquoGenetic diversity of Taenia asiatica from Thailandand other geographical locations as revealed by cytochrome coxidase subunit 1 sequencesrdquoKorean Journal of Parasitology vol50 no 1 pp 55ndash59 2013

[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series no 41 pp 95ndash98 1999

[17] M Nei Molecular Evolutionary Genetics Columbia UniversityPress New York NY USA 1987

[18] F Tajima ldquoThe effect of change in population size on DNApolymorphismrdquo Genetics vol 123 no 3 pp 597ndash601 1989

[19] K Misawa and F Tajima ldquoEstimation of the amount of DNApolymorphism when the neutral mutation rate varies amongsitesrdquo Genetics vol 147 no 4 pp 1959ndash1964 1997

[20] J Rozas J C Sanchez-DelBarrio X Messeguer and R RozasldquoDnaSP DNA polymorphism analyses by the coalescent andother methodsrdquo Bioinformatics vol 19 no 18 pp 2496ndash24972003

[21] L Excoffier P E Smouse and J M Quattro ldquoAnalysis ofmolecular variance inferred from metric distances amongDNA haplotypes application to human mitochondrial DNArestriction datardquo Genetics vol 131 no 2 pp 479ndash491 1992

[22] L Excofer G Laval and S Schneider ldquoArlequin (version30)an integrated software package for population genetics dataanalysisrdquo Evolutionary Bioinformatics Online vol 1 pp 47ndash502005

[23] Y-X Fu ldquoStatistical tests of neutrality of mutations againstpopulation growth hitchhiking and background selectionrdquoGenetics vol 147 no 2 pp 915ndash925 1997

[24] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[25] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[26] H K Jeon and K S Eom ldquoTaenia asiatica and Taenia sag-inata genetic divergence estimated from their mitochondrialgenomesrdquo Experimental Parasitology vol 113 no 1 pp 58ndash612006

[27] H-K Jeon J-Y Chai Y Kong et al ldquoDifferential diagnosis ofTaenia asiatica using multiplex PCRrdquo Experimental Parasitol-ogy vol 121 no 2 pp 151ndash156 2009

[28] P C Fan C Y Lin C C Chen and W C Chung ldquoMorpho-logical description of Taenia saginata asiatica (CyclophyllideaTaeniidae) fromman in Asiardquo Journal of Helminthology vol 69no 4 pp 299ndash303 1995

[29] K S Eom and H J Rim ldquoMorphologic descriptions of Taeniaasiatica sp nrdquo Korean Journal of Parasitology vol 31 no 1 pp1ndash6 1993

[30] L Zhang H Tao B Zhang et al ldquoFirst discovery of Taeniasaginata asiatica infection in Yunnan provincerdquo Zhongguo JiSheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi vol 17 no 2pp 95ndash96 1999

[31] N V De L T Hoa N Q Doanh N B Ngoc and L D CongldquoReport on a new species of Taenia (Taenia asiatica) in HanoiVietnamrdquo Journal ofMalaria and Parasitic Diseases Control vol3 pp 80ndash85 2001

[32] T Wandra A A Depary P Sutisna et al ldquoTaeniasis and cys-ticercosis in Bali and North Sumatra Indonesiardquo ParasitologyInternational vol 55 pp S155ndashS160 2006

[33] M T Anantaphruti H Yamasaki M Nakao et al ldquoSympatricoccurrence of Taenia solium T saginata and T asiaticaThailandrdquo Emerging Infectious Diseases vol 13 no 9 pp 1413ndash1416 2007

[34] L Michelet and C Dauga ldquoMolecular evidence of host influ-ences on the evolution and spread of human tapewormsrdquoBiological Reviews vol 87 no 3 pp 731ndash741 2012


[35] T Li P S Craig A Ito et al ldquoTaeniasiscysticercosis in aTibetan population in Sichuan Province Chinardquo Acta Tropicavol 100 no 3 pp 223ndash231 2006

[36] TWandra P Sutisna N S Dharmawan et al ldquoHigh prevalenceofTaenia saginata taeniasis and status of Taenia solium cysticer-cosis in Bali Indonesia 2002ndash2004rdquo Transactions of the RoyalSociety of TropicalMedicine andHygiene vol 100 no 4 pp 346ndash353 2006

[37] M T Anantaphruti J Waikagul H Yamasaki and A Ito ldquoCys-ticercosis and taeniasis in Thailandrdquo Southeast Asain Journal ofTropical Medicine and Public Health vol 38 no 1 supplementpp 151ndash158 2007

[38] M Ichikawa S Bawn N N Maw et al ldquoCharacterization ofFasciola spp inMyanmar on the basis of spermatogenesis statusand nuclear and mitochondrial DNA markersrdquo ParasitologyInternational vol 60 no 4 pp 474ndash479 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


N

U

K

BangkokΘ

Figure 1 Map of Thailand showing study areas N Nan U UbonRatchathani K Khon Kaen

phylogenetic relationships among taeniid cestodes Distinctintraspecific variations have been detected among variousspecies for example Echinococcus granulosus [9] E multiloc-ularis [10] and T taeniaeformis [11] However little is knownabout cox1 genetic variation within a human Taenia speciesA minor variation was observed in one isolate of T saginatafrom Kenya and Poland [12] which compared mitochondrialcox1 and nuclear rDNA 28S sequences In this study wefocused on the genetic variation ofT saginata among samplescollected from various geographical localities in north andnortheast Thailand where this parasite is highly prevalentamong local inhabitants [13 14] In-depth studies of thegenetic divergence of T saginata specimens inThailand havenever been conducted Indeed the genetic structure amongpopulations of this species and its evolution in Thailandand throughout the world remains limited Our aim usingpartial sequencing of the mitochondrial cox1 gene was toexamine intraspecific variations and the population geneticsof T saginata inThailandThe genetic divergence of the sisterspecies T asiatica fromThailand was also considered

2 Materials and Methods

21 Studied Host Population and Parasites Parasites werecollected during the years 2009-2010 from four sites in northand northeast Thailand The two sites in the northmdashone inthe lowlands the other in the uplandhill tribe communitiesmdashare both in the Thung Chang district Nan province an areaon the northern border with Lao PDR The two sites in thenortheastern region are in two different provinces UbonRatchathani and Khon Kaen Ubon Ratchathani shares bor-ders with both southern Lao PDR and northern CambodiaKhon Kaen lies closer to the center in the upper half ofThailand (Figure 1)

T saginata was collected from both Taenia egg-positivepersons and from persons who spontaneously discharged

gravid proglottids The worms were identified morphologi-cally as T saginata by scolex andor gravid proglottids Maleinfection rateswere almost double females at a ratio of 43 23The ages of the infected individuals ranged between 12 and83 years The worms were fixed in 70 ethanol for molecularanalysis This study was approved by the Ethics Committeesof the Faculty of Tropical Medicine Mahidol University andthe Ministry of Public Health Thailand Informed consentwas obtained prior to subject participation

Twelve T asiatica previously collected from Kan-chanaburi Province [15] were coprocessed with T saginataThe cox1 sequences of 33 T saginata and 18 T asiatica fromvarious different countries in the world deposited in theGenBank database (Table 1) were also analyzed

22 Molecular Studies

221 DNA Analysis Partial proglottid fragments of individ-ual strobila were separated and washed with distilled waterto remove any ethanol remaining from the fixation processThe genomic DNA of each worm was extracted using aGenomic DNA Mini Kit (Geneaid Sijhih City Taiwan) perthe manufacturerrsquos instructions DNA was resuspended in50120583L elution buffer (provided as part of the kit) The PCRamplicons were amplified using two oligonucleotide primerscox1 (forward) 51015840-CATGGAATAATAATGATTTTC-31015840 andcox1 (reverse) 51015840-ACAGTACACACAATTTTAAC-31015840 Theseprimers were designed from the alignment of T saginataand T asiatica mitochondrial cox1 genes (AB533171 andAB533175 resp) PCR amplicons were produced in 50120583L ofreaction mixture containing 10 ng genomic DNA 05120583Mof each primer and 1x TopTaq Master Mix Kit (comprisingTopTaq DNA Polymerase PCR Buffer with 15mM MgCl

2

and 200120583M each dNTP) (QIAGEN Germany) Amplifi-cation conditions were as follows initial heating at 94∘Cfor 3min followed by 30 amplification cycles consisting ofdenaturation at 95∘C for 30 sec annealing at 53∘C for 30 secand elongation at 72∘C for 50 sec PCR products were runinto 12 agarose gel and visualized with a UV illumina-tor The PCR amplicons were purified and sequenced bydideoxyterminationmethod using an ABI3730XL sequencerand BigDye v 31 (Applied Biosystems Foster City CA USA)atMacrogen Inc (Geumcheon-gu Seoul Republic of Korea)DNA sequences were aligned using the BioEdit programversion 70 [16] There is no conflict of interests with thecommercial identities in this paper

222 Population Genetic Analysis The 924 bp cox1 genepopulation genetics ofT saginata samples from four differentlocalities was analyzed Genetic diversity values includingpolymorphic sites between populations (S) haplotype num-bers (h) haplotype diversity (Hd) nucleotide diversity (120587)theta-w (120579119908) and theta-120587 (120579120587) estimators to measure DNApolymorphism [17ndash19] were calculated using DnaSP version40 [20] and the Arlequin computer program version 31[21 22]These programswere also used to evaluate the geneticstructure of the parasites under the population expansioneffect via Tajimarsquos D test and Fursquos 119865

119904test [23]











3 Results





119904value was




ariatio

nsites

ofpartialcox1g

ene(924b

pleng

th)andfre

quency

of73


inthisstu

dy(THAlowast

haplotype

AndashN)and

33fro

m11different

geograph

icalcoun

trieslowastlowast

(Haplotype

OndashW

)

Num

berHaplotype

Positionof

nucle

otidec

hange

Num

bero

find

ividualsperp

opulation

00

11

22

23

34

56

66

77

77

88

99

9NLNH

UB

KKSubtotal

THAlowast

THA

CHN

KOR

JPN

IND

CAM

NEP

ECU

BRA

ETH

BELTo

tal

36

78

13

30

89

72

38

12

69

09

01

29

34

69

17

01

20

19

71

39

86

01

24

1A

CG

GA

CA

CT

AA

AG

AC

TT

AT

CA

CG

T10

175

638

72

11

2mdash

11

21

157

2B


16

36

163

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

193


12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

34


12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

35


11

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

26


mdash2

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

27


mdash1

mdash1

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

28


mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

19


mdashmdash

1mdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110


1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

111


1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

112


mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

113


Tsdotsdot

mdash1

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

114


Tsdotsdot

mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

115


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdash1

mdashmdash

mdash1

mdashmdash

mdashmdash

mdash2


mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdashmdash

mdashmdash

mdashmdash

mdash1

mdashmdash

mdash1

22V


mdashmdash

mdashmdash

mdashmdash

mdashmdash

1mdash

mdash1

23W


mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash2

mdash2

Dotsreprepresenth

omolog

ywith

haplotypeA

sequ

ence

NLNan

lowland

NHN

anhigh

land

UB

Ubo

nRa

tchathaniKK

Kho

nKa

en

lowastlowast

11coun

triesincluding

THATh

ailand

CHNC

hinaK

OR

KoreaJPNJapanINDInd

onesiaC

AMC

ambo

diaNEP

NepalE

CUE

cuadorB

RAB

razilET

HE

thiopiaandBE

LBe

lgium

Then

umberin

gof

nucleotid

eposition

of1ndash924referred

topo

sition40

0ndash1324

ofthec

ompletem

tDNAsequ

ence

(1620b

p)of

Tsaginata

(GenBa

nkacc

noA

B066

495)






4 Discussion




53RH 13BK

66



23

12

37

12BK30PP34JW

30

64

37

5544

61BT

39SK28SP

15KP

36JS

37PP 68SM


05WI

75SW

D

EI

H

JF

KL

C

B

GN

M

A

A

A

A

15

00002

72SS

6320TT11TT22PK

15

19

47SS









Fursquos 119865119904(119875

value)



(0004)minus18798lowast(0000)



THAlowast

THACHNKORJPNIND

NEPECUBRAETHBELCAM

1

A57

CB19 3

R2 E2D3

H1

I1S1

Q1

W2

U1

J1

F2K1

M1N1

O1

G2

P1L1

V1T1












Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











3 Results





119904value was




ariatio

nsites

ofpartialcox1g

ene(924b

pleng

th)andfre

quency

of73


inthisstu

dy(THAlowast

haplotype

AndashN)and

33fro

m11different

geograph

icalcoun

trieslowastlowast

(Haplotype

OndashW

)

Num

berHaplotype

Positionof

nucle

otidec

hange

Num

bero

find

ividualsperp

opulation

00

11

22

23

34

56

66

77

77

88

99

9NLNH

UB

KKSubtotal

THAlowast

THA

CHN

KOR

JPN

IND

CAM

NEP

ECU

BRA

ETH

BELTo

tal

36

78

13

30

89

72

38

12

69

09

01

29

34

69

17

01

20

19

71

39

86

01

24

1A

CG

GA

CA

CT

AA

AG

AC

TT

AT

CA

CG

T10

175

638

72

11

2mdash

11

21

157

2B


16

36

163

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

193


12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

34


12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

35


11

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

26


mdash2

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

27


mdash1

mdash1

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

28


mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

19


mdashmdash

1mdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110


1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

111


1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

112


mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

113


Tsdotsdot

mdash1

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

114


Tsdotsdot

mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

115


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdash1

mdashmdash

mdash1

mdashmdash

mdashmdash

mdash2


mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdashmdash

mdashmdash

mdashmdash

mdash1

mdashmdash

mdash1

22V


mdashmdash

mdashmdash

mdashmdash

mdashmdash

1mdash

mdash1

23W


mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash2

mdash2

Dotsreprepresenth

omolog

ywith

haplotypeA

sequ

ence

NLNan

lowland

NHN

anhigh

land

UB

Ubo

nRa

tchathaniKK

Kho

nKa

en

lowastlowast

11coun

triesincluding

THATh

ailand

CHNC

hinaK

OR

KoreaJPNJapanINDInd

onesiaC

AMC

ambo

diaNEP

NepalE

CUE

cuadorB

RAB

razilET

HE

thiopiaandBE

LBe

lgium

Then

umberin

gof

nucleotid

eposition

of1ndash924referred

topo

sition40

0ndash1324

ofthec

ompletem

tDNAsequ

ence

(1620b

p)of

Tsaginata

(GenBa

nkacc

noA

B066

495)






4 Discussion




53RH 13BK

66



23

12

37

12BK30PP34JW

30

64

37

5544

61BT

39SK28SP

15KP

36JS

37PP 68SM


05WI

75SW

D

EI

H

JF

KL

C

B

GN

M

A

A

A

A

15

00002

72SS

6320TT11TT22PK

15

19

47SS









Fursquos 119865119904(119875

value)



(0004)minus18798lowast(0000)



THAlowast

THACHNKORJPNIND

NEPECUBRAETHBELCAM

1

A57

CB19 3

R2 E2D3

H1

I1S1

Q1

W2

U1

J1

F2K1

M1N1

O1

G2

P1L1

V1T1












Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



ariatio

nsites

ofpartialcox1g

ene(924b

pleng

th)andfre

quency

of73


inthisstu

dy(THAlowast

haplotype

AndashN)and

33fro

m11different

geograph

icalcoun

trieslowastlowast

(Haplotype

OndashW

)

Num

berHaplotype

Positionof

nucle

otidec

hange

Num

bero

find

ividualsperp

opulation

00

11

22

23

34

56

66

77

77

88

99

9NLNH

UB

KKSubtotal

THAlowast

THA

CHN

KOR

JPN

IND

CAM

NEP

ECU

BRA

ETH

BELTo

tal

36

78

13

30

89

72

38

12

69

09

01

29

34

69

17

01

20

19

71

39

86

01

24

1A

CG

GA

CA

CT

AA

AG

AC

TT

AT

CA

CG

T10

175

638

72

11

2mdash

11

21

157

2B


16

36

163

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

193


12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

34


12

mdashmdash

3mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

35


11

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

26


mdash2

mdashmdash

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

27


mdash1

mdash1

2mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

28


mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

19


mdashmdash

1mdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110


1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

111


1mdash

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

112


mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

113


Tsdotsdot

mdash1

mdashmdash

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

114


Tsdotsdot

mdashmdash

mdash1

1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

115


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


1mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdash1

mdashmdash

mdash1

mdashmdash

mdashmdash

mdash2


mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdash1

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash1


mdashmdash

mdashmdash

mdashmdash

mdash1

mdashmdash

mdash1

22V


mdashmdash

mdashmdash

mdashmdash

mdashmdash

1mdash

mdash1

23W


mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash2

mdash2

Dotsreprepresenth

omolog

ywith

haplotypeA

sequ

ence

NLNan

lowland

NHN

anhigh

land

UB

Ubo

nRa

tchathaniKK

Kho

nKa

en

lowastlowast

11coun

triesincluding

THATh

ailand

CHNC

hinaK

OR

KoreaJPNJapanINDInd

onesiaC

AMC

ambo

diaNEP

NepalE

CUE

cuadorB

RAB

razilET

HE

thiopiaandBE

LBe

lgium

Then

umberin

gof

nucleotid

eposition

of1ndash924referred

topo

sition40

0ndash1324

ofthec

ompletem

tDNAsequ

ence

(1620b

p)of

Tsaginata

(GenBa

nkacc

noA

B066

495)






4 Discussion




53RH 13BK

66



23

12

37

12BK30PP34JW

30

64

37

5544

61BT

39SK28SP

15KP

36JS

37PP 68SM


05WI

75SW

D

EI

H

JF

KL

C

B

GN

M

A

A

A

A

15

00002

72SS

6320TT11TT22PK

15

19

47SS









Fursquos 119865119904(119875

value)



(0004)minus18798lowast(0000)



THAlowast

THACHNKORJPNIND

NEPECUBRAETHBELCAM

1

A57

CB19 3

R2 E2D3

H1

I1S1

Q1

W2

U1

J1

F2K1

M1N1

O1

G2

P1L1

V1T1












Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Discussion




53RH 13BK

66



23

12

37

12BK30PP34JW

30

64

37

5544

61BT

39SK28SP

15KP

36JS

37PP 68SM


05WI

75SW

D

EI

H

JF

KL

C

B

GN

M

A

A

A

A

15

00002

72SS

6320TT11TT22PK

15

19

47SS









Fursquos 119865119904(119875

value)



(0004)minus18798lowast(0000)



THAlowast

THACHNKORJPNIND

NEPECUBRAETHBELCAM

1

A57

CB19 3

R2 E2D3

H1

I1S1

Q1

W2

U1

J1

F2K1

M1N1

O1

G2

P1L1

V1T1












Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Fursquos 119865119904(119875

value)



(0004)minus18798lowast(0000)



THAlowast

THACHNKORJPNIND

NEPECUBRAETHBELCAM

1

A57

CB19 3

R2 E2D3

H1

I1S1

Q1

W2

U1

J1

F2K1

M1N1

O1

G2

P1L1

V1T1












Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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