genetic polymorphism study on aedes albopictus of

11
Research Article Genetic Polymorphism Study on Aedes albopictus of Different Geographical Regions Based on DNA Barcoding Yiliang Fang , 1 Jianqing Zhang, 1 Rongquan Wu, 2 Baohai Xue, 1 Qianqian Qian, 3 and Bo Gao 1 1 Fujian International Travel Healthcare Center, Fuzhou, Fujian 350001, China 2 Quanzhou Entry-Exit Inspection and Quarantine Bureau Comprehensive Technical Service Center, Quanzhou 362000, Fujian Province, China 3 Fujian Medical University, Fuzhou, Fujian 350001, China Correspondence should be addressed to Bo Gao; [email protected] Received 30 December 2017; Accepted 19 March 2018; Published 29 May 2018 Academic Editor: Yudong Cai Copyright © 2018 Yiliang Fang et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aedes albopictus is a very important vector for pathogens of many infectious diseases including dengue fever. In this study, we explored the genetic polymorphism of Aedes albopictus strains in different geographical regions using DNA barcoding of mitochondrial COI (MT-COI ) gene. We collected MT-COI sequence of 106 Aedes albopictus mosquitos from 6 provinces in China including Fujian, Guangdong, Hainan, Yunnan, and Taiwan. e length of the sequences is 709bp with the content of A+T (67.7%) greater than that of G+C (32.3%). We identified mutations in 90 (13.68%) loci, of which 57 (63.33%) are transitions, 28 (31.11%) are transversions, and 5 (5.56%) are hypervariable loci. In addition, we obtained 42 haplotypes, 4 (9.52%) of which are shared among different populations. e haplotype diversity of Aedes albopictus is 0.882 and nucleotide diversity is 0.01017. Moreover, the pedigree network diagram shows that most haplotypes are under parallel evolution, suggesting a local expansion of Aedes albopictus in history. Finally, the Neighbor-Joining tree of MT-COI haplotypes reveals a certain correlation between haplotype clusters and geographical distribution, and there are differences among Aedes albopictus in different geographical regions. In conclusion, DNA barcoding of MT-COI gene is an effective method to study the genetic structure of Aedes albopictus. 1. Introduction Aedes albopictus, belonging to the Diptera Nematocera Culi- cidac Aedes genus, are widely distributed all over the world [1–4]. It was originated in Southeast Asia and spread rapidly into many countries in Africa, the Middle East, Europe, and America in the past three decades [1, 5]. International trade, especially that of used tires at ports, has accelerated its transmission [6]. In China, though Aedes albopictus was observed from Hainan island in the south all over to Liaoning province in the north, it was most gathered mostly in the south of 30 north latitude such as the Fujian province [7]. Understanding the population genetic polymorphism of Aedes albopictus is critical to its prevention and control. DNA barcoding is a biometrics based on the mito- chondrial cytochrome C oxidase subunit I (MT-COI ) gene (about 500-600 bp), which was first introduced by Canadian biologist Paul Hebert in 2003 [8, 9]. is technology has received widespread attention since then. Mitochondrial COI gene sequences with strict maternal inheritance, conservative genetic makeup, and moderate evolution rate but higher nuclear DNA characteristics [10] have been widely used in the identification of mammals, fish, insects, birds, and other species [11–15]. However, to our best knowledge, there is no research on genetic polymorphism of Aedes albopictus at species level based on DNA barcoding at present. e reason might be due to the high cost in collecting and sequencing Aedes albopictus strains in a wide region, which might even be infeasible in a small country. In this study, we collected and sequenced the MT-COI gene of 106 Aedes albopictus strains from 6 provinces in China including Fujian, Guangdong, Hainan, Yunnan, and Taiwan. We then performed down-stream bioinformatics and phylogenetic analysis on the sequences. is study reveals Hindawi BioMed Research International Volume 2018, Article ID 1501430, 10 pages https://doi.org/10.1155/2018/1501430

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Research ArticleGenetic Polymorphism Study on Aedes albopictus of DifferentGeographical Regions Based on DNA Barcoding

Yiliang Fang 1 Jianqing Zhang1 RongquanWu2 Baohai Xue1

Qianqian Qian3 and Bo Gao 1

1Fujian International Travel Healthcare Center Fuzhou Fujian 350001 China2Quanzhou Entry-Exit Inspection and Quarantine Bureau Comprehensive Technical Service CenterQuanzhou 362000 Fujian Province China3Fujian Medical University Fuzhou Fujian 350001 China

Correspondence should be addressed to Bo Gao gaobo28083sinacom

Received 30 December 2017 Accepted 19 March 2018 Published 29 May 2018

Academic Editor Yudong Cai

Copyright copy 2018 Yiliang Fang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aedes albopictus is a very important vector for pathogens of many infectious diseases including dengue fever In this studywe explored the genetic polymorphism of Aedes albopictus strains in different geographical regions using DNA barcoding ofmitochondrial COI (MT-COI) gene We collectedMT-COI sequence of 106 Aedes albopictusmosquitos from 6 provinces in Chinaincluding Fujian Guangdong Hainan Yunnan and TaiwanThe length of the sequences is 709bp with the content of A+T (677)greater than that of G+C (323) We identified mutations in 90 (1368) loci of which 57 (6333) are transitions 28 (3111)are transversions and 5 (556) are hypervariable loci In addition we obtained 42 haplotypes 4 (952) of which are sharedamong different populations The haplotype diversity of Aedes albopictus is 0882 and nucleotide diversity is 001017 Moreover thepedigree network diagram shows thatmost haplotypes are under parallel evolution suggesting a local expansion ofAedes albopictusin history Finally the Neighbor-Joining tree of MT-COI haplotypes reveals a certain correlation between haplotype clusters andgeographical distribution and there are differences among Aedes albopictus in different geographical regions In conclusion DNAbarcoding ofMT-COI gene is an effective method to study the genetic structure of Aedes albopictus

1 Introduction

Aedes albopictus belonging to the Diptera Nematocera Culi-cidac Aedes genus are widely distributed all over the world[1ndash4] It was originated in Southeast Asia and spread rapidlyinto many countries in Africa the Middle East Europeand America in the past three decades [1 5] Internationaltrade especially that of used tires at ports has acceleratedits transmission [6] In China though Aedes albopictus wasobserved fromHainan island in the south all over to Liaoningprovince in the north it was most gathered mostly in thesouth of 30∘ north latitude such as the Fujian province[7] Understanding the population genetic polymorphism ofAedes albopictus is critical to its prevention and control

DNA barcoding is a biometrics based on the mito-chondrial cytochrome C oxidase subunit I (MT-COI) gene(about 500-600 bp) which was first introduced by Canadian

biologist Paul Hebert in 2003 [8 9] This technology hasreceivedwidespread attention since thenMitochondrial COIgene sequences with strict maternal inheritance conservativegenetic makeup and moderate evolution rate but highernuclear DNA characteristics [10] have been widely used inthe identification of mammals fish insects birds and otherspecies [11ndash15] However to our best knowledge there isno research on genetic polymorphism of Aedes albopictus atspecies level based on DNA barcoding at present The reasonmight be due to the high cost in collecting and sequencingAedes albopictus strains in a wide region which might evenbe infeasible in a small country

In this study we collected and sequenced the MT-COIgene of 106 Aedes albopictus strains from 6 provinces inChina including Fujian Guangdong Hainan Yunnan andTaiwanWe then performeddown-streambioinformatics andphylogenetic analysis on the sequences This study reveals

HindawiBioMed Research InternationalVolume 2018 Article ID 1501430 10 pageshttpsdoiorg10115520181501430

2 BioMed Research International

Figure 1 Collecting places of different geographical strains of Aedes albopictus

the genetic polymorphism of Aedes albopictus in SouthChina which is important for the prevention and controlof infectious diseases spread by Aedes albopictus like denguefever and yellow fever [16]

2 Materials and Methods

21 Experimental Mosquitos We collected mosquitos fromFujian province (Fuzhou Zhangzhou Jiangling and Wuy-ishan) Guangdong province (Guangzhou) Hainan province(Diaoluoshan Maoyang) Yunnan province (Mengla) Liaon-ing province (Xishan) and Taiwan (Taipei Kaohsiung) (Fig-ure 1)

22 Mosquito DNA Preparation and Amplification Nucleicacid was extracted using the TIANamp Genomic DNA Kitafter removing the head of mosquitos We then used a pairof universal primers including LCO1490 (51015840-GGTCAACAA-ATCATAAAGATATTGG-31015840) and HCO2198 (51015840-TAAACT-TCAGGGTGACCAAAAAAT-CA-31015840) to amplify them [17]The reaction system was set as follows Ex Taq TaKaRa025120583L 10XEx Taq Buffer 5120583L dNTPMixture 4120583L TemplateDNA 3120583L (if the light belt can be properly increased) andupper and lower primer (10 120583M) with each 1120583L making atotal volume of 50120583L by adding ddH

2O In addition the

amplification conditions are set as follows predenaturationat 94∘C for 3 min denaturation at 94∘C for 30 s annealing at55∘C for 30 s extension at 72∘C for 45 sec a total of 30 cyclesand final elongation at 72∘C for 5 min

23 PCR Product Purification and Cloning The PCR prod-ucts were cloned byUniversalDNAPurification kit equippedwith LB liquid medium LB solid medium and 50 mgmlIPTG The PCR products were digested with PCR Identifi-cation Kit for Recombinant pGM-T Clone which were sentout for sequencing

24 Data Analysis

Sequence Verification and Alignment By referring to theNCBI sequence database homology search was performedagainst existing COI gene sequences of Aedes albopictus inGenBank and sequences with identity greater than or equalto 98 were selected [18]

Sequence Analysis Thealigned sequences were input into themultiple sequence alignment tool Clustal X (v18) formultiplesequence alignment [19 20] The MEGA6 software packagewas used to summarize the statistics of sequence charac-teristics including base content the number of transver-sions and transitions and calculate the genetic distanceof the sequences [21] Taking Chironomidae nepeanensis(GenBank KC7503131) as the outgroup we constructedNeighbor-Joining (NJ) tree and Maximum Likelihood (ML)tree according to the genetic distance Kimura 2-parametermethod and bootstrapped 1000 times to test the reliabilityof the branch trees [21ndash23] The DnaSP 50 software [24]was used to identify the polymorphism sites and calculatethe haplotype diversity and nucleotide diversity of each

BioMed Research International 3

Table 1 Haplotype diversity and nucleotide diversity of different geographical strains

Collectingplaces

Number ofsamples

Number ofhaplotypes Haplotype type (n) Polymorphism

sites (s)

Haplotypediversity(Hd)

Nucleotidediversity (Pi)

Fujian 20 12h1(2)h2lowast(7)h3(1)h4(2)h5(1)h6(1)h7(1)h8(1)h9(1)h10(1)

h11(1)h12(1)66 087895 003316

Guangdong 20 7 h2lowast(13)h6lowast(1)h13(1)h14(1)h15(1)h16(1)h17(2) 8 058421 000173

Hainan 19 9h18lowast(5)h19lowast(5)h20(1)h21(1)h22(3)h23(1)h24(1)h25(1)

h26(1)11 086550 000265

Yunnan 20 11h18lowast(4)h19lowast(1)h27(1)h28(6)h29(1)h30(1)h31(1)h32(1)

h33(1)h34(2)h35(1)13 088421 000372

Taiwan 22 8 h2lowast(9)h36(1)h37(7)h38(1)h39(1)h40(1)h41(1)h42(1) 11 075325 000215

Liaoning 5 1 h2lowast(5) 0 000000 000000Note lowast denotes shared haplotypes

geographical population of Aedes albopictus The medianjoiningmethod ofNetwork46 softwarewas used to constructhaplotype pedigree network diagram The analysis of molec-ular variance (AMOVA v31) was used to calculate the geneticdifferentiationwithin and between populations including Fst(F-statistics) and Nm (Nm = (L minus Fst)4Fst) [25] Arlequinwas used to domismatch analysis and neutrality testWe thencalculated sum of squared deviation (SSD) and HarpendingrsquosRaggedness (HR) indices through mismatch analysis andconstructed observation simulation model [26 27] drewbifurcation point distribution and explored the evolutionaryhistory of Aedes albopictus population Using the Tajimarsquos D[28] and Fursquos Fs values [29] of the neutrality test we furtherexplored the population expansion mechanism

The ancestral population size (120579) is calculated as follows120579 = 2Nu where N represents the effective number of femalemosquitos in the population u represents the mutation rateper generation and u = 120583k (120583 is the mutation rate per pointper generation and k is the base number of the analyzedsequences) We used 120591 = 2ut to estimate the approximategeneration number (t) of population expansion [26] and setthemutation rate per point per generation ofAedes albopictuswhich is 1 times 10minus8 [30]

Mantel test was performed by online software IBD WebService (IBDWS) [31] to evaluate the correlation betweengenetic differentiation coefficient and geographical distance

3 Results

31 Sequence Features The mitochondrial COI gene wasobtained with length 709bp According to the sequencealignment ofAedes albopictus inNCBI database the sequenceidentity was 99-100 showing very small difference amongthe 106 sequences

After sequence alignment we took the 658 bp fragment(with the universal primer removed) for subsequent analysisThe overall base composition of the fragment is A (285) T

(392) C (167) and G (156) The A+T content (677)is greater than that of G+C (323) There were 90 (1368)polymorphism sites of which 57 (6333) are transitions 28(3111) are transversions and 5 (556) are hypervariableloci (Table S1)

32 The Relationships among Haplotypes of Different Geo-graphical Strains We obtained 42 haplotypes on 6 geograph-ical strains of Aedes albopictus MT-COI gene (Table 1) 4(952) of which are shared haplotypes among differentpopulations They are h2 h6 h18 and h19 respectively h2is shared by Fujian Guangdong Taiwan and Liaoning h6is shared by Fujian and Guangdong and h18 and h19 areshared by Hainan and YunnanThe result indicates that thereare gene exchanges among the populations However it canbe seen from the exclusive haplotypes that Aedes albopictushas some genetic differentiation Haplotype diversity (Hd)and nucleotide diversity (Pi) were used to indicate the degreeof haplotype differentiation and the degree of nucleotidesequence variation The haplotype diversity of Aedes albopic-tus is 0882 and nucleotide diversity is 001017 The haplotypediversity of geographical strains is ordered as Yunnan gtFujian gt Hainan gt Taiwan gt Guangdong gt Liaoning strainsThe nucleotide diversity is ordered as Fujian gt Yunnan gtHainan gt Taiwan gt Guangdong gt Liaoning strains Fujianstrains had the largest number of polymorphism sites (s = 66)and haplotype number (n = 12) and thus also had the largestnucleotide diversity (Pi = 003316) Its haplotype diversity is087895 only slightly smaller than that of Yunnan strains(Hd = 088421) There were no differences between the 5individuals in Liaoning populations

The median joining method in software Network46was used to construct haplotypes pedigree network of 42haplotypes from 6 geographical strains of Aedes albopictusmtDNA-COI gene (Figure 2) In the network each circlerepresents a haplotype the size of the circle representsthe number of homozygous haplotypes different colors

4 BioMed Research International

Figure 2 The haplotypes pedigree network diagram of different geographical strains of Aedes albopictus MT-COI gene

Table 2 The genetic distances within and between different geographical strains

Collecting places e genetic distanceswithin populations

e genetic distances between populationsFujian Guangdong Hainan Yunnan Taiwan

Fujian 003535Guangdong 000173 002187Hainan 000266 002403 000421Yunnan 000374 002473 000466 000361Taiwan 000216 002252 000223 000462 000504Liaoning 000000 002122 000091 000337 000374 000132

indicate different geographical haplotypes and the blankcircles indicate undetected haplotypes As can be seen fromthe network there is a certain level of parallel evolutionbetween the haplotypes suggesting the expansion of Aedesalbopictus in history Among them unchecked haplotypesare rare (5) but shared haplotypes H2 (34 3208) H18(9 849) and H19 (6 566) are highly distributed in thepopulation which may be the sources of expansion It can beseen that Yunnan + Hainan strains and other geographicalstrains are roughly divided into two categories but there areindividual haplotypes intersecting with each other

MEGA6 was used to calculate the genetic distancesamong MT-COI gene haplotypes of different geographicalstrains and the NJ and ML methods were used to constructthe phylogenetic trees respectively with 1000 times boot-strapping to assess branch confidence The trees constructedfrom the two methods are quite similar so we took NJ treefor illustration (Figure 3) As can be seen part of Fujianhaplotypes was clustered into one branch (H4 H11 andH12) with high confidence and the rest (Hainan + Yunnan)(Guangdong + Taiwan + Liaoning + part of Fujian) were

clustered into their respective branches with low confidenceThe NJ tree roughly coincides with the haplotypes pedigreenetwork suggesting a certain correlation between haplotypeclusters and geographical distribution Further experimentshould be done for Liaoning strains due to the small samplesize and number of single haplotype

33 Population Genetic Structure The MEGA6 softwarepackage was used to calculate the genetic distances of dif-ferent geographical strains (Table 2) Genetic distance is oneof the indicators to measure the genetic diversity within apopulation It can be seen from the table that the geneticdistance within each geographical population is orderedas Fujian gt Yunnan gt Hainan gt Taiwan gt Guangdong gtLiaoning The genetic distance between populations rangesfrom 000091 to 002473 with an average of 001925 Thegenetic distance between Fujian andYunnan strains is largestThe genetic distances between Fujian and other geographicalstrains are more than 002 indicating the significant differ-ence between Fujian and other regions The genetic distancebetween Liaoning and Guangdong strains is the smallest

BioMed Research International 5

Yunnan+Hainan

Fujian+Guandong+Taiwan+Liaoning

Fujian

COh20 COh27 COh19 COh23 COh34 COh35 COh26 COh22 COh30 COh24 COh18 COh29 COh28 COh21 COh33 COh32 COh1 COh5 COh31 COh40 COh25 COh39 COh38 COh36 COh37 COh42 COh13 COh2 COh41 COh3 COh10 COh9 COh15 COh7 COh17 COh14 COh16 COh6 COh8 COh11 COh4 COh12 Cnepeanensis

67100

10068

61

67

65

82

4462

33

44

39

3123

21

25

15

28

53

44

42

3

11

15

13

9

10

1

0

0

1

50

10

0

2

0

09

0

Figure 3 The NJ tree of Aedes albopictus MT-COI gene haplotypes from different geographical strains

Arlequin 31 was used to calculate the genetic differ-entiation coefficients (Fst) among geographical strains andthe results were summarized in Table 3 Fst is an indicatorto measure the genetic differentiation between populationsGreat Fst value means greater genetic differentiation and lessgene exchangeWhenNm is greater than 1 gene exchange canprevent genetic differentiation between populations causedby genetic drift The Nm values between Fujian Guangdongand Taiwan strains are all greater than 1 indicating frequentgenetic exchanges between these geographical populations

The Fst value (011363) and Nm value (195012) betweenHainan and Yunnan strains indicate the smallest geneticdifferentiation and frequent gene exchange between themHowever the Nm values of the two geographical strains withother geographical strains are less than 1 indicating limitedgenetic exchanges

The AMOVA analysis (Table 4) shows that the variationwithin populations (7813) is greater than that betweenpopulations (2187) which suggests that the genetic dif-ferentiation of Aedes albopictus population structure mainly

6 BioMed Research International

Table 3 The Fst (lower triangle) and Nm (upper triangle) values of different geographical strains of Aedes albopictus MT-COI gene

Collecting places FstNmFujian Guangdong Hainan Yunnan Taiwan Liaoning

Fujian 140596 096993 094554 117849 935430Guangdong 015097 027134 035618 171757 minus409084Hainan 020493 047953 195012 027040 029583Yunnan 020911 041242 011363 034867 048844Taiwan 017501 012706 048040 041759 706422Liaoning 002603lowast minus006509lowast 045802 033855 003418lowast

Note lowast represents pgt005

Table 4 The AMOVA analysis of different geographical strains of Aedes albopictus MT-COI gene

Source of variation Degree of freedom Variation components Percentage of variation ()Between populations 5 076116 2187Within population 100 271898 7813Note Fst 021871 Nm 0893

Table 5 The mismatch analysis of different geographical strains of Aedes albopictus

Overall Fujian Guangdong Hainan Yunnan Taiwan LiaoningSSD 0520 000lowast 000lowast 050 020 045 000lowast

HR 0790 100 100 025 030 040 000lowast

Note lowast represents Plt005

comes from the population interior The total of Fst value(021871) and Nm value (0893) indicates that the overallgene flow of Aedes albopictus failed to prevent the populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation in the population

34 Group Dynamics We used the mismatch analysis ofArlequin (Table 5) to assess the reliability of expansionthrough SSD and HR parameters If the difference betweenthe two parameters is not statistically significant (ie P gt005) the assumption of population expansion could not berejected which is in line with the original group expansionhypothesis Overall analysis from Aedes albopictus indicatesthat the p-values of SSD and HR are all greater than 005suggesting an expansion of Aedes albopictus in history Fromthe perspective of geographical strains the p-values of SSDandHR of Hainan Yunnan and Taiwan strains are all greaterthan 005 suggesting an expansion of Aedes albopictus inthese areas in history In Fujian andGuangdong only p-valueof HR parameter is greater than 005 suggesting that Aedesalbopictus in these areas do not have significant expansion inhistory

We mapped the bifurcation distribution of different geo-graphical strains of Aedes albopictus MT-COI gene (Figure 4)and observed a fitting between the expected and the observedvalues which also indicates population expansion Generallyspeaking the population is suggested to be in balancewhen the observed values of bifurcation distribution do notcoincide with the expected ones (ie the figure will showmultipeaks) otherwise (ie single peak) it will indicate apopulation expansion [32] Specifically the observed values

of Hainan Yunnan and Taiwan strains coincide with theexpected values and showed a single peak distributionbut the distributions of Fujian and Guangdong strains arenot consistent The overall bifurcation distribution of Aedesalbopictus presents a single peak distribution indicatingpopulation expansion

Tajimarsquos D value and Fursquos Fs value (Table 6) were cal-culated using the neutrality test of the Arlequin software(Table 6) In theory negative TajimarsquoD and Fursquos Fs valueswill indicate a population expansion in history In our casethe D value (minus1991) and Fs value (minus24983) of overall pop-ulation both reach a significant level indicating a significantpopulation expansion in history As for specific geographicalstrains except for Fujian strains with positive D value andYunnan strains without significant negative D value othergeographical strains are with statistically significant negativeD and Fs values The general results are consistent with themismatch analysis

According to the formula 120579 = 2Nu the effective size offemalemosquito populationwas estimated based on 120579

0and 1205791

before and after expansion When 1205790= 0443 and 120579

1= 99999

the effective number of female mosquitos in the population isestimated to be 34 times 104sim76 times 109 According to 120591 (95 CI)= 2ut = 3943 the population expansion occurred in 30 times 105years ago

35 The Relationship between Genetic Differentiation andGeographical Distance The Mantel test was performed onthe geographical distances between different collection pointsand Aedes albopictus MT-COI gene sequences The correla-tion curve between genetic differentiation coefficients Fst and

BioMed Research International 7

Table 6 The neutrality test of different geographical strains

Collecting places TajimarsquosD Fursquos Fs Tau (95)D value P value FS value P value

Overall minus1991 0002 minus24983 0000 3943Fujian 0703 0280lowast minus6878 0003 1008Guangdong minus1672 0037 minus29191 0000 0000Hainan minus1604 0038 minus27472 0000 2242Yunnan minus1205 0119lowast minus26629 0000 3820Taiwan minus1837 0019 minus27981 0000 2070Liaoning 0000 - infin - 0000Note lowastPgt005

Figure 4 Mismatch distribution of Aedes albopictus MT-COI gene from different geographical regions

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

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Submit your manuscripts atwwwhindawicom

2 BioMed Research International

Figure 1 Collecting places of different geographical strains of Aedes albopictus

the genetic polymorphism of Aedes albopictus in SouthChina which is important for the prevention and controlof infectious diseases spread by Aedes albopictus like denguefever and yellow fever [16]

2 Materials and Methods

21 Experimental Mosquitos We collected mosquitos fromFujian province (Fuzhou Zhangzhou Jiangling and Wuy-ishan) Guangdong province (Guangzhou) Hainan province(Diaoluoshan Maoyang) Yunnan province (Mengla) Liaon-ing province (Xishan) and Taiwan (Taipei Kaohsiung) (Fig-ure 1)

22 Mosquito DNA Preparation and Amplification Nucleicacid was extracted using the TIANamp Genomic DNA Kitafter removing the head of mosquitos We then used a pairof universal primers including LCO1490 (51015840-GGTCAACAA-ATCATAAAGATATTGG-31015840) and HCO2198 (51015840-TAAACT-TCAGGGTGACCAAAAAAT-CA-31015840) to amplify them [17]The reaction system was set as follows Ex Taq TaKaRa025120583L 10XEx Taq Buffer 5120583L dNTPMixture 4120583L TemplateDNA 3120583L (if the light belt can be properly increased) andupper and lower primer (10 120583M) with each 1120583L making atotal volume of 50120583L by adding ddH

2O In addition the

amplification conditions are set as follows predenaturationat 94∘C for 3 min denaturation at 94∘C for 30 s annealing at55∘C for 30 s extension at 72∘C for 45 sec a total of 30 cyclesand final elongation at 72∘C for 5 min

23 PCR Product Purification and Cloning The PCR prod-ucts were cloned byUniversalDNAPurification kit equippedwith LB liquid medium LB solid medium and 50 mgmlIPTG The PCR products were digested with PCR Identifi-cation Kit for Recombinant pGM-T Clone which were sentout for sequencing

24 Data Analysis

Sequence Verification and Alignment By referring to theNCBI sequence database homology search was performedagainst existing COI gene sequences of Aedes albopictus inGenBank and sequences with identity greater than or equalto 98 were selected [18]

Sequence Analysis Thealigned sequences were input into themultiple sequence alignment tool Clustal X (v18) formultiplesequence alignment [19 20] The MEGA6 software packagewas used to summarize the statistics of sequence charac-teristics including base content the number of transver-sions and transitions and calculate the genetic distanceof the sequences [21] Taking Chironomidae nepeanensis(GenBank KC7503131) as the outgroup we constructedNeighbor-Joining (NJ) tree and Maximum Likelihood (ML)tree according to the genetic distance Kimura 2-parametermethod and bootstrapped 1000 times to test the reliabilityof the branch trees [21ndash23] The DnaSP 50 software [24]was used to identify the polymorphism sites and calculatethe haplotype diversity and nucleotide diversity of each

BioMed Research International 3

Table 1 Haplotype diversity and nucleotide diversity of different geographical strains

Collectingplaces

Number ofsamples

Number ofhaplotypes Haplotype type (n) Polymorphism

sites (s)

Haplotypediversity(Hd)

Nucleotidediversity (Pi)

Fujian 20 12h1(2)h2lowast(7)h3(1)h4(2)h5(1)h6(1)h7(1)h8(1)h9(1)h10(1)

h11(1)h12(1)66 087895 003316

Guangdong 20 7 h2lowast(13)h6lowast(1)h13(1)h14(1)h15(1)h16(1)h17(2) 8 058421 000173

Hainan 19 9h18lowast(5)h19lowast(5)h20(1)h21(1)h22(3)h23(1)h24(1)h25(1)

h26(1)11 086550 000265

Yunnan 20 11h18lowast(4)h19lowast(1)h27(1)h28(6)h29(1)h30(1)h31(1)h32(1)

h33(1)h34(2)h35(1)13 088421 000372

Taiwan 22 8 h2lowast(9)h36(1)h37(7)h38(1)h39(1)h40(1)h41(1)h42(1) 11 075325 000215

Liaoning 5 1 h2lowast(5) 0 000000 000000Note lowast denotes shared haplotypes

geographical population of Aedes albopictus The medianjoiningmethod ofNetwork46 softwarewas used to constructhaplotype pedigree network diagram The analysis of molec-ular variance (AMOVA v31) was used to calculate the geneticdifferentiationwithin and between populations including Fst(F-statistics) and Nm (Nm = (L minus Fst)4Fst) [25] Arlequinwas used to domismatch analysis and neutrality testWe thencalculated sum of squared deviation (SSD) and HarpendingrsquosRaggedness (HR) indices through mismatch analysis andconstructed observation simulation model [26 27] drewbifurcation point distribution and explored the evolutionaryhistory of Aedes albopictus population Using the Tajimarsquos D[28] and Fursquos Fs values [29] of the neutrality test we furtherexplored the population expansion mechanism

The ancestral population size (120579) is calculated as follows120579 = 2Nu where N represents the effective number of femalemosquitos in the population u represents the mutation rateper generation and u = 120583k (120583 is the mutation rate per pointper generation and k is the base number of the analyzedsequences) We used 120591 = 2ut to estimate the approximategeneration number (t) of population expansion [26] and setthemutation rate per point per generation ofAedes albopictuswhich is 1 times 10minus8 [30]

Mantel test was performed by online software IBD WebService (IBDWS) [31] to evaluate the correlation betweengenetic differentiation coefficient and geographical distance

3 Results

31 Sequence Features The mitochondrial COI gene wasobtained with length 709bp According to the sequencealignment ofAedes albopictus inNCBI database the sequenceidentity was 99-100 showing very small difference amongthe 106 sequences

After sequence alignment we took the 658 bp fragment(with the universal primer removed) for subsequent analysisThe overall base composition of the fragment is A (285) T

(392) C (167) and G (156) The A+T content (677)is greater than that of G+C (323) There were 90 (1368)polymorphism sites of which 57 (6333) are transitions 28(3111) are transversions and 5 (556) are hypervariableloci (Table S1)

32 The Relationships among Haplotypes of Different Geo-graphical Strains We obtained 42 haplotypes on 6 geograph-ical strains of Aedes albopictus MT-COI gene (Table 1) 4(952) of which are shared haplotypes among differentpopulations They are h2 h6 h18 and h19 respectively h2is shared by Fujian Guangdong Taiwan and Liaoning h6is shared by Fujian and Guangdong and h18 and h19 areshared by Hainan and YunnanThe result indicates that thereare gene exchanges among the populations However it canbe seen from the exclusive haplotypes that Aedes albopictushas some genetic differentiation Haplotype diversity (Hd)and nucleotide diversity (Pi) were used to indicate the degreeof haplotype differentiation and the degree of nucleotidesequence variation The haplotype diversity of Aedes albopic-tus is 0882 and nucleotide diversity is 001017 The haplotypediversity of geographical strains is ordered as Yunnan gtFujian gt Hainan gt Taiwan gt Guangdong gt Liaoning strainsThe nucleotide diversity is ordered as Fujian gt Yunnan gtHainan gt Taiwan gt Guangdong gt Liaoning strains Fujianstrains had the largest number of polymorphism sites (s = 66)and haplotype number (n = 12) and thus also had the largestnucleotide diversity (Pi = 003316) Its haplotype diversity is087895 only slightly smaller than that of Yunnan strains(Hd = 088421) There were no differences between the 5individuals in Liaoning populations

The median joining method in software Network46was used to construct haplotypes pedigree network of 42haplotypes from 6 geographical strains of Aedes albopictusmtDNA-COI gene (Figure 2) In the network each circlerepresents a haplotype the size of the circle representsthe number of homozygous haplotypes different colors

4 BioMed Research International

Figure 2 The haplotypes pedigree network diagram of different geographical strains of Aedes albopictus MT-COI gene

Table 2 The genetic distances within and between different geographical strains

Collecting places e genetic distanceswithin populations

e genetic distances between populationsFujian Guangdong Hainan Yunnan Taiwan

Fujian 003535Guangdong 000173 002187Hainan 000266 002403 000421Yunnan 000374 002473 000466 000361Taiwan 000216 002252 000223 000462 000504Liaoning 000000 002122 000091 000337 000374 000132

indicate different geographical haplotypes and the blankcircles indicate undetected haplotypes As can be seen fromthe network there is a certain level of parallel evolutionbetween the haplotypes suggesting the expansion of Aedesalbopictus in history Among them unchecked haplotypesare rare (5) but shared haplotypes H2 (34 3208) H18(9 849) and H19 (6 566) are highly distributed in thepopulation which may be the sources of expansion It can beseen that Yunnan + Hainan strains and other geographicalstrains are roughly divided into two categories but there areindividual haplotypes intersecting with each other

MEGA6 was used to calculate the genetic distancesamong MT-COI gene haplotypes of different geographicalstrains and the NJ and ML methods were used to constructthe phylogenetic trees respectively with 1000 times boot-strapping to assess branch confidence The trees constructedfrom the two methods are quite similar so we took NJ treefor illustration (Figure 3) As can be seen part of Fujianhaplotypes was clustered into one branch (H4 H11 andH12) with high confidence and the rest (Hainan + Yunnan)(Guangdong + Taiwan + Liaoning + part of Fujian) were

clustered into their respective branches with low confidenceThe NJ tree roughly coincides with the haplotypes pedigreenetwork suggesting a certain correlation between haplotypeclusters and geographical distribution Further experimentshould be done for Liaoning strains due to the small samplesize and number of single haplotype

33 Population Genetic Structure The MEGA6 softwarepackage was used to calculate the genetic distances of dif-ferent geographical strains (Table 2) Genetic distance is oneof the indicators to measure the genetic diversity within apopulation It can be seen from the table that the geneticdistance within each geographical population is orderedas Fujian gt Yunnan gt Hainan gt Taiwan gt Guangdong gtLiaoning The genetic distance between populations rangesfrom 000091 to 002473 with an average of 001925 Thegenetic distance between Fujian andYunnan strains is largestThe genetic distances between Fujian and other geographicalstrains are more than 002 indicating the significant differ-ence between Fujian and other regions The genetic distancebetween Liaoning and Guangdong strains is the smallest

BioMed Research International 5

Yunnan+Hainan

Fujian+Guandong+Taiwan+Liaoning

Fujian

COh20 COh27 COh19 COh23 COh34 COh35 COh26 COh22 COh30 COh24 COh18 COh29 COh28 COh21 COh33 COh32 COh1 COh5 COh31 COh40 COh25 COh39 COh38 COh36 COh37 COh42 COh13 COh2 COh41 COh3 COh10 COh9 COh15 COh7 COh17 COh14 COh16 COh6 COh8 COh11 COh4 COh12 Cnepeanensis

67100

10068

61

67

65

82

4462

33

44

39

3123

21

25

15

28

53

44

42

3

11

15

13

9

10

1

0

0

1

50

10

0

2

0

09

0

Figure 3 The NJ tree of Aedes albopictus MT-COI gene haplotypes from different geographical strains

Arlequin 31 was used to calculate the genetic differ-entiation coefficients (Fst) among geographical strains andthe results were summarized in Table 3 Fst is an indicatorto measure the genetic differentiation between populationsGreat Fst value means greater genetic differentiation and lessgene exchangeWhenNm is greater than 1 gene exchange canprevent genetic differentiation between populations causedby genetic drift The Nm values between Fujian Guangdongand Taiwan strains are all greater than 1 indicating frequentgenetic exchanges between these geographical populations

The Fst value (011363) and Nm value (195012) betweenHainan and Yunnan strains indicate the smallest geneticdifferentiation and frequent gene exchange between themHowever the Nm values of the two geographical strains withother geographical strains are less than 1 indicating limitedgenetic exchanges

The AMOVA analysis (Table 4) shows that the variationwithin populations (7813) is greater than that betweenpopulations (2187) which suggests that the genetic dif-ferentiation of Aedes albopictus population structure mainly

6 BioMed Research International

Table 3 The Fst (lower triangle) and Nm (upper triangle) values of different geographical strains of Aedes albopictus MT-COI gene

Collecting places FstNmFujian Guangdong Hainan Yunnan Taiwan Liaoning

Fujian 140596 096993 094554 117849 935430Guangdong 015097 027134 035618 171757 minus409084Hainan 020493 047953 195012 027040 029583Yunnan 020911 041242 011363 034867 048844Taiwan 017501 012706 048040 041759 706422Liaoning 002603lowast minus006509lowast 045802 033855 003418lowast

Note lowast represents pgt005

Table 4 The AMOVA analysis of different geographical strains of Aedes albopictus MT-COI gene

Source of variation Degree of freedom Variation components Percentage of variation ()Between populations 5 076116 2187Within population 100 271898 7813Note Fst 021871 Nm 0893

Table 5 The mismatch analysis of different geographical strains of Aedes albopictus

Overall Fujian Guangdong Hainan Yunnan Taiwan LiaoningSSD 0520 000lowast 000lowast 050 020 045 000lowast

HR 0790 100 100 025 030 040 000lowast

Note lowast represents Plt005

comes from the population interior The total of Fst value(021871) and Nm value (0893) indicates that the overallgene flow of Aedes albopictus failed to prevent the populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation in the population

34 Group Dynamics We used the mismatch analysis ofArlequin (Table 5) to assess the reliability of expansionthrough SSD and HR parameters If the difference betweenthe two parameters is not statistically significant (ie P gt005) the assumption of population expansion could not berejected which is in line with the original group expansionhypothesis Overall analysis from Aedes albopictus indicatesthat the p-values of SSD and HR are all greater than 005suggesting an expansion of Aedes albopictus in history Fromthe perspective of geographical strains the p-values of SSDandHR of Hainan Yunnan and Taiwan strains are all greaterthan 005 suggesting an expansion of Aedes albopictus inthese areas in history In Fujian andGuangdong only p-valueof HR parameter is greater than 005 suggesting that Aedesalbopictus in these areas do not have significant expansion inhistory

We mapped the bifurcation distribution of different geo-graphical strains of Aedes albopictus MT-COI gene (Figure 4)and observed a fitting between the expected and the observedvalues which also indicates population expansion Generallyspeaking the population is suggested to be in balancewhen the observed values of bifurcation distribution do notcoincide with the expected ones (ie the figure will showmultipeaks) otherwise (ie single peak) it will indicate apopulation expansion [32] Specifically the observed values

of Hainan Yunnan and Taiwan strains coincide with theexpected values and showed a single peak distributionbut the distributions of Fujian and Guangdong strains arenot consistent The overall bifurcation distribution of Aedesalbopictus presents a single peak distribution indicatingpopulation expansion

Tajimarsquos D value and Fursquos Fs value (Table 6) were cal-culated using the neutrality test of the Arlequin software(Table 6) In theory negative TajimarsquoD and Fursquos Fs valueswill indicate a population expansion in history In our casethe D value (minus1991) and Fs value (minus24983) of overall pop-ulation both reach a significant level indicating a significantpopulation expansion in history As for specific geographicalstrains except for Fujian strains with positive D value andYunnan strains without significant negative D value othergeographical strains are with statistically significant negativeD and Fs values The general results are consistent with themismatch analysis

According to the formula 120579 = 2Nu the effective size offemalemosquito populationwas estimated based on 120579

0and 1205791

before and after expansion When 1205790= 0443 and 120579

1= 99999

the effective number of female mosquitos in the population isestimated to be 34 times 104sim76 times 109 According to 120591 (95 CI)= 2ut = 3943 the population expansion occurred in 30 times 105years ago

35 The Relationship between Genetic Differentiation andGeographical Distance The Mantel test was performed onthe geographical distances between different collection pointsand Aedes albopictus MT-COI gene sequences The correla-tion curve between genetic differentiation coefficients Fst and

BioMed Research International 7

Table 6 The neutrality test of different geographical strains

Collecting places TajimarsquosD Fursquos Fs Tau (95)D value P value FS value P value

Overall minus1991 0002 minus24983 0000 3943Fujian 0703 0280lowast minus6878 0003 1008Guangdong minus1672 0037 minus29191 0000 0000Hainan minus1604 0038 minus27472 0000 2242Yunnan minus1205 0119lowast minus26629 0000 3820Taiwan minus1837 0019 minus27981 0000 2070Liaoning 0000 - infin - 0000Note lowastPgt005

Figure 4 Mismatch distribution of Aedes albopictus MT-COI gene from different geographical regions

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

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BioMed Research International 3

Table 1 Haplotype diversity and nucleotide diversity of different geographical strains

Collectingplaces

Number ofsamples

Number ofhaplotypes Haplotype type (n) Polymorphism

sites (s)

Haplotypediversity(Hd)

Nucleotidediversity (Pi)

Fujian 20 12h1(2)h2lowast(7)h3(1)h4(2)h5(1)h6(1)h7(1)h8(1)h9(1)h10(1)

h11(1)h12(1)66 087895 003316

Guangdong 20 7 h2lowast(13)h6lowast(1)h13(1)h14(1)h15(1)h16(1)h17(2) 8 058421 000173

Hainan 19 9h18lowast(5)h19lowast(5)h20(1)h21(1)h22(3)h23(1)h24(1)h25(1)

h26(1)11 086550 000265

Yunnan 20 11h18lowast(4)h19lowast(1)h27(1)h28(6)h29(1)h30(1)h31(1)h32(1)

h33(1)h34(2)h35(1)13 088421 000372

Taiwan 22 8 h2lowast(9)h36(1)h37(7)h38(1)h39(1)h40(1)h41(1)h42(1) 11 075325 000215

Liaoning 5 1 h2lowast(5) 0 000000 000000Note lowast denotes shared haplotypes

geographical population of Aedes albopictus The medianjoiningmethod ofNetwork46 softwarewas used to constructhaplotype pedigree network diagram The analysis of molec-ular variance (AMOVA v31) was used to calculate the geneticdifferentiationwithin and between populations including Fst(F-statistics) and Nm (Nm = (L minus Fst)4Fst) [25] Arlequinwas used to domismatch analysis and neutrality testWe thencalculated sum of squared deviation (SSD) and HarpendingrsquosRaggedness (HR) indices through mismatch analysis andconstructed observation simulation model [26 27] drewbifurcation point distribution and explored the evolutionaryhistory of Aedes albopictus population Using the Tajimarsquos D[28] and Fursquos Fs values [29] of the neutrality test we furtherexplored the population expansion mechanism

The ancestral population size (120579) is calculated as follows120579 = 2Nu where N represents the effective number of femalemosquitos in the population u represents the mutation rateper generation and u = 120583k (120583 is the mutation rate per pointper generation and k is the base number of the analyzedsequences) We used 120591 = 2ut to estimate the approximategeneration number (t) of population expansion [26] and setthemutation rate per point per generation ofAedes albopictuswhich is 1 times 10minus8 [30]

Mantel test was performed by online software IBD WebService (IBDWS) [31] to evaluate the correlation betweengenetic differentiation coefficient and geographical distance

3 Results

31 Sequence Features The mitochondrial COI gene wasobtained with length 709bp According to the sequencealignment ofAedes albopictus inNCBI database the sequenceidentity was 99-100 showing very small difference amongthe 106 sequences

After sequence alignment we took the 658 bp fragment(with the universal primer removed) for subsequent analysisThe overall base composition of the fragment is A (285) T

(392) C (167) and G (156) The A+T content (677)is greater than that of G+C (323) There were 90 (1368)polymorphism sites of which 57 (6333) are transitions 28(3111) are transversions and 5 (556) are hypervariableloci (Table S1)

32 The Relationships among Haplotypes of Different Geo-graphical Strains We obtained 42 haplotypes on 6 geograph-ical strains of Aedes albopictus MT-COI gene (Table 1) 4(952) of which are shared haplotypes among differentpopulations They are h2 h6 h18 and h19 respectively h2is shared by Fujian Guangdong Taiwan and Liaoning h6is shared by Fujian and Guangdong and h18 and h19 areshared by Hainan and YunnanThe result indicates that thereare gene exchanges among the populations However it canbe seen from the exclusive haplotypes that Aedes albopictushas some genetic differentiation Haplotype diversity (Hd)and nucleotide diversity (Pi) were used to indicate the degreeof haplotype differentiation and the degree of nucleotidesequence variation The haplotype diversity of Aedes albopic-tus is 0882 and nucleotide diversity is 001017 The haplotypediversity of geographical strains is ordered as Yunnan gtFujian gt Hainan gt Taiwan gt Guangdong gt Liaoning strainsThe nucleotide diversity is ordered as Fujian gt Yunnan gtHainan gt Taiwan gt Guangdong gt Liaoning strains Fujianstrains had the largest number of polymorphism sites (s = 66)and haplotype number (n = 12) and thus also had the largestnucleotide diversity (Pi = 003316) Its haplotype diversity is087895 only slightly smaller than that of Yunnan strains(Hd = 088421) There were no differences between the 5individuals in Liaoning populations

The median joining method in software Network46was used to construct haplotypes pedigree network of 42haplotypes from 6 geographical strains of Aedes albopictusmtDNA-COI gene (Figure 2) In the network each circlerepresents a haplotype the size of the circle representsthe number of homozygous haplotypes different colors

4 BioMed Research International

Figure 2 The haplotypes pedigree network diagram of different geographical strains of Aedes albopictus MT-COI gene

Table 2 The genetic distances within and between different geographical strains

Collecting places e genetic distanceswithin populations

e genetic distances between populationsFujian Guangdong Hainan Yunnan Taiwan

Fujian 003535Guangdong 000173 002187Hainan 000266 002403 000421Yunnan 000374 002473 000466 000361Taiwan 000216 002252 000223 000462 000504Liaoning 000000 002122 000091 000337 000374 000132

indicate different geographical haplotypes and the blankcircles indicate undetected haplotypes As can be seen fromthe network there is a certain level of parallel evolutionbetween the haplotypes suggesting the expansion of Aedesalbopictus in history Among them unchecked haplotypesare rare (5) but shared haplotypes H2 (34 3208) H18(9 849) and H19 (6 566) are highly distributed in thepopulation which may be the sources of expansion It can beseen that Yunnan + Hainan strains and other geographicalstrains are roughly divided into two categories but there areindividual haplotypes intersecting with each other

MEGA6 was used to calculate the genetic distancesamong MT-COI gene haplotypes of different geographicalstrains and the NJ and ML methods were used to constructthe phylogenetic trees respectively with 1000 times boot-strapping to assess branch confidence The trees constructedfrom the two methods are quite similar so we took NJ treefor illustration (Figure 3) As can be seen part of Fujianhaplotypes was clustered into one branch (H4 H11 andH12) with high confidence and the rest (Hainan + Yunnan)(Guangdong + Taiwan + Liaoning + part of Fujian) were

clustered into their respective branches with low confidenceThe NJ tree roughly coincides with the haplotypes pedigreenetwork suggesting a certain correlation between haplotypeclusters and geographical distribution Further experimentshould be done for Liaoning strains due to the small samplesize and number of single haplotype

33 Population Genetic Structure The MEGA6 softwarepackage was used to calculate the genetic distances of dif-ferent geographical strains (Table 2) Genetic distance is oneof the indicators to measure the genetic diversity within apopulation It can be seen from the table that the geneticdistance within each geographical population is orderedas Fujian gt Yunnan gt Hainan gt Taiwan gt Guangdong gtLiaoning The genetic distance between populations rangesfrom 000091 to 002473 with an average of 001925 Thegenetic distance between Fujian andYunnan strains is largestThe genetic distances between Fujian and other geographicalstrains are more than 002 indicating the significant differ-ence between Fujian and other regions The genetic distancebetween Liaoning and Guangdong strains is the smallest

BioMed Research International 5

Yunnan+Hainan

Fujian+Guandong+Taiwan+Liaoning

Fujian

COh20 COh27 COh19 COh23 COh34 COh35 COh26 COh22 COh30 COh24 COh18 COh29 COh28 COh21 COh33 COh32 COh1 COh5 COh31 COh40 COh25 COh39 COh38 COh36 COh37 COh42 COh13 COh2 COh41 COh3 COh10 COh9 COh15 COh7 COh17 COh14 COh16 COh6 COh8 COh11 COh4 COh12 Cnepeanensis

67100

10068

61

67

65

82

4462

33

44

39

3123

21

25

15

28

53

44

42

3

11

15

13

9

10

1

0

0

1

50

10

0

2

0

09

0

Figure 3 The NJ tree of Aedes albopictus MT-COI gene haplotypes from different geographical strains

Arlequin 31 was used to calculate the genetic differ-entiation coefficients (Fst) among geographical strains andthe results were summarized in Table 3 Fst is an indicatorto measure the genetic differentiation between populationsGreat Fst value means greater genetic differentiation and lessgene exchangeWhenNm is greater than 1 gene exchange canprevent genetic differentiation between populations causedby genetic drift The Nm values between Fujian Guangdongand Taiwan strains are all greater than 1 indicating frequentgenetic exchanges between these geographical populations

The Fst value (011363) and Nm value (195012) betweenHainan and Yunnan strains indicate the smallest geneticdifferentiation and frequent gene exchange between themHowever the Nm values of the two geographical strains withother geographical strains are less than 1 indicating limitedgenetic exchanges

The AMOVA analysis (Table 4) shows that the variationwithin populations (7813) is greater than that betweenpopulations (2187) which suggests that the genetic dif-ferentiation of Aedes albopictus population structure mainly

6 BioMed Research International

Table 3 The Fst (lower triangle) and Nm (upper triangle) values of different geographical strains of Aedes albopictus MT-COI gene

Collecting places FstNmFujian Guangdong Hainan Yunnan Taiwan Liaoning

Fujian 140596 096993 094554 117849 935430Guangdong 015097 027134 035618 171757 minus409084Hainan 020493 047953 195012 027040 029583Yunnan 020911 041242 011363 034867 048844Taiwan 017501 012706 048040 041759 706422Liaoning 002603lowast minus006509lowast 045802 033855 003418lowast

Note lowast represents pgt005

Table 4 The AMOVA analysis of different geographical strains of Aedes albopictus MT-COI gene

Source of variation Degree of freedom Variation components Percentage of variation ()Between populations 5 076116 2187Within population 100 271898 7813Note Fst 021871 Nm 0893

Table 5 The mismatch analysis of different geographical strains of Aedes albopictus

Overall Fujian Guangdong Hainan Yunnan Taiwan LiaoningSSD 0520 000lowast 000lowast 050 020 045 000lowast

HR 0790 100 100 025 030 040 000lowast

Note lowast represents Plt005

comes from the population interior The total of Fst value(021871) and Nm value (0893) indicates that the overallgene flow of Aedes albopictus failed to prevent the populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation in the population

34 Group Dynamics We used the mismatch analysis ofArlequin (Table 5) to assess the reliability of expansionthrough SSD and HR parameters If the difference betweenthe two parameters is not statistically significant (ie P gt005) the assumption of population expansion could not berejected which is in line with the original group expansionhypothesis Overall analysis from Aedes albopictus indicatesthat the p-values of SSD and HR are all greater than 005suggesting an expansion of Aedes albopictus in history Fromthe perspective of geographical strains the p-values of SSDandHR of Hainan Yunnan and Taiwan strains are all greaterthan 005 suggesting an expansion of Aedes albopictus inthese areas in history In Fujian andGuangdong only p-valueof HR parameter is greater than 005 suggesting that Aedesalbopictus in these areas do not have significant expansion inhistory

We mapped the bifurcation distribution of different geo-graphical strains of Aedes albopictus MT-COI gene (Figure 4)and observed a fitting between the expected and the observedvalues which also indicates population expansion Generallyspeaking the population is suggested to be in balancewhen the observed values of bifurcation distribution do notcoincide with the expected ones (ie the figure will showmultipeaks) otherwise (ie single peak) it will indicate apopulation expansion [32] Specifically the observed values

of Hainan Yunnan and Taiwan strains coincide with theexpected values and showed a single peak distributionbut the distributions of Fujian and Guangdong strains arenot consistent The overall bifurcation distribution of Aedesalbopictus presents a single peak distribution indicatingpopulation expansion

Tajimarsquos D value and Fursquos Fs value (Table 6) were cal-culated using the neutrality test of the Arlequin software(Table 6) In theory negative TajimarsquoD and Fursquos Fs valueswill indicate a population expansion in history In our casethe D value (minus1991) and Fs value (minus24983) of overall pop-ulation both reach a significant level indicating a significantpopulation expansion in history As for specific geographicalstrains except for Fujian strains with positive D value andYunnan strains without significant negative D value othergeographical strains are with statistically significant negativeD and Fs values The general results are consistent with themismatch analysis

According to the formula 120579 = 2Nu the effective size offemalemosquito populationwas estimated based on 120579

0and 1205791

before and after expansion When 1205790= 0443 and 120579

1= 99999

the effective number of female mosquitos in the population isestimated to be 34 times 104sim76 times 109 According to 120591 (95 CI)= 2ut = 3943 the population expansion occurred in 30 times 105years ago

35 The Relationship between Genetic Differentiation andGeographical Distance The Mantel test was performed onthe geographical distances between different collection pointsand Aedes albopictus MT-COI gene sequences The correla-tion curve between genetic differentiation coefficients Fst and

BioMed Research International 7

Table 6 The neutrality test of different geographical strains

Collecting places TajimarsquosD Fursquos Fs Tau (95)D value P value FS value P value

Overall minus1991 0002 minus24983 0000 3943Fujian 0703 0280lowast minus6878 0003 1008Guangdong minus1672 0037 minus29191 0000 0000Hainan minus1604 0038 minus27472 0000 2242Yunnan minus1205 0119lowast minus26629 0000 3820Taiwan minus1837 0019 minus27981 0000 2070Liaoning 0000 - infin - 0000Note lowastPgt005

Figure 4 Mismatch distribution of Aedes albopictus MT-COI gene from different geographical regions

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

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Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

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Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Cell BiologyInternational Journal of

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Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

4 BioMed Research International

Figure 2 The haplotypes pedigree network diagram of different geographical strains of Aedes albopictus MT-COI gene

Table 2 The genetic distances within and between different geographical strains

Collecting places e genetic distanceswithin populations

e genetic distances between populationsFujian Guangdong Hainan Yunnan Taiwan

Fujian 003535Guangdong 000173 002187Hainan 000266 002403 000421Yunnan 000374 002473 000466 000361Taiwan 000216 002252 000223 000462 000504Liaoning 000000 002122 000091 000337 000374 000132

indicate different geographical haplotypes and the blankcircles indicate undetected haplotypes As can be seen fromthe network there is a certain level of parallel evolutionbetween the haplotypes suggesting the expansion of Aedesalbopictus in history Among them unchecked haplotypesare rare (5) but shared haplotypes H2 (34 3208) H18(9 849) and H19 (6 566) are highly distributed in thepopulation which may be the sources of expansion It can beseen that Yunnan + Hainan strains and other geographicalstrains are roughly divided into two categories but there areindividual haplotypes intersecting with each other

MEGA6 was used to calculate the genetic distancesamong MT-COI gene haplotypes of different geographicalstrains and the NJ and ML methods were used to constructthe phylogenetic trees respectively with 1000 times boot-strapping to assess branch confidence The trees constructedfrom the two methods are quite similar so we took NJ treefor illustration (Figure 3) As can be seen part of Fujianhaplotypes was clustered into one branch (H4 H11 andH12) with high confidence and the rest (Hainan + Yunnan)(Guangdong + Taiwan + Liaoning + part of Fujian) were

clustered into their respective branches with low confidenceThe NJ tree roughly coincides with the haplotypes pedigreenetwork suggesting a certain correlation between haplotypeclusters and geographical distribution Further experimentshould be done for Liaoning strains due to the small samplesize and number of single haplotype

33 Population Genetic Structure The MEGA6 softwarepackage was used to calculate the genetic distances of dif-ferent geographical strains (Table 2) Genetic distance is oneof the indicators to measure the genetic diversity within apopulation It can be seen from the table that the geneticdistance within each geographical population is orderedas Fujian gt Yunnan gt Hainan gt Taiwan gt Guangdong gtLiaoning The genetic distance between populations rangesfrom 000091 to 002473 with an average of 001925 Thegenetic distance between Fujian andYunnan strains is largestThe genetic distances between Fujian and other geographicalstrains are more than 002 indicating the significant differ-ence between Fujian and other regions The genetic distancebetween Liaoning and Guangdong strains is the smallest

BioMed Research International 5

Yunnan+Hainan

Fujian+Guandong+Taiwan+Liaoning

Fujian

COh20 COh27 COh19 COh23 COh34 COh35 COh26 COh22 COh30 COh24 COh18 COh29 COh28 COh21 COh33 COh32 COh1 COh5 COh31 COh40 COh25 COh39 COh38 COh36 COh37 COh42 COh13 COh2 COh41 COh3 COh10 COh9 COh15 COh7 COh17 COh14 COh16 COh6 COh8 COh11 COh4 COh12 Cnepeanensis

67100

10068

61

67

65

82

4462

33

44

39

3123

21

25

15

28

53

44

42

3

11

15

13

9

10

1

0

0

1

50

10

0

2

0

09

0

Figure 3 The NJ tree of Aedes albopictus MT-COI gene haplotypes from different geographical strains

Arlequin 31 was used to calculate the genetic differ-entiation coefficients (Fst) among geographical strains andthe results were summarized in Table 3 Fst is an indicatorto measure the genetic differentiation between populationsGreat Fst value means greater genetic differentiation and lessgene exchangeWhenNm is greater than 1 gene exchange canprevent genetic differentiation between populations causedby genetic drift The Nm values between Fujian Guangdongand Taiwan strains are all greater than 1 indicating frequentgenetic exchanges between these geographical populations

The Fst value (011363) and Nm value (195012) betweenHainan and Yunnan strains indicate the smallest geneticdifferentiation and frequent gene exchange between themHowever the Nm values of the two geographical strains withother geographical strains are less than 1 indicating limitedgenetic exchanges

The AMOVA analysis (Table 4) shows that the variationwithin populations (7813) is greater than that betweenpopulations (2187) which suggests that the genetic dif-ferentiation of Aedes albopictus population structure mainly

6 BioMed Research International

Table 3 The Fst (lower triangle) and Nm (upper triangle) values of different geographical strains of Aedes albopictus MT-COI gene

Collecting places FstNmFujian Guangdong Hainan Yunnan Taiwan Liaoning

Fujian 140596 096993 094554 117849 935430Guangdong 015097 027134 035618 171757 minus409084Hainan 020493 047953 195012 027040 029583Yunnan 020911 041242 011363 034867 048844Taiwan 017501 012706 048040 041759 706422Liaoning 002603lowast minus006509lowast 045802 033855 003418lowast

Note lowast represents pgt005

Table 4 The AMOVA analysis of different geographical strains of Aedes albopictus MT-COI gene

Source of variation Degree of freedom Variation components Percentage of variation ()Between populations 5 076116 2187Within population 100 271898 7813Note Fst 021871 Nm 0893

Table 5 The mismatch analysis of different geographical strains of Aedes albopictus

Overall Fujian Guangdong Hainan Yunnan Taiwan LiaoningSSD 0520 000lowast 000lowast 050 020 045 000lowast

HR 0790 100 100 025 030 040 000lowast

Note lowast represents Plt005

comes from the population interior The total of Fst value(021871) and Nm value (0893) indicates that the overallgene flow of Aedes albopictus failed to prevent the populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation in the population

34 Group Dynamics We used the mismatch analysis ofArlequin (Table 5) to assess the reliability of expansionthrough SSD and HR parameters If the difference betweenthe two parameters is not statistically significant (ie P gt005) the assumption of population expansion could not berejected which is in line with the original group expansionhypothesis Overall analysis from Aedes albopictus indicatesthat the p-values of SSD and HR are all greater than 005suggesting an expansion of Aedes albopictus in history Fromthe perspective of geographical strains the p-values of SSDandHR of Hainan Yunnan and Taiwan strains are all greaterthan 005 suggesting an expansion of Aedes albopictus inthese areas in history In Fujian andGuangdong only p-valueof HR parameter is greater than 005 suggesting that Aedesalbopictus in these areas do not have significant expansion inhistory

We mapped the bifurcation distribution of different geo-graphical strains of Aedes albopictus MT-COI gene (Figure 4)and observed a fitting between the expected and the observedvalues which also indicates population expansion Generallyspeaking the population is suggested to be in balancewhen the observed values of bifurcation distribution do notcoincide with the expected ones (ie the figure will showmultipeaks) otherwise (ie single peak) it will indicate apopulation expansion [32] Specifically the observed values

of Hainan Yunnan and Taiwan strains coincide with theexpected values and showed a single peak distributionbut the distributions of Fujian and Guangdong strains arenot consistent The overall bifurcation distribution of Aedesalbopictus presents a single peak distribution indicatingpopulation expansion

Tajimarsquos D value and Fursquos Fs value (Table 6) were cal-culated using the neutrality test of the Arlequin software(Table 6) In theory negative TajimarsquoD and Fursquos Fs valueswill indicate a population expansion in history In our casethe D value (minus1991) and Fs value (minus24983) of overall pop-ulation both reach a significant level indicating a significantpopulation expansion in history As for specific geographicalstrains except for Fujian strains with positive D value andYunnan strains without significant negative D value othergeographical strains are with statistically significant negativeD and Fs values The general results are consistent with themismatch analysis

According to the formula 120579 = 2Nu the effective size offemalemosquito populationwas estimated based on 120579

0and 1205791

before and after expansion When 1205790= 0443 and 120579

1= 99999

the effective number of female mosquitos in the population isestimated to be 34 times 104sim76 times 109 According to 120591 (95 CI)= 2ut = 3943 the population expansion occurred in 30 times 105years ago

35 The Relationship between Genetic Differentiation andGeographical Distance The Mantel test was performed onthe geographical distances between different collection pointsand Aedes albopictus MT-COI gene sequences The correla-tion curve between genetic differentiation coefficients Fst and

BioMed Research International 7

Table 6 The neutrality test of different geographical strains

Collecting places TajimarsquosD Fursquos Fs Tau (95)D value P value FS value P value

Overall minus1991 0002 minus24983 0000 3943Fujian 0703 0280lowast minus6878 0003 1008Guangdong minus1672 0037 minus29191 0000 0000Hainan minus1604 0038 minus27472 0000 2242Yunnan minus1205 0119lowast minus26629 0000 3820Taiwan minus1837 0019 minus27981 0000 2070Liaoning 0000 - infin - 0000Note lowastPgt005

Figure 4 Mismatch distribution of Aedes albopictus MT-COI gene from different geographical regions

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 5

Yunnan+Hainan

Fujian+Guandong+Taiwan+Liaoning

Fujian

COh20 COh27 COh19 COh23 COh34 COh35 COh26 COh22 COh30 COh24 COh18 COh29 COh28 COh21 COh33 COh32 COh1 COh5 COh31 COh40 COh25 COh39 COh38 COh36 COh37 COh42 COh13 COh2 COh41 COh3 COh10 COh9 COh15 COh7 COh17 COh14 COh16 COh6 COh8 COh11 COh4 COh12 Cnepeanensis

67100

10068

61

67

65

82

4462

33

44

39

3123

21

25

15

28

53

44

42

3

11

15

13

9

10

1

0

0

1

50

10

0

2

0

09

0

Figure 3 The NJ tree of Aedes albopictus MT-COI gene haplotypes from different geographical strains

Arlequin 31 was used to calculate the genetic differ-entiation coefficients (Fst) among geographical strains andthe results were summarized in Table 3 Fst is an indicatorto measure the genetic differentiation between populationsGreat Fst value means greater genetic differentiation and lessgene exchangeWhenNm is greater than 1 gene exchange canprevent genetic differentiation between populations causedby genetic drift The Nm values between Fujian Guangdongand Taiwan strains are all greater than 1 indicating frequentgenetic exchanges between these geographical populations

The Fst value (011363) and Nm value (195012) betweenHainan and Yunnan strains indicate the smallest geneticdifferentiation and frequent gene exchange between themHowever the Nm values of the two geographical strains withother geographical strains are less than 1 indicating limitedgenetic exchanges

The AMOVA analysis (Table 4) shows that the variationwithin populations (7813) is greater than that betweenpopulations (2187) which suggests that the genetic dif-ferentiation of Aedes albopictus population structure mainly

6 BioMed Research International

Table 3 The Fst (lower triangle) and Nm (upper triangle) values of different geographical strains of Aedes albopictus MT-COI gene

Collecting places FstNmFujian Guangdong Hainan Yunnan Taiwan Liaoning

Fujian 140596 096993 094554 117849 935430Guangdong 015097 027134 035618 171757 minus409084Hainan 020493 047953 195012 027040 029583Yunnan 020911 041242 011363 034867 048844Taiwan 017501 012706 048040 041759 706422Liaoning 002603lowast minus006509lowast 045802 033855 003418lowast

Note lowast represents pgt005

Table 4 The AMOVA analysis of different geographical strains of Aedes albopictus MT-COI gene

Source of variation Degree of freedom Variation components Percentage of variation ()Between populations 5 076116 2187Within population 100 271898 7813Note Fst 021871 Nm 0893

Table 5 The mismatch analysis of different geographical strains of Aedes albopictus

Overall Fujian Guangdong Hainan Yunnan Taiwan LiaoningSSD 0520 000lowast 000lowast 050 020 045 000lowast

HR 0790 100 100 025 030 040 000lowast

Note lowast represents Plt005

comes from the population interior The total of Fst value(021871) and Nm value (0893) indicates that the overallgene flow of Aedes albopictus failed to prevent the populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation in the population

34 Group Dynamics We used the mismatch analysis ofArlequin (Table 5) to assess the reliability of expansionthrough SSD and HR parameters If the difference betweenthe two parameters is not statistically significant (ie P gt005) the assumption of population expansion could not berejected which is in line with the original group expansionhypothesis Overall analysis from Aedes albopictus indicatesthat the p-values of SSD and HR are all greater than 005suggesting an expansion of Aedes albopictus in history Fromthe perspective of geographical strains the p-values of SSDandHR of Hainan Yunnan and Taiwan strains are all greaterthan 005 suggesting an expansion of Aedes albopictus inthese areas in history In Fujian andGuangdong only p-valueof HR parameter is greater than 005 suggesting that Aedesalbopictus in these areas do not have significant expansion inhistory

We mapped the bifurcation distribution of different geo-graphical strains of Aedes albopictus MT-COI gene (Figure 4)and observed a fitting between the expected and the observedvalues which also indicates population expansion Generallyspeaking the population is suggested to be in balancewhen the observed values of bifurcation distribution do notcoincide with the expected ones (ie the figure will showmultipeaks) otherwise (ie single peak) it will indicate apopulation expansion [32] Specifically the observed values

of Hainan Yunnan and Taiwan strains coincide with theexpected values and showed a single peak distributionbut the distributions of Fujian and Guangdong strains arenot consistent The overall bifurcation distribution of Aedesalbopictus presents a single peak distribution indicatingpopulation expansion

Tajimarsquos D value and Fursquos Fs value (Table 6) were cal-culated using the neutrality test of the Arlequin software(Table 6) In theory negative TajimarsquoD and Fursquos Fs valueswill indicate a population expansion in history In our casethe D value (minus1991) and Fs value (minus24983) of overall pop-ulation both reach a significant level indicating a significantpopulation expansion in history As for specific geographicalstrains except for Fujian strains with positive D value andYunnan strains without significant negative D value othergeographical strains are with statistically significant negativeD and Fs values The general results are consistent with themismatch analysis

According to the formula 120579 = 2Nu the effective size offemalemosquito populationwas estimated based on 120579

0and 1205791

before and after expansion When 1205790= 0443 and 120579

1= 99999

the effective number of female mosquitos in the population isestimated to be 34 times 104sim76 times 109 According to 120591 (95 CI)= 2ut = 3943 the population expansion occurred in 30 times 105years ago

35 The Relationship between Genetic Differentiation andGeographical Distance The Mantel test was performed onthe geographical distances between different collection pointsand Aedes albopictus MT-COI gene sequences The correla-tion curve between genetic differentiation coefficients Fst and

BioMed Research International 7

Table 6 The neutrality test of different geographical strains

Collecting places TajimarsquosD Fursquos Fs Tau (95)D value P value FS value P value

Overall minus1991 0002 minus24983 0000 3943Fujian 0703 0280lowast minus6878 0003 1008Guangdong minus1672 0037 minus29191 0000 0000Hainan minus1604 0038 minus27472 0000 2242Yunnan minus1205 0119lowast minus26629 0000 3820Taiwan minus1837 0019 minus27981 0000 2070Liaoning 0000 - infin - 0000Note lowastPgt005

Figure 4 Mismatch distribution of Aedes albopictus MT-COI gene from different geographical regions

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

Table 3 The Fst (lower triangle) and Nm (upper triangle) values of different geographical strains of Aedes albopictus MT-COI gene

Collecting places FstNmFujian Guangdong Hainan Yunnan Taiwan Liaoning

Fujian 140596 096993 094554 117849 935430Guangdong 015097 027134 035618 171757 minus409084Hainan 020493 047953 195012 027040 029583Yunnan 020911 041242 011363 034867 048844Taiwan 017501 012706 048040 041759 706422Liaoning 002603lowast minus006509lowast 045802 033855 003418lowast

Note lowast represents pgt005

Table 4 The AMOVA analysis of different geographical strains of Aedes albopictus MT-COI gene

Source of variation Degree of freedom Variation components Percentage of variation ()Between populations 5 076116 2187Within population 100 271898 7813Note Fst 021871 Nm 0893

Table 5 The mismatch analysis of different geographical strains of Aedes albopictus

Overall Fujian Guangdong Hainan Yunnan Taiwan LiaoningSSD 0520 000lowast 000lowast 050 020 045 000lowast

HR 0790 100 100 025 030 040 000lowast

Note lowast represents Plt005

comes from the population interior The total of Fst value(021871) and Nm value (0893) indicates that the overallgene flow of Aedes albopictus failed to prevent the populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation in the population

34 Group Dynamics We used the mismatch analysis ofArlequin (Table 5) to assess the reliability of expansionthrough SSD and HR parameters If the difference betweenthe two parameters is not statistically significant (ie P gt005) the assumption of population expansion could not berejected which is in line with the original group expansionhypothesis Overall analysis from Aedes albopictus indicatesthat the p-values of SSD and HR are all greater than 005suggesting an expansion of Aedes albopictus in history Fromthe perspective of geographical strains the p-values of SSDandHR of Hainan Yunnan and Taiwan strains are all greaterthan 005 suggesting an expansion of Aedes albopictus inthese areas in history In Fujian andGuangdong only p-valueof HR parameter is greater than 005 suggesting that Aedesalbopictus in these areas do not have significant expansion inhistory

We mapped the bifurcation distribution of different geo-graphical strains of Aedes albopictus MT-COI gene (Figure 4)and observed a fitting between the expected and the observedvalues which also indicates population expansion Generallyspeaking the population is suggested to be in balancewhen the observed values of bifurcation distribution do notcoincide with the expected ones (ie the figure will showmultipeaks) otherwise (ie single peak) it will indicate apopulation expansion [32] Specifically the observed values

of Hainan Yunnan and Taiwan strains coincide with theexpected values and showed a single peak distributionbut the distributions of Fujian and Guangdong strains arenot consistent The overall bifurcation distribution of Aedesalbopictus presents a single peak distribution indicatingpopulation expansion

Tajimarsquos D value and Fursquos Fs value (Table 6) were cal-culated using the neutrality test of the Arlequin software(Table 6) In theory negative TajimarsquoD and Fursquos Fs valueswill indicate a population expansion in history In our casethe D value (minus1991) and Fs value (minus24983) of overall pop-ulation both reach a significant level indicating a significantpopulation expansion in history As for specific geographicalstrains except for Fujian strains with positive D value andYunnan strains without significant negative D value othergeographical strains are with statistically significant negativeD and Fs values The general results are consistent with themismatch analysis

According to the formula 120579 = 2Nu the effective size offemalemosquito populationwas estimated based on 120579

0and 1205791

before and after expansion When 1205790= 0443 and 120579

1= 99999

the effective number of female mosquitos in the population isestimated to be 34 times 104sim76 times 109 According to 120591 (95 CI)= 2ut = 3943 the population expansion occurred in 30 times 105years ago

35 The Relationship between Genetic Differentiation andGeographical Distance The Mantel test was performed onthe geographical distances between different collection pointsand Aedes albopictus MT-COI gene sequences The correla-tion curve between genetic differentiation coefficients Fst and

BioMed Research International 7

Table 6 The neutrality test of different geographical strains

Collecting places TajimarsquosD Fursquos Fs Tau (95)D value P value FS value P value

Overall minus1991 0002 minus24983 0000 3943Fujian 0703 0280lowast minus6878 0003 1008Guangdong minus1672 0037 minus29191 0000 0000Hainan minus1604 0038 minus27472 0000 2242Yunnan minus1205 0119lowast minus26629 0000 3820Taiwan minus1837 0019 minus27981 0000 2070Liaoning 0000 - infin - 0000Note lowastPgt005

Figure 4 Mismatch distribution of Aedes albopictus MT-COI gene from different geographical regions

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

Table 6 The neutrality test of different geographical strains

Collecting places TajimarsquosD Fursquos Fs Tau (95)D value P value FS value P value

Overall minus1991 0002 minus24983 0000 3943Fujian 0703 0280lowast minus6878 0003 1008Guangdong minus1672 0037 minus29191 0000 0000Hainan minus1604 0038 minus27472 0000 2242Yunnan minus1205 0119lowast minus26629 0000 3820Taiwan minus1837 0019 minus27981 0000 2070Liaoning 0000 - infin - 0000Note lowastPgt005

Figure 4 Mismatch distribution of Aedes albopictus MT-COI gene from different geographical regions

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

Figure 5 The relationship between genetic differentiation of Aedesalbopictus MT-COI gene and geographical distance

geographical distances was illustrated in Figure 5 The corre-lation between population Fst and geographical distances (r= 05789 p = 00120) was statistically significant indicatinga positive correlation between genetic differentiation andgeographical distance

4 Discussion

The genetic diversity of insect species can arise from twosources internal and external causes The internal causes aregenetic mutation and adaptability of insect itself while theexternal factors are closely related to ecological environmentof insects The accumulation of genetic difference will lead toreproductive isolation and thus the formation of new speciesA few scholars have studied the diversity of Aedes albopictusby different methods Preliminary studies have confirmedthat there is a certain degree of genetic differentiation amongAedes albopictus populations Therefore further studies ongenetic variation ofAedes albopictus population are needed toprovide valuable information for the prevention and controlof dengue fever Also new mosquito species can become thevector of newdiseases and it is critical to surveil themosquitospecies in a geographical region

Haplotype diversity (Hd) and nucleotide diversity (Pi) aretwo important indicators tomeasure the diversity of a speciespopulation [33] The haplotype diversity and nucleotidediversity of Aedes albopictus in this study (Hd = 0882 Pi =001017) are higher than those in Aedes aegypti (Hd = 0740plusmn 0017 P = 00065 plusmn 00003) indicating high diversity ofAedes albopictus population Aedes albopictus exhibited highHd and low Pi mode probably because of rapid populationexpansion after the bottleneck effect The formation of anew haplotype is due to a single base variation which hasless effect on nucleotide diversity so increasing nucleotidediversity requires more time to accumulate than increasing

haplotype diversity The bottleneck effect can eliminate theaccumulation of nucleotide diversity in the past but can alsocause mutations at the base site resulting in a pattern ofhigh Hd and low Pi The possible reasons for the bottleneckof Aedes albopictus may be as follows on the one hand interms of neutral test of Aedes albopictus (D = minus199 P =0002 Fs =minus2498 P = 0000) and the bifurcation distributionrepresenting a single peak Aedes albopictus has been or is ina state of population expansion On the other hand denguefever has caused great harm to human health since it wasrecognized that controlling the number of media mosquitosis critical for controlling the spread of the disease Thereforea wide range of remediation media mosquitos have a greaterimpact on Aedes albopictus population

In this study the genetic distances between differentgeographical populations ranged from 000091 to 002473which are higher than those of Aedes albopictus in differentregions of Guangzhou about 0000sim0007 in a previous studyAmong them the genetic distances between Fujian and othergeographical strains were greater than 002 (Table 2) whichis greater than the difference of less than 2 of the geneticdistance within 98 of the species proposed by Hebert[10] indicating a large genetic difference between FujianAedes albopictus and other geographical strains Haplotypediversity nucleotide diversity and genetic distance are allindicators to reflect the genetic diversity of population Theresults of the three indicators in this study (Tables 1 and 2)basically showed the largest value of Fujian strains indicatingFujian population with the largest genetic diversity In thepedigree network diagram Haplotypes h2 is found to be ashared haplotype among Fujian Guangdong Taiwan andLiaoning and developed into other haplotypes through director indirect evolution of 1sim5 steps However the NJ treeshows that 3 haplotypes (h4 h11 and h12) of Fujian clusteredindependently into one category with high confidence whichfurther indicates the result that Fujian strains of Aedesalbopictus have obvious genetic structure changes and alsoverified early studies Those studies suggested that thereis a certain degree of genetic differentiation of differentgeographical strains of Aedes albopictus in Fujian provinceDue to the farthest flight distance of Aedes albopictus notexceeding 500 meters it is very difficult to invade from theexterior in short time The reason for this phenomenon maybe related to the ecological and climatic environment ofAedes albopictus in Fujian Fujian is located in a subtropi-cal maritime monsoon climate whose complex topographyforms a variety of local climate thus forming differentecological environments which provide favorable conditionsfor breeding and living Aedes albopictus Fujian geographicalstrains of Aedes albopictus haplotypes diversity the causes ofspecific haplotype and whether there is a special significancein the mosquito-borne disease transmission need furtherstudy

Morton [34] believed that when the gene flow Nm valueis greater than 1 it means that the gene exchange is frequentwhich can prevent the interpopulation differentiation causedby genetic drift When the value is less than 1 it indicatesthat gene exchange is blocked In this study the Aedesalbopictus Nm value is less than 1 (Nm = 0893) indicating

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 9

that the level of gene exchange failed to prevent populationdifferentiation caused by genetic drift and there is a certainlevel of genetic differentiation between populations In termsof Nm value of different geographical strains (Table 3) theNm value between Hainan and Yunnan strains is greaterthan 1 indicating that Aedes albopictus in the two placeshad frequent genetic exchange However the Nm valuesbetween (Yunnan + Hainan strains) and Guangdong Taiwanand Liaoning strains are less than 1 indicating that Aedesalbopictus gene exchanges between (Yunnan + Hainan) andthe four regions are hindered This is consistent with NJtree that Yunnan + Hainan was clustered into one categoryThe NJ tree and gene flow show that the genetic exchangesfrequently happen among Taiwan Fujian and Guangdongmosquitos suggesting that Taiwan strains have the sametype of geographical populations as Fujian and Guangdongstrains No relevant reports have been found yet

Finally this study shows that the genetic differenceswithin Aedes albopictus population are larger than thosebetween populations Due to the different ecological andclimatic conditions in different regions there are variouskinds of natural barriers However Aedes albopictus belongsto the semihabitat mosquito and is closely related to humanactivities the geographical barrier failed to completely pre-vent the gene flow so genetic differentiation mainly whichcomes from internal and genetic differentiation among thepopulations is not high The result of the Mantel test (r =05789 p = 00120) shows that the degree of CO I genesequence variation is positively correlated with geographicaldistance Further experiments will be conducted to confirmthis conclusion Giving more and more genetic sequences ofmosquitos in different regions has been published it wouldbe interesting to study the genetic differences of mosquitopopulations throughout China or across a continent in thefuture

5 Conclusion

In conclusion our results suggest that (1) there are genetic dif-ferences amongAedes albopictus populations in different geo-graphical regions (2) there is a positive correlation betweenthe genetic differences of Aedes albopictus population andtheir geographical distances and (3) DNA barcoding ofMT-COI gene is an effectivemethod to study the genetic structureof Aedes albopictus

Conflicts of Interest

The authors have declared no conflicts of interest

Authorsrsquo Contributions

Bo Gao conceived and designed the experiments YiliangFang and Qianqian Qian performed the experiments YiliangFang analyzed the data Yiliang Fang and Bo Gao wrotethe paper Jianqing Zhang and Baohai Xue contributed tothe discussion and revision of the paper All authors haveapproved the final manuscript

Supplementary Materials

Table S1 variable sites in MT-COI haplotype sequencesof Aedes albopictus in different geographical regionsThe dot ldquordquo denotes consensus nucleotide with coI h1(Supplementary Materials)

References

[1] N G Gratz ldquoCritical review of the vector status of Aedesalbopictusrdquo Medical and Veterinary Entomology vol 18 no 3pp 215ndash227 2004

[2] T Coffinet J R Mourou B Pradines et al ldquoFirst record ofAedes albopictus in Gabonrdquo Journal of the American MosquitoControl Association vol 23 no 4 pp 471-472 2007

[3] D A Yee S A Juliano and S M Vamosi ldquoSeasonal photoperi-ods alter developmental time andmass of an invasive mosquitoAedes albopictus (Diptera Culicidae) across its north-southrange in the United Statesrdquo Journal of Medical Entomology vol49 no 4 pp 825ndash832 2012

[4] L A Ganushkina I V Patraman G Rezza L Migliorini S KLitvinov and V P Sergiev ldquoDetection of Aedes aegypti Aedesalbopictus and Aedes koreicus in the Area of Sochi RussiardquoVector-Borne and Zoonotic Diseases vol 16 no 1 pp 58ndash602016

[5] C E Smith ldquoThe history of dengue in tropical Asia andits probable relationship to the mosquito Aedes aegyptirdquo TheJournal of tropical medicine and hygiene vol 59 no 10 pp 243ndash251 1956

[6] P Reiter ldquoAedes albopictus and the world trade in used tires1988-1995 The shape of things to comerdquo Journal of the Ameri-can Mosquito Control Association vol 14 no 1 pp 83ndash94 1998

[7] X-X Guo C-X Li Y-M Zhang et al ldquoVector competenceof Aedes albopictus and Aedes aegypti (Diptera Culicidae) forthe DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus inFujian Chinardquo Acta Tropica vol 161 pp 86ndash90 2016

[8] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Science vol 270 no 1512 pp313ndash321 2003

[9] F O Costa J R DeWaard J Boutillier et al ldquoBiological iden-tifications through DNA barcodes The case of the CrustaceardquoCanadian Journal of Fisheries and Aquatic Sciences vol 64 no2 pp 272ndash295 2007

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Science vol 270 supplement 1 pp S96ndashS99 2003

[11] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1462 pp 1847ndash1857 2005

[12] A Cywinska F F Hunter and P D N Hebert ldquoIdentifyingCanadian mosquito species through DNA barcodesrdquo Medicaland Veterinary Entomology vol 20 no 4 pp 413ndash424 2006

[13] E L Clare B K Lim M D Engstrom J L Eger andP D Hebert ldquoDNA barcoding of Neotropical bats speciesidentification and discovery within GuyanardquoMolecular EcologyResources vol 7 no 2 pp 184ndash190 2007

[14] K C R Kerr M Y Stoeckle C J Dove L A WeigtC M Francis and P D N Hebert ldquoComprehensive DNA

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

10 BioMed Research International

barcode coverage of North American birdsrdquoMolecular EcologyResources vol 7 no 4 pp 535ndash543 2007

[15] N P Kumar A R Rajavel R Natarajan and P JambulingamldquoDNA barcodes can distinguish species of indian mosquitoes(DipteraCulicidae)rdquo Journal ofMedical Entomology vol 44 no1 pp 1ndash7 2007

[16] C G Moore and C J Mitchell ldquoAedes albopictus in theUnited States ten-year presence and public health implica-tionsrdquo Emerging Infectious Diseases vol 3 no 3 pp 329ndash3341997

[17] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[18] D A Benson I Karsch-Mizrachi D J Lipman J Ostell andD L Wheeler ldquoGenBankrdquo Nucleic Acids Research vol 33 ppD34ndashD38 2005

[19] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[20] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008

[21] S Kumar G Stecher and K Tamura ldquoMEGA7 MolecularEvolutionary Genetics Analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[22] J Yang S Grunewald and X-F Wan ldquoQuartet-net A quartet-basedmethod to reconstruct phylogenetic networksrdquoMolecularBiology and Evolution vol 30 no 5 pp 1206ndash1217 2013

[23] J Yang S Grunewald Y Xu and X-F Wan ldquoQuartet-basedmethods to reconstruct phylogenetic networksrdquo BMC SystemsBiology vol 8 article 21 2014

[24] 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 2496-24972003

[25] 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

[26] A R Rogers and H Harpending ldquoPopulation growth makeswaves in the distribution of pairwise genetic differencesrdquoMolecular Biology and Evolution vol 9 no 3 pp 552ndash569 1992

[27] H C Harpending ldquoSignature of ancient population growth ina low-resolution mitochondrial DNA mismatch distributionrdquoHuman Biology vol 66 no 4 pp 591ndash600 1994

[28] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

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

[30] J R Powell A Caccone G D Amato and C Yoon ldquoRates ofnucleotide substitution in Drosophila mitochondrial DNA andnuclear DNA are similarrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 83 no 23 pp9090ndash9093 1986

[31] J L Jensen A J Bohonak and S T Kelley ldquoIsolation bydistance web servicerdquo BMC Genetics vol 6 2005

[32] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable amp exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

[33] R C Vrijenhoek ldquoGenetic diversity and fitness in small popu-lationsrdquo Conservation Genetics pp 37ndash53 1994

[34] N E Morton ldquoIsolation by distance in human populationsrdquoAnnals of Human Genetics vol 40 no 3 pp 361ndash365 1977

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

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