Genetic Diversity of Aedes Aegypti (Diptera: Culicidae) Isolated from Five Cities in North Coast Area of Central Java, Indonesia
Total Page：16
File Type：pdf, Size：1020Kb
AEDES AEGYPTI GENETIC DIVERSITY IN JAVA GENETIC DIVERSITY OF AEDES AEGYPTI (DIPTERA: CULICIDAE) ISOLATED FROM FIVE CITIES IN NORTH COAST AREA OF CENTRAL JAVA, INDONESIA Benediktus Yohan1, Yulvica Fauziah1,2, Sayono Sayono2, Hidayat Trimarsanto1,3 and R Tedjo Sasmono1 1Eijkman Institute for Molecular Biology, Jakarta; 2Faculty of Public Health, Universitas Muhammadiyah Semarang, Semarang; 3The Agency for the Assessment and Application of Technology, Ministry of Research, Technology and Higher Education of the Republic of Indonesia, Jakarta, Indonesia Abstract. The tropical mosquito Aedes aegypti is the major vector in the transmission of human arboviruses, including chikungunya, dengue and yellow fever viruses. All provinces in the Indonesian archipelago have reported incidences of dengue. In order to study the genetic diversity and the dispersal of Ae. aegypti in Central Java, genetic analyses based on mitochondria cytochrome oxidase 1 gene (mtCOI) were performed on Ae. aegypti isolated in five cities along the national North Coast road of Central Java. Seventeen representative mtCOI fragments from either larvae or adult mosquitoes were PCR-amplified, sequenced and analyzed for sequence polymorphism, haplotype and genetic differentiation. A phylogenetic tree was constructed using maximum likelihood method and general time reversible model. There were seven haplotypes and the presence of different haplotypes in the cities was indicative of the heterogeneity of Ae. aegypti in Central Java. Gene flow and genetic differentiation analyses revealed no differentiation among populations from the cities. The possible gene flow between populations may reflect an active dispersal of Ae. aegypti among the cities as a result of movement of traffic along the national North Coast road of Central Java. Keywords: Aedes aegypti, haplotype, mitochondria COI, Central Java, Indonesia INTRODUCTION the world (Gubler, 2011). The species was introduced to Southeast Asia and the Aedes (Stegomyia) aegypti (L.) is the Pacific islands during World War II. The principal vector for urban yellow fever dramatic global geographic expansion (YF) and dengue (DENV) viruses. Origi- of this species along with the increase in nating from Africa, this mosquito has human population growth, urbanization expanded geographically throughout and globalization have increased the risk the tropical and subtropical regions of for outbreaks of dengue epidemics. Although there is available dengue Correspondence: R Tedjo Sasmono, Eijkman Institute for Molecular Biology, Jl Diponegoro vaccine, it is registered for widespread 69, Jakarta 10430, Indonesia. use only in a limited number of Southeast Tel: +62 21 3917131; Fax: +62 21 3147982 Asian countries (Wichmann et al, 2017) E-mail: [email protected] and thus, dengue prevention strategy Vol 49 No. 2 March 2018 217 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH still need to be combined with mosquito MATERIALS AND METHODS vector controls. Control measures should target dengue-competent mosquito popu- Study sites and sample collection lations to interrupt DENV transmission. Mosquito specimens were collected Knowledge of the genetic structure and as larvae or reared-adult mosquitoes gene flow amongAe . aegypti populations from five regions/cities along the North provides useful clues to help track and Coast part of Central Java, namely, Brebes, even prevent movements of associated Demak, Jepara, Kudus, and Semarang genetic traits such as vector competence (Fig 1) during August-September 2013. (Urdaneta-Marquez and Failloux, 2011). Larvae were collected from artificial Mitochondrial (mt)DNA is commonly water-containers in households surveyed. used for molecular evolution studies in Identification of Ae. aegypti isolates was insects and has proved to be particularly performed by assessment of morphologi- useful for detecting mosquito genetic cal features (Chan et al, 2014). Collected divergence and dispersal history (Kamb- samples were frozen and stored at -20°C hampati and Rai, 1991; Kambhampati, until used. 1995; Mousson et al, 2005). In particular, MtCOI amplification and sequencing mitochondrial cytochrome oxidase 1 gene Individual larvae or adult mosquitoes (mtCOI) has been found useful for genetic representing each breeding container in studies of Anopheles and Aedes mosquitoes each city were macerated using MagNA (Cook et al, 2005; Walton et al, 2000). Mt- Lyser Green Beads (Roche, Mannheim, COI has also been reported to be useful Germany) and 350 µl aliquot of lysis buffer for DNA barcoding, which complements in a MagNA Lyser homogenizer (Roche) taxonomy-based identification of mos- at 6,000 rpm for 15 minutes. Following quito species (Chan et al, 2014). centrifugation (4,000g for 5 minutes), Indonesia has been witnessing a 200 µl aliquot of the homogenate was dramatic spread of Ae. aegypti in the archi- extracted using MagNA Pure LC DNA pelago resulting in dengue disease hyper- Isolation Kit I (Roche) in an automated endemicity (Kraemer et al, 2015; Rašić et al, MagNA Pure LC 2.0 extraction system. 2015). The four DENV serotypes were DNA in 50 µl of elution buffer was stored shown to be circulating in all provinces of at -20°C until used. The 5′ segment of the country (Setiati et al, 2006). The clini- mtCOI was PCR amplified as described cal, virology and demographical features by Beebe et al (2005). The forward primer of dengue in Semarang, Central Java, and (5’-TAGTTCCTTTAATATTAGGAGC its surrounding regions have previously -3’) was designed to lie 245 bp into the been reported (Fahri et al, 2013). Here, COI 5’ region and the reverse primer we investigated the genetic diversity of (5’-TAATATAGCATAAATTATTCC-3’) Ae. aegypti isolated in five cities in coastal 813 bp into COI, generating a 590-bp COI areas of Central Java. This should aid in fragment. PCR mixture (50 µl) contained tracking the spread of this dengue virus 10X Expand Hi-fi PCR buffer (Roche), 25 vector from this region into other areas mM MgCl2 (Promega, Madison, WI), 10 of Java, which could be facilitated by mM dNTP Mix (Promega), Expand High vehicles using the national North Coast Fidelity DNA polymerase (Roche), and road leading to cities and provinces in 50-100 ng of DNA template. Thermocy- Java Island. cling was conducted in a GeneAmp PCR 218 Vol 49 No. 2 March 2018 AEDES AEGYPTI GENETIC DIVERSITY IN JAVA System 9700 instrument (Applied Biosys- populations. The relationship among tems, Foster City, CA) as follows: initial haplotypes was computed as a distance denaturation step at 95°C for 2 minutes; map based on pairwise difference to gen- 40 cycles of 95°C for 30 seconds, 50°C for erate a minimum spanning tree (MST) 1 minute; and 72°C for 1.5 minutes; with and network (MSN) among haplotypes. final step at 72°C for 10 minutes and stor- Distance map data were prepared using age at 4°C. Amplicons were separated by Arlequin v.3.5 software (Excoffier and 2% agarose gel-electrophoresis, visualized Lischer, 2010). MST and MSN were gen- by staining with ethidium bromide (EtBr), erated using HapStar v.0.7 (Teacher and purified from gel using QIAquick Gel Griffiths, 2011). Extraction Kit (Qiagen, Hilden, Germany) MtCOI phylogenetic analysis and sequenced in both directions using Multiple sequence alignment was BigDye Dideoxy Terminator sequencing performed using MUSCLE (Edgar, 2004) kits v3.1 (Applied Biosystems) in a 3130xl to generate sequence alignments of the Genetic Analyzer (Applied Biosystems) at mtCOI fragments with reference se- the Eijkman Institute, Jakarta. Sequences quences downloaded from GenBank. were assembled using SeqScape v.2.5 Further editing, trimming and alignment software (Applied Biosystems) with ad- generated sequence alignment lengths of ditional manual adjustments when neces- 503 nt. Phylogenetic relationships among sary and deposited to GenBank (Table 1). specimens were determined using the MtCOI haplotyping and population genetic maximum-likelihood (ML) method em- structure and gene flow analysis ploying MEGA5 (Tamura et al, 2011). A The mtCOI sequences were identi- test run was performed to identify the fied as Ae. aegypti using BOLD Systems best-fit model using jModelTest v.2.1.4 origin querying database (Ratnasingham software (Posada, 2009). Trees were in- and Hebert, 2007) and then analyzed ferred using general time reversible (GTR) for polymorphisms and haplotypes. Se- model with Gamma distributed (4 discrete quences were grouped according to their categories) rates (GTR+G). Branch sup- collection locations (except for the single ports were estimated by bootstrapping sample from Demak) and analyzed for with 1,000 replicates. their population genetic structure differ- entiation and gene flow. Sequence poly- RESULTS morphism analysis, haplotyping, gene flow, and genetic differentiation among Seventeen mtCOI gene sequences populations were performed using DnaSP obtained from larvae/mosquitoes col- software v.5.10 (Rozas and Rozas, 1995; lected from five cities in the north coastal Librado and Rozas, 2009). The Fixation region of Central Java Province could be index measured by F-statistics (FST) values divided into seven different haplotypes were calculated to estimate genetic dif- (Table 1) with 18 sites of nucleotide poly- ferentiation between populations. Popula- morphisms (Table 2). Two sequences of tion differentiation based on differences Ae. albopictus samples from Jakarta were in allele frequencies were estimated using used as outgroups. Haplotype H1 was the Nei’s GST values (Nei, 1973). Significant most predominant haplotype