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Acta Tropica 136 (2014) 27–31

Contents lists available at ScienceDirect

Acta Tropica

jo ur nal home p age: www.elsev ier .com/ locate /ac ta t ropica

hylogenetic analysis of the GST family in AnophelesNyssorhynchus) darlingi

ilson Martins de Azevedo-Júniora, Giselle Moura Guimarães-Marquesa,eticia Cegatti Bridia, Ketlen Christine Ohseb, Renato Vicentini c,

anderli Tadeid, Míriam Silva Rafaeld,∗

Programa de Pós-Graduac ão em Genética, Conservac ão e Biologia Evolutiva–PPG GCBEv, Instituto Nacional de Pesquisas da Amazônia–INPA, Manaus,mazonas, BrazilPrograma de Pós-Graduac ão em Biotecnologia e Recursos Naturais–PPG MBT, Universidade do Estado do Amazonas–UEA, Manaus, Amazonas, BrazilLaboratório de Biologia de Sistemas, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas–UNICAMP, Campinas, Sãoaulo, BrazilCoordenac ão de Sociedade, Ambiente e Saúde–CSAS, Laboratório de Vetores da Malária e Dengue/INPA, Manaus, Amazonas, Brazil

r t i c l e i n f o

rticle history:eceived 19 July 2013eceived in revised form 24 March 2014ccepted 26 March 2014vailable online 5 April 2014

eywords:nopheles darlingilutathione S-transferase

a b s t r a c t

Anopheles darlingi Root, 1926 and Anopheles gambiae (Diptera: Culicidae) are the most important humanmalaria vectors in South America and Africa, respectively. The two species are estimated to have diverged100 million years ago. Studies on the phylogenetics and evolution of gene sequences, such as glutathioneS-transferase (GST) in disease-transmitting mosquitoes are scarce. The sigma class GST (KC890767) fromthe transcriptome of An. darlingi captured in the Brazilian Amazon was studied by in silico hybridiza-tion, and mapped to chromosome 3 of An. gambiae. The sigma class GST of An. darlingi was used forphylogenetic analyses to understand the GST base composition of the most recent common ancestorbetween An. darlingi, Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. The GST (KC890767)

igma class of An. darlingi was studied to generate the main divergence branches using a Neighbor-Joining and boot-strapping approaches to confirm confidence levels on the tree nodes that separate the An. darlingi andother mosquito species. The results showed divergence between An. gambiae, Ae. Aegypti, Cx. quinquefas-ciatus, and Phlebotomus papatasi as outgroup, and the homology relationship between sigma class GST ofAn. darlingi and GSTS1 1 gene of An. gambiae was valuable for phylogenetic and evolutionary studies.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Anopheles (Nyssorhynchus) darlingi Root 1926 may has origi-ated in the Jurassic (Bertone et al., 2008). The family Culicidaeiverged into the subfamilies Anophelinae and Culicinae approx-

mately 120 million years ago, in the early Cretaceous (Raind Black, 1999). The subfamily Culicinae comprises 3045osquito species (http://mosquito-taxonomic-inventory.info/

ubfamily-culicinae-meigen-1818), and includes mosquito vec-ors of yellow fever and dengue (Ae. aegypti) and lymphatic

lariasis (Culex quinquefasciatus), whose vectorial capac-

ty evolved independently. The subfamily Anophelinae withbout 478 species (http://mosquito-taxonomic-inventory.info/

∗ Corresponding author at: Instituto Nacional de Pesquisas da Amazônia, Av. Andréraújo, 2.936, Petrópolis, CEP 69067-375, Manaus-AM, Brasil. Tel.: +55 92 3643 3066.

E-mail address: msrafael@inpa.gov.br (M.S. Rafael).

ttp://dx.doi.org/10.1016/j.actatropica.2014.03.027001-706X/© 2014 Elsevier B.V. All rights reserved.

subfamily-anophelinae-grassi-1900) includes the main vectorsof human malaria, An. darlingi and An. gambiae (Deane, 1986;Mirabello et al., 2008). Anopheles darlingi has a high susceptibilityto infection by Plasmodium falciparum and Plasmodium vivax(Zimmerman, 1992). Its marked preference for feeding on humanblood is one of the key biological factors for An. darlingi, whichis the main vector of malaria in the Amazon region (Tadei et al.,1998).

Malaria causes the death of about 700,000–1 million people intropical regions annually (Schwenk and Richie, 2011). In Brazil,malaria transmission is concentrated in the Amazon region (>99%).The resistance of mosquitoes to chemical insecticides contributesto the increasing incidence of malaria. Resistance has increased invarious mosquito populations: An. gambiae in East and West Africa

(Elissa et al., 1993), Anopheles funestus in South Africa (Hargreaveset al., 2000), Anopheles arabiensis in Tanzania and Mozambique(Kulkarni et al., 2006), and Anopheles stephensi from India and Dubai(Hodjati and Cutis, 1997).

2 . / Acta

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8 G.M.d. Azevedo-Júnior et al

GST, cytochrome P450 (CYP) and carboxyl esterase (COE)upergene families are primarily involved in chemical insecticideetabolism (Ranson et al., 2002). Insecticide resistance is mainly

ssociated with high enzyme levels involved in cellular detoxifi-ation (Scott and Kasai, 2004), such as Glutathione S-transferasesGSTs), a multifunctional family of enzymes found in all aero-ic organisms, which is involved in detoxification of xenobioticompounds (Gunasekaran et al., 2011). The GST enzyme familyonsists of seven classes of enzymes that metabolize exogenousnd endogenous compounds (Wongtrakul et al., 2010) known aselta, epsilon, sigma, theta, zeta, and omega classes (Ding et al.,003; Lumjuan et al., 2007). Delta and epsilon classes are foundxclusively in insects (Kostaropoulos et al., 2001; Vontas et al.,001; Ranson et al., 2004). Omega and theta classes of GSTs wereound in An. gambiae (Ding et al., 2003). The theta class found in Ae.egypti is not related to the resistance to the insecticide Dichloro-iphenyl-Trichloroethane (DDT) (Lumjuan et al., 2005). The omegalass found in Anopheles cracens is associated with oxidative stressWongtrakul et al., 2010). In An. darlingi, 89 P450s, 20 CCEs and0 GSTs were found and four classes of GSTs were identified:heta, zeta, delta and epsilon (Marinotti et al., 2013). Resistanceo DDT was observed in An. darlingi populations from four loca-ions in the Quibdo locality, Colombia (Quinones et al., 1987; Suárezt al., 1990; Fonseca-González et al., 2009) bioassayed five An. dar-ingi populations from Colombia, in the town of Amer-Beth, andetected strains resistant to the insecticides lambda-cyhalothrinnd DDT, with a mortality rate of 65-75%. In a recent study ofn. darlingi (Coari city, State of Amazonas, Brazil) samples weressayed for differential expression of a GST gene family (cDNAequence) by quantitative Real Time–Polymerase Chain ReactionqRT-PCR), after exposure to different concentrations of the insec-icide deltamethrin. The GST gene family was a good indicator ofesistance to deltamethrin, because of its high level of expressionn relation to the housekeeping glyceraldehyde-3-phosphate dehy-rogenase (GAPDH) gene in An. darlingi (Naice and collaborators,npublished data).

The rDNA (ITS2) is a useful molecular marker to study tax-nomic and phylogenetic relationships. The ITS2 regions of An.arlingi differed in length with 410 bp (Marrelli et al., 2005) and26 bp in Afrotropical members of the An. (Cellia) gambiae complexPaskewitz et al., 1993). The ITS2 marker was used to construct

neighbor-joining bootstrap tree that showed nucleotide diver-ences in 23 samples of 16 Anopheles species, including An. darlingirom the Brazilian Amazon region (Walton et al., 1999; Sallum et al.,002).

Analyses of microsatellite markers and the mitochondrialytochrome oxidase subunit I (COI) showed low and moderateenetic differentiation of An. darlingi in Colombia, respectivelyGutiérrez et al., 2010). COI sequences of An. darlingi from 19 locali-ies of Central and South America indicated population structuringMirabello and Conn, 2006). However, a comparison of all mtDNAenes within An. darlingi populations from Manaus, Brazil and theentral Cayo District of Belize, Moreno et al. (2010) did not detectny speciation processes in this taxon.

Because phylogenetic studies have provided valuable infor-

ation on the evolutionary divergence between biosequences in

everal organisms, we compared sequences of the sigma class GSTKC890767) from cDNA libraries of larvae and adults of An. darlingiRafael et al., 2010) by in silico mapping and comparison of global

able 1rthology reciprocal Blast and in silico hybridization mapping of GSTS1 1 gene between A

Mosquito species Base pars length Access number Gene

Anopheles darlingi 1060 KC890767 Sigma class GlAnopheles gambiae 1343 AGAP010404 Glutathione S

Tropica 136 (2014) 27–31

alignment among homologous An. darlingi sigma class GST. We alsoperformed a phylogenetic analysis using sigma class GST of An. dar-lingi (KC890767), An. gambiae (Q8MUR9), Ae. aegypti (Q16P78), Cx.quinquefasciatus (B0WFX0), with Ph. papatasi (A8CAE7) as the out-group to understand phylogenetic and evolutionary relationships.

2. Materials and methods

2.1. In silico differential expression

cDNA libraries from larvae and adults of An. darlingi (568 uni-Genes) were bioinformatically analyzed by Rafael et al. (2010) atEmbrapa Genetic Resources and Biotechnology-CENARGEN (http://valine.cenargen.embrapa.br), the Center for Molecular Biologyand Genetic Engineering, CBMEG/Unicamp (http://sysbiol.cbmeg.unicamp.br/adarlingi/) and the National Institute of AmazonianResearch, INPA (http://inpa.gov.br). A total of 347 contigs con-structed from libraries of larvae and adults of An. darlingi wereanalyzed for their in silico differential expression (Audic andClaverie, 1997), with Bonferroni (1936) correction. Homologoussequences, with a cut off e-value set at e-10, were retrieved withBLASTX using the Blast2go software (Conesa et al., 2005).

2.2. In silico chromosome mapping

In silico hybridization chromosome mapping of the sigmaclass GST from cDNA libraries of An. darlingi was performedagainst An. gambiae sequences (www.vectorbase.com.br) onthe local server (http://sysbiol.cbmeg.unicamp.br/adarlingi), usingthe SIM4 (Florea et al., 1998) software package program.We also confirmed the sigma class GST in the An. darlingigenome (scaffold 446), according to https://www.vectorbase.org/organisms/anopheles-darlingi.

2.3. ClustalW and phylogenetic analysis

A comparison of global alignment between homologous sigmaclass GST in An. darlingi, An. gambiae, Ae. Aegypti, Cx. quinquefascia-tus, and Ph. papatasi (outgroup) was performed with the ClustalWsoftware program (Thompson et al., 1994). Sigma class GST ofAn. darlingi (KC890767), An. gambiae, Ae. Aegypti, Cx. quinquefas-ciatus, and orthologous genes of Ph. papatasi were compared andphylogenetically analyzed using genetic distances implemented inNeighbor Joining (Saitou and Nei, 1987). The Mega4.0 (Tamura et al.,2007) Bootstrap analysis (Mega4.0 Software (Felsenstein, 1985)was used to calculate statistical reliability of the nodes of the phy-logenetic tree.

3. Results and discussion

3.1. In silico hybridization mapping of sigma class glutathioneS-transferase

Expressed Sequence Tags (ESTs) obtained from libraries of An.

darlingi larvae and adults (Rafael et al., 2010) were mapped by insilico hybridization to An. gambiae chromosomes, using the soft-ware SIM4 (Florea et al., 1998). The sigma class GST obtained fromcDNA libraries was mapped to the left arm of chromosome 3 in An.

n. darlingi and An. gambiae.

Anopheles gambiae polytene chromosome 3

utathione S-transferase Left arm of Chromosome 3: 2781203-2783301-transferase 1-1 (GSTS1-1) Left arm of Chromosome 3: 2779188-2784335

G.M.d. Azevedo-Júnior et al. / Acta Tropica 136 (2014) 27–31 29

Table 2Homology of sigma class GSTs from An. gambiae, Ae. aegypti and Cx. quinquefasciatus with sigma class GST of An. darlingi, showing access numbers in the UniProt alignment,e-value rates, similarity and ontologies.

Species Access numbers e-value Similarity (%) Ontology Uniprot

Aedes aegypti EAT36156.1 3.9E-103 96.00 GO:0016740-IEA Q16P78

gG

o3mrb

3sm

tshCUg

g(ti2(s(td

(cGiemetGAd2

3

acbvcot2s

Anopheles gambiae AAM53611.1 8.6E-103

Culex quinquefasciatus EDS26505.1 2.5E-102

ambiae (Table 1), and orthology relationship between sigma classST and GSTS1 1 was established by reciprocal best Blast hit.

Genomic organization of sigma class GST is based on homol-gous regions of An. darlingi and An. gambiae on chromosomeL. Because the GSTS1 1 is orthologous in both Anophelineosquitoes, these results were to be expected, and open new

esearch fields for comparative GSTs and evolutionary studies inoth species.

.2. Homology elucidation based on genomic organization ofigma class GST of An. darlingi to three disease-transmittingosquitoes

Sigma class GST (KC890767) of An. darlingi was identified inhe Molecular Function ontology using the blast2go software. Ithowed transferase activity of sigma class and a high degree ofomology with the sigma class of Ae. aegypti, An. gambiae andx. quinquesfasciatus. Alignment of these GSTs according to theniProt protein database (Table 2) returned high similarities ofene sequences (>95%) with highly significant e-values (Table 2).

GST, cytochrome P450 (CYP) and carboxyl esterase (COE) super-ene families are primarily involved in insecticide metabolismRanson et al., 2002) with GSTs being the most conserved amonghese three superfamilies (Wilding et al., 2009). The GST gene fam-ly is found in most insects (Kostaropoulos et al., 2001; Vontas et al.,001; Ranson et al., 2004). It is a family of multifunctional enzymesSrivastava et al., 2010), which detoxify xenobiotic compounds,uch as chemicals and pesticides that pollute the environmentRanson and Hemingway, 2005) and also enable resistance to insec-icides, such as DDT (Prapanthadara et al., 1993), temephos andeltamethrin (Naice and collaborators, unpublished data).

Six GST classes were identified in insects: delta (GSTd), epsilonGSTe), omega (GSTo), sigma (GSTs), theta (GST) and zeta (GSTz)lasses (Tang and Tu, 1994; Huang et al., 1998; Ortelli et al., 2003).enes of the epsilon class GSTs (agGSTe2) are highly expressed

n An. gambiae strains resistant to DDT, with an approximatelyight-fold expression level. The agGSTe2 gene is important foretabolization and detoxification of DDT, similar to carboxyl

sterase (COE) in An. gambiae, and can be 350 times more effectivehan the delta class agGST1-6 gene (Wang et al., 2008). GSTd1 andSTs1 (sigma and delta classes) are orthologous in An. gambiae ande. Aegypti. Their difference in alternative splicing sites predatesivergence between the genera Aedes and Anopheles (Lumjuan et al.,007).

.3. Phylogeny of glutathione S-transferase sigma class

We studied the sigma class GST to construct a phylogenetic treemong the Anophelini and Culicini groups analyzed. Taxonomi-ally, the Anophelinae and Culicinae subfamilies diverged at theeginning of the Cretaceous. Anopheles darlingi is the main malariaector in the Neotropical Region, and its importance as a vectorhanges continuously due to ecological changes and adaptations

f this mosquito (Manguin et al., 1999). Its geographic distribu-ion ranges from Eastern Mexico to Northern Argentina (Forattini,002). In the Brazilian Amazon region, deforestation, highway con-truction, agricultural and mineral activities and the presence of

97.00 GO:0016740-IEA Q8MUR995.00 GO:0016740-IEA B0WFX0

Anophelinae vectors may contribute to an increase of malariaincidence (Deane, 1986). Anopheles darlingi has been observed fre-quently at the modified sites, especially in and around houses,confirming its high degree of association with human dwellings(Tadei et al., 1998). The eradication of malaria has been a chal-lenge for years, and it is important to increase the knowledge aboutcontrol strategies, such as reinforcing the allelic selection for insec-ticide resistance in disease-transmitting mosquitoes.

The Culicidae family diverged into the subfamilies Anophelinaeand Culicinae approximately 120 million years ago (Rai and Black,1999). Different approaches have been used to determine specia-tion and divergence times within An. darlingi, including Amazonianand southern South American populations. Microsatellite markersshowed isolation-by-distance (IBD) in An. darlingi from Amazonia(Conn et al., 2006; Scarpassa and Conn, 2007; Mirabello et al., 2008;Lima et al., 2010). Using COI, population structuring was detected,but did not confirm IBD (Mirabello and Conn, 2006). Instead, proofof IBD was observed using nuclear markers (Mirabello and Conn,2006; Scarpassa and Conn, 2007; Mirabello et al., 2008) suggestingthat the ancestor of modern An. darlingi originated in Amazonianand/or central Brazil. Later, COI gene studies suggested an ances-tral distribution of An. darlingi populations in central Amazoniaduring the late Pleistocene (Pedro and Sallum, 2009). In a recentstudy based on all mtDNA genes, Moreno et al. (2010) suggestedAn. darlingi and An. gambiae to have diverged 100 mya. Further,they suggest that the time of divergence between Anopheles andthe Culicinae dates to the Cretaceous, with the radiation of An.gambiae, An. funestus, An. quadrimaculatus and An. darlingi, occur-ring approximately 79 million years ago. In addition, the authorsfound no evidence for speciation within An. darlingi. The phylo-genetic position of this species highlights the importance of ourstudy.

Here, we analysed the evolutionary relationship and divergencetime of the sigma class GSTs of An. darlingi in comparison to ortho-logous genes of the related species Ae. aegypti, An. gambiae andCx. quinquefasciatus. The GST of Ph. papatasi (outgroup), was usedfor rooting the phylogenetic tree. The global alignment betweenthe homologous sequences to generate the phylogenetic tree wasperformed using the CLUSTALW program (Fig. 1-A and B).

Phylogenetic analysis of the GSTs by Neighbor Joining – NJ(Saitou and Nei, 1987), using the software MEGA 4.0 (Tamuraet al., 2007), generated two main branches, separating Ph. pap-atasi (UniProt: A8CAE7) from the second group that contained Cx.quinquefasciatus (UniProt: B0WFX0), An. darlingi (KC890767), An.gambiae (Q8MUR9) and Ae. aegypti (Q16P78). GSTs of Ae. aegypti,An. gambiae and An. darlingi had a common ancestor, while GSTsof Cx. quinquefasciatus diverged before those of Ae. aegypti, An.gambiae and An. darlingi. The sigma class of An. darlingi was mostclosely related to that of An. gambiae, and these two species werealso grouped as sister species in the same branch. Bootstrap values(Felsenstein, 1985) were equal to or greater than the 95% confi-dence level and are shown on the nodes of the tree that connectthe gene sequences or OTUs (operational taxonomic units).

The glutathione-S-transferase GST gene family is known to beprimarily involved in oxidative stress metabolism. In the An. dar-lingi genome, four classes of GSTs were identified: theta (5 genes),zeta (one gene), delta (3 genes) and epsilon (6 genes) classes, in

30 G.M.d. Azevedo-Júnior et al. / Acta Tropica 136 (2014) 27–31

F quinqut amino

avw

2ibilmaA2Gohig

A

g

ig. 1. Comparison of Sigma class GSTs of An. darlingi, An. gambiae, Ae. aegypti, Cx.

ree obtained by Neighbor Joining – NJ (Saitou and Nei, 1987). The bar indicates 2%

ddition to 89 P450s, 20 CCEs and 30 GSTs genes. Putative conser-ative orthologs in An. darlingi and An. gambiae also were identifiedith >70% sequence identity (Marinotti et al., 2013).

The sigma class of cDNA libraries of An. darlingi (Rafael et al.,010) was clustered with An. gambiae on the same branch, suggest-

ng that the sequence evolved from a common ancestor, as showny the comparison between An. gambiae and An. darlingi with an

dentity of sigma class GSTs greater than 70%. Inspite of ∼100 mil-ion years of evolutionary divergence between two distantly related

embers of the Anopheles genus (An. darlingi and An. gambiae),nd the fact that the most recent ancestor of Nyssorhynchus andnopheles and Cellia lived ∼94 million years ago (Moreno et al.,010), our result suggests an adaptive evolution shared betweenSTS1-1 gene of both species. Furthermore, a robust phylogeny ofther GST classes (in addition to the sigma class gene presentedere) will help to develop malaria control strategies by elucidat-

ng evolutionary relationships of GST classes in An. darlingi and An.ambiae.

cknowledgements

We are grateful to Dr. Michel Vincentz, Centro de Biolo-ia Molecular e Engenharia Genética, Universidade Estadual

efasciatus and Ph. papatasi. (A) Global alignment using Clustal W. (B) Phylogeneticacid divergence.

de Campinas, SP, Brasil, and Embrapa Genetic Resources andBiotechnology - CENARGEN for providing technical support forautomatic bioinformatic data analysis. This work was sup-ported by Coordenac ão de Aperfeic oamento de Pessoal de NívelSuperior–CAPES (PROCAD–Process no.023/2006), the Fundac ão deAmparo à Pesquisa do Estado do Amazonas–FAPEAM (PIPT, trans-criptome projects, and PPP–process no. 2680/2009), Financiadorade Estudos e Projetos - FINEP (CTPETRO Amazon network–processno. 01.08.0282.00), and Conselho Nacional de DesenvolvimentoCientífico e Tecnológico–CNPq (Malaria Network–Process no.555665/2009-7).

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