Phylogenetic analysis of the GST family in Anopheles (Nyssorhynchus) darlingi

Acta Tropica 136 (2014) 27–31

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Phylogenetic analysis of the GST family in Anopheles (Nyssorhynchus) darlingi Gilson Martins de Azevedo-Júnior a , Giselle Moura Guimarães-Marques a , Leticia Cegatti Bridi a , Ketlen Christine Ohse b , Renato Vicentini c , Wanderli Tadei d , Míriam Silva Rafael d,∗ a Programa de Pós-Graduac¸ão em Genética, Conservac¸ão e Biologia Evolutiva–PPG GCBEv, Instituto Nacional de Pesquisas da Amazônia–INPA, Manaus, Amazonas, Brazil b Programa de Pós-Graduac¸ão em Biotecnologia e Recursos Naturais–PPG MBT, Universidade do Estado do Amazonas–UEA, Manaus, Amazonas, Brazil c Laboratório de Biologia de Sistemas, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas–UNICAMP, Campinas, São Paulo, Brazil d Coordenac¸ão de Sociedade, Ambiente e Saúde–CSAS, Laboratório de Vetores da Malária e Dengue/INPA, Manaus, Amazonas, Brazil

a r t i c l e

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Article history: Received 19 July 2013 Received in revised form 24 March 2014 Accepted 26 March 2014 Available online 5 April 2014 Keywords: Anopheles darlingi Glutathione S-transferase Sigma class

a b s t r a c t Anopheles darlingi Root, 1926 and Anopheles gambiae (Diptera: Culicidae) are the most important human malaria vectors in South America and Africa, respectively. The two species are estimated to have diverged 100 million years ago. Studies on the phylogenetics and evolution of gene sequences, such as glutathione S-transferase (GST) in disease-transmitting mosquitoes are scarce. The sigma class GST (KC890767) from the transcriptome of An. darlingi captured in the Brazilian Amazon was studied by in silico hybridization, and mapped to chromosome 3 of An. gambiae. The sigma class GST of An. darlingi was used for phylogenetic analyses to understand the GST base composition of the most recent common ancestor between An. darlingi, Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. The GST (KC890767) of An. darlingi was studied to generate the main divergence branches using a Neighbor-Joining and bootstrapping approaches to confirm confidence levels on the tree nodes that separate the An. darlingi and other mosquito species. The results showed divergence between An. gambiae, Ae. Aegypti, Cx. quinquefasciatus, and Phlebotomus papatasi as outgroup, and the homology relationship between sigma class GST of An. darlingi and GSTS1 1 gene of An. gambiae was valuable for phylogenetic and evolutionary studies. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Anopheles (Nyssorhynchus) darlingi Root 1926 may has originated in the Jurassic (Bertone et al., 2008). The family Culicidae diverged into the subfamilies Anophelinae and Culicinae approximately 120 million years ago, in the early Cretaceous (Rai and Black, 1999). The subfamily Culicinae comprises 3045 mosquito species (http://mosquito-taxonomic-inventory.info/ subfamily-culicinae-meigen-1818), and includes mosquito vectors of yellow fever and dengue (Ae. aegypti) and lymphatic filariasis (Culex quinquefasciatus), whose vectorial capacity evolved independently. The subfamily Anophelinae with about 478 species (http://mosquito-taxonomic-inventory.info/

∗ Corresponding author at: Instituto Nacional de Pesquisas da Amazônia, Av. André Araújo, 2.936, Petrópolis, CEP 69067-375, Manaus-AM, Brasil. Tel.: +55 92 3643 3066. E-mail address: [email protected] (M.S. Rafael). http://dx.doi.org/10.1016/j.actatropica.2014.03.027 0001-706X/© 2014 Elsevier B.V. All rights reserved.

subfamily-anophelinae-grassi-1900) includes the main vectors of human malaria, An. darlingi and An. gambiae (Deane, 1986; Mirabello et al., 2008). Anopheles darlingi has a high susceptibility to infection by Plasmodium falciparum and Plasmodium vivax (Zimmerman, 1992). Its marked preference for feeding on human blood is one of the key biological factors for An. darlingi, which is the main vector of malaria in the Amazon region (Tadei et al., 1998). Malaria causes the death of about 700,000–1 million people in tropical regions annually (Schwenk and Richie, 2011). In Brazil, malaria transmission is concentrated in the Amazon region (>99%). The resistance of mosquitoes to chemical insecticides contributes to the increasing incidence of malaria. Resistance has increased in various mosquito populations: An. gambiae in East and West Africa (Elissa et al., 1993), Anopheles funestus in South Africa (Hargreaves et al., 2000), Anopheles arabiensis in Tanzania and Mozambique (Kulkarni et al., 2006), and Anopheles stephensi from India and Dubai (Hodjati and Cutis, 1997).

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GST, cytochrome P450 (CYP) and carboxyl esterase (COE) supergene families are primarily involved in chemical insecticide metabolism (Ranson et al., 2002). Insecticide resistance is mainly associated with high enzyme levels involved in cellular detoxification (Scott and Kasai, 2004), such as Glutathione S-transferases (GSTs), a multifunctional family of enzymes found in all aerobic organisms, which is involved in detoxification of xenobiotic compounds (Gunasekaran et al., 2011). The GST enzyme family consists of seven classes of enzymes that metabolize exogenous and endogenous compounds (Wongtrakul et al., 2010) known as delta, epsilon, sigma, theta, zeta, and omega classes (Ding et al., 2003; Lumjuan et al., 2007). Delta and epsilon classes are found exclusively in insects (Kostaropoulos et al., 2001; Vontas et al., 2001; Ranson et al., 2004). Omega and theta classes of GSTs were found in An. gambiae (Ding et al., 2003). The theta class found in Ae. aegypti is not related to the resistance to the insecticide DichloroDiphenyl-Trichloroethane (DDT) (Lumjuan et al., 2005). The omega class found in Anopheles cracens is associated with oxidative stress (Wongtrakul et al., 2010). In An. darlingi, 89 P450s, 20 CCEs and 30 GSTs were found and four classes of GSTs were identified: theta, zeta, delta and epsilon (Marinotti et al., 2013). Resistance to DDT was observed in An. darlingi populations from four loca˜ et al., 1987; Suárez tions in the Quibdo locality, Colombia (Quinones et al., 1990; Fonseca-González et al., 2009) bioassayed five An. darlingi populations from Colombia, in the town of Amer-Beth, and detected strains resistant to the insecticides lambda-cyhalothrin and DDT, with a mortality rate of 65-75%. In a recent study of An. darlingi (Coari city, State of Amazonas, Brazil) samples were assayed for differential expression of a GST gene family (cDNA sequence) by quantitative Real Time–Polymerase Chain Reaction (qRT-PCR), after exposure to different concentrations of the insecticide deltamethrin. The GST gene family was a good indicator of resistance to deltamethrin, because of its high level of expression in relation to the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in An. darlingi (Naice and collaborators, unpublished data). The rDNA (ITS2) is a useful molecular marker to study taxonomic and phylogenetic relationships. The ITS2 regions of An. darlingi differed in length with 410 bp (Marrelli et al., 2005) and 426 bp in Afrotropical members of the An. (Cellia) gambiae complex (Paskewitz et al., 1993). The ITS2 marker was used to construct a neighbor-joining bootstrap tree that showed nucleotide divergences in 23 samples of 16 Anopheles species, including An. darlingi from the Brazilian Amazon region (Walton et al., 1999; Sallum et al., 2002). Analyses of microsatellite markers and the mitochondrial cytochrome oxidase subunit I (COI) showed low and moderate genetic differentiation of An. darlingi in Colombia, respectively (Gutiérrez et al., 2010). COI sequences of An. darlingi from 19 localities of Central and South America indicated population structuring (Mirabello and Conn, 2006). However, a comparison of all mtDNA genes within An. darlingi populations from Manaus, Brazil and the Central Cayo District of Belize, Moreno et al. (2010) did not detect any speciation processes in this taxon. Because phylogenetic studies have provided valuable information on the evolutionary divergence between biosequences in several organisms, we compared sequences of the sigma class GST (KC890767) from cDNA libraries of larvae and adults of An. darlingi (Rafael et al., 2010) by in silico mapping and comparison of global

alignment among homologous An. darlingi sigma class GST. We also performed a phylogenetic analysis using sigma class GST of An. darlingi (KC890767), An. gambiae (Q8MUR9), Ae. aegypti (Q16P78), Cx. quinquefasciatus (B0WFX0), with Ph. papatasi (A8CAE7) as the outgroup 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 uniGenes) were bioinformatically analyzed by Rafael et al. (2010) at Embrapa Genetic Resources and Biotechnology-CENARGEN (http:// valine.cenargen.embrapa.br), the Center for Molecular Biology and Genetic Engineering, CBMEG/Unicamp (http://sysbiol.cbmeg. unicamp.br/adarlingi/) and the National Institute of Amazonian Research, INPA (http://inpa.gov.br). A total of 347 contigs constructed from libraries of larvae and adults of An. darlingi were analyzed for their in silico differential expression (Audic and Claverie, 1997), with Bonferroni (1936) correction. Homologous sequences, with a cut off e-value set at e-10, were retrieved with BLASTX using the Blast2go software (Conesa et al., 2005). 2.2. In silico chromosome mapping In silico hybridization chromosome mapping of the sigma class GST from cDNA libraries of An. darlingi was performed against An. gambiae sequences (www.vectorbase.com.br) on the local server (http://sysbiol.cbmeg.unicamp.br/adarlingi), using the SIM4 (Florea et al., 1998) software package program. We also confirmed the sigma class GST in the An. darlingi genome (scaffold 446), according to https://www.vectorbase.org/ organisms/anopheles-darlingi. 2.3. ClustalW and phylogenetic analysis A comparison of global alignment between homologous sigma class GST in An. darlingi, An. gambiae, Ae. Aegypti, Cx. quinquefasciatus, and Ph. papatasi (outgroup) was performed with the ClustalW software program (Thompson et al., 1994). Sigma class GST of An. darlingi (KC890767), An. gambiae, Ae. Aegypti, Cx. quinquefasciatus, and orthologous genes of Ph. papatasi were compared and phylogenetically analyzed using genetic distances implemented in Neighbor 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 phylogenetic tree. 3. Results and discussion 3.1. In silico hybridization mapping of sigma class glutathione S-transferase Expressed Sequence Tags (ESTs) obtained from libraries of An. darlingi larvae and adults (Rafael et al., 2010) were mapped by in silico hybridization to An. gambiae chromosomes, using the software SIM4 (Florea et al., 1998). The sigma class GST obtained from cDNA libraries was mapped to the left arm of chromosome 3 in An.

Table 1 Orthology reciprocal Blast and in silico hybridization mapping of GSTS1 1 gene between An. darlingi and An. gambiae. Mosquito species

Base pars length

Access number

Gene

Anopheles gambiae polytene chromosome 3

Anopheles darlingi Anopheles gambiae

1060 1343

KC890767 AGAP010404

Sigma class Glutathione S-transferase Glutathione S-transferase 1-1 (GSTS1-1)

Left arm of Chromosome 3: 2781203-2783301 Left arm of Chromosome 3: 2779188-2784335

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

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Table 2 Homology 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 Anopheles gambiae Culex quinquefasciatus

EAT36156.1 AAM53611.1 EDS26505.1

3.9E-103 8.6E-103 2.5E-102

96.00 97.00 95.00

GO:0016740-IEA GO:0016740-IEA GO:0016740-IEA

Q16P78 Q8MUR9 B0WFX0

gambiae (Table 1), and orthology relationship between sigma class GST and GSTS1 1 was established by reciprocal best Blast hit. Genomic organization of sigma class GST is based on homologous regions of An. darlingi and An. gambiae on chromosome 3L. Because the GSTS1 1 is orthologous in both Anopheline mosquitoes, these results were to be expected, and open new research fields for comparative GSTs and evolutionary studies in both species. 3.2. Homology elucidation based on genomic organization of sigma class GST of An. darlingi to three disease-transmitting mosquitoes Sigma class GST (KC890767) of An. darlingi was identified in the Molecular Function ontology using the blast2go software. It showed transferase activity of sigma class and a high degree of homology with the sigma class of Ae. aegypti, An. gambiae and Cx. quinquesfasciatus. Alignment of these GSTs according to the UniProt protein database (Table 2) returned high similarities of gene sequences (>95%) with highly significant e-values (Table 2). GST, cytochrome P450 (CYP) and carboxyl esterase (COE) supergene families are primarily involved in insecticide metabolism (Ranson et al., 2002) with GSTs being the most conserved among these three superfamilies (Wilding et al., 2009). The GST gene family is found in most insects (Kostaropoulos et al., 2001; Vontas et al., 2001; Ranson et al., 2004). It is a family of multifunctional enzymes (Srivastava et al., 2010), which detoxify xenobiotic compounds, such as chemicals and pesticides that pollute the environment (Ranson and Hemingway, 2005) and also enable resistance to insecticides, such as DDT (Prapanthadara et al., 1993), temephos and deltamethrin (Naice and collaborators, unpublished data). Six GST classes were identified in insects: delta (GSTd), epsilon (GSTe), omega (GSTo), sigma (GSTs), theta (GST) and zeta (GSTz) classes (Tang and Tu, 1994; Huang et al., 1998; Ortelli et al., 2003). Genes of the epsilon class GSTs (agGSTe2) are highly expressed in An. gambiae strains resistant to DDT, with an approximately eight-fold expression level. The agGSTe2 gene is important for metabolization and detoxification of DDT, similar to carboxyl esterase (COE) in An. gambiae, and can be 350 times more effective than the delta class agGST1-6 gene (Wang et al., 2008). GSTd1 and GSTs1 (sigma and delta classes) are orthologous in An. gambiae and Ae. Aegypti. Their difference in alternative splicing sites predates divergence between the genera Aedes and Anopheles (Lumjuan et al., 2007). 3.3. Phylogeny of glutathione S-transferase sigma class We studied the sigma class GST to construct a phylogenetic tree among the Anophelini and Culicini groups analyzed. Taxonomically, the Anophelinae and Culicinae subfamilies diverged at the beginning of the Cretaceous. Anopheles darlingi is the main malaria vector in the Neotropical Region, and its importance as a vector changes continuously due to ecological changes and adaptations of this mosquito (Manguin et al., 1999). Its geographic distribution ranges from Eastern Mexico to Northern Argentina (Forattini, 2002). In the Brazilian Amazon region, deforestation, highway construction, agricultural and mineral activities and the presence of

Anophelinae vectors may contribute to an increase of malaria incidence (Deane, 1986). Anopheles darlingi has been observed frequently 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 challenge for years, and it is important to increase the knowledge about control strategies, such as reinforcing the allelic selection for insecticide resistance in disease-transmitting mosquitoes. The Culicidae family diverged into the subfamilies Anophelinae and Culicinae approximately 120 million years ago (Rai and Black, 1999). Different approaches have been used to determine speciation and divergence times within An. darlingi, including Amazonian and southern South American populations. Microsatellite markers showed 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, proof of IBD was observed using nuclear markers (Mirabello and Conn, 2006; Scarpassa and Conn, 2007; Mirabello et al., 2008) suggesting that the ancestor of modern An. darlingi originated in Amazonian and/or central Brazil. Later, COI gene studies suggested an ancestral distribution of An. darlingi populations in central Amazonia during the late Pleistocene (Pedro and Sallum, 2009). In a recent study based on all mtDNA genes, Moreno et al. (2010) suggested An. darlingi and An. gambiae to have diverged 100 mya. Further, they suggest that the time of divergence between Anopheles and the Culicinae dates to the Cretaceous, with the radiation of An. gambiae, An. funestus, An. quadrimaculatus and An. darlingi, occurring approximately 79 million years ago. In addition, the authors found no evidence for speciation within An. darlingi. The phylogenetic position of this species highlights the importance of our study. Here, we analysed the evolutionary relationship and divergence time of the sigma class GSTs of An. darlingi in comparison to orthologous genes of the related species Ae. aegypti, An. gambiae and Cx. quinquefasciatus. The GST of Ph. papatasi (outgroup), was used for rooting the phylogenetic tree. The global alignment between the homologous sequences to generate the phylogenetic tree was performed 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 (Tamura et al., 2007), generated two main branches, separating Ph. papatasi (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 GSTs of Cx. quinquefasciatus diverged before those of Ae. aegypti, An. gambiae and An. darlingi. The sigma class of An. darlingi was most closely related to that of An. gambiae, and these two species were also grouped as sister species in the same branch. Bootstrap values (Felsenstein, 1985) were equal to or greater than the 95% confidence level and are shown on the nodes of the tree that connect the gene sequences or OTUs (operational taxonomic units). The glutathione-S-transferase GST gene family is known to be primarily involved in oxidative stress metabolism. In the An. darlingi genome, four classes of GSTs were identified: theta (5 genes), zeta (one gene), delta (3 genes) and epsilon (6 genes) classes, in

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Fig. 1. Comparison of Sigma class GSTs of An. darlingi, An. gambiae, Ae. aegypti, Cx. quinquefasciatus and Ph. papatasi. (A) Global alignment using Clustal W. (B) Phylogenetic tree obtained by Neighbor Joining – NJ (Saitou and Nei, 1987). The bar indicates 2% aminoacid divergence.

addition to 89 P450s, 20 CCEs and 30 GSTs genes. Putative conservative orthologs in An. darlingi and An. gambiae also were identified with >70% sequence identity (Marinotti et al., 2013). The sigma class of cDNA libraries of An. darlingi (Rafael et al., 2010) was clustered with An. gambiae on the same branch, suggesting that the sequence evolved from a common ancestor, as shown by the comparison between An. gambiae and An. darlingi with an identity of sigma class GSTs greater than 70%. Inspite of ∼100 million years of evolutionary divergence between two distantly related members of the Anopheles genus (An. darlingi and An. gambiae), and the fact that the most recent ancestor of Nyssorhynchus and Anopheles and Cellia lived ∼94 million years ago (Moreno et al., 2010), our result suggests an adaptive evolution shared between GSTS1-1 gene of both species. Furthermore, a robust phylogeny of other GST classes (in addition to the sigma class gene presented here) will help to develop malaria control strategies by elucidating evolutionary relationships of GST classes in An. darlingi and An. gambiae.

Acknowledgements We are grateful to Dr. Michel Vincentz, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual

de Campinas, SP, Brasil, and Embrapa Genetic Resources and Biotechnology - CENARGEN for providing technical support for automatic bioinformatic data analysis. This work was supported by Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior–CAPES (PROCAD–Process no.023/2006), the Fundac¸ão de Amparo à Pesquisa do Estado do Amazonas–FAPEAM (PIPT, transcriptome projects, and PPP–process no. 2680/2009), Financiadora de Estudos e Projetos - FINEP (CTPETRO Amazon network–process no. 01.08.0282.00), and Conselho Nacional de Desenvolvimento Científico e Tecnológico–CNPq (Malaria Network–Process no. 555665/2009-7).

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