Identification of novel Glutathione S-Transferases epsilon 2 mutation in Anopheles maculipennis s.s. (Diptera: Culicidae)

Abstract

Anopheles maculipennis complex comprises some important malaria vectors in Iran, Middle East, and Europ. The principal way to control of malaria remains on the use of chemical insecticides against its vectors because there is no vaccine for malaria prevention. Extensive use of organophosphate compounds has caused to emergence and distribution of insecticide resistance in Anopheles species in Asia. The current study aimed to the detection of three well-known amino acid substitutions (I114T, L119F, and F120L) in the Glutathione S-Transferases epsilon 2 (GSTe2) gene are associated with DDT and organophosphate insecticides resistance in an Anopheles maculipennis population collected from Iran. Adult samples of An. maculipennis were collected by hand and Total catch in Animal and Human Shelters from Azerbaijan-Gharbi and Zanjan provinces. Following morphological identification, DNA was extracted by YTA Genomic DNA Extraction Mini Kit for amplification of rDNA-ITS2 and GSTe2 fragments. ∼500 bp fragment was amplified using F rDNA-ITS2 and GSTe2 primers. rDNA-ITS2 sequence analysis showed 100% similarity with An. maculipennis. GSTe2 nucleotide sequence similarity within species was 99-100%, while, it was 95-96 % when compared with Anopheles sacharovi GSTe2 sequences available in GenBank. Amino acid sequence comparisons showed a novel amino acid substitution in N148D position with 15.79% frequency. The current study reports new GSTe2 amino acid substitution in An. maculipennis s.s., for the first time. The function of the mutation N148D and its association with resistance phenotype need to validate. However, the integration of these data into the malaria control program still remains a challenge.

Keywords: Anopheles maculipennis; Bioinformatics; DNA sequencing; Genetics; Genomics; Glutathione S-Transferases epsilon 2; Molecular biology; Novel mutation; Polymorphism; Proteins.

Fig. 1

Multiple nucleotide sequence alignment of 19 GSTe2 open reading frames partial sequences (MK421369-MK421387) of An. maculipennis collected from Iran. Alignment was generated in MEGA6 program. A dot indicates identity with the relevant sequence. Transitions and transversion are highlighted in green and blue colores, respectively. Alighnments with 100% similarity was not shown.

Fig. 2

Multiple nucleotide sequence alignment of 19 GSTe2 open reading frames partial sequences (MK421369-MK421387) of An. maculipennis collected from Iran (exons highlighted in grey and white colors), and available nucleotide sequences of An. sacharovi, An. stephensi, An. fluviatilis, An. gambiae, An. funestus and An. plumbeus in GenBank. Related accession numbers are included after the species name. Alignment was generated in MEGA6 program. A dash indicates deletion and a dot indicates identity with the relevant sequence.

Fig. 3

The alignment of amino acid sequences of a partial sequence of the GSTe2 gene of An. maculipennis specimens collected from Iran. D148N mutation are highlighted in green colore. Alignment was generated in MEGA6 program. Alighnments with 100% similarity was not shown. A dot indicates identity with the relevant sequence.

Fig. 4

The alignment of amino acid sequences of a partial sequence of the GSTe2 gene of An. maculipennis specimens are collected from Iran and available sequences of An. sacharovi, An. stephensi, An. fluviatilis, An. gambiae, An. funestus and An. plumbeus in GenBank. Related accession numbers are included after the species name. Alignment was generated in MEGA6 program. A dash indicates deletion and a dot indicates identity with the relevant sequence. A dash indicates deletion and a dot indicates identity with relevant sequence.

Fig. 5

Maximum likelihood tree based on GSTe2 amino acid (A) and nucleotide (B) sequences for 19 Iranian An. maculipennis specimens and available sequences of An. sacharovi, An. stephensi, An. fluviatilis, An. gambiae, An. funestus and An. plumbeus in GenBank. Bootstrap values (1000 replicates) >50 have been shown above the lines.