Elsevier

Gene Reports

Volume 5, December 2016, Pages 70-74
Gene Reports

Identification and evaluation expression level of arrestin 1 gene during the development stage of Anopheles stephensi

https://doi.org/10.1016/j.genrep.2016.09.002Get rights and content

Abstract

Olfaction and visual play are an important role in many insect behaviors, including host-seeking behavior, and blood-feeding. Identification of molecular mechanism of these kinds of behaviors is a crucial issue that should be considered to find new methods in controlling vector-born diseases. Previous studies on arrestin 1 from Anopheles gambiae (main malaria vector in Iran, Indian subcontinent and China) have shown that this gene plays a prominent role in the visual process of this mosquito. Despite its importance, this gene and its protein have not yet been identified and characterized in Anopheles stephensi (main malaria vector in Iran, Indian subcontinent and China). In this study, we used Rapid Amplification of cDNA Ends (RACE) technique to identify total cDNA of this gene, applied real time PCR to evaluate expression level of this gene in different stages of mosquito development, and characterized it using bioinformatics tools. The result showed that arrestin 1 from Anopheles stephensi (arr1As) gene and its protein are remarkably similar to that of the arrestin 1 gene from Anopheles gambiae (arr1Ag) and is expressed during all stages of mosquito development in Anopheles stephensi except for the embryo stage. Although it needs to further test the determining function of arrestin 1, based on the similarity of sequences (cDNA and amino acid) and expression pattern of this gene between Anopheles gambiae and Anopheles gambiae, it seems that it has the same function in Anopheles stephensi.

Introduction

Malaria is a main public health problem in third world countries and is the top ranked priority tropical disease of the World Health Organization (Hay and Snow 2006). Despite of the global effort to eradicate malaria disease by different methods from chemical to DNA vaccine, it remains a significant epidemic that is responsible for hundreds of thousands of deaths every year. Therefore, finding new targets and novel methods in controlling this disease should be considered at priority of new research.

Olfaction and visual play is a crucial role in most insect behaviors among mosquito vectors and other insects (Zwiebel and Takken 2004). Several studies have shown that arrestins are key elements in both of visual and olfactory processes (Pippig et al., 1993, Mombaerts, 1999, Pilpel and Lancet, 1999, Tian et al., 2012).

In the olfaction process, joining transmembrane-domain G-protein-coupled receptors (GPCRs) and heterotrimeric G proteins leads to the activation of downstream effector enzymes, producing several messengers to induce the depolarization or hyperpolarization of olfactory neurons, leading to start an olfaction process (Pilpel et al., 1998, Mombaerts, 1999). For interruption olfactory process, coupling between GPCRs and heterotrimeric G proteins should be separated. Desensitization of GPCRs by arrestins separates coupling between GPCRs and heterotrimeric G proteins, leading to an interrupted visual process (Pippig et al. 1993).

In the visual process, the coupling between rhodopsin, light-sensitive receptor protein, and a heterotrimeric G-protein leads to activation of reactions, leading to visual process. To terminate visual process, visual arrestins are coupled to phosphorylated rhodopsin and separate it from reactions.

Several arrestins, including arr1, arr2 and Krz, have been characterized in insects, especially in Drosophila melanogaster (Krishnan and Ganguly, 1990, Yamada et al., 1990, Roman et al., 2000, Merrill et al., 2003, Manzini and Schild, 2010). Insects have two visual arrestins (arrestin 1 and arrestin 2) and one non-visual arrestin (Krz) (Gurevich and Gurevich 2006). In Drosophila melanogaster arr1 is considered as a minor arrestin due to low expression level compared with arr2 gene, involving in visual signaling process. Also, arrestin 1 from Drosophila melanogaster recognizes phosphorylated rhodopsin and several regulating rhodopsin level of the membrane (Dolph et al., 1993, Satoh and Ready, 2005, Shieh et al., 2014). One of the arrestins in the visual process of Anopheles gambiae and Drosophila melanogaster is arr1 (Merrill et al. 2002). Arrestin 1 recognizes phosphorylated rhodopsin and several regulating rhodopsin level of the membrane in Drosophila melanogaster and Anopheles gambiae (Dolph et al., 1993, Satoh and Ready, 2005, Shieh et al., 2014). Despite the importance of arr1 gene in visual and olfactory processes of mosquito vectors, this gene and its protein have not yet been characterized in Anopheles stephensi (main malaria vector in Iran, Indian subcontinent and China). In this study, we identified the full cDNA of arr1As, characterized it at bioinformatics level, and evaluated expression levels of this gene both in male and female mosquitoes at different stages of mosquito development. Accordingly, although it needs further biological tests, with regard to high similarity in sequences (nucleotide and protein) and expression pattern of arr1As and other insects, especially Anopheles gambiae, it may be concluded that this gene has the same function in visual and olfactory processes as Anopheles gambiae.

Section snippets

Sample collection

The Anopheles adult were collected using an aspirator from the malaria endemic areas of Iran, including Iranshahr, Chabahar, Khash, Zabol, Zahedan, Sarbaz, Saravan, and Nikshahr districts that are located in Sistan and Baluchistan province, south-east of Iran (Fig. 1).

Morphological and molecular identification

First, morphological studies were carried out based on identification key for the female Anopheles of Southwestern Asia and Egypt as previously described (Glick 1992). In the next step, molecular identification of Anopheles was

Result

Total RNA was extracted from Anopheles stephensi, and then cDNA was synthesized by random primer. As previous study has shown, A. stephensi and A. gambiae have high genetic similarity (Jiang et al. 2014). Accordingly, in the first step to identify full cDNA of arr1As, the CDNA of arr1Ag (GenBank accession no. XM_319289.3) was blasted in Vector-Base database to find similar sequences of arr1As. After analysis, it was found that nucleotides 14041–16210 of accession number (KB664461) from A.

Discussion

In this study full cDNA of arr1As was identified by using RACE technique. The identified nucleotides reported to Gene bank (accession number KX015963) as full length of cDNA arr1As. Results of blast showed that identified sequences have 90% and 92% identity similarity with arr1Ag at nucleotide and amino acids levels, respectively. Additionally, it has 73% and 68% similarity at nucleotides and protein with arr1 from Drosophila melanogaster, charactering as key protein in photoreceptors during

Acknowledgments

We wish to thank Iranian CDC, Malaria Control Program, especially Dr. A. Raeisi for his kind support in the coordination for field work and Public Health Department in Zahedan University of Medical Sciences for their kind hospitality in the sample collection.

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