Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights

Zeeshan Zafar; Sidra Fatima; Muhammad Faraz Bhatti; Farooq A Shah; Zack Saud; Tariq M Butt

doi:10.1371/journal.pone.0265896

. 2022 Mar 22;17(3):e0265896. doi: 10.1371/journal.pone.0265896

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights

Zeeshan Zafar ¹, Sidra Fatima ¹, Muhammad Faraz Bhatti ^1,^*, Farooq A Shah ², Zack Saud ³, Tariq M Butt ^3,^*

Editor: Joseph Clifton Dickens⁴

PMCID: PMC8939812 PMID: 35316281

Abstract

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti’s. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species’ OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.

Introduction

Anopheles stephensi, is vector of several Plasmodium species causing malaria [1,2]. Despite advances in prophylactic treatments, malaria continues to impose heavy burdens on global healthcare systems. An. stephensi has a wide geographic distribution throughout the South Asia, the Middle East and recently East Africa [3–7]. An. stephensi predominately transmits malaria in urban areas. Thus, control of An. stephensi can significantly reduce the malaria transmission. Olfaction mechanisms are central to insects including An. stephensi for host-seeking, oviposition and feeding [8,9]. Olfactory sensing mechanisms are located within the antennal sensillum of the An. stephensi [10,11]. Excitatory behaviour modifying compounds or semiochemicals enter sensilla through pores and cross the sensillum lymph with the aid of odorant binding proteins (OBPs) to induce olfactory sensing in insects [12].

The odorant binding proteins (OBPs) are globular in nature and specifically transport semiochemicals through sensillum lymph to odorant receptors (ORs) that ultimately send electrical signals to the insect brain [13,14]. OBPs also have an additional role of protecting the odorants from odorant degrading enzymes [15]. In Antheraea polyphemus (polyphemus moth), first OBP was identified 40 years ago [16]. Binding of semiochemicals to OBPs is pH dependent wherein binding takes place at pH6.5 whereas bound chemicals are released from OBPs near ORs where the pH measures around 4.5 [17–20].

The number of OBPs varies between insect species, with OBPs being highly expressed in sensillum lymph [21–23]. The number of OBPs identified in Drosophila melanogaster, Aedes aegypti and Anopheles gambiae are 51, 66 and 57, respectively [24–27]. There are several classes of OBPs: classic, atypical, plus-C, and minus-C OBPs [21]. Classification of OBPs is based on the presence of conserved cysteine residues that are considered as motifs. Six conserved cysteines are present in the classic OBPs. In each class, cysteine 2 (C2) and cysteine 3 (C3), being three amino acid residues apart, are conserved. Similarly, cysteine 5 (C5) and cysteine (C6) are conserved having eight amino acid residues between them. However, number of amino acid residues varies between C1-C2, C3-C4 and C4-C5. Classic and atypical OBPs contain same number of conserved cysteines but the number of amino acids between conserved cysteine residues vary. However, plus-C OBPs contain the two extra cysteines 4a and 6a along with a conserved proline immediately after the C6. Most of insect OBPs belong to classic group of OBPs [28]. In An. stephensi, two OBPs and one OR that is OR8 have been identified [29–31].

Odorant receptors (ORs) are transmembrane proteins containing seven helices like G-protein coupled receptors (GPCRs). ORs have been previously identified based on in situ hybridization using RNA probes and transcriptomic data from different organisms [32]. Similarly, ORs have also been identified by analysing the genomic data as in An. gambiae [33]. The number of ORs varies with the organisms. ORs are coupled with the Odorant Receptor co-receptors (Orco). Orco are highly conserved across all the insect species having been originally named as OR83b in D. melanogaster [34]. Insect olfaction relies on the interplay of OBPs that carry odorant molecules to the ORs and Orco, which results in electrical signals being sent to the insect brain.

In this study, genome wide analysis of the An. stephensi OBPs was performed to identify and classify the odorant binding proteins (OBPs) using the custom motifs and odorant receptors (ORs). Phylogenetic analyses were conducted to establish the evolutionary relationship of the OBPs and ORs with the closely related organisms Ae. aegypti, An. gambiae, and D. melanogaster. Further, we investigated synteny between the OBPs and ORs to identify the syntenic regions between these insects. This study has analyzed the gene structure and predicted the physicochemical properties and subcellular localization of the OBPs and ORs as well for confirmation of the OBPs and ORs. Being a vital component of insect olfaction, OBPs and ORs of An. stephensi play crucial roles in the mosquito life cycle and therefore, are likely to have a major influence on the potential for the mosquito to transmit malarial diseases. This study serves as the basis for the structural and functional characterization of the OBPs and ORs of An. stephensi. Identified OBPs and ORs will help in understanding the pathways sensitive to attractants and repellents in the olfactory mechanism and vector control strategies. It provides the foundation for comparative studies based on olfactory mechanisms in other insect species as well.

Methodology

The methodology is summarized as a flow chart shown in Fig 1.

Identification of the OBPs and ORs

Protein FASTA file of the Anopheles stephensi Indian strain, sequenced by UC Irvine, was downloaded from the NCBI database (Refseq: GCF_013141755.1). Retrieved protein FASTA file was used as a protein database in BLAST+ software for local BLASTp using the OBP sequences of Ae. aegypti, An. gambiae and D. melanogaster as query sequences. Custom motifs were designed on earlier studies according to the ScanProsite input format. These motifs are based on the conserved cysteine residues present in three classes of OBPs: classic [Cx(15,39)Cx(3)Cx(21,44)Cx(7,12)Cx(8)C], atypical [Cx(26,27)Cx(3)Cx(36,38)Cx(11,15)Cx(8)C], and plus-C [Cx(8,41)Cx(3)Cx(39,47)Cx(17,29)Cx(9)Cx(8)CPx(9,11)C]. BLASTp output was used to detect the presence of these motifs using ScanProsite (https://prosite.expasy.org/scanprosite/) to extract OBPs sequences based on their classification [35]. The retrieved sequences were further analyzed to detect the presence of the PBP/GOBP domain (Pfam: PF01395) using Pfam web server (http://pfam.xfam.org/). Pfam is a large collection of the protein families and domains [36]. Retrieved OBPs sequences were used to perform a BLASTp search against protein database of An. stephensi to search for the missing OBPs sequences in. OBPs were named according to their position on the chromosomes as AsteOBP. Positions of the OBP encoding genes on An. stephensi chromosomes were visualized using phenogram tool (http://visualization.ritchielab.org/phenograms/plot) [37]. Whereas open reading frame (ORF) were predicted using the ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/).

Like OBPs, OR sequences of the Ae. aegypti, An. gambiae and D. melanogaster were used as the query sequences for the local BLASTp [38]. The protein FASTA file of An. stephensi was used as the database. BLASTp output sequences were further checked for the presence of the 7tm_6 domain (Pfam ID: PF02949) using the Pfam web server. Further, redundant sequences having 100% sequence identity were removed. Similarly, positions of OR encoding genes on An. stephensi chromosomes were visualized using phenogram.

Multiple sequence alignment and phylogenetic tree analyses

Multiple sequence alignment for the OBPs was done using Clustal-W in Mega X-V10.2 with gap opening penalty 10. The gap extension penalty was set to 0.1 for pairwise alignment and 0.2 for the multiple sequence alignment [39]. Multiple sequence alignment was performed separately for the classic, atypical and plus-C OBPs to visualize the motif regions in each OBP subtype. Multiple sequence alignments were visualized using Jalview [40].

Multiple sequence alignments of the OBPs of the An. stephensi, An. gambiae, Ae. aegypti and D. melanogaster were performed using the Clustal-W on the Galaxy server (https://usegalaxy.org/). This alignment was used for the generation of the Maximum-Likelihood tree using the FastTree 2 on the galaxy webserver [41]. Jones-Taylor-Thornton 1992 model (JTT-Model) was used as evolutionary model that uses protein sequences for the faster generation of mutation metrices of proteins. The tree was further modified using the iTOL web server [42]. Similarly, Clustal W and FastTree 2 were used for the construction of phylogenetic tree of the Odorant Receptors (ORs).

Synteny prediction of the OBPs and ORs

Synteny analysis was performed using TBTools [43]. TBTools uses MCScanX to find the syntenic regions between the chromosomes of two organisms [44]. MCScanX is an algorithm that is used to scan the multiple genomes and identify putative homologous chromosomal regions by aligning those using genes as anchors. OBPs of the An. stephensi were analyzed against the genome of An. gambiae (Genbank: GCA_000005575.1), Ae. aegypti (Genbank: GCA_002204515.1) and D. melanogaster (Genbank: GCA_000001215.4) to identify the collinear blocks between their genomes. Likewise, syntenic regions were also predicted in the ORs of An. stephensi with the ORs of An. gambiae, Ae. aegypti and D. melanogaster.

Gene structure analysis of OBPs and ORs

Gene structures of the OBPs and ORs were visualized using the TBTools [43]. A Genome Feature File (GFF) file of the genomic sequences of An. stephensi was used for the identification of the Untranslated Regions (UTRs) and Coding DNA Sequences (CDS) in OBPs and ORs genes. MEME web server (https://meme-suite.org/meme/tools/meme) was used for the presence of conserved motifs in the peptides sequences of the OBPs and ORs [45]. Conserved Domain Database (CDD) was used to visualize the conserved Pfam domains in peptide sequences of OBPs and ORs [46]. Phylogenetic tree of OBPs and ORs of An. stephensi was constructed using the Neighbor Joining Method in Mega X-V10.2 for the use of cladogram in the gene structure representation.

Physiochemical properties and sub-cellular localization prediction of OBPs and ORs

Physiochemical properties of the OBPs and ORs including molecular weight and isoelectric points were predicted using ProtParam in the Expasy webserver (https://web.expasy.org/protparam/). Sub-cellular localization of the OBPs and ORs was predicted using the WoLF PSORT (https://wolfpsort.hgc.jp/) and CELLO web server (http://cello.life.nctu.edu.tw/cgi/main.cgi).

Results

Identification of OBPs and ORs

A total 44 OBPs were identified in the An. stephensi having complete conserved motifs. There were 27 classic, 10 atypical, and 7 plus-C OBPs as their NCBI peptide accessions and gene IDs are represented in the Table 1. Genomic and protein sequences of putative OBPs are given in S1 and S2 Data, respectively. Similarly, ORF length and number of amino acids in the OBPs have been provided in the S1 Table.

Table 1. Classification and NCBI accessions of identified OBPs.

OBP	Protein Accession	Gene ID	Classes of OBPs	Chromosome	Molecular weight	Isoelectric Point
AsteOBP1	XP_035917841.1	LOC118504557	Classic	X	18669	4.7
AsteOBP2	XP_035891203.1	LOC118504570	Classic	X	16593	6.5
AsteOBP3	XP_035891204.1	LOC118510310	Classic	X	16564	5.6
AsteOBP4	XP_035891207.1	LOC118510318	Classic	X	16813	5.6
AsteOBP5	XP_035891740.1	LOC118510325	Classic	X	15898	4.2
AsteOBP6	XP_035891741.1	LOC118510331	Classic	X	14940	6.2
AsteOBP7	XP_035892015.1	LOC118502745	Classic	2	15105	8.4
AsteOBP8	XP_035892546.1	LOC118502746	Atypical	2	50208	8.2
AsteOBP9	XP_035892547.1	LOC118502748	Atypical	2	39006	5.1
AsteOBP10	XP_035893080.1	LOC118503023	Classic	2	17788	4.9
AsteOBP11	XP_035894183.1	LOC118503024	Plus-C	2	22832	4.7
AsteOBP12	XP_035894335.1	LOC118503161	Classic	2	15761	4.3
AsteOBP13	XP_035894381.1	LOC118503418	Classic	2	16437	6.5
AsteOBP14	XP_035895118.1	LOC118503419	Atypical	2	31279	8.4
AsteOBP15	XP_035895684.1	LOC118503662	Classic	2	15844	6.3
AsteOBP16	XP_035895818.1	LOC118504157	Classic	2	17738	8.9
AsteOBP17	XP_035895821.1	LOC118504227	Classic	2	15028	4.8
AsteOBP18	XP_035897326.1	LOC118504259	Atypical	2	24233	6.6
AsteOBP19	XP_035897725.1	LOC118504578	Classic	2	16412	5.2
AsteOBP20	XP_035898485.1	LOC118504803	Classic	2	16227	8.1
AsteOBP21	XP_035898834.1	LOC118504874	Classic	2	19676	8.9
AsteOBP22	XP_035898836.1	LOC118504876	Classic	2	16709	8.7
AsteOBP23	XP_035899548.1	LOC118505522	Classic	2	17444	5.8
AsteOBP24	XP_035900970.1	LOC118505693	Classic	2	13982	8.4
AsteOBP25	XP_035902367.1	LOC118506007	Classic	2	16094	4.8
AsteOBP26	XP_035903187.1	LOC118506171	Classic	2	14920	4.7
AsteOBP27	XP_035903789.1	LOC118506172	Classic	2	18610	4.6
AsteOBP28	XP_035905778.1	LOC118506477	Classic	2	17744	9.3
AsteOBP29	XP_035907589.1	LOC118507089	Plus-C	2	21790	8.5
AsteOBP30	XP_035907591.1	LOC118507680	Plus-C	2	21161	5.9
AsteOBP31	XP_035907600.1	LOC118507965	Plus-C	2	22467	8.8
AsteOBP32	XP_035908250.1	LOC118508265	Plus-C	2	23618	7.5
AsteOBP33	XP_035908252.1	LOC118509362	Plus-C	3	19688	4.9
AsteOBP34	XP_035908253.1	LOC118510176	Plus-C	3	19627	4.9
AsteOBP35	XP_035909003.1	LOC118510178	Atypical	3	36926	6
AsteOBP36	XP_035910841.1	LOC118510185	Classic	3	17618	8.4
AsteOBP37	XP_035914753.1	LOC118510478	Atypical	3	31775	5.3
AsteOBP38	XP_035917598.1	LOC118510479	Classic	3	15299	4.8
AsteOBP39	XP_035895071.1	LOC118510480	Classic	3	20736	5.2
AsteOBP40	XP_035895083.1	LOC118510798	Classic	3	15612	5.3
AsteOBP41	XP_035907843.1	LOC118511635	Atypical	3	35623	5.8
AsteOBP42	XP_035907857.1	LOC118513288	Atypical	3	33435	5.2
AsteOBP43	XP_035907868.1	LOC118514663	Atypical	3	33188	5.2
AsteOBP44	XP_035907882.1	LOC118515263	Atypical	Unknown	31914	5.4

Open in a new tab

Classification of the identified odorant binding proteins (OBPs) along with their NCBI peptide accession and gene IDs are presented in the table. Molecular weight and isoelectric point of OBPs are also given.

Further, putative ORs identified in the genome of the An. stephensi were 45. These genes were renamed according to their respective position on the chromosomes of An. stephensi. The name, NCBI protein accessions and gene IDs of the sequences are presented in the Table 2. Genomic and peptide sequences of putative ORs are given in S3 and S4 Data, respectively. Similarly, ORF length and number of amino acids in the ORs have been provided in the S2 Table.

Table 2. Renaming of odorant receptor (ORs) and chromosome.

ORs	NCBI Protein Accession	NCBI Gene ID	Chromosome	Molecular weight	Isoelectric Point
AsteOR1	XP_035892088.1	LOC118502993	1	47.484	9.49
AsteOR2	XP_035917717.1	LOC118514681	1	47.46	9.51
AsteOR3	XP_035900263.1	LOC118506771	1	11.761	9.51
AsteOR4	XP_035918152.1	LOC118516006	1	29.498	8.69
AsteOR5	XP_035893454.1	LOC118503850	2	50.216	9.17
AsteOR6	XP_035893486.1	LOC118503863	2	30.798	7
AsteOR7	XP_035901553.1	LOC118507296	2	45.165	8.73
AsteOR8	XP_035899159.1	LOC118506317	2	97.083	9.08
AsteOR9	XP_035898901.1	LOC118506200	2	49.355	8.23
AsteOR10	XP_035898518.1	LOC118506022	2	45.771	8.7
AsteOR11	XP_035901800.1	LOC118507433	2	46.713	6.22
AsteOR12	XP_035903051.1	LOC118507903	2	46.521	6.55
AsteOR13	XP_035891168.1	LOC118502720	2	46.096	5.9
AsteOR14	XP_035899865.1	LOC118506605	2	52.647	8.83
AsteOR15	XP_035893072.1	LOC118503656	2	39.869	9.08
AsteOR16	XP_035893066.1	LOC118503653	2	47.581	9.26
AsteOR17	XP_035895309.1	LOC118504650	2	14.085	6.7
AsteOR18	XP_035890596.1	LOC118502471	2	43.074	7.17
AsteOR19	XP_035890597.1	LOC118502472	2	48.388	8.52
AsteOR20	XP_035897380.1	LOC118505546	2	43.686	7.6
AsteOR21	XP_035890600.1	LOC118502475	2	51.037	9.41
AsteOR22	XP_035903785.1	LOC118508262	2	53.873	8.11
AsteOR23	XP_035903780.1	LOC118508261	2	55.875	8.31
AsteOR24	XP_035890608.1	LOC118502484	2	46.469	8.91
AsteOR25	XP_035896260.1	LOC118505078	2	46.133	7.55
AsteOR26	XP_035914390.1	LOC118513096	3	46.464	9.36
AsteOR27	XP_035913964.1	LOC118512920	3	48.938	6.37
AsteOR28	XP_035910189.1	LOC118511343	3	53.323	6.59
AsteOR29	XP_035904929.1	LOC118509018	3	42.07	8.72
AsteOR30	XP_035904961.1	LOC118509031	3	43.592	9.29
AsteOR31	XP_035904965.1	LOC118509034	3	43.903	5.75
AsteOR32	XP_035907269.1	LOC118509993	3	44.707	8.63
AsteOR33	XP_035907274.1	LOC118509997	3	17.289	4.97
AsteOR34	XP_035907276.1	LOC118509999	3	43.583	5.65
AsteOR35	XP_035904780.1	LOC118508959	3	10.478	8.73
AsteOR36	XP_035907277.1	LOC118510000	3	32.545	6.1
AsteOR37	XP_035910820.1	LOC118511624	3	48.887	8.47
AsteOR38	XP_035908757.1	LOC118510691	3	43.658	8.99
AsteOR39	XP_035908756.1	LOC118510690	3	43.438	8.56
AsteOR40	XP_035908759.1	LOC118510692	3	43.242	9.03
AsteOR41	XP_035908990.1	LOC118510790	3	15.858	5.27
AsteOR42	XP_035907332.1	LOC118510050	3	32.201	8.45
AsteOR43	XP_035907338.1	LOC118510056	3	45.4	9.52
AsteOR44	XP_035916306.1	LOC118513983	3	33.276	5.81
AsteOR45	XP_035919246.1	LOC118517353	Unknown	31.278	6.55

Open in a new tab

Identified odorant receptors (ORs) along with their NCBI peptide accession, chromosome and gene IDs are presented in the table. Molecular weight and isoelectric points of ORs are also given.

Chromosomal location of the OBPs and ORs

Chromosomal location of the OBPs was depicted using the Phenogram web server as shown in the Fig 2. The highest numbers of the OBPs were present on chromosome 2 containing 26 OBPs, whereas chromosome X and chromosome 3 contained 6 and 11 OBPs, respectively. Eight OBPs, starting from AsteOBP7 to AsteOBP14, were clustered on chromosome 2. Whilst on chromosome X, AsteOBP3, AsteOBP4, AsteOBP5 and AsteOBP6 were clustered together. Similarly, AsteOBP33-AsteOBP35 and AsteOBP36-AsteOBP38 were clustered on the chromosome 3. Chromosome X only contained the classic OBPs whereas chromosome 2 and 3 contained all three types. AsteOBP44 gene had not been placed on the chromosomes to date due to limitations of genomic assembly.

ORs were clustered on chromosome 2 of An. stephensi as there were 21 ORs’ gene located on it as represented in Fig 3. Chromosome X contained the least OR gene as it contained only 4 genes. Chromosome 3 contained 19 OR genes. Most of the OR genes were scattered except a few that were clustered together. AsteOR2-AsteOR4 were clustered together on the chromosome X. Similarly, some genes were clustered in pairs at the chromosome 2. But on chromosome 3, only three clusters of the OR genes were present where each cluster had three OR genes. AsteOR45 was present on a scaffold that was not placed on the chromosomes the genome assembly.

Multiple sequence alignment and phylogenetic analysis of the OBPs and ORs

Alignments are presented in Fig 4. Clear motifs patterns depicting the conserved cysteines were visible in all three types of OBPs. Phylogenetic tree was constructed using the maximum-likelihood method. OBP sequences of the An. stephensi, An. gambiae, D. melanogaster, and Ae. aegypti were used for the construction of phylogenetic tree in Mega-X as shown in Fig 5. Most of the OBPs showed close relationship with OBPs of An. gambiae but, none of the OBPs showed close relationship with the OBPs of D. melanogaster. Similarly, only AsteOBP1, AsteOBP5, AsteOBP21, AsteOBP27, and AsteOBP31 were closely related to the AaegOBP20, AaegOBP15, AaegOBP4, AaegOBP59 and AaegOBP5 of the Ae. aegypti, respectively. It confirms the close relationship of the An. stephensi with the An. gambiae whereas D. melanogaster was distantly rooted in the phylogenetic tree among the compared organisms.

Fig 5 — Phylogenetic analysis of the OBPs of *Anopheles stephensi*, *Anopheles gambiae*, *Aedes aegypti* and *Drosophila melanogaster* were carried out using FastTree 2. An. *stephensi* OBPs showed closer relationship to An. *gambiae* OBPs. The bootstrap values have been represented in the figure.

ORs showed close phylogenetic relationship with the ORs of An. gambiae as represented in Fig 6. However, only two AsteORs: AaegOR28 and AaegOR23 were closely related to AaegORs: AaegOR5 and AaegOR7, respectively. None of the An. stephensi ORs were closely rooted to the D. melanogaster ORs.

Synteny analysis of OBPs and ORs

Synteny analysis was performed between the An. stephensi and Ae. aegypti. Total 11 OBPs of An. stephensi had the syntenic region with the OBPs of the Ae. aegypti as shown in the Fig 7A. OBPs present on the chromosome X of the An. stephensi and Ae. aegypti did not share any orthologues. Syntenic regions were present between the OBPs of the chromosome 2 of An. stephensi and the OBPs present on the chromosome 3 of the Ae. aegypti whereas chromosome 3 OBPs of An. stephensi contained collinear with the OBPs of chromosome 2 of Ae. aegypti.

Synteny analysis were also performed between the An. stephensi and An. gambiae genome as represented in Fig 7B. A total of 29 OBPs of the An. stephensi contained the collinear blocks with the OBPs of An. gambiae. Four syntenic regions were present between the chromosome X of both organisms. Whereas others were distributed between chromosome 2 and chromosome 3 of the An. stephensi and chromosome 2L, 2R, 3L and 3R of the An. gambiae. In comparison to An. gambiae OBPs, OBPs of Ae. aegypti had less synteny with the OBPs of An. stephensi.

Syntenic analyses of the ORs of An. stephensi and Ae. aegypti are shown in Fig 8A. A total of 12 ORs of An. stephensi shared syntenic regions with 13 OR genes of the Ae. aegypti. Only one OR2 gene present on the chromosome X of An. stephensi showed homologous region with the OR41 of the Ae. aegypti. OR genes present on the chromosome 2 of An. stephensi showed close homology with the ORs present on the Chromosome X and 3 of Ae. aegypti. However, OR15 on chromosome 2 shared homology with the two ORs: OR8 and OR37 of Ae. aegypti. Whereas OR genes present on the chromosome 3 of An. stephensi were homologous to ORs of chromosome 2 of Ae. aegypti.

Synteny analysis of An. stephensi with An. gambiae ORs is shown in Fig 8B. A total of 32 ORs of An. stephensi shared homology with 34 ORs of An. gambiae. OR1 gene present on the chromosome X of An. stephensi showed homology with the OR36 and OR52 genes present on the chromosome X of An. gambiae. However, ORs present on the chromosome 2 of the An. stephensi were homologous to the ORs present on chromosome 2R and 3L of An. gambiae. But ORs present on the chromosome 3 of the An. stephensi shared syntenic region with the ORs of chromosome 2L and 3R in An. gambiae. None of the An. stephensi OBPs and ORs shared homology with the OBPs and ORs of D. melanogaster.

Gene structure analysis of OBPs and ORs

Gene structure analysis of the OBPs was carried out using the TBTool as depicted in the Fig 9. Conserved motifs present in the OBPs were identified using the MEME web server. Motif 1 was present in all the OBPs. Whereas motifs 2, 3 and 4 were present in the most of the OBPs. Conserved domains were identified using the Conserved Domain Database (CDD). CDD results indicated OBPs 11, 30, 31, 32, 33 and 34 had insignificant similarity with the Pfam PBP/GOBP domain but these were Plus-C OBPs having complete motifs. Whereas all other OBPs contained the PBP/GOBP domains.

Gene structure of the ORs of An. stephensi has been represented in the Fig 10. 7tm_6 domain was present in all the sequences. CDS were present in the sequences whereas some sequences lacked UTRs. Motif 2 and Motif 3, predicted by MEME, were present in most of the sequences except OR36. Whereas motif 1, 4, and 5 were present in few sequences.

Prediction of physicochemical properties and subcellular localization of the OBPs and ORs

The molecular weight of the atypical OBPs varied from the 24.2 kDa to 39.0 kDa except for the OBP8 that had 50.2 kDa as presented in Table 1. In classic OBPs, molecular weight ranged between the 14 kDa to 20.7 kDa. Plus-C OBPs had the molecular weight in the range of 19.7–23.6 kDa. Isoelectric point, a pH at which molecule is neutral, ranged from 4.2 to 8.9 for all the OBPs as given in Table 1.

The molecular weight of the ORs varied from 30.798 kDa to 97.083 kDa for OR6 and OR8 respectively as shown in Table 2. However, some sequences that had partial 7tm_6 (Odorant Receptor Domain) had less molecular weight. Isoelectric point ranged between the 4.97 and 9.52 for OR33 and OR43, respectively. Isoelectric points of the OBPs and ORs in relation to the molecular weight are shown in the Fig 11.

Fig 11 — Molecular weight and isoelectric point of the OBPs and ORs are shown as these are important indicators of the OBPs and ORs functions. Atypical OBPs had highest molecular weight among other classes whereas ORs have higher molecular weight than OBPs.

CELLO and WoLF PSORT predicted extra-cellular localization of the OBPs indicating that OBPs are secretory proteins whereas ORs were predicted to be membrane embedded that proved their transmembrane propensity.

Discussion

In An. stephensi, 44 OBPs are identified. There are 27 classic, 10 atypical and 7 plus-C OBPs. Similarly, a total of 66 OBP encoding genes were identified in the Ae. aegypti, 66 in An. gambiae, and 49 in D. melanogaster [47–49]. Like An. stephensi, classic OBPs are abundant than atypical and plus-C OBPs in other insect species including Ae. aegypti and An. gambiae as well [14,50,51]. There were total 41, 29, and 30 classic OBPs identified in Ae. aegypti, An. gambiae, and D. melanogaster respectively [25,26,52]. Atypical OBPs are more abundant than the plus-C OBPs in insects. In Ae. aegypti, there were more plus-C OBPs than the atypical contradicting with other insects. There were 10, 16 and 12 atypical OBPs identified in Ae. aegypti, An. gambiae, and D. melanogaster, respectively. However, plus-C OBPs were 16, 12 and 12 in Ae. aegypti, An. gambiae, and D. melanogaster, respectively. Odorant Receptors (ORs) also vary in their number between insect species. A total of 45 ORs have been identified in the An. stephensi. Like An. stephensi, the numbers of identified ORs were 75 in An. gambiae, 61 in D. melanogaster, and 131 in Ae. aegypti [33,53,54]. However, a total of 226 ORs were identified in Aenasius bambawalei belonging to order Hymenoptera [55]. Some species can contain less ORs, for instance, only 8 ORs have been identified in Tomicus yunnanensis that belongs to order Coleoptera [56].

Most of the OBP genes are localized on chromosome 2 of An. stephensi. Whereas Chromosome X contains six OBP genes. Similarly, the least number of OBP genes were found on chromosome X whereas most were present on chromosome 3L in An. gambiae [52]. Clusters of OBP encoding genes are present on all chromosomes whereas some of these genes are scattered all over the chromosomes in An. stephensi. A similar pattern of clustering has been reported for D. melanogaster that have four clusters of OBPs genes [25]. Like OBPs, ORs are also abundant on chromosome 2 whereas chromosome X contains only 4 OR genes in An. stephensi. This indicates a pattern that odorant genes are abundant on the chromosome 2 whereas chromosome X contains only few odorant genes in the insects.

Phylogenetic analysis showed a close relationship of the OBPs and ORs of An. stephensi with An. gambiae as both are closely related organisms. Whereas few OBPs and ORs of An. stephensi showed relationship with the OBPs and ORs of Ae. aegypti. However, OBPs and ORs were distantly related to the OBPs and ORs of the D. melanogaster among compared organisms. Because An. stephensi shares same genus with An. gambiae whereas Ae. aegypti belong to different genus. However, D. melanogaster belonged to different family as compared to An. stephensi. Like Anopheles stephensi, OBPs and ORs of the Ae. aegypti also showed close relationship with An. gambiae OBPs and ORs because of the closer relationship between both organism than the distantly related D. melanogaster [26]. Likewise, Culex quinquefasciatus OBPs and ORs showed the closer relationship with OBPs and ORs of Ae. aegypti as compared to D. melanogaster [57]. Syntenic analysis showed more homologous OBPs and ORs between An. stephensi and An. gambiae genomes. Whereas less syntenic regions of An. stephensi OBPs and ORs are predicted with the OBPs and ORs of Ae. aegypti. None of the OBPs and ORs of An. stephensi shared homology with the D. melanogaster OBPs and ORs, syntenic analysis revealed. Similarly, in Ae. aegypti, OBPs were more related to An. gambiae than D. melanogaster. It would have been interesting to compare An. stephensi, a hematophagous species, with a non-hematophagous mosquito species but genome assemblies of the later, such as like Toxorhynchites splendens are currently yet to be sequenced.

PBP/GOBP domain (Pfam ID: PF01395) is conserved in all the OBPs despite of their subclasses. Like OBPs of other insects, classic and atypical OBPs of An. stephensi had significant similarity with PBP/GOBP domain and conserved motifs as well. Whereas plus-C OBPs had insignificant similarity to PBP/GOBP domain even though conserved plus-C OBP motif is present in these OBPs as checked with multiple sequence alignment. Similarly, ORs contain 7tm_6 domain (Pfam ID: PF02949) that is a transmembrane protein domain having 7 helices [54,58].

Physicochemical analysis of the OBPs predicted that atypical OBPs have the highest molecular weight in An. stephensi that is followed by the plus-C and classic OBPs. Similar results have been found in the OBPs of Ae. aegypti and An. gambiae as classic OBPs had molecular weight less than 15.5 kDa [47,49]. Whereas atypical OBPs of Ae. aegypti and An. stephensi had molecular weight between 27 and 38 kDa along with plus-C OBPs having molecular weight between 25–35 kDa [47,49]. However, ORs are high molecular weight proteins as their molecular weight varies from 30 kDa to 97 kDa in An. stephensi. The isoelectric point of the OBPs of dipteran species is found between 4 and 10 as is the case with Ae. aegypti OBPs [26]. Similarly, pI was between 4 and 10 for the An. stephensi OBPs. ORs also had pI ranged between 4 and 10 but it has not reported previously in any insect species.

With advancements in computation biology, new techniques are being devised for the functional role of the OBPs. New attractants and repellents are being discovered on the basis of molecular docking, quantitative structure-activity-relationship (QSAR), and molecular dynamic simulations [59,60]. QSAR has been used extensively for the identification of new repellents and for the discovery of novel attractants [61,62]. Similarly, molecular docking is a fast and reliable method to screen multiple ligands for the identification of OBP-semiochemicals interactions [63]. Molecular simulations helps in the retention of ligand-receptor interactions for an extended period to induce behavioral response, thus helping to predict novel attractants [64,65]. Identification of OBPs is the first step towards the computational biology-based discovery of the novel attractants and repellents. It will not only be cost efficient but also time efficient for the in-silico screening of large chemical libraries. So, the identification of the OBPs and ORs of An. stephensi, given their vital role in olfaction process, will help in further understand mosquito olfactory system. It will help in the identification of novel attractants and repellents to control human malaria vector, An. stephensi.

Conclusion

Odorant Binding Proteins (OBPs) are the first responder in the insect olfactory mechanism delivering the semiochemicals to the Odorant Receptors (ORs) in insects including An. stephensi. Total of the 44 OBPs and 45 ORs have been identified in the An. stephensi by this study. OBPs were further classified into the classic (27), atypical (10), and plus-C OBPs (7) based on the presence of the conserved motifs. Phylogenetic analysis revealed close relationship of OBPs and ORs of An. stephensi with An. gambiae. However, very few OBPs and ORs were related to Ae. aegypti OBPs and ORs but no relationship was established with D. melanogaster OBPs and ORs. Syntenic analysis showed close homology with An. gambiae OBPs. Physicochemical properties predicted the molecular weight and isoelectric point of the OBPs and ORs within previously reported range of OBPs and ORs. This study revealed the OBPs and ORs in the Anopheles stephensi which can be further characterized by NMR and crystallographic studies and help in the identification of novel compounds to control the spread of An. stephensi.

Supporting information

S1 Data. Nucleotide sequences of identified 44 AsteOBP genes in FASTA format.

(TXT)

Click here for additional data file.^{(82KB, txt)}

S2 Data. Protein sequences of identified 44 AsteOBPs in FASTA format.

(TXT)

Click here for additional data file.^{(9KB, txt)}

S3 Data. Nucleotide sequences of identified 45 AsteOR genes in FASTA format.

(TXT)

Click here for additional data file.^{(105.8KB, txt)}

S4 Data. Protein sequences of identified 45 AsteORs in FASTA format.

(TXT)

Click here for additional data file.^{(16.6KB, txt)}

S1 Table. Nucleotide length of the open reading frame (ORF) and protein length in the OBPs have been provided along with their complete and partial status.

ORFs were predicted by ORF Finder. Similarly, OBP’s names and accessions have also been given.

(DOCX)

Click here for additional data file.^{(15.5KB, docx)}

S2 Table. Nucleotide length of the open reading frame (ORF) and protein length in the ORs have been provided along with their complete and partial status.

ORFs were predicted by ORF Finder. Similarly, OR’s names and accessions have also been given.

(DOCX)

Click here for additional data file.^{(16.3KB, docx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Rickman LS, Jones TR, Long GW, Paparello S, Schneider I, Paul CF, et al. Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans. NAVAL MEDICAL RESEARCH INST BETHESDA MD; 1990. [DOI] [PubMed] [Google Scholar]
2.Tadesse FG, Ashine T, Teka H, Esayas E, Messenger LA, Chali W, et al. Anopheles stephensi Mosquitoes as Vectors of Plasmodium vivax and falciparum, Horn of Africa, 2019. 2021;27(2):603. doi: 10.3201/eid2702.200019 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Ghosh SK, Tiwari S, Raghavendra K, Sathyanarayan TS, Dash APJJob. Observations on sporozoite detection in naturally infected sibling species of the Anopheles culicifacies complex and variant of Anopheles stephensi in India. 2008;33(3):333–6. doi: 10.1007/s12038-008-0052-5 [DOI] [PubMed] [Google Scholar]
4.Djadid ND, Gholizadeh S, Aghajari M, Zehi AH, Raeisi A, Zakeri SJAt. Genetic analysis of rDNA-ITS2 and RAPD loci in field populations of the malaria vector, Anopheles stephensi (Diptera: Culicidae): implications for the control program in Iran. 2006;97(1):65–74. doi: 10.1016/j.actatropica.2005.08.003 [DOI] [PubMed] [Google Scholar]
5.Seyfarth M, Khaireh BA, Abdi AA, Bouh SM, Faulde MKJPr. Five years following first detection of Anopheles stephensi (Diptera: Culicidae) in Djibouti, Horn of Africa: populations established—malaria emerging. 2019;118(3):725–32. doi: 10.1007/s00436-019-06213-0 [DOI] [PubMed] [Google Scholar]
6.Faulde MK, Rueda LM, Khaireh BAJAt. First record of the Asian malaria vector Anopheles stephensi and its possible role in the resurgence of malaria in Djibouti, Horn of Africa. 2014;139:39–43. doi: 10.1016/j.actatropica.2014.06.016 [DOI] [PubMed] [Google Scholar]
7.Takken W, Lindsay SJEid. Increased threat of urban malaria from Anopheles stephensi mosquitoes, Africa. 2019;25(7):1431. doi: 10.3201/eid2507.190301 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Gakhar S, Sharma R, Sharma A. Population genetic structure of malaria vector Anopheles stephensi Liston (Diptera: Culicidae). 2013. [PubMed] [Google Scholar]
9.Gholizadeh S, Djadid ND, Nouroozi B, Bekmohammadi MJAt. Molecular phylogenetic analysis of Anopheles and Cellia subgenus anophelines (Diptera: Culicidae) in temperate and tropical regions of Iran. 2013;126(1):63–74. doi: 10.1016/j.actatropica.2012.11.003 [DOI] [PubMed] [Google Scholar]
10.Healy T, Copland M, Cork A, Przyborowska A, Halket JJM, entomology v. Landing responses of Anopheles gambiae elicited by oxocarboxylic acids. 2002;16(2):126–32. doi: 10.1046/j.1365-2915.2002.00353.x [DOI] [PubMed] [Google Scholar]
11.Dekker T, Steib B, Cardé R, Geier MJM, entomology v. L‐lactic acid: a human‐signifying host cue for the anthropophilic mosquito Anopheles gambiae. 2002;16(1):91–8. doi: 10.1046/j.0269-283x.2002.00345.x [DOI] [PubMed] [Google Scholar]
12.Hoffman SA, Aravind L, Velmurugan SJP, vectors. Female Anopheles gambiae antennae: increased transcript accumulation of the mosquito-specific odorant-binding-protein OBP2. 2012;5(1):1–6. doi: 10.1186/1756-3305-5-27 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Sun JS, Xiao S, Carlson JRJRSOB. The diverse small proteins called odorant-binding proteins. 2018;8(12):180208. doi: 10.1098/rsob.180208 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Venthur H, Zhou J-JJFip. Odorant receptors and odorant-binding proteins as insect pest control targets: a comparative analysis. 2018;9:1163. doi: 10.3389/fphys.2018.01163 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Chertemps T, Maïbèche M. Odor degrading enzymes and signal termination. Insect Pheromone Biochemistry and Molecular Biology: Elsevier; 2021. p. 619–44. [Google Scholar]
16.Vogt RG, Riddiford LMJN. Pheromone binding and inactivation by moth antennae. 1981;293(5828):161–3. doi: 10.1038/293161a0 [DOI] [PubMed] [Google Scholar]
17.Mazumder S, Chaudhary BP, Dahal SR, Al-Danoon O, Mohanty SJB. Pheromone Perception: Mechanism of the Reversible Coil–Helix Transition in Antheraea polyphemus Pheromone-Binding Protein 1. 2019;58(45):4530–42. doi: 10.1021/acs.biochem.9b00737 [DOI] [PubMed] [Google Scholar]
18.Wang J, Murphy EJ, Nix JC, Jones DNJSr. Aedes aegypti Odorant Binding Protein 22 selectively binds fatty acids through a conformational change in its C-terminal tail. 2020;10(1):1–15. doi: 10.1038/s41598-020-60242-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Yu G-Q, Li D-Z, Lu Y-L, Wang Y-Q, Kong D-X, Wang M-QJSr. Deciphering the Odorant Binding, Activation, and Discrimination Mechanism of Dhelobp21 from Dastarus Helophoroides. 2018;8(1):1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Damberger FF, Michel E, Ishida Y, Leal WS, Wüthrich KJPotNAoS. Pheromone discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding protein. 2013;110(46):18680–5. doi: 10.1073/pnas.1317706110 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Zhou J-JJV, Hormones. Odorant-binding proteins in insects. 2010;83:241–72. [DOI] [PubMed] [Google Scholar]
22.He P, Zhang J, Liu N-Y, Zhang Y-N, Yang K, Dong S-LJPO. Distinct expression profiles and different functions of odorant binding proteins in Nilaparvata lugens Stål. 2011;6(12):e28921. doi: 10.1371/journal.pone.0028921 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Brito NF, Moreira MF, Melo ACJJoIP. A look inside odorant-binding proteins in insect chemoreception. 2016;95:51–65. doi: 10.1016/j.jinsphys.2016.09.008 [DOI] [PubMed] [Google Scholar]
24.Hekmat-Scafe DS, Scafe CR, McKinney AJ, Tanouye MAJGr. Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster. 2002;12(9):1357–69. doi: 10.1101/gr.239402 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Graham LA, Davies PLJG. The odorant-binding proteins of Drosophila melanogaster: annotation and characterization of a divergent gene family. 2002;292(1–2):43–55. doi: 10.1016/s0378-1119(02)00672-8 [DOI] [PubMed] [Google Scholar]
26.Zhou JJ, He XL, Pickett J, Field LJImb. Identification of odorant‐binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses. 2008;17(2):147–63. doi: 10.1111/j.1365-2583.2007.00789.x [DOI] [PubMed] [Google Scholar]
27.Vogt RGJJoce. Odorant binding protein homologues of the malaria mosquito Anopheles gambiae; possible orthologues of the OS-E and OS-F OBPs of Drosophila melanogaster. 2002;28(11):2371–6. doi: 10.1023/a:1021009311977 [DOI] [PubMed] [Google Scholar]
28.Pelletier J, Leal WSJPo. Genome analysis and expression patterns of odorant-binding proteins from the Southern House mosquito Culex pipiens quinquefasciatus. 2009;4(7):e6237. doi: 10.1371/journal.pone.0006237 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Sengul M, Tu ZJImb. Characterization and expression of the odorant‐binding protein 7 gene in Anopheles stephensi and comparative analysis among five mosquito species. 2008;17(6):631–45. doi: 10.1111/j.1365-2583.2008.00837.x [DOI] [PubMed] [Google Scholar]
30.Sengul MS, Tu ZJImb. Identification and characterization of odorant‐binding protein 1 gene from the Asian malaria mosquito, Anopheles stephensi. 2010;19(1):49–60. doi: 10.1111/j.1365-2583.2009.00929.x [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Speth Z, Kaur G, Mazolewski D, Sisomphou R, Siao DDC, Pooraiiouby R, et al. Characterization of Anopheles stephensi Odorant Receptor 8, an Abundant Component of the Mouthpart Chemosensory Transcriptome. Insects. 2021;12(7):593. doi: 10.3390/insects12070593 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Gao Q, Chess AJG. Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. 1999;60(1):31–9. doi: 10.1006/geno.1999.5894 [DOI] [PubMed] [Google Scholar]
33.Rinker DC, Zhou X, Pitts RJ, Rokas A, Zwiebel LJ. Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae. BMC genomics. 2013;14(1):1–15. doi: 10.1186/1471-2164-14-749 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Vosshall LB, Hansson BSJCs. A unified nomenclature system for the insect olfactory coreceptor. 2011;36(6):497–8. doi: 10.1093/chemse/bjr022 [DOI] [PubMed] [Google Scholar]
35.De Castro E, Sigrist CJ, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. 2006;34(suppl_2):W362–W5. doi: 10.1093/nar/gkl124 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths‐Jones S, et al. The Pfam protein families database. 2004;32(suppl_1):D138–D41. doi: 10.1093/nar/gkh121 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Wolfe D, Dudek S, Ritchie MD, Pendergrass SAJBm. Visualizing genomic information across chromosomes with PhenoGram. 2013;6(1):1–12. doi: 10.1186/1756-0381-6-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Hall T, Biosciences I, Carlsbad CJGBB. BioEdit: an important software for molecular biology. 2011;2(1):60–1. [Google Scholar]
39.Kumar S, Stecher G, Li M, Knyaz C, Tamura KJMb, evolution. MEGA X: molecular evolutionary genetics analysis across computing platforms. 2018;35(6):1547. doi: 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJJB. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. 2009;25(9):1189–91. doi: 10.1093/bioinformatics/btp033 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Price MN, Dehal PS, Arkin APJPo. FastTree 2–approximately maximum-likelihood trees for large alignments. 2010;5(3):e9490. doi: 10.1371/journal.pone.0009490 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Letunic I, Bork PJNar. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. 2019;47(W1):W256–W9. doi: 10.1093/nar/gkz239 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Chen C, Chen H, He Y, Xia RJB. TBtools, a toolkit for biologists integrating various biological data handling tools with a user-friendly interface. 2018:289660. [Google Scholar]
44.Wang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. 2012;40(7):e49–e. doi: 10.1093/nar/gkr1293 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Bailey TL, Johnson J, Grant CE, Noble WSJNar. The MEME suite. 2015;43(W1):W39–W49. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, et al. CDD: a Conserved Domain Database for the functional annotation of proteins. 2010;39(suppl_1):D225–D9. doi: 10.1093/nar/gkq1189 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Xu P, Zwiebel L, Smith D. Identification of a distinct family of genes encoding atypical odorant‐binding proteins in the malaria vector mosquito, Anopheles gambiae. Insect molecular biology. 2003;12(6):549–60. doi: 10.1046/j.1365-2583.2003.00440.x [DOI] [PubMed] [Google Scholar]
48.Hekmat-Scafe DS, Scafe CR, McKinney AJ, Tanouye MA. Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster. Genome research. 2002;12(9):1357–69. doi: 10.1101/gr.239402 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Zhou JJ, He XL, Pickett J, Field L. Identification of odorant‐binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses. Insect molecular biology. 2008;17(2):147–63. doi: 10.1111/j.1365-2583.2007.00789.x [DOI] [PubMed] [Google Scholar]
50.Pelosi P, Maida RJCB, Biochemistry PPB, Biology M. Odorant-binding proteins in insects. 1995;111(3):503–14. doi: 10.1016/0305-0491(95)00019-5 [DOI] [PubMed] [Google Scholar]
51.Pelosi P, Zhu J, Knoll WJS. Odorant-binding proteins as sensing elements for odour monitoring. 2018;18(10):3248. doi: 10.3390/s18103248 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Li ZX, Pickett JA, Field LM, Zhou JJJAoIB, America PPiCwtESo. Identification and expression of odorant‐binding proteins of the malaria‐carrying mosquitoes Anopheles gambiae and Anopheles arabiensis. 2005;58(3):175–89. doi: 10.1002/arch.20047 [DOI] [PubMed] [Google Scholar]
53.Shiao M-S, Fan W-L, Fang S, Lu M-YJ, Kondo R, Li W-H. Transcriptional profiling of adult Drosophila antennae by high-throughput sequencing. Zoological Studies. 2013;52(1):1–10. [Google Scholar]
54.Bohbot J, Pitts R, Kwon HW, Rützler M, Robertson H, Zwiebel LJImb. Molecular characterization of the Aedes aegypti odorant receptor gene family. 2007;16(5):525–37. doi: 10.1111/j.1365-2583.2007.00748.x [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Nie X, Li Q, Xu C, Li D, Zhang Z, Wang MQ, et al. Antennal transcriptome and odorant binding protein expression profiles of an invasive mealybug and its parasitoid. 2018;142(1–2):149–61. [Google Scholar]
56.Liu N-Y, Li Z-B, Zhao N, Song Q-S, Zhu J-Y, Yang BJCB, et al. Identification and characterization of chemosensory gene families in the bark beetle, Tomicus yunnanensis. 2018;25:73–85. doi: 10.1016/j.cbd.2017.11.003 [DOI] [PubMed] [Google Scholar]
57.Manoharan M, Ng Fuk Chong M, Vaïtinadapoulé A, Frumence E, Sowdhamini R, Offmann BJGb, et al. Comparative genomics of odorant binding proteins in Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. 2013;5(1):163–80. doi: 10.1093/gbe/evs131 [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Ray A, Van Naters WVDG, Carlson JRJJob. Molecular determinants of odorant receptor function in insects. 2014;39(4):555–63. doi: 10.1007/s12038-014-9447-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Basak SC, Bhattacharjee AKJCmc. Computational approaches for the design of mosquito repellent chemicals. 2020;27(1):32–41. doi: 10.2174/0929867325666181029165413 [DOI] [PubMed] [Google Scholar]
60.Oliferenko PV, Oliferenko AA, Poda G, Pillai GG, Bernier UR, Agramonte NM, et al. Insect olfactory system as target for computer-aided design of mosquito repellents. Computational Design of Chemicals for the Control of Mosquitoes and their Diseases: CRC Press; 2017. p. 39–64. [Google Scholar]
61.Devillers JJS, Research QiE. 2d and 3d structure–activity modelling of mosquito repellents: A review. 2018;29(9):693–723. doi: 10.1080/1062936X.2018.1513218 [DOI] [PubMed] [Google Scholar]
62.Bhadoriya KS, Sharma MC, Jain SV, Raut GS, Rananaware JRJMCR. Three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis and molecular docking-based combined in silico rational approach to design potent and novel TRPV1 antagonists. 2013;22(5):2312–27. [Google Scholar]
63.Gopal JV, Kannabiran KJISCLS. Studies on interaction of insect repellent compounds with odorant binding receptor proteins by in silico molecular docking approach. 2013;5(4):280–5. doi: 10.1007/s12539-013-0152-2 [DOI] [PubMed] [Google Scholar]
64.Bezerra-Silva PC, Dutra KA, Santos GK, Silva RC, Iulek J, Milet-Pinheiro P, et al. Evaluation of the activity of the essential oil from an ornamental flower against Aedes aegypti: electrophysiology, molecular dynamics and behavioral assays. 2016;11(2):e0150008. doi: 10.1371/journal.pone.0150008 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Gupta KK, Sethi G, Jayaraman MJJovbd. Molecular docking and simulation studies of gustatory receptor of Aedes aegypti: A potent drug target to distract host-seeking behaviour in mosquitoes. 2016;53(2):179. [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0265896.r001

Decision Letter 0

Joseph Clifton Dickens

12 Oct 2021

PONE-D-21-25203Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and Comparative InsightsPLOS ONE

Dear Dr. Bhatti,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

This is an important study as it describes proteins involved in odorant reception in an important malaria vector. However both reviewers suggest ways in which the presentation could be improved including enhancement of the figures, language and an updating of the references. The authors should respond to these comments in an effort to improve their submission.

Please submit your revised manuscript by Nov 26 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Joseph Clifton Dickens

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have compiled and analyzed amino acid sequences corresponding to all potential Odorant Receptors (ORs) and Odorant Receptor Proteins (OBPs) in Anopheles stephensi. They have used these sequences to reveal within species and comparative evolutionary relationships. The main value of this work is to serve as a complete reference for other researchers beginning functional studies related to neuroethology of this important species, which vectors malaria in severely at-risk human populations. Comparative analyses of these sequences shed light on the potential function of A. stephensi genes that may have evolved as adaptations to feeding in urban environments. The main weakness of this manuscript is the lack of context in the introduction and discussion. The datasets are quite useful as presented but need better framing as to their value to other researchers and relation to other anopheline species.

Introduction

The introduction would benefit from a few more statements describing the value of your data to other researchers, namely that it will be useful when embarking on functional characterization of the various olfactory sensitivities of this species or identifying the pathways most sensitive to effective repellents and attractants (as mentioned at the end of the discussion section). This could help identify novel control strategies in the future. Additionally, these genes serve as the basis for comparative studies that could explain the preference of certain subtypes for urban environments. It may be important to specify which sub-species was used in this study, as there are multiple morphological subtypes belonging to A. stephensi.

Methods

The methods are adequately described and well-chosen.

Results

Fig1 font is small relative to size of figure, making it hard to read.

Figures are pretty straightforward and helpful.

Interpretation of results

The first sentence of the discussion seems unnecessary since gene expression is not being measured here and many ORs are expressed at very low levels. Some sections of the discussion read more like extended results, offering little interpretation of the findings in a broader context. To be clear, this is a rich dataset, worthy of publication but in need of more consideration in the context of literature on other anopheline species. For example, the observation described in lines 310-311 is interesting, but there is no discussion of why this is relevant. There are other instances of data with no corresponding discussion of relevance in the discussion, like the paragraph at line 332.

Discussion is needed that compares the number of genes identified here with those found in anopheline species. The number of ORs identified is somewhat less than would be expected based on the number found in A. gambiae. This may be from difficulty of prediction or simply there are less present in this species. Some discussion is needed to qualify the significance of OR gene number in terms of how this gene family’s expansion/retraction may relate to physiology or behavior on an evolutionary scale. The same would be helpful for the OBPs.

Suggested edits by line:

Line 327 – reword sentence

Reviewer #2: The authors present an in-silico descriptive genomic analysis of Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) in the malaria vector mosquito Anopheles stephensi. Report of these gene families in this species is novel aside from a few studies that have characterized a couple of individual genes. The report is strictly an in silico analysis, there is a lack of any “wet lab” experimental validation. The authors provide interspecies comparisons to other dipterans both with respect to gene phylogeny and chromosomal synteny. Within-gene-family motif analysis is also examined for each gene family.

Considering that this is a major malaria vector in some parts of the world, it is surprising that there has not been a comprehensive report on olfactory genes in this species, as has been done for other mosquito disease vectors. So this manuscript would most certainly be worthy of publication, however major revisions to the text and figures are required before this manuscript would be suitable for publication. Many of the figures are low resolution. I am uncertain if this is a consequence of assembly of the images in a PDF, or of low-resolution images were initially provided by the authors, but it is difficult to fully assess especially the OR phylogeny relative to some of the claims made by the authors, so a better resolution image would be needed, as it is now, the text in this figure is too blurry to read.

The manuscript will also need to be thoroughly edited for language after all revisions have been made. There are numerous errors throughout the text. Furthermore, the language is not always clear nor well-structured. Careful revisions with respect to grammar, structure/organization and word choice needs be considered.

Description in the methodology is not always clear. Specifically, in the first section “Identification of the OBPs and ORs”, it is mentioned that proteome fasta files are examined for OBPs and ORs (Lines 96 and 114). Then on Lines 110-112 it is mentioned:

-OBPs were renamed according to their position on the chromosomes. Chromosomal positions of the OBP encoding genes were visualized…-

However, it has never been made clear in the manuscript that an An. stephensi genome has been examined. There is no citation or reference to a genome for this species anywhere, and it is clear that the authors have conducted some studies of these genes in the genome, so this basic information on the source of the genome would have to be clearly indicated in the appropriate section(s). This is also apparent in that in the supplemental materials file, in which nucleotide as well as protein sequences are presented for the ORs and OBPs of An. stephensi, however it is not at all clear where the nucleotide sequences have been derived from, considering that the authors only mention about proteome fasta files, and it is not clear where these proteome files are derived from. It is not always the case that proteomes are directly connected to sequenced genomes.

The authors mention in the introduction about the odorant receptor co-receptor (Orco), but it is not clear in Table 2 nor any of the figures dealing with the ORs (Fig 3, 6, 8 and 10) that they are reporting on the Orco orthologue for this species. Has this critically important gene been omitted in this report?

There are no statistical measurements provided for phylogenetic relationships in Figures 5 and 6, such as bootstrap values of maximum likelihood support values. These values are essential to ensure strength of relationship between different genes and nodes in the tree.

Table 1 and Table 2 both show basic information on the OBPs and ORs identified in An. stephensi, however this information as shown is not critically useful, especially the Protein Accession and Gene ID, which is more suitable to be included as a supplemental info. For a main-text table, such as these, it would be better to include other metrics such as completion status of the open reading frame (complete/incomplete) as well as the size of the protein (number of amino acids, molecular weight) and also isoelectric values. It is also common to show the geneID of the best blast hit from other species for each novel gene identified.

In fact, the isoelectric values and molecular weight values are summarized in Figure 11, but the actual result metrics are not presented anywhere in the report. At the very least, these values should be included as supplemental info, but could also be incorporated into a revised version of Tables 1 and 2.

With respect to the Isoelectric and Molecular Weight values, are only ORs and OBPs with complete open reading frames being considered? Because those are the only ones which could usefully be compared. It would be essential to clarify on this in the appropriate Materials and Methods section.

Specific minor comments for each section also follow:

Abstract:

Line 26. “from protein sequences of the An. stephensi” Again here it is not clear what is the source of these protein sequences. This needs to be clarified according to my previous comment, throughout the text and at the appropriate sections.

Line 35. “whereas only three OBPs and two ORs were related to the Ae. aegypti OBPs and ORs.”

Are you sure about that? Aside from difficulties in clearly reading the phylogenetic trees due to low figure resolution, it is not accurate to suggest that genes are related only if they are directly next to each other with most recent common ancestry on the phylogenetic tree. All genes within a subfamily may indeed be related to each other provided statistical support for the clustering, even if they are not the most closely related genes within the cluster.

Several times throughout the text there is statements about relatedness or lack of relatedness. Please ensure clarifications or corrections are made to these kinds of statements throughout the entirety of the manuscript.

Introduction

Line 72. “In An. stephensi, two OBPs have been identified but no ORs as of yet.”

This needs to be updated, as a publication this year, Speth et al., 2021, Insects, has reported on the characterization of an OR in An. stephensi.

Line 76-77 “ORco” should be written as “Orco” according to convention established by Vosshall and Hansson, 2011, Chemical Senses.

Materials and Methods

Line 133. “between the chromosomes of the two genomes”.

Which two genomes? In the appropriate methods section, it should be clearly written out which versions of which genomes are being analyzed for each species, and some kind of source information (accession number, literature reference, etc.) for each genome, in order to provide clear indication of which datasets are being analyzed.

Line 151. “were predicted were predicted” needs to be fixed.

Results

Line 169. “Renaming of ORs and chromosome”.

What is meant here by “renaming”? This is apparently the first description of ORs in this species, so how is it they are being renamed? Should say something like “Description or Classification of ORs” as is written for Table 1 on Line 161.

Line 183/Line193. “Chromosome 2 is saturated with…”

This is incorrect scientific terminology. “Saturated” has a very specific meaning in chemistry and its usage here is most certainly incorrect, in that the chromosomes probably could contain more OR/OBP genes than they already have. In this sense, they would not be saturated. Please consider another word choice for these descriptions.

Line 253-254. “None of the An. stephensi OBPs and ORs shared homologous regions with the OBPs and ORs of D. melanogaster.”

It is not clear what this is referring to. Homologous regions of the protein sequence? Or referring to genomic synteny? This should be more clearly written to accurately describe what is being compared.

Line 260. “Contained insignificant domains”.

What is an insignificant domain? If such descriptions are going to be made, they need to be clearly defined in the appropriate Materials and Methods section.

Line 287. “CELLO and WoLF PSORT predicted…” In the materials and methods section these softwares are mentioned but it is not clear how these determinations have been made. It would be more useful to indicate that identified OBPs contain Signal Peptides and that ORs contain appropriate number of transmembrane domains. There are specific softwares that analyze these features, but if that is what the indicated softwares have done, it would be important to clarify this in the Materials and Methods section.

Discussion.

Line 291-292. “OBPs and ORs are expressed at very high level in insects”.

This is not necessarily correct. ORs are not generally expressed at very high levels (with sometimes exceptions for Orco and sometimes pheromone receptors), and Some OBPs are indeed expressed at very high levels, but many of them are not. This needs to be corrected.

Line 301 and 302. “Aenasius bambawaei” and “Tomicus yunnanensis”.

What kind of species are these? Are they mosquitoes or other Dipterans? It should be indicated at the very least what Insect Order they belong to since the information being presented here is comparative.

Line 304-305. “which are less than the other two chromosomes in An. gambiae”.

Why is An. gambiae mentioned here. Shouldn’t this be referencing the other two chromosomes in An. stephensi?

Line 308-309. “Like OBPs, ORs are also concentrated on chromosome 3.”

This statement is logically inconsistent. On Line 303, it is mentioned that most of the OBP genes are localized on chromosome 2, not chromosome 3. So this is not a correct “like…also” statement of logic, since the patterns being compared are different. Consider to re-word this.

Line 318-319. “Likewise, Culex quinquefasciatus OBPs and ORs showed the closer relationship with OBPs and ORs of Ae. aegypti”

Closer compared to what? What exactly is the reference point? An. gambiae? D. melanogaster? Some other species?

Line 328/329. Again here with the “significant PGP domain…insignificant PGP-OBP domain”.

Significance is usually indicated to refer to statistical significance. So it is not clear what is meant by significance versus insignificance in this sense.

Line 333-334, Line 335-336. Here it says “average molecular weight of the OBPs is 18 kDA” first time and then “Average molecular weight of the OBPs is also reported to be around 50 kDA the second time.

Cannot be correct for both as written. Do you mean to say ORs for the second instance?

Figures and Tables

Figure 6, in-figure legend it says OBPs, but this figure is depicting phylogeny of the ORs. This needs to be corrected.

Figure 9B. In the in-figure legend it shows a PFAM domain indicated as PBP/GOBP. However, the PBP/GOBP subfamily of OBPs is known to be specific only to Lepidopterans, however this report is focused on a Dipteran mosquito. So it is not clear how a PBP/GOBP domain would be relevant to OBPs of this species. It is cautioned against relying too much on PFAM domains due to inaccuracies such as this. It gives the impression that all of these proteins are members of the PBP/GOBP subfamily, which they are not.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: William B. Walker III

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Mar 22;17(3):e0265896. doi: 10.1371/journal.pone.0265896.r002

Author response to Decision Letter 0

25 Nov 2021

We have made all the changes recommended by reviewers. I hope our manuscript will be considered for potential publication in this journal.

Profound regards

Attachment

Submitted filename: Response to Reviewer 2.docx

Click here for additional data file.^{(22.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0265896.r003

Decision Letter 1

Joseph Clifton Dickens

4 Jan 2022

PONE-D-21-25203R1Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and Comparative InsightsPLOS ONE

Dear Dr. Bhatti,

While the manuscript has been substantially improved, one of the reviewers recommends additional modifications. Please respond to the reviewer's comments.

Please submit your revised manuscript by Feb 18 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Joseph Clifton Dickens

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

While the manuscript has been substantially improved, one of the reviewers recommends additional modifications. Please respond to the reviewer's comments.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The revised manuscript is much more precise and the figures/tables greatly improved. All reviewer comments have been addressed.

Reviewer #2: The authors have substantially improved the manuscript, however some minor revisions are requested before this manuscript can be approved for publication. Importantly, greater clarity of documentation in some of the figure legends is required. Any symbolics in the figures should be clearly defined in the figure legends where appropriate.

1. In Figures 5 and 6, the authors have added bootstrap values. In the figure legends for these figures, it should be indicated that these numbers represent bootstrap values. In these figures some of the sequence IDs are different colors, including those that are in black font. The representations of the different colors should also be indicated in the appropriate figure legends. Additionally, the presentation of the bootstrap values in these figures is somewhat disorganized, sometimes with the numbers being overlapping and obscured by the phylogenetic relationship lines. This should be avoided, and numbers should be positioned such that they are not overlapping with other elements of the figure and it should be clear which phylogenetic relationships they are referring to.

2. In Figures 7 and 8, it is not clear to the reader what the grey synteny lines (figures 7 and 8) represent relative to the green (figure 7), blue (figure 7) or red ones (figure 8). These representations should be clarified in the appropriate figure legends

3. It was previously requested for the authors to update the tables to include ORF completion status for each OR/OBP as well as the size of the ORF, in terms of number of amino acids. This information is typically standard to include in these kinds of reports, though it has not been provided here yet. It is again requested to provide this information, either as an expansion of tables 1 and 2, appended to Figures 9 and 10, or else included as supplementary material.

4. On lines 75,76, it is mentioned that “ORs have been previously identified based on in situ hybridization using RNA probes and transcriptomic data from different organisms”. However, it is also the case that ORs have been previously identified through analysis of genomic data, as with An. gambiae and numerous other species. This approach should also be mentioned here.

5. In the first paragraph of the Discussion, from Lines 308 to 323, total number of OBPs and ORs are presented. Number of OBPs are compared to total numbers from An. gambiae, Ae. aegypti, and D. melanogaster (Lines 310-311), however a similar comparison of ORs between An. stephensi and these three species is not included towards the end of this paragraph. It would seem relevant to also compare total number of ORs across these four species.

6. On Line 377-378, “Molecular simulations helps in the retention of ligand-receptor interactions for an extended period to induce behavioral response, thus helping to identify novel attractants.” This statement is unclear. It is unclear what is meant how molecular simulations help in the retention of ligand-receptor interactions? Do the authors mean to say that the molecular simulations help to identify/predict extended ligand-receptor interactions that are retained sufficiently long enough to possibly induce behavioral response? Or is something else meant? As written it is not clear how simulations could help in retention of anything.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: William B. Walker

PLoS One. 2022 Mar 22;17(3):e0265896. doi: 10.1371/journal.pone.0265896.r004

Author response to Decision Letter 1

17 Jan 2022

Dear Reviewer,

All modifications have been incorporated in the manuscript as per your suggestion.

Thanks

Attachment

Submitted filename: Response to Reviewer 2.docx

Click here for additional data file.^{(14.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0265896.r005

Decision Letter 2

Joseph Clifton Dickens

3 Feb 2022

PONE-D-21-25203R2Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and Comparative InsightsPLOS ONE

Dear Dr. Bhatti,

Please respond to the reviewer's comments, especially those regarding Table S2.

Please submit your revised manuscript by Mar 20 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Joseph Clifton Dickens

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments:

Please respond to the reviewer's comments, especially those regarding Table S2.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: All previous concerns have been addressed, however two very minor issues remain that need to be addressed.

One is a typo found on line 272 of the untracked version. There should be a space in between "homology" and "with". In present version it is written as one word.

More substantially, in the new Supplementary Tables, specifically the Table S2 with the ORs, the complete/incomplete status for the ORs seems not to be filled in (as was the case for OBPs in Table S1), for column 3. This is critical information to include because it appears several of the ORs are incomplete. Also in the figure legend for Table S2, it is written that "Similarly, OBP's names and accessions have also be given". This should be changed to indicate that it is the OR's names.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: William B Walker

PLoS One. 2022 Mar 22;17(3):e0265896. doi: 10.1371/journal.pone.0265896.r006

Author response to Decision Letter 2

15 Feb 2022

Dear Editor,

All changes have been incorporated in the manuscript as per reviewer's suggestion.

Regards,

Attachment

Submitted filename: Response to Reviewer 2.docx

Click here for additional data file.^{(12.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0265896.r007

Decision Letter 3

Joseph Clifton Dickens

10 Mar 2022

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and Comparative Insights

PONE-D-21-25203R3

Dear Dr. Bhatti,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Joseph Clifton Dickens

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0265896.r008

Acceptance letter

Joseph Clifton Dickens

14 Mar 2022

PONE-D-21-25203R3

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and Comparative Insights

Dear Dr. Bhatti:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Joseph Clifton Dickens

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Data. Nucleotide sequences of identified 44 AsteOBP genes in FASTA format.

(TXT)

Click here for additional data file.^{(82KB, txt)}

S2 Data. Protein sequences of identified 44 AsteOBPs in FASTA format.

(TXT)

Click here for additional data file.^{(9KB, txt)}

S3 Data. Nucleotide sequences of identified 45 AsteOR genes in FASTA format.

(TXT)

Click here for additional data file.^{(105.8KB, txt)}

S4 Data. Protein sequences of identified 45 AsteORs in FASTA format.

(TXT)

Click here for additional data file.^{(16.6KB, txt)}

S1 Table. Nucleotide length of the open reading frame (ORF) and protein length in the OBPs have been provided along with their complete and partial status.

ORFs were predicted by ORF Finder. Similarly, OBP’s names and accessions have also been given.

(DOCX)

Click here for additional data file.^{(15.5KB, docx)}

S2 Table. Nucleotide length of the open reading frame (ORF) and protein length in the ORs have been provided along with their complete and partial status.

ORFs were predicted by ORF Finder. Similarly, OR’s names and accessions have also been given.

(DOCX)

Click here for additional data file.^{(16.3KB, docx)}

Attachment

Submitted filename: Response to Reviewer 2.docx

Click here for additional data file.^{(22.5KB, docx)}

Attachment

Submitted filename: Response to Reviewer 2.docx

Click here for additional data file.^{(14.5KB, docx)}

Attachment

Submitted filename: Response to Reviewer 2.docx

Click here for additional data file.^{(12.3KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0265896.ref001] 1.Rickman LS, Jones TR, Long GW, Paparello S, Schneider I, Paul CF, et al. Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans. NAVAL MEDICAL RESEARCH INST BETHESDA MD; 1990. [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref002] 2.Tadesse FG, Ashine T, Teka H, Esayas E, Messenger LA, Chali W, et al. Anopheles stephensi Mosquitoes as Vectors of Plasmodium vivax and falciparum, Horn of Africa, 2019. 2021;27(2):603. doi: 10.3201/eid2702.200019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref003] 3.Ghosh SK, Tiwari S, Raghavendra K, Sathyanarayan TS, Dash APJJob. Observations on sporozoite detection in naturally infected sibling species of the Anopheles culicifacies complex and variant of Anopheles stephensi in India. 2008;33(3):333–6. doi: 10.1007/s12038-008-0052-5 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref004] 4.Djadid ND, Gholizadeh S, Aghajari M, Zehi AH, Raeisi A, Zakeri SJAt. Genetic analysis of rDNA-ITS2 and RAPD loci in field populations of the malaria vector, Anopheles stephensi (Diptera: Culicidae): implications for the control program in Iran. 2006;97(1):65–74. doi: 10.1016/j.actatropica.2005.08.003 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref005] 5.Seyfarth M, Khaireh BA, Abdi AA, Bouh SM, Faulde MKJPr. Five years following first detection of Anopheles stephensi (Diptera: Culicidae) in Djibouti, Horn of Africa: populations established—malaria emerging. 2019;118(3):725–32. doi: 10.1007/s00436-019-06213-0 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref006] 6.Faulde MK, Rueda LM, Khaireh BAJAt. First record of the Asian malaria vector Anopheles stephensi and its possible role in the resurgence of malaria in Djibouti, Horn of Africa. 2014;139:39–43. doi: 10.1016/j.actatropica.2014.06.016 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref007] 7.Takken W, Lindsay SJEid. Increased threat of urban malaria from Anopheles stephensi mosquitoes, Africa. 2019;25(7):1431. doi: 10.3201/eid2507.190301 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref008] 8.Gakhar S, Sharma R, Sharma A. Population genetic structure of malaria vector Anopheles stephensi Liston (Diptera: Culicidae). 2013. [PubMed] [Google Scholar]

[pone.0265896.ref009] 9.Gholizadeh S, Djadid ND, Nouroozi B, Bekmohammadi MJAt. Molecular phylogenetic analysis of Anopheles and Cellia subgenus anophelines (Diptera: Culicidae) in temperate and tropical regions of Iran. 2013;126(1):63–74. doi: 10.1016/j.actatropica.2012.11.003 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref010] 10.Healy T, Copland M, Cork A, Przyborowska A, Halket JJM, entomology v. Landing responses of Anopheles gambiae elicited by oxocarboxylic acids. 2002;16(2):126–32. doi: 10.1046/j.1365-2915.2002.00353.x [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref011] 11.Dekker T, Steib B, Cardé R, Geier MJM, entomology v. L‐lactic acid: a human‐signifying host cue for the anthropophilic mosquito Anopheles gambiae. 2002;16(1):91–8. doi: 10.1046/j.0269-283x.2002.00345.x [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref012] 12.Hoffman SA, Aravind L, Velmurugan SJP, vectors. Female Anopheles gambiae antennae: increased transcript accumulation of the mosquito-specific odorant-binding-protein OBP2. 2012;5(1):1–6. doi: 10.1186/1756-3305-5-27 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref013] 13.Sun JS, Xiao S, Carlson JRJRSOB. The diverse small proteins called odorant-binding proteins. 2018;8(12):180208. doi: 10.1098/rsob.180208 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref014] 14.Venthur H, Zhou J-JJFip. Odorant receptors and odorant-binding proteins as insect pest control targets: a comparative analysis. 2018;9:1163. doi: 10.3389/fphys.2018.01163 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref015] 15.Chertemps T, Maïbèche M. Odor degrading enzymes and signal termination. Insect Pheromone Biochemistry and Molecular Biology: Elsevier; 2021. p. 619–44. [Google Scholar]

[pone.0265896.ref016] 16.Vogt RG, Riddiford LMJN. Pheromone binding and inactivation by moth antennae. 1981;293(5828):161–3. doi: 10.1038/293161a0 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref017] 17.Mazumder S, Chaudhary BP, Dahal SR, Al-Danoon O, Mohanty SJB. Pheromone Perception: Mechanism of the Reversible Coil–Helix Transition in Antheraea polyphemus Pheromone-Binding Protein 1. 2019;58(45):4530–42. doi: 10.1021/acs.biochem.9b00737 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref018] 18.Wang J, Murphy EJ, Nix JC, Jones DNJSr. Aedes aegypti Odorant Binding Protein 22 selectively binds fatty acids through a conformational change in its C-terminal tail. 2020;10(1):1–15. doi: 10.1038/s41598-020-60242-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref019] 19.Yu G-Q, Li D-Z, Lu Y-L, Wang Y-Q, Kong D-X, Wang M-QJSr. Deciphering the Odorant Binding, Activation, and Discrimination Mechanism of Dhelobp21 from Dastarus Helophoroides. 2018;8(1):1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref020] 20.Damberger FF, Michel E, Ishida Y, Leal WS, Wüthrich KJPotNAoS. Pheromone discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding protein. 2013;110(46):18680–5. doi: 10.1073/pnas.1317706110 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref021] 21.Zhou J-JJV, Hormones. Odorant-binding proteins in insects. 2010;83:241–72. [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref022] 22.He P, Zhang J, Liu N-Y, Zhang Y-N, Yang K, Dong S-LJPO. Distinct expression profiles and different functions of odorant binding proteins in Nilaparvata lugens Stål. 2011;6(12):e28921. doi: 10.1371/journal.pone.0028921 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref023] 23.Brito NF, Moreira MF, Melo ACJJoIP. A look inside odorant-binding proteins in insect chemoreception. 2016;95:51–65. doi: 10.1016/j.jinsphys.2016.09.008 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref024] 24.Hekmat-Scafe DS, Scafe CR, McKinney AJ, Tanouye MAJGr. Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster. 2002;12(9):1357–69. doi: 10.1101/gr.239402 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref025] 25.Graham LA, Davies PLJG. The odorant-binding proteins of Drosophila melanogaster: annotation and characterization of a divergent gene family. 2002;292(1–2):43–55. doi: 10.1016/s0378-1119(02)00672-8 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref026] 26.Zhou JJ, He XL, Pickett J, Field LJImb. Identification of odorant‐binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses. 2008;17(2):147–63. doi: 10.1111/j.1365-2583.2007.00789.x [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref027] 27.Vogt RGJJoce. Odorant binding protein homologues of the malaria mosquito Anopheles gambiae; possible orthologues of the OS-E and OS-F OBPs of Drosophila melanogaster. 2002;28(11):2371–6. doi: 10.1023/a:1021009311977 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref028] 28.Pelletier J, Leal WSJPo. Genome analysis and expression patterns of odorant-binding proteins from the Southern House mosquito Culex pipiens quinquefasciatus. 2009;4(7):e6237. doi: 10.1371/journal.pone.0006237 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref029] 29.Sengul M, Tu ZJImb. Characterization and expression of the odorant‐binding protein 7 gene in Anopheles stephensi and comparative analysis among five mosquito species. 2008;17(6):631–45. doi: 10.1111/j.1365-2583.2008.00837.x [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref030] 30.Sengul MS, Tu ZJImb. Identification and characterization of odorant‐binding protein 1 gene from the Asian malaria mosquito, Anopheles stephensi. 2010;19(1):49–60. doi: 10.1111/j.1365-2583.2009.00929.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref031] 31.Speth Z, Kaur G, Mazolewski D, Sisomphou R, Siao DDC, Pooraiiouby R, et al. Characterization of Anopheles stephensi Odorant Receptor 8, an Abundant Component of the Mouthpart Chemosensory Transcriptome. Insects. 2021;12(7):593. doi: 10.3390/insects12070593 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref032] 32.Gao Q, Chess AJG. Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. 1999;60(1):31–9. doi: 10.1006/geno.1999.5894 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref033] 33.Rinker DC, Zhou X, Pitts RJ, Rokas A, Zwiebel LJ. Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae. BMC genomics. 2013;14(1):1–15. doi: 10.1186/1471-2164-14-749 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref034] 34.Vosshall LB, Hansson BSJCs. A unified nomenclature system for the insect olfactory coreceptor. 2011;36(6):497–8. doi: 10.1093/chemse/bjr022 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref035] 35.De Castro E, Sigrist CJ, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. 2006;34(suppl_2):W362–W5. doi: 10.1093/nar/gkl124 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref036] 36.Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths‐Jones S, et al. The Pfam protein families database. 2004;32(suppl_1):D138–D41. doi: 10.1093/nar/gkh121 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref037] 37.Wolfe D, Dudek S, Ritchie MD, Pendergrass SAJBm. Visualizing genomic information across chromosomes with PhenoGram. 2013;6(1):1–12. doi: 10.1186/1756-0381-6-18 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref038] 38.Hall T, Biosciences I, Carlsbad CJGBB. BioEdit: an important software for molecular biology. 2011;2(1):60–1. [Google Scholar]

[pone.0265896.ref039] 39.Kumar S, Stecher G, Li M, Knyaz C, Tamura KJMb, evolution. MEGA X: molecular evolutionary genetics analysis across computing platforms. 2018;35(6):1547. doi: 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref040] 40.Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJJB. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. 2009;25(9):1189–91. doi: 10.1093/bioinformatics/btp033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref041] 41.Price MN, Dehal PS, Arkin APJPo. FastTree 2–approximately maximum-likelihood trees for large alignments. 2010;5(3):e9490. doi: 10.1371/journal.pone.0009490 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref042] 42.Letunic I, Bork PJNar. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. 2019;47(W1):W256–W9. doi: 10.1093/nar/gkz239 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref043] 43.Chen C, Chen H, He Y, Xia RJB. TBtools, a toolkit for biologists integrating various biological data handling tools with a user-friendly interface. 2018:289660. [Google Scholar]

[pone.0265896.ref044] 44.Wang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. 2012;40(7):e49–e. doi: 10.1093/nar/gkr1293 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref045] 45.Bailey TL, Johnson J, Grant CE, Noble WSJNar. The MEME suite. 2015;43(W1):W39–W49. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref046] 46.Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, et al. CDD: a Conserved Domain Database for the functional annotation of proteins. 2010;39(suppl_1):D225–D9. doi: 10.1093/nar/gkq1189 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref047] 47.Xu P, Zwiebel L, Smith D. Identification of a distinct family of genes encoding atypical odorant‐binding proteins in the malaria vector mosquito, Anopheles gambiae. Insect molecular biology. 2003;12(6):549–60. doi: 10.1046/j.1365-2583.2003.00440.x [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref048] 48.Hekmat-Scafe DS, Scafe CR, McKinney AJ, Tanouye MA. Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster. Genome research. 2002;12(9):1357–69. doi: 10.1101/gr.239402 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref049] 49.Zhou JJ, He XL, Pickett J, Field L. Identification of odorant‐binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses. Insect molecular biology. 2008;17(2):147–63. doi: 10.1111/j.1365-2583.2007.00789.x [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref050] 50.Pelosi P, Maida RJCB, Biochemistry PPB, Biology M. Odorant-binding proteins in insects. 1995;111(3):503–14. doi: 10.1016/0305-0491(95)00019-5 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref051] 51.Pelosi P, Zhu J, Knoll WJS. Odorant-binding proteins as sensing elements for odour monitoring. 2018;18(10):3248. doi: 10.3390/s18103248 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref052] 52.Li ZX, Pickett JA, Field LM, Zhou JJJAoIB, America PPiCwtESo. Identification and expression of odorant‐binding proteins of the malaria‐carrying mosquitoes Anopheles gambiae and Anopheles arabiensis. 2005;58(3):175–89. doi: 10.1002/arch.20047 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref053] 53.Shiao M-S, Fan W-L, Fang S, Lu M-YJ, Kondo R, Li W-H. Transcriptional profiling of adult Drosophila antennae by high-throughput sequencing. Zoological Studies. 2013;52(1):1–10. [Google Scholar]

[pone.0265896.ref054] 54.Bohbot J, Pitts R, Kwon HW, Rützler M, Robertson H, Zwiebel LJImb. Molecular characterization of the Aedes aegypti odorant receptor gene family. 2007;16(5):525–37. doi: 10.1111/j.1365-2583.2007.00748.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref055] 55.Nie X, Li Q, Xu C, Li D, Zhang Z, Wang MQ, et al. Antennal transcriptome and odorant binding protein expression profiles of an invasive mealybug and its parasitoid. 2018;142(1–2):149–61. [Google Scholar]

[pone.0265896.ref056] 56.Liu N-Y, Li Z-B, Zhao N, Song Q-S, Zhu J-Y, Yang BJCB, et al. Identification and characterization of chemosensory gene families in the bark beetle, Tomicus yunnanensis. 2018;25:73–85. doi: 10.1016/j.cbd.2017.11.003 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref057] 57.Manoharan M, Ng Fuk Chong M, Vaïtinadapoulé A, Frumence E, Sowdhamini R, Offmann BJGb, et al. Comparative genomics of odorant binding proteins in Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. 2013;5(1):163–80. doi: 10.1093/gbe/evs131 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref058] 58.Ray A, Van Naters WVDG, Carlson JRJJob. Molecular determinants of odorant receptor function in insects. 2014;39(4):555–63. doi: 10.1007/s12038-014-9447-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref059] 59.Basak SC, Bhattacharjee AKJCmc. Computational approaches for the design of mosquito repellent chemicals. 2020;27(1):32–41. doi: 10.2174/0929867325666181029165413 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref060] 60.Oliferenko PV, Oliferenko AA, Poda G, Pillai GG, Bernier UR, Agramonte NM, et al. Insect olfactory system as target for computer-aided design of mosquito repellents. Computational Design of Chemicals for the Control of Mosquitoes and their Diseases: CRC Press; 2017. p. 39–64. [Google Scholar]

[pone.0265896.ref061] 61.Devillers JJS, Research QiE. 2d and 3d structure–activity modelling of mosquito repellents: A review. 2018;29(9):693–723. doi: 10.1080/1062936X.2018.1513218 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref062] 62.Bhadoriya KS, Sharma MC, Jain SV, Raut GS, Rananaware JRJMCR. Three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis and molecular docking-based combined in silico rational approach to design potent and novel TRPV1 antagonists. 2013;22(5):2312–27. [Google Scholar]

[pone.0265896.ref063] 63.Gopal JV, Kannabiran KJISCLS. Studies on interaction of insect repellent compounds with odorant binding receptor proteins by in silico molecular docking approach. 2013;5(4):280–5. doi: 10.1007/s12539-013-0152-2 [DOI] [PubMed] [Google Scholar]

[pone.0265896.ref064] 64.Bezerra-Silva PC, Dutra KA, Santos GK, Silva RC, Iulek J, Milet-Pinheiro P, et al. Evaluation of the activity of the essential oil from an ornamental flower against Aedes aegypti: electrophysiology, molecular dynamics and behavioral assays. 2016;11(2):e0150008. doi: 10.1371/journal.pone.0150008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265896.ref065] 65.Gupta KK, Sethi G, Jayaraman MJJovbd. Molecular docking and simulation studies of gustatory receptor of Aedes aegypti: A potent drug target to distract host-seeking behaviour in mosquitoes. 2016;53(2):179. [PubMed] [Google Scholar]

PERMALINK

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights

Zeeshan Zafar

Sidra Fatima

Muhammad Faraz Bhatti

Farooq A Shah

Zack Saud

Tariq M Butt

Roles

Abstract

Introduction

Methodology

Fig 1. Pipeline of the methodology.

Identification of the OBPs and ORs

Multiple sequence alignment and phylogenetic tree analyses

Synteny prediction of the OBPs and ORs

Gene structure analysis of OBPs and ORs

Physiochemical properties and sub-cellular localization prediction of OBPs and ORs

Results

Identification of OBPs and ORs

Table 1. Classification and NCBI accessions of identified OBPs.

Table 2. Renaming of odorant receptor (ORs) and chromosome.

Chromosomal location of the OBPs and ORs

Fig 2. Chromosomal location of Odorant Binding Proteins (OBPs).

Fig 3. Chromosomal location of Odorant Receptors (ORs).

Multiple sequence alignment and phylogenetic analysis of the OBPs and ORs

Fig 4. Multiple sequence alignment of the OBPs.

Fig 5. Phylogenetic analysis of OBPs.

Fig 6. Phylogenetic analysis of ORs.

Synteny analysis of OBPs and ORs

Fig 7. Synteny analysis of OBPs.

Fig 8. Synteny analysis of ORs.

Gene structure analysis of OBPs and ORs

Fig 9. Gene structure of the OBPs.

Fig 10. Gene structure of the ORs.

Prediction of physicochemical properties and subcellular localization of the OBPs and ORs

Fig 11. Physicochemical properties of OBPs and ORs.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Joseph Clifton Dickens

Roles

Author response to Decision Letter 0

Decision Letter 1

Joseph Clifton Dickens

Roles

Author response to Decision Letter 1

Decision Letter 2

Joseph Clifton Dickens

Roles

Author response to Decision Letter 2

Decision Letter 3

Joseph Clifton Dickens

Roles

Acceptance letter

Joseph Clifton Dickens

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases