An odorant receptor from Anopheles sinensis in China is sensitive to oviposition attractants

Background Anopheles sinensis is an important vector for the spread of malaria in China. Olfactory-related behaviours, particularly oviposition site seeking, offer opportunities for disrupting the disease-transmission process. Results This is the first report of the identification and characterization of AsinOrco and AsinOR10 in An. sinensis. AsinOrco and AsinOR10 share 97.49% and 90.37% amino acid sequence identity, respectively, with related sequences in Anopheles gambiae. A functional analysis demonstrated that AsinOrco- and AsinOR10-coexpressing HEK293 cells were highly sensitive to 3-methylindole, but showed no significant differences in response to other test odorants when compared to DMSO. Conclusions AsinOrco was characterized as a new member of the Orco ortholog subfamily. AsinOR10, which appears to be a member of the OR2-10 subfamily, is directly involved in identification of oviposition sites. This finding will help to elucidate the molecular mechanisms underlying olfactory signaling in An. sinensis and provide many more molecular targets for eco-friendly pest control. Electronic supplementary material The online version of this article (10.1186/s12936-018-2501-4) contains supplementary material, which is available to authorized users.


Background
Malaria is one of the most important infectious diseases seriously endangering human health and safety. The World Health Organization (WHO) lists malaria with AIDS and tuberculosis as the top three public health problems globally. Malaria is also one of the most important mosquito-borne diseases in China. To respond proactively to the global action to eliminate malaria, China launched the Malaria Action Plan [1] in 2010, which clearly states that "by 2015, the country except for some border areas of Yunnan and other areas have no local malaria cases"; "by 2020, the national malaria elimination. " Currently, most counties (districts) in China have completed an assessment of malaria elimination. However, conditions are still favourable for the spread of malaria in some regions; even if the source of infection can be discovered and cleared in a timely, there is still a risk of local transmission and epidemic rebound. With rapid globalization and implementation of the national "Belt and Road" initiative, the number of people visiting areas of high malaria transmission, such as Africa and Southeast Asia, for business, employment and tourism purposes has increased significantly. As a result, the proportion of overseas imported cases, which reached 99.9% (3317/3321) in 2016, shows an increasing trend [2]. Such an increase poses a potential risk to relatively stable malaria-endemic areas. For example, a short-term and large-scale clustered imported outbreak occurred in Guangxi Province in 2013 [3]. In addition, malarianonendemic areas lack diagnostic awareness of imported malaria cases, and severe illness and death can occur.
Anopheles sinensis, with a wide distribution and a large population, is an important vector for the spread of malaria in China. The main strategy for the elimination of malaria by the WHO is the timely and effective removal of infection sources and preventing spread among epidemic sites. Given the resistance of An. sinensis populations to commonly used insecticides, alternative control methods are crucially needed. Researchers have combined Bacillus thuringiensis var. israelensis with oviposition attractants in "attract-andkill" strategies [4] to collect more gravid females [5] and [6] eggs than with control traps. As mosquitoes use their olfactory system to search for oviposition sites, research on these systems is of key importance.
The olfactory system of insects mainly includes olfactory receptors (ORs), odorant-binding proteins (OBPs) and olfactory receptor neurons (ORNs). Previous studies have demonstrated that ORs can convert odourstimulating chemical signals into electrical signals and transmit nerve impulses to the dendrites of olfactory neurons [7]. Accordingly, ORs are involved in mating, blood sucking, oviposition site searching and other important life activities of mosquitoes.

Mosquito rearing and blood feeding
Anopheles sinensis (laboratory-susceptible strain) larvae and pupae were reared on yeast powder, and adults were maintained on a 10% sugar solution at 25-27 °C and 70-80% relative humidity with a photoperiod of 12:12 h. Three-day-old adult females were blood-fed on a human volunteer arm using standard protocols [17].

Sequence analysis
Amino acid sequences of ORs were aligned using the program ClustalW, and the neighbor-joining tree was built using the MEGA 5.0 program [19]. The membrane topology of the OR sequences was predicted using the HMMTOP (version 2.0) and TMHMM (version 2.0) [20] servers.

Expression of AsinORs in HEK293 cells
The full-length coding sequences (CDSs) of AsinORs were cloned into the pME18s mammalian expression plasmid [9] using specific primers (Additional file 1: Table S1). The DsRed coding sequence was amplified from pIRES2-DsRed plasmids (Clontech, Mountain View, CA, USA) using primers containing the appropriate restriction sites. AsinORs were cloned into the pME18s plasmid in-frame with the DsRed coding sequence [8]. HEK293 (human embryo kidney 293) cells (purchased from the Chinese Academy of Sciences) were cultured in an incubator at a constant temperature of 37 °C with 5% CO 2 and transiently transfected with AsinORs using Lipofectamine ® 2000 Reagent (Invitrogen, Carlsbad, CA) [21]. Expression of ORs was confirmed by RT-PCR after 24 h; subcellular location analysis and western blotting were performed after 48 h. The two-step RT-PCR primers and nested RT-PCR primers are provided in Additional file 1: Table S1. Cells were lysed with RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 0.1% Triton X-100, 1% Nonidet P-40). The lysates were mixed with in SDS-PAGE buffer (62.5 mM Tris, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue), heated at 95 °C for 5 min, separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Immobilon TM -P, Millipore). The blot was washed with TBST, incubated with 5% skim milk for 60 min, and incubated overnight with an anti-RFP antibody (Abcam, Cambridge, US) raised in mice at a dilution ratio of 1:1000 in 1 × PBS or anti-GAPDH antibody (Abcam, Cambridge, US) at 1:3000 dilution at 4 °C. The blot was then incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:4000) (Bethyl Laboratories, Montgomery, TX, USA) at room temperature for 90 min.
Fluorescence images were acquired using a laser scanning confocal microscope (Olympus, Japan). The Ca 2+ level is represented as relative fluorescence change (ΔF/ F 0 ), where ΔF is the difference in peak fluorescence caused by stimulation and F 0 is the baseline fluorescence [22,23]. Baseline fluorescence was measured 100 s prior to adding the chemicals. Responses were quantified by the mean values of the maximal elevations (ΔF/F 0 ) [8]. Each odorant was assayed in triplicate per dish, and at least seven cells per dish were selected randomly. All assays were performed in triplicate.

Statistical analysis
Statistical analyses of differences in the cellular experimental results were conducted with one-way ANOVA followed by post hoc Tukey HSD tests (homogeneity of variance: P > 0.05).

Identification of putative AsinOR genes
Full-length coding sequences for AsinOrco and AsinOR10 were successfully obtained based on bioinformatics and homologous genes. AsinOrco, which is 1437 bp in length and encodes 479 amino acids, exhibits 96.66% sequence identity with predicted AsinOrco (98.96%) and AgamOrco (90.61%). Similarly, AsinOR10, which is 1125 bp in length and encodes 375 (93.35%) amino acids, shares 100% and 80.59% identity with predicted AsinOR10 and AgamOR10, respectively. An alignment of AsinOrco (97.49%) and AsinOR10 (90.37%) amino acid sequences with related sequences in An. gambiae is shown in Figs. 1, 2. In general, ORs display a high level of divergence [24]. An interesting phenomenon is that the ORs from different species have very high sequence conservation.

Sequence analysis
To explore relationships among ORs from different species, phylogenetic tree analysis was carried out using similar OR sequences, mainly including AgamORs, Aae-gORs and CquiORs. The results revealed the existence of different subgroups (Fig. 3). For this study, AsinOrco and AgamOrco, AfunOrco, AalbOrco, AaegOrco, CquiOrco and CppOrco were found to be clustered together. This finding indicates that AsinOrco belongs to the coreceptor subfamily, whereas AsinOR10, which is identified as a conventional odorant receptor, clusters with the OR2-10 subgroup. Among them, AsinOrco and AsinOR10 display the highest identity with AgamOrco/AfunOrco and AgamOR10/AsteOR10, respectively. Significantly, with the exception of OR7 orthologs, OR2 and OR10 are the most conserved ORs in the phylogenetic tree. This sequence conservation suggests that OR10 may show an odorant-induced response profile similar to that of OR2. These interesting phenomena encouraged us to examine the odorant response profile of AsinOR10.
Membrane topology predictions for AsinOrco and AsinOR10 revealed that these receptors belong to the seven-transmembrane (TM) protein family with an intracellular amino-terminus (Fig. 4). Analysis of the primary amino acid sequence of AsinOrco shows that it contains a putative calmodulin (CaM)-binding site ( 328 SAIKY-WVER 336 ) identified in DmelOrco ( 336 SAIKYWVER 344 ) and in AalbOrco ( 329 SAIKYWVER 337 ) [8]; in contrast, AsinOR10 does not have this putative CaM-binding site or channel gate sequences. The observed sequence conservation supports our hypothesis that AsinOrco may form a channel gate, as reported for DmelOrco [25,26], and that it may form complexes involved in odour signal transduction [20,25].

Discussion
This study is the first report of the identification and characterization of AsinOrco and AsinOR10. Although ORs typically display a high level of divergence [24], AsinOrco and AsinOR10 share 97.49% and 90.37% amino acid sequence identity with the coreceptor and OR2-10 subfamilies, respectively. This study utilized the nomenclature for Orco [27] and found that AsinOrco exhibits at least 50% sequence identity with orthologs from other insect species, and the predicted protein size is larger than that of conventional ORs. Membrane topology predictions show that AsinOrco and AsinOR10 belong to the TM7 protein family and have an intracellular amino-terminus. In addition, AsinOrco has the putative CaM-binding site ( 328 SAIKYWVER 336 ) identified in DmelOrco ( 336 SAIKYWVER 344 ) and in AalbOrco ( 329 SAIKYWVER 337 ) [8]. This conservation of structure may also account for functional similarity. Overall, identification and functional validation of Orco orthologs are hot research topics. In the previous study, AalbOrco was demonstrated to transmit olfactory signaling, but did not recognize odorants [8]. In fact, Orco forms a complex with conventional odorant receptors and is essential for odour signal transduction [20]. Indeed, silencing ORs are in blue, and Aedes albopictus ORs are in yellow. AsinOrco was grouped into the coreceptor subfamily, and AsinOR10 was grouped into the OR2-10 subgroup or mutation of Orco [8,28,29] damages normal odorant responses. Notably, the function of Orco is so similar that some researchers [21] have even used Drosophila melanogaster Orco as a heterodimerization partner to examine the function of AalbORs. In this study, AsinOrco was characterized as a new member of the Orco ortholog subfamily. Furthermore, HEK293 cells coexpressing AsinOrco and AsinOR10 responded to odorants.
In contrast to DMSO, 3-methylindole elicits a fluorescence reaction (measured as relative fluorescence change, ΔF/F0). This finding is similar to previous results showing that CquiOR10 [13,30], AalbOR10 [8] and AgamOR10 [12] orthologs respond sensitively to 3-methylindole and thus further confirm the functional conservation of OR10 orthologs. Regardless, CquiOR10 [13,30], AalbOR10 [8] and AgamOR10 [12] responded to a set of aromatic compounds, including each of the methylindoles, 1-octen-3-ol and indole, using the Xenopus Oocyte System or the Drosophila melanogaster "empty neuron" system, whereas AsinOR10 showed no significant differences in responses to indole, 1-octen-3-ol and 1-methylindole compared to DMSO in HEK293 cells. These results might be due to differences in the intracellular epitope tags of these systems, which may influence the selectivity of the receptor, or this OR might not be responsive to the chemicals tested. Despite the use of a heterogeneous expression system, the results indicate that AsinOR10 is directly involved in oviposition site-seeking behaviour.

Conclusions
In summary, AsinOrco was characterized as a new member of the Orco ortholog subfamily, and AsinOR10 was found to be a member of the OR2-10 subfamily. AsinOR10 is directly involved in oviposition site identification. These results will help in exploration of the molecular mechanism underlying the olfactory signal transduction pathway in An. sinensis and provide more molecular targets for eco-friendly pest control.