Cloning,Characterization And Functional Analysis Of Genes Encoding Odorant Receptors In Aedes Albopictus

Background:Aedes albopictus (Skuse) (Diptera:Culicidae), the Asian tiger mosquito, is an important vector of arboviruses including Chikungunya, dengue, Zika virus and yellow fever. The species has a remarkable capacity for invading new habitats worldwide, and climatic adaptation, diapause, and ability to shelter in microhabitats make it an increasingly important vector in dengue outbreaks. Aedes albopictus was the primary vector of recent dengue outbreaks in Europe, the Indian Ocean islands, Central Africa, Hawaii and southern China. In particular, an unprecedented outbreak occurred in China in 2014 in which more than 40,000 dengue cases were reported. At present, the most effective means of curbing dengue transmission is to control the vector. Aedes albopictus transmits dengue viruses during blood feeding, a behavior mediated in part by olfaction, and this offers opportunities to disrupt the transmission process.Odorant receptors (ORs) in insect olfactory sensory neurons (OSNs) include a co-receptor designated Oreo (OR7) and conventional ligand-binding odorant receptors (ORX). Orco is expressed in most OSNs in both larvae and adults, and is conserved highly among insect species. The conservation of its structure and expression in mosquitoes support the conclusion that Orco plays an important role in olfactory functions. The spatial and temporal expression profiles and functions of conventional odorant receptors, which are highly divergent and species-specific, correlate with some olfactory-mediated behavioral roles. Multiple roles have been proposed for Orco, the first of which is that it forms a heteromeric complex with conventional ORs (ORX+Orco). For example, An. gambiae Orco, as with its Drosophila ortholog DOR83b, forms heterodimeric complexes with conventional ORs in a heterologous HEK293 expression system, and this increases the activity of the complexed conventional ORs. Orco may have a second role in which it forms a homodimer that acts as an ion channel.ORs may have a distinct range of odor selectivity or may respond narrowly to a salient odorant. These findings along with developmental and tissue-specific expression profiles led to the hypothesis that ORs genes expressed differentially in mosquitoes were likely to be involved in host seeking and host preference. For example, AgOrl is expressed specifically in female An. gambiae and has a role in host-seeking behavior. AaOR4, expressed in’domestic’Aedes aegypti, plays an important role in responding to human odors. The identification and function of OR families in Ae. albopictus is limited at this time to AalOR2, which responds to indole, a volatile in human sweat. Therefore, there is an urgent need to identify the full complement of OR genes in this species, especially the Orco gene, and initiate investigations into the behaviors they drive.Objective:Here we identify the OR genes of Ae. albopictus using bioinformatic tools. We proposed that those OR genes transcribed abundantly in olfactory organs may play important roles in behaviors such as host seeking and oviposition. Two heterologous expression systems, HEK293 cells and transgenic D. melanogaster, were used to investigate the functions of the highly-expressed AalOR genes, AalOR7, AalOR10 and AalOR88. AalORs were expressed individually and in combination to discover their independent response as well as interactions with odor stimulation. Furthermore, RNA interference and behavioral assays were used to test whether host-seeking and preference might be associated with specific AalORs.Methods:1. Sequences of Ae. albopictus odorant receptors(AalORs) were identified according to their homologues of Ae. aegypti in the Ae. albopictus genome data by NCBI Blast and were named with the reference to sequence similarities to Ae. aegypti and Culex quinquefasciatus.2. Expression profiles in different tissues (larvae, pupasa, ntennae, proboscis, maxillary palp, leg and body) were performed using RT-PCR.3. Amplification of full-length AalORs cDNA (including 5’-or 3’-UTR) using RACE-PCR.4. Sequence analysis:Amino acid sequences of AalORs were aligned using the program ClustalW and the neighbour-joining tree built using the MEGA 5.0 program.5. Transmembrane regions of AalORs were predicted using HMMTOP version 2.0 and TMHMM server version 2.0.6. Plasmid construction of pME18s-AalOR7-eGFP, pME18s- AalOR10- DsRed and pME18s- AalOR88- DsRed, which was indentified by PCR and sequence.7. Calcium Imaging Assay:we analysed the differences in single AalORs and complexes in responding to the same odorant.8. The φC31 recombination system was used to clone Aa1ORx (UAS-AalORx) to the upstream activating sequence (UAS) in the "empty neuron" (△halo) D. melanogaster strain. △halo strain lacks its endogenous odorant receptor and does not respond to stimulation. UAS responder (UAS-AalORx) and GAL4 driver (22a-GAL4) lines were crossed and F1 progeny (w/w/y; Ahalo/Ahalo; (UAS-ORx)/22a-Ga14) screened for EAG experiments.9. EAG was performed using antennae of transgenic fly.10. Behaviour experiment was performed to detect the effect of different compounds on transgenic fly.11. RNAi was carried out and the expressional level of AalOR7, AalORlOand AalOR88 was detected by qPCR.12. The biting assay was performed to compare the effect of seeking a host on Ae. Albopictus mosquitoes post-RNAi-treatment.13. Host preference experiments was performed to compare the effect of host preference onAe. Albopictus mosquitoes post-RNAi-treatment.14. EAG experiment in control and RNAi-treatment group was performed to compare the effect of odors on Ae. albopictus mosquitoes post-RNAi-treatment.Results:1. We predicted 158 putative odorant receptors in the Ae. albopictus genome and transcriptome data, and we named all ORs with the reference to sequence similarities to An.gambiae and Ae. aegypti.2. Tissue-specific expressional profile showed that 29 AalORs were expressed in different tissues and 7 AalORs (7,10,14,45,59,88 and 105) were detected in female olfactory tissues.3. We got full-length of AalOR7, AalOR10, and AalOR88, containing 5’-and 3’-UTR.4. The co-receptor subfamily (AalOR7, AaOR7, AgOR7, CquiOR7, DOR83b) is clustered in one branch with clear orthologous relationships in different species. AalOR7～99%、AalOR 1096% and AalOR 8881%)。AalOR7, AaOR7, AgOR7, CquiOR7, DOR83b belongs to co-receptor. AalOR10 and AalOR88 belongs to conventional odorant receptors。5. Membrane topology predictions of AalOR7 and AalOR10 show that they belong to the TM7 class while AalOR88 is a TM6 protein. Analysis of the primary amino acid sequence of AalOR7 shows that it shares the highly-conserved ICL3, TMH6 and TMH7 regions with other Orco proteins and a putative calmodulin (CaM) binding site (329SAIKYWVER337) identified in DmelOrco (336SAIKYWVER344), in the ICL2 domain. AalOR10 and AalOR88 do not have the putative CaM binding site and channel gate sequences.6. Successful construction of expressional plasmid, pME 18s-AalOR7-eGFP, pME18s-AalOR10-DsRed and pME18s-AalOR88-DsRed.7. Calcium imaging experiments showed no significant differences (measured as relative fluorescence changes, △F/Fo) compared to DMSO in intracellular calcium concentration in HEK293 cells expressing only AalOR7, AalOR10 or AalOR88 stimulated with the test chemicals. Cells co-expressing AalOR7 and AalOR10 responded strongly to indole, l-octen-3-ol,3-methyindole and DEET (F=76.961, P< 0.05). Similarly, cells co-expressing AalOR7 and AalOR88 also are activated by the tested odors (F=47.871, P< 0.05).8. Successful crossed and screened transgenic fruit flies for EAG experiments.9. EAG results showed that:transgenic fruit fly antennae only expressing AalOR7, AalOR10 and AalOR88 showed no significantly different responses to tested odors when compared with controls. Antennae co-expressing AalOR7 and AalOR10 respond specifically to 1-octen-3-ol (F=28.254, P< 0.05). Antennae co-expressing AalOR7 and AalOR88 responded to indole and 1-octen-3-ol (F=8.281,P<0.05).10. Fruit flies co-expressing AalOR7and AalORlO can attract to 1-octen-3-ol (F=10.387, P< 0.05), while those co-expressing AalOR7and AalOR88 showed a preference for indole (F=14.807, P< 0.05) and 1-octen-3-ol in the modified Y-tube olfactometer.11. EAG response in RNAi-treatment group was significantly lower than that of normal one (AalOR7-siRNA-treated mosquitoes:t=13.191, P< 0.05; AalOR10-siRNA-treated mosquitoes:t=12.490, P< 0.05 AalOR88-siRNA-treated mosquitoes:t=18.275, P< 0.05).12. AalOR7-siRNA injected mosquitoes showed a significantly lower blood-fed rate compared to the control (F=32.183, P< 0.05). AalOR10-siRNA-and AalOR88-siRNA-injected mosquitoes showed no significant differences.13. Mosquitoes treated with AalOR7-siRNA have a statistically-significant lower bias for humans (F=9.738, P< 0.05).14. Electroantennograms of mosquito antennae stimulated with odorants. AalOR7-siRNA-treated mosquitoes did not respond to any odorant. Water injected mosquitoes showed significantly responses to odours in EAG assay.Conclusion:1. We predicted 158 putative Ae. albopictus odorant receptors. Cloning and characterization successfully of AalOR7, AalOR10 and AalOR88.2. Transmembrane regions of AalORs were predicted using HMMTOP version 2.0 and TMHMM server version 2.0.5. Membrane topology predictions of AalOR7 and AalOR10 show that they belong to the TM7 class while Aa1OR88 is a TM6 protein. AalOR7 shows that it shares the highly-conserved ICL3, TMH6 and TMH7 regions with other Orco proteins and a putative calmodulin (CaM) binding site (329SAIKYWVER337) identified in DmelOrco (336SAIKYWVER344), in the ICL2 domain. AalOR10 and AalOR88 do not have the putative CaM binding site and channel gate sequences.3. Expressional pattern of AalORs showed special and temporal specific manner and 7 AalORs (7,10,14,45,59,88 and 105) were detected in female olfactory tissues.4. Individual conventional (ORXs) and Orco can form heteromeric complexes to recognize odorants and respond to components of human volatiles.5. Expressional levels of AalORs were successfully knowed down by RNAi.6. The reduction of AalOR7 transcript levels led to a significant decrease in host seeking and confusion in host preference.