IMMUNOLOCALIZATION AND ROLE OF AN OLFACTORY CO-RECEPTOR IN CHEMOSENSORY ORGANS OF AEDES ALBOPICTUS

An olfactory co-receptor gene AalbOrco was isolated and described from Aedes albopictus using the RACE technique. AalbOrco contained typical structures of seven transmembrane domains and Nin-Cout. Sequence analysis demonstrated that AalbOrco is very similar to Orco orthologs from other dipterans, especially in the C-terminus. AalbOrco was expressed at significantly higher levels in embryos compared with larvae, however, AalbOrco transcripts were found to greatly increase in number from the larval to the pupal stage and were at the highest level at the adult stage. Since AalbOrco is abundantly expressed in adults and is distributed in chemosensory organs such as the antenna, maxillary palps and proboscis suggests that AalbOrco may play an important role in locating hosts. In addition, AalbOrco is also highly expressed in male abdomens as compared to female abdomens, however, no expression was detected in the ovipositor and the copulatory organ. SEM (scanning electron microscopy) and immunolocalization showed that AalbOrco is located in the cell bodies of olfactory sensory neurons (OSNs) under antennal cuticula and the dendrites of neurons in sensilla trichodea. Moreover, fluorescence signals were detected in some neuronal cell bodies and sensilla T2 from the labellum but were scarcely detected in male maxillary palps. The transcription level of AalbOrco in female adults decreased by 55.7% after the larvae continued to ingest exogenous AalbOrco dsRNA expressed bacterially until eclosion. Blood-sucking behavior was impaired by 44.7% compared to the control groups, although pupation and emergence rate were not significantly different between the control and treatment groups. We conclude that AalbOrco has the general characteristics of Orco orthologs and plays an important role in host-locating behavior. AalbOrco performs the olfactory function in antennae as well as in female maxillary palps.


INTRODUCTION
The olfactory system in mosquitoes, similar to other insects, plays an important role in host orientation behavior.The interference of key molecules such as olfactory receptors (ORs) may provide new avenues for mosquito control.Unlike the conventional ORs (cORs), the olfactory co-receptor (Orco) was highly conserved (Hallem et al., 2004;Vosshall and Hansson, 2011).Orco may direct the cORs located on the dendritic membrane of olfactory sensory neurons (OSNs) and enhance their interaction with odor molecules, although the efficiency of cORs was low without Orco (Larsson et al., 2004;Benton et al., 2006).In signal transduction, Orco associated with cORs forms a heterodimeric complex, a ligand-gate ion channel, on the dendritic membrane of OSNs (Sato et al., 2008;Wicher et al., 2008;Smart et al., 2008).In addition, a separate Orco could be directly activated by the level of intracellular cAMP (cyclic adenosine monophosphate) or cGMP (cyclic guanosine monophosphate) independent of a G-protein (Wicher et al., 2008;Yao and Carlson, 2011;Jones et al., 2011).
Currently, research on olfactory-related genes in Aedes albopictus has involved cORs and odorant binding proteins (OBPs) (Hao et al., 2013;Scialò et al., 2012;Deng et al., 2013;Zhao and Zheng, 2013); however, there are still no studies of Orco in Ae. albopictus.The functions of Orco from other insects have primarily been inferred from RNA interference (RNAi) through injection or feeding of dsRNA and the construction of heterogeneous expression systems (Larsson et al., 2004;Jones et al., 2005;Zhao et al., 2011).Considering the key role of Orco in olfactory signaling pathways, in this paper we describe an Orco gene from Ae. albopictus (AalbOrco), along with its anatomic sites of expression and developmental expression patterns.The functions of AalbOrco were indirectly observed through larval ingestion of dsRNA expressed in bacteria, and the blood-sucking behavior of adults was assessed.

Mosquitoes, RNA isolation and conversion to cDNA
Ae. albopictus was collected, and colonies were formed at Xiamen University, China.After breeding three generations, total RNA from different stages and tissues was extracted using the HP Total RNA Kit (Omega, USA) according to the manufacturer's instructions.First-strand cDNA was synthesized from 1 μg total RNA of female antenna in a 20-μL reaction volume using a PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, China) under the conditions recommended by the manufacturer.

Molecular cloning of the Orco gene from Ae. albopictus
A short fragment was obtained using the primer pairs Aalb-1F (ACTGGGTSGAGMGGCACAAG) and Aalb-1R (ATGAAGTAGGTSACRACBGCT). PCR assays were performed in a final reaction volume of 25 μL that consisted of 1 μL of the product of reverse transcription, 12.5 μL of 2 × Premix EX Taq (TaKaRa, China), 1 μL of each primer (10 μM), ddH 2 O 9.5 μL.Amplifications were performed under the following conditions: 94°C for 5 min; 36 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min; and 72°C for 7 min.One pair of nested primers was designed: GSP (ACTGGGTGGAGAGGCACAAGCAC) and NGSP (TCGGTGTAACCTACGGTGCTGCC) for 3'RACE using the SMARTer TM RACE cDNA Amplification Kit (CLONTECH, USA).The 5'-terminal coding region of cDNA was amplified using a degenerate forward primer Aalb-2F (ATGMAMGTCCARC-CGACMAAG) and a specific reverse primer Aalb-2R (CGAAAATGCAAAACAGGAACAC).PCR amplifications were performed as described above but the extension was performed at 72°C for 1 min 40 s and 72°C for 10 min.A full-length Orco gene from Ae. albopictus was obtained by assembling two fragments from the 3' and 5' termini.
Specific primers (AalbOrco-F: ATGCACGTC-CAGCCGAC, AalbOrco-R: TTATTTCAACTGCAC-CAACACCA) were designed to detect the coding sequence of Orco.The conditions of the PCR amplifications were the same as above: 94°C for 5 min; 36 cycles at 94°C for 40 s, 63°C for 30 s and 72°C for 1 min 50 s; and 72°C for 10 min.All PCR products were checked on 2.0% (w/v) agarose gels and purified with a Gel Extraction Kit (Omega, USA).The purified DNA was subcloned into a pMD19-T vector (TaKa-Ra, China), and then transformed into competent Escherichia coli DH5α.Products were bidirectionally sequenced (BGI, Guangzhou) and blasted in the GenBank Database.The TMHMM Sever v1.0 (http:// www.cbs.dtu.dk/services/TMHMM/) was used to predict the transmembrane domain.Sequences were aligned using the ClustalX2 (Larkin et al., 2007).The neighbor-joining tree was constructed using MEGA6 (Tamura et al., 2013).

Expression patterns and tissue specificity of Orco in Ae. albopictus
Specific primers (across an intron) for Orco and β-actin were designed as follows: qAalb-F (ACAGTGCCAGAAAGCGATGA), qAalb-R (CTGCACCAACACCATGAAGTAG); actin-F (AACTTCCCGACGGACAGGT), and actin-R (TAC-CGCAGGCTTCCATACCC). First-strand cDNA was synthesized from 1 μg of total RNA of different stages and tissues in a 20-μL reaction volume using the PrimeScript RT reagent Kit with a gDNA Eraser (Perfect Real Time) (TaKaRa, China).Quantitative real-time PCR (qRT-PCR) assays were performed on the Bio-Rad CFX96 at a final reaction volume of 10 μL that consisted of 1 μL cDNA diluted at 1:10, 2 × SYBR Premix EX Taq II (TaKaRa, China) 5 μL, each specific primer (10 μM) for 0.35 (Orco) or 0.4 μL (β-actin), ddH 2 O up to 10 μL.The reactions without cDNA templates served as negative controls; reactions were run in triplicate for each sample.the qRT-PCR program was carried out with a two-step method as follows: 95°C for 30 s; 40 cycles at 95°C for 5 s, followed by 60°C at 30 s, and fluorescence acquisition at 80°C for 10 s.The expression of the Orco gene at specific stages and in different tissues was presented as 2 -△△Ct (Schmittgen et al., 2000;Livak and Schmittgen, 2001;Schmittgen, 2001).

Antibody production and immunolocalization
The steps of the paraffin section of the olfactory and gustatory organs were performed according to the method of Yan et al. (2011), but the dehydration time was shortened to 10-15 min.SEM was also performed as described previously (Pitts et al., 2004).
The peptide (LNEEKDPDTKDFDL) located in the second intracellular loop (IC2) was synthesized (Fig. 2), coupled to the keyhole limpet hemocyanin (KLH), and used to generate sequence-specific, peptide-affinity purified polyclonal antibodies in mice (Kunming strain) as described by Yu et al. (2014).The immunized mice were maintained in the Laboratory Animal Center, Xiamen University, and the studies were conducted in accordance with the guidelines for animal husbandry and approved by the Ethics Committee from Xiamen University according to the Regulations for the Administration of Affairs Concerning Experimental Animals (approved by the State Council of the People's Republic of China) (Permit Number: XUMLAC2012-0122).
After tissue processing, the slides were treated in 3% H 2 O 2 for 10 min and washed with distilled water 3 times (2 min each).Citrate buffer (92-98°C) was processed for 10-15 min and naturally cooled to room temperature.After rinsing for 5 min and soaking in Tris-buffered saline (contained 0.1% Tween-20) (TBST, pH 7.2-7.6)for 5 min, the slides were incubated in a block solution (TBST containing 5% skim milk) for 1 h at 37°C, then washed in TBST (pH 7.2-7.6) 3 times (5 min each).The slides were incubated in primary antibodies (1:1000 diluted in 3% skim milk) overnight at 4°C, followed by washing in TBST 3 times (15 min each).The slides were stained with secondary antibodies for 2 h (Cy3-conjugated goat anti-mouse IgG (Beyotime, China), 1:1000 in 3% skim milk) at room temperature, followed by washing in TBST 3 times (10 min each).Hoechst's dye 0.1% (diluted with 3% skim milk) was added for 30 min and TBST was checked 4 times (10 min each).Next, 60% of the glycerol was mounted and inspected immediately under fluorescence microscopy (Nikon 80i).Mouse pre-immune serum was used as a negative control.

Vector construction and dsRNA preparation
The fragment of AalbOrco (578 bp) was amplified and subcloned into a pMD19-T vector (forward primer dsAalbOrco-F: CGAGCTCTGTTTCGCTACGT-GAGT and reverse primer dsAalbOrco-R: CCGCTC-GAGCCAGGAGCAGAACATCA, with restriction enzymes of Sac I and Xho I (underlined), respectively).PCR assays were performed in 40-μL reaction volumes containing 1 μL of template, 20 μL of 2 × Premix EX Taq (TaKaRa, China), 2 μL of each primer (10 μM), and ddH 2 O 15 μL.Amplification conditions were as follows: 94°C for 5 min; 36 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 40 s; and 72°C for 5 min.The recombinant pMD19-T vector and plasmid L4440 was digested with Sac I and Xho I, and the purpose fragments were linked by T4 ligase (TransGen, China).After sequencing, the correct recombinant vector was named L4440-AalbOrco and transformed into E. coli HT115 (DE3), and then the single colonies were grown overnight in LB (Amp + ).The required amount of bacterial solution in fresh LB media (Amp + ) was taken and allowed to grow to OD 595 = 0.4.Synthesis of T7 polymerase was induced by IPTG, and bacteria were incubated with shaking for an additional 4 h at 37°C.An unrelated fragment gene from EGFP (enhanced green fluorescent protein) plasmid was used as a control dsRNA.The recombinant vector L4440-EGFP was constructed similarly to the L4440-AalbOrco.The fragment of EGFP (574 bp) was amplified with primers: dsEGFP-F (CGAGCTCCAAGTTCAGCGTGTCCG) and dsEG-FP-R (CCGCTCGAGATCGCGCTTCTCGTTGG), with underlined restriction enzyme of Sac I and Xho I, respectively.The expressed dsRNA was extracted and detected as described previously (Timmons et al., 2001).

Feeding bioassay and gene silencing evaluation
To confirm the effect of RNA interference on different concentrations of bacterial-expressed dsRNA, bacterial cells were collected from 30 mL (10 × dsAalbOrco) and 15 mL (5 × dsAalbOrco) IPTG-induced culture by centrifugation at 10 000 g for 2 min, resuspended in 1 mL 2% pre-melted agarose gel solution mixed with 0.1 g rat food at 60°C.After cutting into small pieces (2 mm thick), the solidified gels were fed to 20 mosquito larvae in water (and repeated in three groups).FD&C Sunset Yellow was used to indicate that the bacteria were colored and ingested by larvae.In control groups, the artificial diet contained ddH 2 O, 5 × dsEGFP and 10 × dsEGFP separately.The rate of pupation and emergence was counted.Three days after eclosion, 10 heads of female mosquitoes were cut and the transcription level of the Orco gene was detected by qRT-PCR (repeated three times).Blood-sucking behavior (the most impacted group and its control groups after RNAi) was carried out according to the method of De-Gennaro et al. ( 2013).Here we counted the engorged mosquitoes within 5 min in a group of 20 mosquitoes; the test was repeated three times.

Statistics
All values were expressed as mean ± standard deviation (SD).Significant differences between means from the data were tested using one-way analysis of variance (ANOVA) followed by Duncan's multiple comparison test using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA).A P value <0.05 was considered to be significant.
The cloned full length AalbOrco ORF had 1440 bp and encoded a peptide with 479 amino acid residues, which showed a sequence similarity to the Orco of various insects across different orders, such as Aedes aegypti (97.9%),Tribolium castaneum (66.1%),Heliothis virescens (65.5%) and Locusta migratoria (52.0%).The extremely conservative region located in the Cterminus (corresponding to the final 87 amino aacids) (Fig. 2) was aligned in the AalbOrco sequence with other 6 insect species of dipterans using Clus-talX.The predicted topology structure showed that AalbOrco had 7 transmembrane domains (TM) containing an intracellular N-terminus and an extracellular C-terminus (Fig. 2).The result also showed that the longest loop among the 6 loops was connected to TM4 and TM5 with IC2.The result of phylogenetic analysis showed that the AalbOrco clustered with other Diptera members to form a clade and that the mosquitoes might be separated earlier from other members of Diptera (Fig. 3).

Immunostaining of AalbOrco in chemosensory organs
As shown in the SEM, noninnervated microtrichia were distributed on the surface of the proboscis and maxillary palps, which were covered with scales.Indeed, a number of sensilla T2 and sensilla trichodea (ST) were observed on the labellum and proboscis (Fig. 5A, B, D), and mechanosensory sensilla chaetica (SC) on the maxillary palps (Fig. 5C-F).The capitate peg sensilla (CP) in the apical part of the female maxillary palps were also recorded (Fig. 5C).Three types of sensilla, ST, SC and sensilla basiconica (SB) were found in antennae (Fig. 5G, H).
Immunostaining revealed that native AalbOrco signals were localized and expressed at various levels in the cross sections of the three chemosensory organs (Fig. 6).Intense staining of AalbOrco was observed under the cuticulae of the antennae (Fig. 6B-1), whereas it was spottily distributed in the proboscis (Fig. 6A-1) and limited in the maxillary palps (♂) (Fig. 6C-1, C-2, NBM).
In contrast to the white light control (Fig. 6A-2, NBL), the red signal spots in the proboscis were overlapped with neuron cell bodies (Fig. 6A-1, NBL), indicating that the expression of AalbOrco was within these neuronal cells.As shown in Fig. 6A-1, A-2 (yel-  low arrow), the AalbOrco protein is likely expressed in the dendrites of neurons of sensilla T2.Likewise, AalbOrco was located in the dendrites of neurons of ST (Fig. 6B-1, B-2, yellow arrow) and in the cell bodies of OSNs under the antennal cuticula, respectively.Nevertheless, no positive fluorescence was detected in the SB (Fig. 6B-1, B-2, green arrow).

Feeding dsRNA reduces AalbOrco mRNA level and influences blood-sucking behavior
AalbOrco fragments were inserted into the plasmid L4440 within the Sac I and Xho I sites (Fig. 7A), and AalbOrco dsRNA (dsAalbOrco) was produced with the bacterial strain HT115 (DE3) containing a recombinant plasmid L4440-AalbOrco (Fig. 7C).Similarly, L4440-EGFP was constructed using a control for EGFP dsRNA (dsEGFP) expression (Fig. 7B, C).As shown in Fig. 7C, the production of nucleic acid extracted from HT115 (DE3) induced by IPTG showed the same size as that of AalbOrco and the EGFP fragment.In addition, the results of digestion using RNase A (Fig. 7D, lane 1, 3) and DNase I (Fig. 7D, lanes 2, 4) indicated that the targeted fragments were dsRNA from AalbOrco (578 bp) and EGFP (574 bp).
The digestive tracts of larvae were colored after feeding by the colored bacteria delivered with the artificial diet (Fig. 7E), indicating that dsRNA expressed bacterially was introduced into the larvae through ingestion.No significant difference was observed in the pupation rates of larvae (Fig. 8A, P = 0.355 ANO-VA) and eclosion rates of pupa (Fig. 8B, P = 0.412 ANOVA) between the treatment and control groups.However, the transcription level of AalbOrco from the head of females significantly reduced after larvae fed on dsRNA until pupation compared to the controls (Fig. 8C, P = 0.01 ANOVA).The transcription level of the treatment groups decreased by approximately 55.7%, but the different dsAalbOrco concentrations did not differ significantly according to Duncan's   5) bacteria carried blank plasmid L4440, lane (2), ( 3), (4) HT115 carried L4440-AalbOrco was induced by different concentrations of IPTG (0, 0.4, 0.8 mM), lane ( 6), ( 7), ( 8 multiple comparison test.In addition, the bloodsucking behavior of mosquitoes was significantly affected by RNA interference with AalbOrco (Fig. 8D, P = 8.44 × 10 −5 ANOVA): the rate of blood-sucking behavior decreased

DISCUSSION
The encoded protein (AalbOrco) belongs to the Orco family receptors, which may co-express with conventional ORs to detect odor molecules from hosts.The long IC2 in AalbOrco was another feature of Orco family proteins (Sato et al., 2008;Wicher et al., 2008) and the conserved threonines and tyrosines in the Cterminus might be important for regulating protein function (Ohguro et al., 1996;Nakagawa et al., 2012).
The antibody was difficult to administer inside the sensilla because of the cuticular layers; as a result, positive signals could only be detected with ease from damaged sensilla.Signals could only be detected from the broken ST; however, no signal was located in the broken SB.In the light of results described in previous research (Pitts et al., 2004;Melo et al., 2004;Xia and Zwiebel, 2006), we speculated that the expression pattern of Orco in mosquito antennae is primarily located in ST but is lacking in SB.In contrast, Orco was expressed in ST, SB, and sensilla placodea in other insects, such as drosophila spp., Stomoxys calcitrans, Apis cerana cerana, and Haematobia irritans irritans (Larsson et al., 2004;Zhang et al., 2012;Olafson, 2013).These results suggest that the expression patterns of Orco in mosquitoes possess their own unique features.Although there was a high level of transcription of AalbOrco in the male maxillary palps, expression was only rarely detected in part of the neuron cell body.This limited detection may be associated with the section position observed.Previous studies in mosquitoes indicated that Orco was only expressed in capitate peg sensilla (CP) of female maxillary palps  (Pitts et al., 2004;Melo et al., 2004;Xia and Zwiebel, 2006).However, unlike in females, no CP were observed in male the maxillary palps of Ae. albopictus based on the results of SEM.This discrepancy may account for the lower amount of AalbOrco detected in male maxillary palps, and the most likely reason is that this organ lacks the AalbOrco translation related factors that may exist in CP.These phenomena suggest that the male maxillary palps may lose their ability to smell.Similar to Ae. aegypti and Anopheles gambiae (Pappas and Larsen, 1976;Melo et al., 2004;Pitts et al., 2004), AalbOrco was also located in the neuronal cell body and dendrites of sensilla T2 in the labellum, suggesting that AalbOrco might have a gustatory function in the proboscis.
Similar to other mosquitoes (Pitts et al., 2004;Melo et al., 2004;Xia and Zwiebel, 2006), Orco in Ae. albopictus (AalbOrco) is highly expressed in adults and primarily distributed in chemosensory organs, suggesting that it may play an important role in locating hosts.The higher levels of transcription of Aal-bOrco in female adults were in line with our expectations.Interestingly, a higher level of expression in embryos was observed .This finding may be related to the development of OSNs in antenna based on the result that AalbOrco was highly expressed in the cell bodies of OSNs, but details of the mechanism remain unknown.
In comparison, RNAi, through ingestion of bacteria expressing dsRNA, appears to be a potentially useful method in functional genomics and pest control research (Zhang et al., 2010;Zhou et al., 2014;Singh et al., 2013;Tian et al., 2009;Zhu et al., 2011;Wynant et al., 2014).A specific downregulation of transcript levels for AalbOrco and a low efficiency for females in locating hosts after feeding on dsRNA which downregulate AalbOrco during the larval stage was observed, suggesting that silencing AalbOrco via RNAi impaired the blood-sucking ability of Ae. albopictus.

Fig. 3 .
Fig. 3. Neighbor-joining tree of Orco orthologs from seven insect orders.Species and accession numbers are shown in the figure.Numerical values indicated bootstrap support for each node.Bootstrap support values were based on 1000 replicates.