Identification and Cloning of AipsDopEcR in A. Ipsilon Males

By taking advantage of the high degree of protein sequence conservation of DopEcR receptors, we first cloned a full-length cDNA named AipsDopEcR via a degenerate RT-PCR reaction from A. ipsilon brain mRNA combined with a strategy of 5′/3′ RACE PCR (Fig. S1). This cDNA of 2126 bp deposited in the Genbank database with accession number (KC715734) contains a putative coding region of 1037 bp, a 220 bp 5′-untranslated region (5′-UTR) and a 837 bp 3′-UTR, with a polyadenylation signal upstream of the poly(A) (Fig. S1). The open reading frame encodes 345 amino acids, predicting a 39 kDa protein as determined by using MWCALC (Infobiogen). The deduced amino acid sequence of AipsDopEcR displays the putative seven transmembrane-spanning domains that characterize the GPCR family (Fig. S1). AipsDopEcR shows 67% identity with the predicted isoform A of DmDopEcR, 68% identity with Tribolium castaneum DopEcR, and 68% with Apis mellifera isoform 1 β 2-adrenergic receptor (accession numbers in Materials and Methods).

AipsDopEcR is Predominantly Expressed in the Central Nervous System

To evaluate the tissue distribution of AipsDopEcR, its expression was determined in the antenna, brain, AL, thorax, leg, wing and abdomen of 5-day-old males. RT-PCR analysis revealed the amplification of only one AipsDopEcR cDNA fragment of expected size (198 bp) whose amount was high in the brain and the AL. Only traces were detected in the antennae, and AipsDopEcR was not amplified in the other investigated tissues (Fig. 1A).

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Figure 1. AipsDopEcR gene expression and protein detection in the brain of A. ipsilon males.

A) Tissue-related AipsDopEcR gene expression. AipsDopEcR was detected mainly in the brain. B–D) 3D projections of confocal optical sections showing immunocytochemical AipsDopEcR protein detection in brains of 5-day-old males. B) Frontal view of the central brain parts. C) Protocerebrum with mushroom body calyx. D) Antennal lobe. Insets show manual reconstructions of labeled cell bodies in the respective series of sections. (MBC) mushroom body calyces; (CC) central complex; (AL) antennal lobes.

https://doi.org/10.1371/journal.pone.0072785.g001

In whole brain immunostaining experiments with a custom-made antibody we confirmed the presence of the AipsDopEcR protein in the central nervous system in 5-day-old sexually mature males. AipsDopEcR protein expression seems to be restricted to a few areas within the A.ipsilon brain (Fig. 1B). We observed labeled cell bodies predominantly around the mushroom body calyces (MBC) (Fig. 1C) and in the lateral cell body cluster of the AL (Fig. 1D). We counted 30±7 labeled cells (n = 11 brains) in each MBC, and 38±8 labeled cell bodies (n = 10 brains) in each AL.

The Expression Level of AipsDopEcR in the Brain is Age-dependent

In order to investigate if the expression of AipsDopEcR increases with age, we performed real time quantitative PCR (qPCR) and Western blot experiments on brain extracts collected from day-1 to day-5. The transcriptional level of AipsDopEcR was low during the first three days of adult life, and significantly higher at day-4 and day-5 (Fig. 2A).

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Figure 2. Age-related AipsDopEcR transcriptional activity and protein level from brains of 1- to 5-day-old A. ipsilon males.

A) Transcriptional activity of AipsDopEcR. The ribosomal gene RpL8 was used as reference. B) Relative protein expression of AipsDopEcR based on Western blot analysis as shown in (C). C) Protein expression of AipsDopEcR. The control used was the rabbit α-Tubulin protein. AipsDopEcR transcript and protein expression increase with age. Bars represent means ± s.e.m of 6 biological repetitions. Bars with same letters are not significantly different (ANOVA; Tukey test; α = 0.05).

https://doi.org/10.1371/journal.pone.0072785.g002

Western blot results show that the AipsDopEcR protein is detected throughout adult life in the brain (Fig. 2B and 2C). The increased AipsDopEcR transcriptional activity is associated to a significant increase in brain protein synthesis at 4 and 5 days (Fig. 2B), which is correlated with the increased behavioral responses to sex pheromone [13].

AipsDopEcR-dsRNA Injection Results in Reduced AipsDopEcR Expression in the Brain

To explore the role of AipsDopEcR in age-dependent pheromone-guided behavior, we used RNAi-mediated gene-silencing technology. In a first step, we evaluated the efficiency of AipsDopEcR gene knockdown in the brain at transcriptional and protein levels through the injection of double-stranded RNA into males (see Materials and Methods for details).

AipsDopEcR-dsRNA administration in 1-day-old sexually immature males induced a significant decline in the amount of AipsDopEcR transcript 4 days after injection as compared to the three control insect groups (Saline-, Bacterial LacZ-dsRNA-, and non-injected males) (Fig. 3A). This transcriptional silencing was accompanied by a drastic suppression of AipsDopEcR protein synthesis in AipsDopEcR-dsRNA-injected males as compared with controls (Fig. 3B and 3C).

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Figure 3. Efficiency of double-stranded RNA-mediated AipsDopEcR silencing in A. ipsilon males.

1-day-old sexually immature males received an injection of saline solution, bacterial LacZ-dsRNA or AipsDopEcR-dsRNA, or no injection (non-injected). For each treatment, the AipsDopEcR mRNA and protein amounts in the brain were evaluated 4 days after the injection by real time qPCR and Western blot respectively. A) Transcriptional activity of AipsDopEcR. The ribosomal gene RpL8 was used as reference. B) Relative protein expression of AipsDopEcR based on Western blot analysis as shown in (C). C) Protein expression of AipsDopEcR. The control used was the rabbit α-Tubulin protein. Bars represent means ± s.e.m of 3 biological repetitions. Bars with same letters are not significantly different (ANOVA; Tukey test; α = 0.05).

https://doi.org/10.1371/journal.pone.0072785.g003

AipsDopEcR-dsRNA Injection Results in Reduced Behavioral Response to Sex Pheromone

In a second step, the behavioral response to sex pheromone of 5-day-old sexually mature males that were injected at day-1 with AipsDopEcR-dsRNA or with one of the three controls as cited above, was tested in the wind tunnel (see Materials and Methods for details). There was no significant difference in the oriented behavioral response among the three control insect groups (G-test: G = 5. 28; df = 2; p = 0.07) (Fig. 4A). However, the behavioral response of AipsDopEcR-dsRNA-injected males was significantly decreased as compared to that of the three control insect groups (G = 18.05; 11.18; 4.59; p<0.0001; p = 0.0008; p = 0.032; df = 1 between AipsDopEcR-dsRNA-injected males and non-injected, Saline-, and Bacterial LacZ-dsRNA-injected males respectively) (Fig. 4A). Detailed analysis shows that there is no statistical difference in the proportions of the 3 different behavioural sequences of the oriented response (partial flight, complete flight, and landing) between the four groups of insects (non-injected, saline-, lacZ-dsRNA- and DopEcR-dsRNA-injected males) (G test : G = 2.80; df = 3; p = 0.42). General flight activity was high (>90%) and statistically similar for all tested insect groups (G test: G = 2.14; df = 3; p = 0.54), thus showing that the injections did not disturb the flight performances of the moths (Fig. 4B).

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Figure 4. Effect of AipsDopEcR silencing by injection of dsRNA on the oriented responses to the sex pheromone (A) and on general flight activity (B) in A. ipsilon males.

1-day-old sexually immature males were injected with saline solution, bacterial LacZ-dsRNA or AipsDopEcR-dsRNA, or received no injection (non-injected). For each treatment, the percentage of oriented or random flight activity was evaluated 4 days after injection in wind tunnel experiments. A) Oriented responses (partial flight+complete flight+landing). AipsDopEcR-dsRNA injection induces a significant inhibition of oriented upwind flight towards the sex pheromone in sexually mature male moths. B) General flight activity (oriented responses+random flights). Locomotor behavior is not affected by AipsDopEcR-dsRNA injection. Numbers in brackets indicate numbers of tested males. Bars with same letters are not significantly different (G-test; P≤0.05).

https://doi.org/10.1371/journal.pone.0072785.g004

Insects and Tissue Collection

Adults of A. ipsilon originated from a laboratory colony established in Bordeaux and transferred to Versailles. The colony is based on field catches in southern France and wild insects are introduced each spring. Insects were reared on an artificial diet [51] in individual cups until pupation. Pupae were sexed, and males and females were kept separately in an inversed light/dark cycle (16 h light: 8 h dark photoperiod) at 22°C. Newly emerged adults were removed from the hatching containers every day and were given access to a 20% sucrose solution ad libitum. The day of emergence was considered as day-0.

All tissue dissections were performed at mid-scotophase when males respond maximally to the sex pheromone [49]. For AipsDopEcR cloning and tissue/age-related expression, legs, wings, brains, ALs, antennae, thorax and abdomen of males were dissected under Ringer’s solution, then immediately deep frozen in liquid nitrogen and then stored at −80°C until treatment. For the collection of ALs, brains were first dissected, then ALs were cut from the protocerebrum with a pair of fine scissors under saline solution, and immediately dipped in Eppendorf vials kept in liquid nitrogen, then stored at −80°C.

RNA Isolation and cDNA Synthesis

Total RNAs were extracted with TRIzol reagent (TRI Reagent®, Euromedex) according to the manufacturer’s instructions, and were quantified by spectrophotometry at 260 nm. DNase treatment was performed (2 Units TURBO™ DNase, Ambion) during 30 min at 37°C followed by an inactivation of 10 min at 75°C. After DNase treatment, single stranded cDNAs were synthesized from total RNAs (1 µg) with SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions.

Cloning of A. ipsilon DopEcR

Degenerate DNA primers were designed on the basis of conserved amino acid sequences of Apis mellifera (accession number XP_396491.1), Drosophila melanogaster isoform A (accession number NP_647897.2) and Tribolium castaneum (accession number XP_968380.1) DopEcRs.

PCRs were carried out with 200 ng of brain cDNA with 2.5 units of High Expand Fidelity DNA polymerase (Roche). The degenerate primers DAEcd dir (5′- TGGAGGCBCTCAYSMAGGC-3′) and DAEcd rev (5′- GCCATRTTSCCCCASTCSAGCAT-3′) were added thereafter at 0.4 µM and each dNTP at 0.25 mM. Following an initial 5 min denaturation at 94°C, the thermal amplification procedure included 35 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, elongation at 72°C for 30 s and then final elongation at 72°C for 10 min.

The 5′ and 3′ regions of the corresponding cDNA were obtained by 5′- and 3′-RACE (SMART RACE cDNA Amplification Kit, Clontech) following the manufacturer’s instructions. For 5′-RACE, we used a specific reverse primer DopEcR5′-RACE (5′- CCCCAGTCCAGCATGCATATAAAC-3′) and Universal Primer Mix (UPM, Clontech) as the forward anchor primer. The 3′-RACE amplification was carried out with UPM as the reverse primer and a specific forward primer DopEcR 3′-RACE (5′- GGAAAATGTCTTTCGACGGAAGTC-3′). Touchdown PCR was performed using hot start as follows: after 1 min at 94°C, five cycles of 30 s at 94°C and 3 min at 68°C, then five cycles of 30 s at 94°C, 30 s at 66°C and 3 min at 72°C, then 25 cycles of 30 s at 94°C, 30 s at 64°C and 3 min at 72°C, then 10 min at 72°C.

PCR products were purified by agarose gel electrophoresis (NucleoSpin® Extract II, Macherey-Nagel GmbH & Co. KG, Düren, Germany) and cloned into PCRII -Topo plasmid (Invitrogen, Carlsbad, CA, USA). After colony isolation, DNA minipreps were prepared (NucleoSpin® Plasmid DNA Purification, Macherey-Nagel GmbH & Co. KG, Düren, Germany) and the DNA clone containing the proper insert was then sequenced (GATC Biotech SARL, Marseille, France). By merging the overlapping sequences obtained from the 5′- and 3′-RACE, a putative full-length cDNA of 2126 bp was generated and named AipsDopEcR.

PCR and qPCR

PCR was performed on 75 ng of cDNA preparations from various tissues with High Expand Fidelity DNA polymerase (Roche). Specific primers DopEcR dir (5′-CGTTGACCGTTATCTAGCGTTCG-3′) and DopEcR rev (5′-TTATATGCATGCTGGACTGGGG -3′) were added at 0.2 µM, and each dNTP at 0.2 mM to perform PCR reactions. Following an initial 3 min denaturation at 94°C, the thermal amplification procedure included 35 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, elongation at 72°C for 30 s, and then final elongation at 72°C for 10 min. Amplification products (198 pb) were loaded on 1.5% agarose gels and visualized with Gel Red.

Real-time qPCR was done on cDNA from brains using the SYBR green detection system and LightCycler 480 Real-Time PCR System (Roche) in the technical platform of The Integrative Biology Institute (University Pierre et Marie Curie, France). Tissues were isolated from 1-day- to 5-day-old males, and RNA was extracted as described previously. Data were analyzed with Light Cycler iQ software and geNORM Visual Basic application for Microsoft Excel [52]. The standard curve was analyzed for all primers and gave amplification efficiencies of 90–100%. The control used was the A. ipsilon ribosomal protein RpL8 (accession number JX975720.1) whose expression was previously analyzed to be invariant considering the age of the moth. The expression of AipsDopEcR was normalized to geometric means of this reference and the normalized gene expression was then calculated with Q-Gene software [53].

The sequences for specific primers of Aips-RpL8 are: Aips-RpL8dir: 5′-CCAGTTTGTCTACTGCGGCAA-3′, and Aips-RpL8rev: 5′-GCTTAACCCTAGTACGCTTGGCA-3′.

Protein Extraction and Western Analysis

Tissues were ground with a Polytron in 100 µl of lysis buffer: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) supplemented with Complete Mini Protease Inhibitors (1 tablet per 10 ml of lysis buffer, Roche). The homogenate was centrifuged during 10 min, 10,000 rpm at 4°C, and the supernatant, which contained the protein extract was quantified using the BCA assay method [54]. Samples with equal protein contents (10 µg) were adjusted to an equal volume with 50 mM Tris (pH 6.8), containing 2% SDS, 8% glycerol and 2% 2-mercaptoethanol with Bromophenol Blue as a marker, and then boiled for 3 min. Protein extracts were separated by 10% SDS-PAGE and were transferred to nitrocellulose membranes (Whatman® Protran® BA85, pore size 0.45 µm), using an electrophoretic transfer system (BioRad). Membranes were saturated 1 h at room temperature with TBS-T (Tris 1 M pH 7.4, NaCl 5 M, Tween 20) 5% Milk powder (Régilait) then incubated overnight at 4°C and 1 h at room temperature with the following primary antibodies: polyclonal anti-DopEcR raised in rabbit against the C-terminal peptide motif CD18: CRSKGRLAAELLGLDNDD of A. ipsilon (1∶10,000, Proteogenix) or monoclonal mouse anti-α-Tubulin (1∶640,000, Sigma T5168 isotype IgG-1). Two washes of 20 min were performed with TBS-T and the respective secondary antibody diluted in TBS-T containing 2.5% Milk powder 1 h at room temperature followed by two washes. Immune complexes were detected using a horseradish peroxidase-conjugated secondary antibody (1∶10,000 anti-rabbit IgG for DopEcR (Sigma A0545) and 1∶10,000 anti-mouse IgG for anti-α-Tubulin (Sigma A2304)). The reaction was visualized, and revealed in a dark room by 6 min exposure on photosensitive film (GE Healthcare ECL Amersham Hyperfilm™). For each Western blot α -Tubulin was used as control.

Immunostaining

Paraformaldehyde-fixed (4%) whole brains were incubated 5 days in primary AipsDopEcR antibody diluted 1∶500 in phosphate buffered saline containing Triton X (PBST) at room temperature. After washing in PBST, the secondary antibody (Alexa fluor 555 goat anti-rabbit IgG) diluted 1∶150 in PBST was added for 4 days at 4°C. Finally samples were washed in PBST and cleared and mounted in Vectashield mounting medium (Vector Laboratories, ABCYS, France). Whole-mount fluorescent preparations were scanned with a confocal laser scanning microscope (Leica TCS SPE, Leica Microsystems, Heidelberg, Germany) using a HC PL APO CS 10.0×objective under an excitation wavelength of 555 nm to generate an emission peak at 565 nm. Partial 3D projections of optical sections were prepared using ImageJ (National Institute of Health, Bethesda, MD, USA) software. Numbers of immuno-stained cell bodies were determined manually by scrolling through stacks of optical sections.

dsRNA Synthesis, Injection and Validation

For preparation and injections of dsRNA, and for the choice of the time delay between injections and dissections/tests, we followed the protocol used to prepare a dsRNA targeting a neuropeptide present in another moth species, Spodoptera frugiperda [55]. The double stranded RNA designed against DopEcR (586 bp) or LacZ (372 bp) were produced with MEGAscript® T7 High Yield Transcription Kit (Ambion) according to manufacturer’s instructions. A PCR was performed on 1 µL of plasmid (50 ng/ µL) with specific primers of each target gene DopEcR T7 dir/DopEcR T7 rev and LacZ T7 dir/LacZ T7 rev under the following program: 35 cycles of 95°C 30 s, 60°C 30 s, 72°C 1 min. The sequences of the four specific primers are:

DopEcR T7 dir: 5′-taatacgactcactatagggTAGCAGTTCGGAAGCCTCTC-3′;

DopEcR T7 rev: 5′-taatacgactcactatagggTATGTCTGACGGCGTGTTGT-3′;

LacZ T7 dir: 5′-taatacgactcactatagggATGACCATGATTACGCCAAGC-3′, and.

LacZ T7 rev: 5′-taatacgactcactatagggCCATTCGCCATTCAGGCTGCG-3′.

PCR products were purified with Nucleospin® extract II kit (Macherey Nagel) and quantified by spectrometry. Then the transcription reaction was realized at 37°C overnight on 1 µg of PCR product in a reaction mix containing 2 µL of each nucleotide, 2 µL of T7 RNA polymerase enzyme and 2 µL T7 10X Buffer for a final volume of 20 µL. After spectrometry quantification and gel dock analysis, the reaction mix was incubated during 15 min at 37°C with 2 units of Turbo DNase (Ambion). dsRNA precipitation was performed by addition of 30 µL DEPC water and 20 µL LiCl, stored at−20°C during 2 h, and centrifuged 30 min at 16,000 g. The pellet was washed with 1 mL ethanol 75%, centrifuged and dried before elution in 11 µL DEPC water. Samples were denaturated at 95°C during 5 min followed by a rehybridization step of 1 h 30 min at room temperature. Then, dsRNA integrity was checked by loading on agarose gel. Before injection, dsRNA was diluted at 0.5 µg/ µL in saline solution (CaCl2 7.1 mM, Na2 β-glycerophosphate 22 mM, MgSO4 13.5 mM, MgCl2 26.9 mM, KCl 29.5 mM, Glucose 23.9 mM, pH 6.8). qPCR and Western blot analysis were performed on treated samples in order to validate silencing efficiency.

A first series of 1-day-old adult males were injected with 1 µg dsRNA into the abdomen, and brains were dissected at day-5 for cDNA synthesis and protein extraction to validate treatment efficiency by qPCR and Western blot analysis. A second series of 1-day-old males were injected as described above, and their behavioral response to sex pheromone was tested at day-5 in a wind tunnel. For both series of experiments, control groups consisted of bacterial LacZ-dsRNA-, Saline-, and non-injected males.

Wind Tunnel Experiments

Behavioral tests were performed using a 2 m-long flight tunnel during the middle of the scotophase (4–7 h after lights off) under red light illumination as previously described [49]. Environmental conditions during the bioassay were held constant: 22°C, 50±10% relative humidity, wind speed of 0.3 ms^–1. A cage containing a single experimental male was introduced in the wind tunnel. After 30 s, during which the male adjusted to the airflow, a filter paper containing the stimulus was placed 160 cm upwind from the cage. Pheromone stimulation was performed with an artificial pheromone blend containing (Z)-7-dodecen-1-yl acetate (Z7–12:OAc), (Z)-9-tetradecen-1-yl acetate (Z9–14:OAc), and (Z)-11-hexadecen-1-yl acetate (Z11–16:OAc) (Sigma Aldrich, Saint-Quentin Fallavier, France) at a ratio of 4∶1∶4 [56], [57]. Ten ng of pheromone blend were used for all behavioral tests as this dose was shown to give the best behavioral results with sexually mature virgin males [49]. The behavior of the moths (5-day-old AipsDopEcR-dsRNA-injected and control males) was observed for 3 min, and partial flight (half of the distance between the source and the cage), complete flight (within 2 cm of the source) and landing on the pheromone source were considered as an oriented response towards the pheromone. Oriented as well as random flights were counted altogether in order to quantify the general flight activity of insects.

Statistical Analysis

AipsDopEcR expression level means were compared using one-way ANOVA followed by the post hoc Tukey test if results were significant with the significance level α = 0.05. For behavioural experiments, statistical differences between groups of injected experimental males were evaluated using a R X C test of independence by means of a G-test and applying the Williams’s correction [58]. Regarding the statistical comparison of behavioral sequences of the 4 groups of insects, we grouped two behavioural sequences (complete flight+landing) of the AipsDopEcR-dsRNA-treated group because there was a 0 value for “landing” for this group.