Materials and methods

Insects

Green budworm moth Hedya nubiferana Haworth (dimidioalbaRetzius) (Lepidoptera, Tortricidae) (Figure 1) is a polyphagous leafroller on Rosacean trees and shrubs and co-occurs with codling mothCydia pomonella on apple, throughout the Northern hemisphere. The larvae feed on fruit in autumn and on flower buds in the spring (Bradley et al. 1979).
For pheromone analysis, last-instar larvae were field-collected in apple orchards in Scania (Sweden) during May. Larvae were fed with apple leaves and a semisynthetic agar-based diet (Rauscher et al. 1984). Pupae and adults were kept under a 18:6 h light-dark cycle in screen cages and were supplied with fresh apple branches and sucrose solution. For transcriptomic studies, H. nubiferana males were captured in pheromone traps baited with a 10:1:5-blend of (E ,E )-8,10-dodecadienyl acetate (E 8,E 10-12Ac), (E )-8-dodecenyl acetate (E 8-12Ac) and Z 8-12Ac. Live males were taken to the laboratory and used for antennal dissection.

Pheromone gland extraction and chemical analysis

Female abdominal sex pheromone glands were dissected at the onset of the calling period, towards the end of the scotophase. Glands of 2- to 4-d old females were extracted in batches of 5 to 15 in 7 µL of redistilled hexane for 1 min (Bäckman et al. 1997). Identification of female gland compounds by coupled gas chromatography-mass spectrometry (GC-MS) was done on a Hewlett Packard 5970 B instrument, with electron impact ionization (70 eV), interfaced with a Hewlett Packard 5890 GC. Helium was used as carrier gas on a 30 m x 0.25 mm DB-Wax column (J&W Scientific, Folsom, CA, USA), programmed from 80°C (hold 2 min) at 10°C/min to 230°C. The compounds were identified by comparing retention times and mass spectra of natural and synthetic compounds. Double bond position was determined by co-injection with synthetic samples and by evaluation of mass spectra.

Field trapping

The geometric isomers of E 8,E 10-12Ac andE 8,E 10-12OH were synthesized (Witzgall et al . 1993). All other compounds were purchased from S. Voerman (Institute for Pesticide Research, Wageningen, The Netherlands). Purity of synthetic pheromone compounds was ≥96.2 % (chemical) and ≥99.7 % (isomeric). Compounds in hexanic solution were formulated on red rubber septa (Merck ABS, Dietikon, Switzerland), which were replaced every 2 weeks. Tetra traps (Arn et al. 1979) were hung in apple trees at eye level, and were ca. 5 m apart within one replicate. Traps were placed in untreated apple orchards at Alnarp, Scania (Sweden) and at Halásztelek, Pest county (Hungary) and checked twice a week.
Further traps were placed in orchards treated with commercial pheromone dispensers for mating disruption of codling moth. These dispensers were polyethylene tubes containing 87 mg E 8,E 10-12OH, 49 mg 12OH and 10 mg 14OH (Shin-Etsu Chemical Co., Tokyo), they were applied at a rate of 1000/ha.
For statistical analysis, trap captures were transformed to log(x+1) and submitted to a 2-way ANOVA, followed by Tukey’s test.

Wind tunnel

The wind tunnel had a flight section of 63 × 90 × 200 cm (Witzgall et al. 2001). Air was blown by a horizontal fan onto an array of activated charcoal cylinders. The wind tunnel was lit diffusely from above at 6 lux, the wind speed was 30 cm/s, and the temperature ranged from 22 to 24°C. Two-day-old males were transferred to glass tubes (2.5 × 12.5 cm) stoppered with gauze before testing. Males were flown individually, in batches of 15, to one test stimulus. Two batches of 15 males were tested on one day, 1 to 3 h after onset of the, each blend was tested four times (n = 60 males), on different days. The following types of behaviour were recorded: taking flight, flying upwind over 100 cm towards the source, and landing at the source.

Dissection of antennae and RNA extraction

Antennae of 100 adult males were dissected with forceps and transferred into a 1.5-mL microcentrifuge tube (Eppendorf, Hamburg, Germany) held in liquid nitrogen. Thereafter, 500 µL of Trizol were added to the excised antennae.
Total RNA was extracted and purified following Trizol-based extraction protocol and spin column purification with the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). Briefly, antennae held in the Eppendorf tube with Trizol were manually homogenized with a pestle. The tube was placed in liquid nitrogen and then allowed to thaw at room temperature. The sample was then homogenized again with a pestle and another 500 µL of Trizol were added to the tube. The tube was vortexed and incubated at room temperature for 5 min, 200 µL of chloroform (Riedel de Haen, Seelze, Germany) was added to the sample and the tube was vortexed again for 20 s and incubated at room temperature for 15 min. Samples were centrifuged at 4°C for 15 min at maximum speed. The aqueous upper phase was transferred to a clean 1.5-mL centrifuge tube and an equal amount of 100% isopropanol (Sigma Aldrich, Saint Louis, MO, USA) was added along with 3 µL of 5 mg/mL of glycogen (Life Technologies, Carlsbad, Ca, USA). Samples were mixed by inversion a couple of times and stored at -20°C overnight.
The next day, the sample was centrifuged at 4°C for 15 min at maximum speed. The supernatant was decanted and the excess of liquid extracted with a pipette without disturbing the pellet, 1 mL of cold 70% ethanol was added to the pellet sample and centrifuged at 4°C for 10 min at 7500 RCF. Supernatant was discarded and 100 µL of RNAse free water (Life Technologies, Carlsbad, Ca, USA) was added to the tube. Extracted RNA was then purified with the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands); 350 µL of Buffer RLT and 250 µL of 100% ethanol were added to the sample. The sample was transferred to RNeasy spin columns and the RNA was fixed to the filter membrane via centrifugation at room temperature for 15 s at 10000 RCF. According to manufacturers recommendation, RNA purification was completed with the RNA Cleanup Protocolol, including an on-column DNase digestion, performed with the RNase Free DNase system (Qiagen). Total RNA was quantified with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

RNA sequencing and bioinformatics

RNA sequencing at the National Genomics Infrastructure (NGI, Uppsala, Sweden) followed the standard protocols for Illumina Sequencing (Illumina, CA, USA), sequence read files were sent to UPPMAX Computational Science Server (Uppsala, Sweden). Two .fq files were produced, one containing all left-pair reads and another containing all right-pair reads.
The .fq files were used as a starting point to assemble the transcriptome, annotate the genes and calculate their expression (see Walker et al. 2016). Quality control analysis was performed using the software Trimmomatic (version 0.32), and all reads with a PHRED score lower than 20 were removed. Processed reads were then assembled,de novo , into one transcriptome using Trinity (version r2014717; Grabherr et al. 2011). Cd-hit-est (version 4.5.4-2011-03-07), was used to identify and remove redundant sequences that share 98% or greater identity with other sequences (Li and Godzik 2006). The processed transcriptome was used to compare and annotate gene transcripts according to their homology to protein sequences of C. pomonella(Walker et al. 2016), using blast (version 2.2.29). Top blast hit transcript clusters with similarity to putative pheromone receptors ofC. pomonella were extracted and translated into protein sequence with the ExPASY web translate tool (Artimo et al. 2012). Translated sequences with open reading fragments (ORFs) shorter than 50% of the average length of a OR (428 amino acids) were excluded from analysis. Sequences were aligned to putative PRs from C. pomonella (Walker et al. 2016) and all new putative PRs from H. nubiferana were named according to the closest homolog of C. pomonella .
To estimate the expression of these putative PRs in the antennae the RSEM software package (version 1.2.12; Li and Dewey 2013), including Bowtie (version 0.12.6; Langmead et al. 2009) and Samtools (version 0.1.19; Li et al. 2009) were used, allowing measurement of transcript abundance estimates as fragments per kilobase of transcript per million mapped reads (FPKM) (Trapnell 2010).

Phylogenetic analysis

Sequences of predicted pheromone receptors from C. pomonella(Walker et al. 2016), Epiphyas postvittana (Corcoran et al. 2015), Grapholita molesta (Li et al. 2015) and Bombyx mori(Krieger et al. 2005), were used for comparison with putative PRs ofH. nubiferana . All amino acid sequences were aligned using MAFFT online (version 7.220; http://mafft.cbrc.jp/alignment/server/phylogeny.html) with the FFT-NS-i iterative refinement method, with JTT200 scoring matrix, and default parameters. Aligned sequences were used to calculate the best fitting model for comparison in MEGA6 software (Tamura et al. 2013). Then, a Maximum Likelihood Tree was constructed using the JTT+F+G model with bootstrap support inferred from 500 replicates.