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.