Mosquito rearing
Experiments utilised a mixed colony of Cx. pipiens biotypes originating from an allotment area in Brookwood, Surrey, UK established in 2011 and identified as the ‘Brookwood’ line [11]. Five to seven days old adults from this colony line were offered defibrinated horse blood (TCS Biosciences, UK) overnight using a Hemotek™ membrane feeding system (Hemotek™ Ltd., UK). Five days after blood feeding, egg rafts were collected from oviposition cups and left to hatch in approximately 500 ml of fresh tap water. Larvae were reared at a density of 200 larvae/litre (L) water and maintained on a diet of 1 milligram (mg) of guinea pig pellets/larva (Pets at Home, UK) on alternate days. Larvae were reared in an environmentally controlled incubator maintained at a temperature of 25oC ± 1oC and relative humidity of 50% ± 1% and were exposed to a lighting regime of 16:8 hours light: dark. Conditions in the incubator were monitored using a HOBO™ U12-012 temperature/relative humidity/light data logger (Measurement Systems Limited, UK). Daylight period light intensity was approximately 3500 lux whilst light intensity during the night-time period was 3.5 lux. Dusk and dawn were simulated by either an increase or decrease in light intensity, respectively, each over a one-hour period.
Pupal exuviae sample collection and preparation
Following the onset of pupation, pupae were collected daily and placed into separate 2 ml tubes containing 1 ml of water. To compare the efficacy between DNA extraction methods, pupal exuviae and emerged adult mosquitoes were placed in 70% ethanol immediately following eclosion and stored individually at room temperature prior to DNA extraction. To assess the effect of time following eclosion on DNA extraction efficiency, adult mosquitoes and their corresponding pupal exuviae were placed individually into 70% ethanol at defined time points, post-eclosion (0, 1, 6, 12, 18 and 24 hours). Pupal exuviae and adult mosquitoes were then removed from ethanol and left to air dry for approximately 10 minutes prior to DNA extraction. Comparisons of DNA quantity between different processing times post-eclosion were assessed using Kruskal-Wallis with Bonferroni correction for multiple comparisons. For all statistical tests, the statistical significance level was set at P < 0.05 and analyses were computed in R studio version 1.2 [39].
Use of pupal exuviae as a non-invasive method of sampling field populations
Two periods of pupal exuviae collection were undertaken in the field, each spanning two weeks. The first collections were performed in August 2020 and used for comparison between individual and pooled processing approaches. The second collection period was between June to July 2021 and used to validate processing methods in field collected samples. The oviposition site in the field was created using a 20 L polypropylene black bucket of 28.3 cm x 47.8 cm x 33.0 cm (HxWxD), filled with 10 L of tap water and seeded with 5 g crushed guinea pig pellets (Pets at Home, UK) to attract gravid female Cx. pipiens. The bucket was placed in a residential garden in Guildford, Surrey (51°240'N; -0°578'W). Pupal exuviae floating on the surface were collected and placed into 70% ethanol each morning over a period of two weeks and transferred to the laboratory for processing. Samples were either processed individually or in pools containing five exuviae using the ethanol precipitation method described below. Water temperature and light intensity in the bucket were measured using a HOBO™ temperature/light weatherproof pendant data logger (Measurement Systems Limited, UK). Mann-Whitney U tests were used to assess the effect of pooling or year of collection on nucleic acid concentrations from field collected pupal exuviae.
DNA extraction method comparison
Three extraction methods and a direct real-time PCR processing method were chosen to compare DNA yield and subsequent PCR amplification success: an ethanol precipitation method and two commercially available kits, DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK), hereafter referred to as DNeasy® and Wizard® respectively. For each of the extraction methods, total genomic DNA was isolated from 20 individual pupal exuviae alongside a distilled water negative control, with ten males and ten females processed for each method. DNA extracted using the same method from the corresponding adult heads were additionally included as positive controls.
Ethanol Precipitation: DNA was extracted as follows: samples were placed into 200 µl of digestion solution comprised of 100 mM UltraPure™ 1 M Tris-HCL (pH 8.0) (Invitrogen™ by Thermo Fisher Scientific, UK), 200 mM NaCl (Invitrogen™, UK), 0.2% (w/v) SDS (Merck Life Science UK Limited, UK), 5 mM UltraPure™ 0.5 M EDTA (pH8.0) (Invitrogen™, UK), 200 µg/ml proteinase K (Qiagen, UK) made up to a total volume of 200 µl per sample using UltraPure™ water (Invitrogen™, UK). Samples were incubated overnight at 37oC before proceeding with DNA extraction. Immediately following incubation, 500 µl of ice-cold 100% ethanol, 20 µl 3 M NaOAc pH 5.5 (Invitrogen™, UK) and 2 µl of GlycoBlue™ coprecipitant (15 mg/ml) (Invitrogen™, UK) were added to each sample and incubated at -20oC for 1 hour. DNA was pelleted by centrifugation at 14,000 rpm at 4oC for 30 minutes and supernatant was removed. Pellets were then washed in 400 µl 70% ethanol, re-pelleted by centrifugation under the same conditions for 15 minutes and supernatant was removed. Pellets were air-dried for 20 minutes to allow for the evaporation of excess ethanol and resuspended in either 15 µl or 200 µl nuclease free water for pupal exuviae and adult samples respectively.
Commercially available kits
DNA was extracted according to the manufacturer’s instructions with the following modifications to enhance DNA yield [40, 41]. Mosquito exuviae were subjected to a homogenisation stage within the digestion solution detailed in each of the kit’s instructions using 3 mm stainless steel homogenisation beads (Qiagen, UK) for 1 minute at 30 hertz using a Qiagen tissue lyser (Qiagen, UK). Samples were then incubated overnight at 56oC prior to DNA extraction. To maximise DNA yield from exuviae samples, 30 µl of elution buffer was incubated on the spin column membrane at room temperature for 5 minutes prior to elution. For DNA extraction from mosquito heads, elution was performed twice in 30 µl for a total elution volume of 60 µl.
Direct real-time PCR
samples were prepared according to Thongjued et al. [42] with minor adjustments to sample preparation. Briefly, pupal exuviae were removed from ethanol and left to air-dry for approximately 10 minutes before being placed in 20 µl PBS (pH 7.4) (Gibco™ by Thermo Fisher Scientific, UK). Samples were vortexed before incubating at 98oC for 4 minutes and 2 µl of supernatant was added directly to the PCR mix.
In all samples, prior to PCR processing, nucleic acid concentrations were measured for all extraction methods using the Qubit® dsDNA HS assay kit (Invitrogen™, UK) and read by a Qubit® 3.0 fluorometer (Invitrogen™, UK). For all samples, 2 µl of DNA template were used in 198 µl of the dsDNA HS assay. Variation in DNA yield between processing methods for both colony and field collected exuviae were assessed using a Kruskal-Wallis with Bonferroni correction for multiple comparisons.
Differentiation of Culex pipiens biotypes
Mosquitoes were simultaneously assigned to species and biotype level using a real-time PCR assay originally designed by Rudolf et al. [9] with minor adaptations to primer concentrations. Reactions were performed in 10 µl reaction volume consisting of 5 µl TaqMan™ multiplex master mix (2x) (Applied Biosystems™ by Thermo Fisher Scientific, UK), 0.3 µM CxPipF, 0.4 µM CxPipR, 0.2 µM CxPipP, 0.2 µM CxPipPipP, 0.2 µM CxPipMolP, 0.15 µM CxTorrF, 0.15 µM CxTorrR R, 0.1 µM CxTorrP, 0.16 µl BSA (20 mg/ml) (Merck Life Science UK Limited, UK), 1.14 µl UltraPure™ water (Invitrogen™, UK) and 2 µl DNA extract. Sequences for the primers and probes are shown in Table 1. The thermal profile started with an initial activation step of 95oC for 20 seconds, followed by 40 cycles of 95oC for 3 seconds and 60oC for 1 minute using a QuantStudio™ 7 Flex Real-Time PCR machine (Applied Biosystems™, UK). All samples were run alongside positive controls consisting of pure f. pipiens, f. molestus, hybrid and Cx. torrentium DNA as well as negative controls including extraction and PCR negative controls. Real-time PCR amplification success was defined by the number of samples for which the quantification cycle (Cq) value was below the assay cutoff of 39. Any samples not producing a Cq value were assigned a value of 40 for analysis. Cq values obtained from each of the probes for each sample were averaged for use in analysis. Variation in PCR amplification success between processing time post-eclosion or processing method was assessed with pairwise chi-squared tests for independence with Bonferroni adjustment for multiple comparisons.
Table 1
Primer and probe sequences for the simultaneous differentiation of Culex pipiens and Culex torrentium species through amplification of the ACE-2 gene as well as the Culex pipiens biotypes by the CQ11 microsatellite locus. Letters in the primer/probe names identify whether they are forward (F) primers, reverse (R) primers or probes (P).
Primer/probe name | Primer/probe sequence |
CxPipF | 5’- GCGGCCAAATATTGAGACTT-3’ |
CxPipR | 5’-CGTCCTCAAACATCCAGACA-3’ |
CxTorrF | 5’-GACACAGGACGACAGAAA-3’ |
CxTorrR | 5’-GCCTACGCAACTACTAAA-3’ |
CxPipP | 5’-VIC- GGAACATGTTGAGCTTCGG-QSY-3’ |
CxPipPipP | 5’-ABY-GCTTCGGTGAAGGTTTGTGT-QSY-3’ |
CxPipMolP | 5’-JUN-TGAACCCTCCAGTAAGGTATCAACTAC-QSY-3’ |
CxTorrP | 5’-FAM-CGATGATGCCTGTGCTACCA-QSY-3’ |
DNA extraction cost and handling time
The cost of the extraction method for each sample was estimated based on the retail price of the chemicals or kits used in the UK. The handling time for each sample was calculated as the time required to complete all processing, starting from dehydration of the pupal exuviae, to obtaining a DNA extract ready for PCR. This was replicated three times with each round containing 10 individuals and averaged.
Wing length measurements
Wing length was measured as a proxy for body size to control for specimen size variation for the method comparison and time trial experiments [43]. Wings from sampled individuals were transferred to a piece of paper towel dampened with 70% ethanol, flattened, and left to air dry for approximately 5 minutes. Wings were then transferred to a strip of Scotch Magic Tape™ with both wings from one adult placed together and placed on a glass microscope slide for imaging. Wing images were visualised using a Leica EZ4HD microscope (Leica Microsystems, Germany) alongside a stage micrometer for scale where one division is equal to 0.01 mm. Images were subsequently processed for size measurement using ImageJ [44]. Wing length was measured from the axillary incision to the apical margin, excluding the fringe (Fig. 1) [45]. Measurements were taken from both wings of the same adult and averaged for use in analysis. Correlations between concentration, wing measurement and Cq values were assessed using Spearman’s correlation.
Creation of single biotype colony lines
Adults from the Brookwood colony line (F97) were offered defibrinated horse blood (TCS Biosciences, UK) overnight using a Hemotek™ membrane feeding system sealed with parafilm (Hemotek™ Ltd., UK). Approximately five days after blood-feeding, egg rafts were collected and separated into individual 25 ml pots in approximately 15 ml of dechlorinated tap water for hatching. Larvae from each egg raft were reared separately under the same conditions described above. Following the onset of pupation, pupae were collected daily into individual 2.0 ml tubes and monitored for eclosion. Pupal exuviae were collected into 70% ethanol at least every twelve hours and stored prior to processing. Exuviae from emerging adults were processed for DNA extraction by ethanol precipitation as described and biotype identified daily. Adult mosquitoes were subsequently allocated to separate colony cages (Bugdorm, Watkins and Doncaster, UK) according to biotype with approximately 80–100 individuals per biotype used to create single biotype colonies. The colony lines were subsequently maintained according to Manley et al. [11]. After 10 generations, 40 adults from each line were tested to confirm the lines remained homologous or heterozygous as determined by the CQ11 microsatellite marker. Heads of adult mosquitoes were used to obtain genomic DNA using the Wizard® SV Genomic DNA Purification System (Promega, UK) and biotype characterised, as described above.