Field collections of adult Culicoides were conducted across five sites in the UK (Figure 1) at different times of the year (Additional File 1: Table S1). In South East England, three equine holdings (site 1: 51°08’46.5”N, 0°36’51.6”W; site 2: 51°17'26.2"N, 0°39'09.0"W; site 3: 51°17'00.3"N, 0°36'24.6"W) were used for collections in June – August 2017 (South East England summer cohort; SES) and again for collections in September – October 2017 (South East England autumn cohort; SEA). All three sites were predominately used for grazing, with animal stabling and muck heaps present at each site. In North East England, collections were made from a forest campsite (site 4: 55°14'13.1"N, 2°35'12.1"W) during July 2018 (North East England summer; NES). During the same month, a collection was also made at a mixed farm in the Scottish Borders (site 5: 55°15'44.5"N, 2°41'16.4"W) (Scottish Borders summer cohort; SBS).
Insects were collected using down-draught miniature blacklight (UV) Centers for Disease Control (CDC), model 912 traps (John W Hock Co, Gainesville, FL, USA). Each CDC trap uses a 4W UV tube and is powered by a 12V lead acid sealed battery (Yuasa, Japan). At each site, between four and eight traps were used in order to catch sufficient Culicoides for the trials and were positioned at least 50m apart . Traps were suspended at a height of approximately 1.5m above ground. Traps were set up at least two hours before sunset and run overnight before collection within two hours of sunrise the following day. Insects were collected live into a 340ml cardboard collection cup containing a cotton pad soaked in 10% sucrose to provide a sugar source and paper towel cut into long, thin strips to provide shelter from the trap fan downdraft. Cardboard collection cups were secured to the CDC trap using a CDC sleeve and elastic bands (Additional File 2: Figure S1). The following morning, sleeves from each trap were removed and tied with a clip-lock tie to contain the insects within the collection cup and sleeve.
Flight activity experiments
Insects collected were transferred and released into a large plastic, black box (42cm (l) x 35cm (w) x 32cm (h)) with a translucent funnel (14cm in length) attached on one side to allow for insects to exit. Clear plastic tubing (6cm in length) was secured to the translucent funnel to visualise and count approximately 150-250 active, phototactic adult Culicoides exiting the box and entering a ‘flight activity pot’ (Figure 2). The flight activity pots were modified 340ml cardboard collection cups with a small plastic funnel (66mm top diameter; 7.4cm in length) covered in aluminium foil fitted at the top, and a fine mesh covering the bottom of the pot. The total height of the flight activity pot from the base of the cardboard cup to the tip of the funnel was 14cm. A cotton wool bung within the funnel retained all insects within the flight activity pot until the start of the experiments.
Once eight flight activity pots had been filled, they were numbered at random from one to eight. Pots one to six were placed into a ‘test’ temperature-controlled incubator (MIR-254 Panasonic, UK) at an initial temperature of 20±1°C, while pots seven and eight were placed in a second ‘maintenance’ incubator also set at 20±1°C as a control for activity of Culicoides in the sample. Both incubators contained a UV light source (4W blue-blacklight tube; John W Hock Co, Gainesville, FL, USA) suspended within the incubator that acted as an attractant and no other light was provided within the incubators. The test incubator was then set to the desired temperature (2°C – 14°C) and insects were allowed to acclimatise for two hours. Following the acclimatisation period, the cotton wool bung at the top of the flight activity pot funnel was removed and a 7.5cm diameter pill box (Watkins and Doncaster, UK) with a fine mesh lid was secured on top of the funnel allowing active insects to fly towards the UV light stimulus into the attached pill box (Figure 2E). All flight activity pots were placed with the mesh lid faced downwards onto a tray to ensure darkness, so that the UV light was the only light source available above the flight activity pots. A small cotton pad soaked in 10% sucrose was placed underneath each flight activity pot on the tray, rather than within the pot, so that insects could still access the sugar-meal through the fine mesh but were prevented from sticking to the pad.
At regular intervals the collection pill boxes were replaced (0.5, 1, 2, 4, 6, 8, 10- and 24-hour intervals) and the collected Culicoides were killed by freezing at -20°C for a minimum of 2 hours. All remaining inactive individuals in the flight activity pots were also killed by freezing after 24 hours. The percentage of Culicoides flying after 24 hours was determined by calculating the mean cumulative proportion of active Culicoides across the six flight activity pots maintained at each test temperature. In each temperature trial, collections were also made at the same intervals from pots seven and eight, which were maintained at 20±1°C throughout the experiment to ensure the population tested were active without the constraint of cold temperatures. A flow diagram of the full experimental design is provided in Additional File 3: Figure S2. In the SES cohort, five temperatures were tested (6, 8, 10, 12 and 14°C) and the SEA cohort a further five temperatures were tested (2, 4, 6, 8 and 10°C). Four temperatures were tested in the NES cohort (8, 10, 12 and 14°C) and only one temperature of 12°C was tested in the SBS cohort.
Only individuals that were tested in activity experiments were identified, all surplus insects that remained in the initial black plastic sorting box were not used for experimental study. Culicoides from the activity experiments were sorted morphologically under a dissecting microscope using characteristic wing patterns with the aid of an identification key . Adult Culicoides were grouped into six categories: subgenus Avaritia, Culicoides pulicaris Linnaeus, Culicoides punctatus Meigen, Culicoides achrayi Kettle and Lawson, C. impunctatus and other Culicoides. Female Culicoides were also identified to physiological state by examination of the abdomen  and assigned to one of the following categories: unpigmented, pigmented, gravid and blood-fed.
For both South East England cohorts (SES and SEA), a sub-sample of females within the subgenus Avaritia, were identified further to species level (C. obsoletus and C. scoticus only) using an adapted multiplex polymerase chain reaction (PCR) method targeting the internal transcribed spacer (ITS) 1-5.8S-ITS2 region . All individuals belonging to the subgenus Avaritia from one flight pot from each temperature trial was chosen at random for molecular analysis and used as a representative sample for each population at each temperature trial. Culicoides were transferred to individual reaction tubes with 200µl of tissue digest solution containing 100nM Tris-HCl pH8 (Thermo Fisher, UK), 200mM NaCl (Sigma-Aldrich, UK), 0.2%(w/v) SDS (Thermo Fisher), 5mM EDTA (Thermo Fisher), 200µg/mL proteinase K (Thermo Fisher), and nuclease-free water (Thermo Fisher). Following an overnight incubation in tissue digest solution at 37°C, individual Culicoides specimens were transferred to tubes containing 70% ethanol for storage. The Culicoides DNA was then extracted from 100µl of tissue digest solution and eluted into 100µl buffer using the KingFisher Flex automated extraction platform and the MagMAX™ CORE Nucleic Acid Purification Kit (Thermo Fisher) according to manufacturer’s instructions.
Two microliters of each sample DNA was added to each well on a PCR plate (Life Technologies, UK) each containing 8µl of mastermix which consisted of 1x TaqMan Fast Advanced MasterMix (Thermo Fisher), 0.3µM of each primer , 0.2µM of each probe  and diluted to a total reaction volume of 10µl using nuclease free water (Thermo Fisher). Negative extraction controls consisted of elution from wells which did not contain any Culicoides specimen in the extraction plate and negative PCR controls contained just nuclease free water. At least three negative controls and at least three positive controls, using DNA extracted from males from the same study morphologically identified as either C. obsoletus or C. scoticus, were added to each plate.
The PCR thermal profile used consisted of 2 mins at 50°C for activation of uracil-DNA-glycosylases (UDG), an initial denaturation step of 2 mins at 95°C, followed by 40 cycles of 95°C for 3 secs and 60°C for 30 secs and was carried out using an Applied Biosystems 7500 Fast instrument (Thermo Fisher). Each plate was analysed using the ViiA7 Real Time PCR system software (Thermo Fisher). Determination of species for each individual specimen was based on the cycle threshold (Ct) value for each species-specific primer-probe pairing. Negative samples were defined as having a Ct ≥35 and positive samples were defined as having a Ct ≤25. Samples with a Ct between >25 and <35 were regarded as undetermined and were repeated. If samples remained undetermined following re-examination, samples were defined as unknown and were removed from analysis.
Generalised linear mixed models (GLMMs) were used to investigate the relationship between temperature and the proportion of Culicoides flying and how this relationship differed amongst cohorts. Specifically, a binomial family GLMM with a logit link function was constructed with the proportion of Culicoides flying as the response variable. Model selection proceeded by stepwise deletion of non-significant (P>0.05) terms (as judged by likelihood ratio tests), starting from a model including temperature (°C), cohort and an interaction between them as fixed effects and pot as a random effect (to allow for between-pot variation). The models were implemented using the lme4 package  in R (version 3.6.1) .
Separate models were constructed for total Culicoides (all Culicoides, unpigmented females and pigmented females), the Avaritia subgenus (all Culicoides, unpigmented females and pigmented females) and C. impunctatus (all Culicoides and pigmented females). Sample sizes were insufficient to examine relationships for: (i) C. pulicaris, C. achrayi or other Culicoides; or (ii) blood fed females, gravid females or males for any species/groups. In addition, the SBS cohort was excluded from this analysis as activity was only assessed at a single temperature (12°C) for this cohort.
Further models were constructed for Culicoides flight activity at 12°C to compare activity amongst the SES cohort, the NES cohort and the SBS cohort. A similar approach to that described above was used, except the model included cohort as a fixed effect and pot as a random effect. Flight activity in the cohorts were compared using Tukey multiple comparisons.
For the SES and SEA cohorts, there was a sufficient number of Culicoides caught to allow two further analyses. First, to compare flight activity of unpigmented and pigmented females of the Avaritia subgenus a GLMM was constructed including pigmentation state (i.e. unpigmented or pigmented) as a fixed effect, as well as two- and three-way interactions between it and the other fixed effects (i.e. temperature and cohort). Model selection was carried out as described above. Second, to compare flight activity of C. obsoletus and C. scoticus, a binomial family generalised linear model (GLM) with a logit link function was constructed. The proportion of Culicoides flying was the response variable and temperature, cohort and species were fixed effects. Model selection proceeded as described above.