We experimentally examined the effect of blood meal digestion time and storage method on the PCR amplification success of host DNA in two species of biting midges (Culicoides nubeculosus and C. sonorensis) using novel vertebrate PCR primers. Individuals from each species were fed fresh horse blood, killed and preserved in 95% ethanol at 13 specific time points, ranging from freshly fed to 96 h post-feeding, in 8-h intervals. At each time point, 30 individuals from each species were knocked down by freezing and stored in ethanol to minimise DNA degradation. An additional 10 individuals at time point 0 were collected to investigate long-term storage effects, thus resulting in 40 individuals at time point 0. In total, 800 midges were collected for blood meal analysis (Table 1).
To examine the effect of storage method, 15 samples from each species at every time point were preserved in 95% ethanol and stored in a − 20 °C freezer, whilst the remaining 15 were stored in ethanol and left at room temperature (22–24 °C). To minimise biases, samples were extracted and underwent PCR amplification in a randomised order across all time points and storage methods. To examine the impact of long-term storage on DNA integrity, two sets of five individuals of each species from the first time point were preserved in ethanol and stored under the two temperature conditions (− 20 °C freezer and at ambient room temperature), respectively, and left for 9 months before extraction and amplification. Extraction and amplification of all samples, excluding the long-term storage sets, took place across a 3-month period following sample preparation.
Midge rearing
Culicoides sonorensis and C. nubeculosus specimens were obtained from lines maintained in existing closed colonies at the Pirbright Institute. The C. nubeculosus colony was established in 1969 from pupae collected in Hertfordshire, UK. The C. sonorensis colony was established from eggs provided by Dr. H. Jones who initiated a laboratory colony in Colorado in 1957. The colonies were maintained following previously developed protocols [16] with adult females fed on commercially supplied horse blood (TCS Biosciences, UK) using a Hemotek blood feeder (Hemotek, UK). Midges were not sugar fed during the 96-h period as a trial conducted prior to the commencement of the study indicated that unfed midges survived beyond the 96-h period (unpublished data). Furthermore, this most accurately represented the behaviour of females in the field following feeding, whereby they rest until oogenesis is complete and then find a suitable habitat to lay [17].
Blood meal analysis
DNA was extracted from individual blood meals using a Qiagen DNeasy Blood and Tissue kit, using the standard protocol with the following minor alterations. Prior to lysis, individuals were homogenised using an MP Biomedicals FastPrep-24 5G homogeniser to release the blood meal from the abdomen. Prior to homogenisation, a single sterile 2.3-mm zirconia/silica agitating ball was placed in each 1.5-ml microcentrifuge tube with 180 µl of Buffer ATL. To minimise Buffer ATL foaming, which may reduce the homogenisation efficiency, 2 µl of Reagent DX was added to each microcentrifuge tube. To increase final DNA concentration, prior to elution, 60 µl of Buffer AE was pipetted onto the Dneasy spin column membrane and allowed to incubate at room temperature for 5 min. Final DNA concentration for all samples was quantified using a Qubit 3.0 fluorometer.
In the absence of a priori host assumptions and the potential presence of mixed blood meals (derived from different host species), the PCR amplification of host genomic DNA from blood meals of wild-caught Culicoides and other haematophagous Diptera requires the use of general vertebrate primers [18]. To facilitate the identification of horse-derived blood meals used in this study and to determine the likelihood of successful amplification in future blood meal metabarcoding studies, we designed primers intended to amplify vertebrate templates on the 16S rRNA gene, whilst excluding invertebrate templates. We downloaded 128 mitogenomes belonging to the classes Aves and Mammalia from the NCBI Genbank database. As these primers are designed for blood meal analysis of a wide range of biting Diptera, we also downloaded five biting Diptera mitogenomes (Anopheles gambiae, Aedes albopictus, Aedes aegypti, Culicoides arakawae and Culicoides imicola). The sequences were MAFFT aligned using the bioinformatics software Geneious Prime (Biomatters Limited, New Zealand). Where DNA is highly degraded, primer pairs targeting short amplicons are preferable. Consequently, this alignment was used to identify two potential 18–22-bp primer binding sites that contained primer-invertebrate annealing site mismatches, which produced a 200-base pair (bp) amplicon. Initial binding site selection was informed by previous work [19]. To ensure binding across all mammal and bird mitogenomes, two degenerate bases were inserted into the forward primer to account for single base mismatches. Primer protocol selection and validation were conducted using gradient PCRs with a range of known mammalian and avian DNA templates (Additional file 1: Text S1.). As the PCR primers were designed for use with a wide range of biting Diptera species, examination of blood meal amplification success and identification of non-specific binding was conducted on genomic DNA from blood fed and unfed C. sonorensis and An. gambiae. I. Fed and unfed C. sonorensis were obtained from the colonies at the Pirbright Institute. Anopheles gambiae individuals were wild caught and stored in 95% ethanol at field sites in Burkina Faso as part of ongoing research by members of the Target Malaria research consortium (see Acknowledgements). The PCR product of blood-fed C. sonorensis and An. gambiae were Sanger sequenced (Source Bioscience) to confirm successful target amplification.
The presence of sufficient concentrations of non-degraded host DNA template for PCR amplification was assessed using end-point PCR and our novel primer set, 16smbF (5′-GGT TGG GGY GAC CTY GGA-3′) and 16sbbR (5′–CTG ATC CAA CAT CGA GGT CGT A-3′-). PCR amplification was carried out in 25 μl reactions that contained 12.5 μl HotStarTaq Master mix (Qiagen, Germany), 10 μM of each primer, 8.5 μl H2O and 2 μl DNA template. The PCR protocol consisted of an initial denaturation step of 15 min at 95 °C followed by 35 cycles of 94 °C for 45 s, 58 °C for 45 s and 72 °C for 30 s, followed by a final extension step of 72 °C for 10 min. Two negative controls, containing nuclease-free water (ThermoFisher Scientific, USA), were included in every set of reactions to monitor for contamination.
PCR products were electrophoresed and visualised on a gel red (SYBR™ Safe, ThermoFisher Scientific, USA) stained 2% agarose gel. The presence of a band of the expected amplicon size was taken as a positive result, indicating that sufficient host DNA remained viable for blood meal identification. To confirm successful target amplification, a subset of PCR products from across the range of time steps that yielded bands were Sanger sequenced (Source Bioscience, England) and the sequence’s origin was identified using the basic alignment search tool (BLAST).
Statistical analysis
We modelled the effect of digestion time, storage method and species on a binary measure of amplification success (1 or 0) using a multivariate binomial logistic regression. Model fit was evaluated with a likelihood ratio test and the significance of the overall effect of each variable was determined using the chi-squared statistic [5]. Data were analysed using R (version 4.1.2) and visualised using the package ggplot2 [20].
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