Design and optimisation of rapid real-time PCR assays for the detection of key Culicoides species

I n e x t e n s i v e s u r v e i l l a n c e p r o g r a m m e s o f Culicoides biting midges (Diptera: Cerat opogonidae), morphological identification can be time-consuming and difficult, while DNA barcoding, although highly accur ate, may not be cost-eff ective or suitable for rapid analysis, as it requir es individual specimen processing. T o address these limi t ations, we developed a r apid scr eening method using real-time PCR assay s with either SYBR Gr een or hydroly sis probe-based det ection chemistries. Species-specific primer s and, where necessary , h ydrolysis probes wer e designed based on the updat ed sequences of the ITS2 region of seven Culicoides species. The specificity and efficiency of these assay s were validat ed both in silico and through real-time PCR t esting on t arget and non-target Culicoides .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint species, t ested individually and as mix ed-species samples. The new real-time PCR assay s detect vector species including C. obsoletus , C. scoticus , C. chiopterus , C. dewulfi , C. pulicaris , C. punctatus , and C. impunctatus in pools of individual specimens, with single-specimen sensitivity . The molecular techniques developed in this study provide a valuable tool for accur ate and high-throughput Culicoides surveillance, which can be used for year-round monitoring of adult midges in tr aps and larvae in environment al samples, pot entially revealing novel insights into the spatial and t emporal turnover of Culic oides spe cie s. The se

can be applied to large-scale vector scr eening programmes involving pooled samples, addressing the limitations of pr eviously described methods used in midge surveillance.

Culicoides , Real-time PCR, Species-specific primer s, ITS2 gene region

Molecular-based species identification is often a crucial step in ecological surveillance programmes, whether for biodiversity monitoring (Curran et al. 2022) , surveillance of protected species (Harrington et al. 2019), invasive species (Browett et al. 2020), or species of interest from a One Health perspective, such as vectors of disease (Isiye et al. 2025). Novel real-time PCR assays have been developed and applied across a range of ecological contexts in Ireland and Britain. Applications include species and sex identification of Eurasian otters (Lutra lutra ) from non-invasive samples (O’Neill et al. 2013), large-scale monitoring of native bat species (Harrington et al. 2019), and discrimination between native red squirrels ( Sciurus vulgaris ) and invasive grey squirrels ( Sciurus carolinensis ) (O’Meara et al. 2012). Real-time PCR has also been applied to detect target species from indirect sources of DNA, such as faecal or environmental DNA (eDNA) sources (O’Meara et al. 2014). The versatility and accuracy of real-time PCR detection methods can be useful in various ecological surveillance contexts, including vector surveillance. In Europe, Culicoides biting midges are well recognised as both disease vectors and nuisance pests across various regions (Carpenter et al. 2013; Prudhomme et al. 2025). In Ireland, the majority of commonly occurring Culicoides species are of significant veterinary importance, as they are well-known vectors of bluetongue virus (BTV) and Schmallenberg virus (SBV) (McCarthy et al. 2009; Collins et al. 2018; Jess et al. 2018; Isiye et al. 2025). The .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint identification of Culicoides species has traditionally relied on morphological characteristics, including wing patterns, head structures, and genitalia (Mathieu et al. 2012), a method that has served as the foundation for the identification and classification of insect species. In recent years, DNA barcodin g of mitochondrial and ribosomal gene regions has been increasingly used, providing high accuracy in distinguishing closely related species (Mignotte et al. 2020) and identifying new species (Nielsen et al. 2015; Sarvašová et al. 2017). In large- scale vector surveillance programs, both these methods are too labour-intensive and costly, as they require processing of very high numbers of individual specimens and significant expertise to identify the exact species within the sample collection. On the other hand, DNA metabarcoding, although capable of detecting a broad range of species in complex mixtures or pooled samples (Browett et al. 2021; Curran et al. 2022), often identifies numerous non- target organisms and depends on extensive reference databases, which are still limited for group s su ch a s Culicoides midges. While genus-specific primers have been successfully used to distinguish Culicoides from other Ceratopogonidae (Cêtre-Sossah et al. 2004), species- specific tests would provide a robust approach for simplifying their identification and allowing for targeted surveillance. The first conventional species-specific PCR tests to be developed targeted Culicoides imicola Kieffer, 1913, as it was considered to be the primary vector of BTV and African horse sickness virus (AHSV) in southern Eur ope and endemic regions of Africa (Mellor & Boorman, 1995; Miranda et al. 2003; Carpenter et al. 2009; Jacquet et al. 2015; Leta et al. 2019). This involved the design of species-specific primers to develop multiplex PCR assays for the detection of C. imicola in insect pools (Cêtre-Sossah et al. 2004; Casati et al. 2009). However, as BTV spread further north and SBV emerged, beyond the range of C. imicola , additional competent vector species within the subgenera Avaritia and Culicoides were identified, including Culicoides obsoletus (Meigen, 1818) , Culicoides scoticus Downes & Kettle, 1952 , Culicoides dewulfi Goetghebuer, 1936 , Culicoides chiopterus (Meigen, 1830) , Culicoides pulicaris , and Culicoides punctatus (Meigen, 1804) (Nolan et al. 2007; Gloster et al. 2008; Carpenter et al. 2009; Wilson & Mellor, 2009). This expansion has led to the development of conventional diagnostic PCR tests for the detection of these vector species. Species-specific multiplex PCR assays have been developed for Culicoides species identification, utilising both cytochrome c oxidase subunit 1 (CO1) and the internal transcribed spacer (ITS) .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint barcodes (Nolan et al. 2007; Pagès et al. 2009; Stephan et al. 2009; Mathieu et al. 2011; Lehmann et al. 2012). A species-specific real-time PCR test targeting the ITS-1 gene region of C. imicola sensu stricto significantly improved the surveillance of this species in France, as it can be used to detect target species in mixed samples (Cêtre-Sossah et al. 2008). Subsequently, a quantitative duplex real-time TaqMan PCR assay was developed to determine the relative abundance of C. obsoletus a n d C. sco t icus in pooled samples (Mathieu et al. 2011). Around the same period, Wenk et al. (2012) designed 11 real-time PCR assays targeting the mitochondrial COI gene for the specific identification of both vector and non-vector Culicoides species, including C. chiopterus, Culicoides deltus Edwards, 1939, C. de wulfi , Culicoides grisescens Edwards, 1939, C. imicola , Culicoides lupicaris Downes & Kettle, 1952, C. obsoletus , C. pulicaris, C. scoticus, and Culicoides sp. However, cross-species reactivity was observed for C. chiopterus , C. scoticus , C. del tus , C. grisescens , and C. obsoletus . Dähn et al. (2025) later developed TaqMan quantitative real-time PCR assays targeting six members of the Culicoides s ubgenus Avaritia : C. obsoletus clades O1, O2, and O3, C. scoticus c l a d e 1 , C. chiopterus , and C. dewulfi . Although these assays were designed for use in both singleplex and multiplex PCR formats, the study highlighted cross-reactivity in the multiplex format, which was attributed to overlapping primer-probe interactions. Several Culicoides s p e c i e s and distinct haplotypes have been identified in the Palearctic region using a combination of morphological and molecular characterisation. The presence of different haplotypes poses challenges for species-specific assay design, as some may be inadvertently excluded from detection or may lead to cross-reactivity with existing assays when degenerate primers are used to account for sequence variation (Wenk et al. 2012). These issues are particularly problematic when analysing unsorted field specimens. In Ireland, although morphological and molecular characterisation have facilitated the development and validation of the Culicoides species list (Collins et al. 2018; Isiye et al. 2025), no species-specific assays have been developed to target the important Culicoides species. The development of such assays would significantly advance the large-scale monitoring of these species, greatly supporting surveillance efforts and improving our understanding of their ecology and role as vectors, particularly of BTV and SBV. This stud y aimed to develop a high-thr oughput species-specific real-time PCR method targeting the .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint internal transcribed spacer 2 (ITS2) region for the surveillance of important Culicoides species captured in light tr aps on farmlands in Ir eland.

Pr i m er and probe des ig n A nucleotide BLAST (BLASTn) search was performed using the ITS sequences obtained from seven Culicoides biting midge species: C. obsoletus , C. scoticus , C. chiopterus, C. dewulfi, C. pulicaris , C. punctatus , and Culicoides impunctatus Goetghebuer, 1920, which were generated through DNA barcoding analysis by Isiye et al. (2025). The corresponding

sequences from GenBank with the highest similarity to the Irish Culicoides sequences were selected, and multiple sequence alignment (MSA) was performed using the EMBL-EBI Clustal Omega program (Madeira et al., 2022). The alignment included the following sequences: C. obsoletus (MK893033), C. scoticus (MK893045), C. chiopterus (MK893002), C. dewulfi (MK893005), C. pulicaris (DQ371264), C. punctatus (DQ371247), and C. impunctatus (EU908207) (Gomulski et al., 2006; Mathieu et al., 2020). Conserved and variable regions were identified within the alignment, and the forward primer, reverse primer, and, for certain tests, a probe was designed based on interspecies variable regions to ensure test specificity, using Primer Express™ Software v3.0.1. Primer specificity was evaluated in silico by initially performing a Primer-BL AST analysis using the NCBI Primer Design Tool. Efficiency t esting T h e e f f i c i e n c y o f t h e n o ve l r e al - ti m e P C R p r i m e r s w a s e va l u a t e d u si n g a 5 - l o g d il u t i o n se r i e s of target species DNA obtained from a previous study by Isiye et al. (2025). Each reaction c o n si s t e d o f 5 µ L o f 2 x F a st St a r t U n i v e r s a l SY B R Gr e e n M a s t e r m ix ( R OX ) ( Si gma -Aldr ich ), 0.4 µL of forward and reverse primer mix with a final concentration of 200 nM, 3.6 µL of molecular-grade water, and 1 µL of extracted DNA as the template. The negative control reactions included 1 µL of molecular-grade water instead of the DNA template. Reactions were prepared in a final volume of 10 µL and performed in triplicate using a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). The real-time PCR profile consisted of an initial incubation at 50 °C for 2 min, followed by denaturation at 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Primer specificity and sensitivity were assessed using .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint amplification and melt curve analyses. A standard curve was generated, and the primer efficiency (E) was calculated, with the recommended efficiency range being 90–110%. The curve was visualised using the ggplot2 package in R version 4.4.1 (R Core Team, 2025). Cro s s- spec ie s reactivity t e s t ing To determine cross-species amplification, all primer sets were tested against 0.25 ng DNA of each non-target species, as described above. All assays were first tested as SYBR-based assays; however, for assays that amplified the target and non-target species, a probe was introduced to increase the specificity of the tests. Each probe-based reaction contained 5 µL TaqMan™ Fast Advanced Master Mix (Applied Biosystems), 0.2 µL of forward and reverse primers, probe mix wi th a final concentration of 250 nM, 3.8 µL of molecular-grade water, and 1 µL of extracted DNA. Negative controls were prepared by replacing the DNA template with 1 µL molecular-grade water. App li cation to mixed fiel d t ra p samples Culicoides specimens collected in June 2023 from 13 study sites were pooled into groups of individual midges, resulting in 13 pooled samples representing field trap collections. The pooled midge samples were crushed in extraction buffer using a pipette tip, DNA was extracted from each pool, and its concentration was measured using a NanoDrop™ 8000 spectrophotometer. Each extracted DNA sample was subjected to a 1:10 dilution and used as template DNA. All reactions were performed in triplicate for both SYBR-based and hydrolysis probe-based assays. The reaction conditions followed the respective protocols previously established during cross-species reactivity testing, and the average Ct values and melting temperatures (Tm) were recorded. To visualise the presence or absence of each species within the mixed samples, a dot plot was generated in R version 4.4.1 (R Core Team, 2025) using the ggplot2 package.

Pr i m er and probe des ig n Seven real-time PCR primer sets wer e designed targeting the Culicoides s p e c i e s : C. pulicaris , C. scoticus , C. dewulfi, C. chiopterus , C. obsoletus, C. punctatus , and C. impunctatus (Table 1). .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint T a bl e 1: Pr im e rs a n d pr o b es de si gne d f or th e s p e c i fi c det ecti on of C u lic oi de s s p e c ies , al ong with t he s t an d ar d c u rv e re su l t s and e f fi c ien cie s fo r t he sp e c ie s -s pe c i fi c p rime r se t s s e le ct ed fo r e a c h ta rge t s pe ci e s . Species Primer /Probe (5' t o 3') Slope R 2 Ef ficie n c y Am plicon length (bp ) C. chiopt eru s CCHIOF – AAACT A TCTT A T GT GTT AAC GCGCA CCHIOR - GCA CAAGT ACC GTT AACCA T A TCCTT -3.49 0.999 93.434 102 C. pulic aris CPULF - AGGTCT GT GT GGGGT ACACA T G CPULR - CCA T A T CA T CA T A GCA T ACACTT AGAGGT -3.344 0.99 99.103 103 C. de wulfi CDEWF – ACA GAGTCT GT GT GGGGT AC ACA T CDEWR - GCAC TT A T AA T GCCACA T AACT GACA C -3.391 0.998 97.192 90 C. impunct at us CIMPF - GT GT GTTT GAA T AAACA TT GT AAGT GCA CIMPR - GGA T GGT A T GTT AAGGT AAGT AACAAAAACT A T - 3.448 0.997 94.999 192 C . s co ti c us CSC OF – GCA T GA T GT GA T CA T A T GGA T GT AAC CSC OR - AGTTT ACT AAA T GCAGAGCA CA TTT GA -3.387 0.996 97.357 149 C. punct at us CPUNF – GGT ACACA TGGT TGAGTGTCGTT A T CPUNR – GCTT AA T A T A TTT GTT A CCACCACACTT F AM- GT GTTT GAAAGAACA TTT AAGT G-MGBEQ -3.459 0.996 94.57 141-143 C. obsoletu s COBSOF – TCT T A T GT GTCTT T AACGGGCA TC COBSOR – TGCACACAAAGT AGCGT T AACCA T FA M - TG TG A T C A T A TG G A TG T A A C A - M G B E Q -3.512 0.998 92.647 105 E fficiency t e st ing .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint Figu r e 1 : A mp lific atio n o f Culic oides p ulic aris target D NA using CPUL p r imers. (a) A mp lifi c ation p lot s howi n g a 1:1 0 se r ial dilution seri e s with c orr es po nd ing a mplific ation curv es a nd (b ) stan d ard c urve sh ow ing the lin ear re gres sio n of Ct v alue s ag ains t D NA c on c en tration, d emo ns tr atin g ass ay effic ie nc y and s ens i tivity . .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint F i g ur e 2 : Amp li fi c a ti on o f C u li co ide s o b so le t u s targe t DNA u s in g COBS O pr imer s and a T aq Man® p robe . (a ) Ampli fic ati o n plot disp la yin g a 1 :1 0 s eri al diluti o n s eri e s with c o r r es p o n di ng a m plifi c atio n cur v e s a nd ( b) St an da rd cur ve s h owin g t he li nea r r eg r e s si on o f Ct v alu es agai ns t DN A c onc e n tr ati o n , de mo ns tr ati ng ass a y eff ici enc y and se ns iti v ity . When tested on the bench, the selected primer sets exhibited a well-defined dynamic range of amplification, producing five distinct amplification curves (Figures 1 and 2). Melt curve analysis further validated the specificity of amplification by generating distinct peaks at specific melting temperatures (Tm) (Supplemental Information). Additionally, standard curve analysis (Figures 1 and 2) confirmed that the primer efficiency remained within the optimal range of 90-100% (Table 1). Cro s s- spec ie s reactivity t e s t ing Figu re 3 : Am pl ific ati o n o f C. c hi o pt e r u s a nd n on -t a rg et s pec ies u si ng s p eci e s -s pec if i c pr i m ers , ens u ri ng sel e c ti ve am pl ific ation with out c r oss- r eacti v ity . As shown in Figure 3, the assays effectively differentiated the target species while minimising non-specific amplification. Setting a cutoff Ct value of 34 ensured that false positives were not included, as any amplification beyond this value was not considered indicative of target detection. Furthermore, no cross-reactivity was detected in the assays developed for C. pulicaris (CPUL) , C. impunctatus ( C I M P ) , C. scot icu s ( C S C O ) , C. dewulfi (CDEW) , and C. chiopterus (CCHIO). The designed SYBR-based assays lacked specificity for .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint two species. These included C. punctatus , which was successfully amplified using the primers CPUNF and CPUNR at a Ct of 25. However, C. impunctatus was also amplified (at a Ct of 30), indicating some cross-reactivity. To enhance specificity, a probe was introduced, which improved the assay by amplifying C. punctatus a t a C t v a l u e o f 2 3 w h i l e e l i m i n a t i n g cross-reactivity with C. impunctatus . The second species was C. obsoletus , which was successfully amplified using COBSOF and COBSOR with a Ct of 21 and Tm of 71°C. However, non-target species, including C . pulicaris, C. chiopterus , C. dewul fi , C. impunctatus , C. scoticus, and C. punctatus were also amplified, though at Ct values >34. To improve specificity, a probe was introduced to eliminate non-target amplification, and the resulting Ct values are presented in Table 2. During cross-species reactivity testing of the C. obsoletus assay, occasional late amplification was observed at Ct values ranging f rom 35.6 to 38.6. These amplifications were inconsistent, occurring in only one or two of the three technical replicates, and were detected in some non-target species despite the inclusion of a species- specific probe, which reduced but did not fully eliminate these non-specific signals. No amplification was observed in any of the no-template control (NTC) assays. The average Ct and Tm values for all the assays are presented in Table 2. T a bl e 2 : Th e a v e r ag e c y c le th res h o l d (Ct ) an d melti ng t e mp er atu r e ( T m) v alu es fo r each sp eci e s -s pec if ic a s s ay usi n g S Y BR G r e e n- bas ed a n d p ro b e -b as e d r e a l-ti m e PCR. N ot e : T m va lues a re n o t r epo rte d f o r C . o bs ol e tu s an d C. p u nct atus bec a us e p r obe- bas ed a ssa ys d o n ot util iz e me lti n g t e mp er atu re a n alysis f or t ar g e t de te ct i o n. Ass a y name Spec ies A verage Ct A verag e T m ( ° C) St anda rd de viatio n CCHIO C. chiopt erus 21 69 0.07 CPUL C. pulicaris 21 70 0.09 CDEW C. dewulfi 23 70 0.06 CIMP C. impunctatus 27 68 0.48 CCSO C. s coticus 26 69 0.07 CPUN C. punctatus 23 - 0.21 CO BS O C. obsoletus 22 - 0.07 App li cat ion t o mix ed field trap sampl es .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint Figu re 4: A d ot p l ot dis pl ayi ng t he p r e s en c e o r abs e nc e of Cul ic o ides sp ec ie s acr oss 1 3 pool e d fi el d s am p l e s , p1 to p1 3 . A total of 840 Culicoides specimens, pooled into 13 samples from 13 distinct field sites (OVI 1 to OVI 13), wer e scr eened using the species-specific assay s developed in this study (see Supplemental Information). DNA concentrations fr om the pooled extr actions ranged from 3.0 to 27.3 ng/µL. Figur e 4 display s a presence/absence dot plot indicating the detection of the target Culicoides species across the 13 pooled field samples. The corresponding cy cle thr eshold (Ct) values for each assay are pr ovided in the Supplemental Information.

Advances in molecular t echniques have significantly improved biodiversity monitoring, enhancing the efficiency of vector surveillance thr ough accur at e species discrimination and genetic comparative analyses across diff erent geogr aphic r egions (Mignotte et al. 2021; On yango et al. 2015; T a y et al. 2016). T o aid in molecular surveillance, diagnostic tests have been developed by designing primers that tar get highly variable regions, ensuring high specificity and amplification efficiency . T ools such as Primer 3 and Primer Express™ Software are used to select primers, considering fact ors lik e melting temperatur e (T m), primer length, GC cont ent, and secondary structure (R ozen and Skaletsky , 2000). Species-specific r eal-time PCR assay s ar e designed for high sensitivity , enabling the detection of a single Culicoides .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint species within a mixed insect pool. Some assay s have demonstr at ed the ability to detect a single C. imicola in a pool of 3,200 Culicoides or within 65 mg of material from a light trap (Cêtre-Sossah et al., 2004), while others can detect specific species in pools of up to 1,000 Culicoides (W enk et al. 2012). The mitochondrial COI gene has been commonly used in the design of species-specific assays because of its low intraspecific variability , which accounts for haplotype diversity , and high interspecific variation (Aguilar-V ega et al. 2021; Balczun et al. 2009; Dallas et al. 2003; Pagès et al. 2009). However , for closely r elat ed or cryptic species, the more variable ITS r egion is oft en pref erred (Dallas et al. 2003, Pages et al. 2009; Augot et al 2010; Curr an et al. 2025). The ITS1 region was initially used t o develop species-specific assa y s for C. imicola (Cêtre-Sossah et al. 2008), while later studies tar get ed the ITS2 region to design real-time PCR assays for C. obsoletus , C. scoticus, and Culicoides montanus Wir th & Blanton, 1969, demonstrating impr oved sensitivity compared to conventional assay s (Monaco et al. 2010). The entire ITS1-5.8S-ITS2 region has also been used in quantitative duplex real-time PCR assa ys for detecting and quantifying C. obsoletus and C. scoticus (Mathieu et al. 2011). In this study , the ITS2 r egion was selected for primer design because it has been successfully used for the molecular characterisation of Cul icoides species in Ireland, confirmed thr ough both morphological and DNA barcoding of individual specimens (Isiye et al. 2025). This provided us with verified, high-quality sequences and ample genetically scored positive control samples for assa y development and validation. In this study , we developed five SYBR-based and two probe-based r eal-time PCR pr otocols for identifying C. obsoletus, C. scoticus, C. chiopt erus, C. dewulfi, C. pulicaris, C. punctatus , and C. impunctatus , species commonly found acro ss Ireland (McCarthy et al. 2009; Jess et al. 2018; Collins et al. 2018; Isiye et al. 2025). Primers wer e designed to account for haplotype diversity , while degenerate bases were avoided to minimise non-specific amplification and enhance PCR efficiency . Sequence alignments of the seven Culicoides species wer e used to design species-specific primers, following approaches similar to those described in previous studies (Nolan et al. 2007; Stephan et al. 2009; Mathieu et al. 2011). While primer design aimed t o balance specificity and amplification efficiency (Dieff enbach et al. 1993), in silico predictions did not always align with results achieved at the bench; theref ore, sever al primers were designed, and alternative primer s were used in case of non-specificity . For species such as C. impunctatus, the r ever se primer was designed to be longer than most .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint typical real-time PCR primers to ensure adequat e specificity owing to the lack of a potential variable r egion in the alignment. The primary aim was to design SYBR assa y s, as they are mor e economical than T aqMan® probes. While this was successfully achieved for five species (C. pulicaris , C. scoticus , C. impunctatus, C. dewulfi, and C. chiopterus ), for C. obsoletus and C. punctatus, specificity was increased through the inclusion of T aqMan® probes. All primers used in this study accounted for haplotype diversity within each Culicoides species, as demonstr ated by the Primer-BL AST results. Specifically , CPUL matched eight C. pulicaris and f ive C. lupicaris haplotypes; CIMP matched two C. impunctatus ; CDEW matc hed 20 C. dewulfi; CCHIO matched seven C. chiopterus ; CPUN matc he d eight C. punctatus ; and CSC O matched 14 C. scoticus . How ever , the COBSO primer s matched a wider range of haplotypes, including 21 haplotypes of C. obsoletus , 15 of C. mont anus , two of Culicoides sinanoensis T okunga, 1937, and one of C. scoticus (Supplemental Information). Further analysis by sequence alignment confirmed that the COBSO pr obe successfully excluded most non-t arget species; however , it could likely bind t o one haplotype each of C. sinanoensis (MK893047), C. scoticus (FN263316), and C. mont anus (MK893025). Repr esentative specimens f or these three specific haplotypes were not available, pr eventing experimental validation of this limitation. The C. sinanoensis haplotype identified in Russia (Mathieu et al., 2020) belongs to the eastern Palear ctic obsoletus complex. Primer-BLAST results detected four SNP s, three on the forward primer and one on the r ever se primer , potentially increasin g the Ct values if amplified. The C. scoticus haplotype, deposited by Kiehl et al. (2009), was collect ed in Germany (2007–2008) after the 2006 B TV outbr eak. In their study , this C. scoticus haplotype was consider ed a C. obsoletus variant rather than a distinct species. However , r ecent studies have confirmed it as a distinct cryptic species (Mathieu et al. 2020; Mignot te et al. 2020). Finally , C. mont anus is a rare species with a limited distribution in the Palearctic region (Delécolle et al. 2002; Mathieu et al. 2007). It has been observed in Mor occo, ar eas with high land surface temper atures in Italy , and in small numbers in Fr ance, Norway , and Spain (Mathieu et al. 2012; Balenghien et al. 2018; Antoine et al. 2020; Aguilar- V ega et al. 2021). Morphologically , C. montan us is part of the obsoletus complex and is characterised by a deep sensory pit and hypertrophied trichoid sensilla (hair-lik e structures of diff erent lengths) (Mathieu et al. 2011). Genetically , C. montanus and C. obsoletus f o r m a .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint monophyletic clade with a low genetic divergence between them (Garr os et al., 2014; Mathieu et al. 2011). Despite efforts to diff erentiate between them using species-specific assa y s, complete discrimination between C. obsoletus a n d C. montanus r emains elusive. Therefore, the accur acy of PCR assa y s is hindered by unresolved phylogenetic relationships and the high genetic similarity shared between the two species (Aguilar-V ega et al. 2021; Dähn et al. 2025). C. montanus h a s n o t b e e n r e p o r t e d i n I r e l a n d , a n d i t s v e ct o r c o m p e t e n ce and public health significance in Europe ar e lar gely unstudied, so it was not prioritised in this study . However , primer s designed by Monaco et al. (2010) for C. montanus based on the ITS2 r egion could be useful in regions wher e C. montanus and C. obsoletus occur sympatricall y . A Ct cut-off value of 34 was used in all assay s t o impr ove the t est accur acy , ensuring good sensitivity and specificity ( Gr einer , 1995; Caraguel et al. 2011). This ensur ed that only reliable amplification signals were interpreted as positive . The cut-off was based on primer efficiency testing, balancing the det ection of low DNA concentrations, and minimising false positives/negatives. For assa ys such as that developed for C. obsoletus , the application of this Ct cut-off value eff ectively excluded the high Ct signals observed during cross- species reactivity testing (Supplement al Information), which lik ely reflect low-level, non-specific amplification or background noise. Implemen ting this threshold also mi nimises the risk of false positives when the assa y is applied to mixed-species field tr aps. Overall, the results indicate that cr oss-species amplification is minimal and unlik ely t o compromise the accuracy or reliability of the assa y . When used to analyse tr ap samples, our real-time PCR assa ys can provide a semi-quantit ative assessment of the number of t arget species in individual light tr ap catches, with lower Ct values indicating higher tar get DNA concentrati ons. Cêtre-Sossah et al. (2008) emphasised that det ecting higher numbers of species, like C. imicola , is more critical for risk assessment than low number s of individuals. In this study , Ct values were used to estimate the relative abundance of each Culicoides species within the mixed-species pools. Lower Ct values were interpreted as indicative of higher DNA concentr ations and, ther efore, a greater number of individuals of the corresponding species. This appr oach provides an appr oximat e estimation of species prevalence in the pooled samples. However , it is import ant t o consider that interspecific variations in body size, such as the r elatively .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint larger size of C. pulicaris compared t o other species, may influence DNA yield and thus aff ect Ct-based abundance estimates. In addition t o facilitating the rapid identification of adult midges in trap samples, species- specific real-time PCR assay s should also be able t o detect larvae in environmental samples. V ector control efforts for disease pr evention typically focus on managing breeding sit es, a strategy that has been extensively applied to mosquito species. While di f ferent species of Culicoides larvae are thought t o occupy diff erent niches within the same site (Ket tle and Lawson, 1952), little is known about their species-specific occupation in breeding sites. While conventional PCR has been used to identify larval samples (Y anase et al., 2013), real- time PCR is recommended for large-scale studies, as it is mor e high-thr oughput, rapid, and eff ective for identifying breeding sites in farming envir onments, which are usually link ed to the spread of vect or species and disease tr ansmission. The assay s can also be used for biodiversity monit oring by enabling precise detection of Culicoides s p e c i e s d i v e r s i t y a c r o s s habitats, supporting fine-scale habitat association studies, and revealing ecological pref er ences and potential shifts in emergence patterns driven by climate change or land use changes. Over all, this molecular approach can be used t o enhance the ecological under standing of Culicoides communities in the Palear ctic region, supporting both conservation efforts and the risk managemen t of vect or-borne diseases. Although this method is highly efficient, designing species-specific primers for all species that occur in Ireland was not possible because of the high number of species present. Therefore, this study focused on designing species-specific real-time PCR assa y s for the most common Culicoides species, as well as those most likely to drive B TV /SBV transmission in Ireland, to improve year-round monitoring of Culicoides biodiversity .

The assa y s developed in this study successfully detected and discriminated all seven target Culicoides species, providing a valuable diagnostic tool for monitoring species composition in mix ed fi eld collections. These species-specific assa ys off er a high-throughput, rapid, cost- eff ective, and reliable method for tracking the distribution of C. obsoletus, C. scoticus, C. chiopterus, C. dewulfi, C. pulicaris, C. punctatus, a n d C. impunctatus adults in trap samples. Although their ability to detect Culicoides larvae in envir onmental samples has yet t o be .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 28, 2025. ; https://doi.org/10.1101/2025.09.26.678746doi: bioRxiv preprint tested, this application would gr eatly aid tar get ed surveillance and contr ol efforts. Over all, the PCR tools developed in this study can help impr ove our under st anding of Culicoides biodiversity and vect or-pathogen dynamics.

This r esear ch was conducted as part of the Network of Insect V ect ors (NetV ec) Ireland project ( https://www .ucd.ie/netvecireland/ ), funded by the Department of Agriculture, Food, and the Marine. The authors sincer ely thank the NetV ec Ir eland team and the midge survey volunt eers across Ir eland for their efforts in specimen collection. The invaluable support and cooperation of these collaborator s were instrumental in the successful completion of this study.

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