Investigating the Role of Stable Flies (Stomoxys calcitrans) and Biting Midges (Culicoides spp.) as Potential Mechanical Vectors of African Swine Fever Virus in Lithuania, Poland and Romania.

Investigating the Role of Stable Flies (Stomoxys calcitrans) and Biting Midges (Culicoides spp.) as Potential Mechanical Vectors of African Swine Fever Virus in Lithuania, Poland and Romania. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigating the Role of Stable Flies (Stomoxys calcitrans) and Biting Midges (Culicoides spp.) as Potential Mechanical Vectors of African Swine Fever Virus in Lithuania, Poland and Romania. Sofie Dhollander, Oana-Maria Balmoș, Eleonora Cattaneo, José Abrahantes Cortiñas, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5942596/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Parasites & Vectors → Version 1 posted 13 You are reading this latest preprint version Abstract Background Since its emergence in Georgia in 2007, the seasonal pattern of African swine fever virus (ASFV) genotype II outbreaks in European pig populations has been evident. It is hypothesized that summer-related farming practices, along with the increased activity and abundance of arthropod vectors during warmer months, contribute to the increased incidence of these outbreaks during this period. This study investigated the potential role of stable flies ( Stomoxys calcitrans ) and biting midges ( Culicoides spp.) as mechanical vectors of ASFV. Methods From August 2021 to August 2023, vector surveillance was conducted as part of a case-control study on 42 outbreak farms and 70 control farms across Romania, Lithuania, and Poland. Collected insect specimens were pooled and tested for ASFV DNA using real-time PCR. Results A total of 8,604 biting midges’ specimens and 742 S. calcitrans flies were collected, with ASF DNA detected in 27 out of 1,219 insect pools. Positive pools were predominantly observed in Culicoides punctatus , C. newsteadi , and the Obsoletus complex, with most detections occurring in August. However, ASFV isolation was unsuccessful. Statistical analyses revealed no significant association between farm status (outbreak vs. control) and ASFV DNA detection in biting midges’ pools, likely due to limited sample size. Additionally, two ASFV-positive S. calcitrans pools were identified, supporting their potential role as mechanical vehicles for ASFV. The findings highlight the affinity of C. punctatus , C. newsteadi , and the Obsoletus complex and S. calcitrans for pig farms, which may increase the likelihood of ASFV acquisition and dissemination, assuming viral infectivity is maintained in these insects. Environmental factors, such as pig farm density and proximity to other hosts, likely influence this risk. Conclusions These results emphasize the importance of vector control strategies, including insect netting, to mitigate ASFV transmission risks. Further research is needed to understand the dynamics of ASFV infection in arthropods. Notably, this study also reports the first identification of Culicoides riethi and Culicoides salinarius in Lithuania. Stomoxys calcitrans Culicoides Mechanical transmission African Swine Fever Virus pigs Figures Figure 1 Figure 2 Figure 3 Introduction African swine fever (ASF) is a highly contagious and severe viral disease of domestic and wild suids, caused by a double-stranded DNA virus of the Asfarviridae family, genus Asfivirus . The virus spreads through direct contact with infected animals or their secretions, and indirectly via contaminated feed or fomites [ 1 , 2 ]. During the last 10 years. ASF has caused devastating economic losses globally, affecting both backyard and commercial pig farmers. The disease has disrupted pork supply chains, led to the culling of millions of pigs, and significantly increased pork prices. Overall, the global pork industry has faced losses running into billions of dollars due to ASF outbreaks [ 3 ]. In Europe, the epidemiological situation worsened in 2023, with ASF virus (ASFV) genotype II affecting 14 EU Member States, including new outbreaks in Croatia and a resurgence in Romania. The number of outbreaks among domestic pigs in 2023 was five times higher than in 2022, causing substantial disruptions to the EU’s pork production and trade. ASF outbreaks in domestic pigs were clearly seasonal in all countries, with 88% of outbreaks reported between July and October [ 4 ]. The seasonal nature of ASFV genotype II outbreaks in pigs in Europe has been evident since its introduction in Georgia in 2007. Summer-related husbandry practices and the transmission by arthropod vectors, abundant and active during the warmer months of the year, have been hypothesized to contribute to the increased risk of virus introduction on farms in the summer [ 5 – 11 ]. The soft tick from the Ornithodoros erraticus complex (Ixodida; Argasidae) is currently the only confirmed biological vector of ASFV transmission in Europe. The biological transmission cycle with pigs has been demonstrated using field strains of ASFV collected during the epidemic in the Iberian Peninsula ([ 12 – 14 ]). However, with the currently circulating Georgia 2007/1 ASFV strain, only oral transmission by feeding pigs with infected ticks has been demonstrated [ 15 ], despite infectious virus being detectable in ticks up to 8 months after exposure no transmission by feeding ticks was observed [ 16 ]). Thanks to its capacity to harbour infectious ASFV for several months, and its nidicolous behaviour (i.e. inhabiting burrows or cracks and feeding for short periods), O. erraticus is considered a reservoir of ASFV in outdoor farms. In such environments, these ticks can thrive and remain close to pigs. However, they are not considered to play a relevant role in spreading the virus between farms [ 17 ] Furthermore, O. erraticus and other potential soft tick vector species have not been observed in most areas currently affected by ASFV Georgia 2007/1 in Europe, making it highly unlikely that they are responsible for the peak in incidence during the summer months in these regions. However, mechanical transmission by arthropod vectors that are abundant during the warmer months has been the focus of several field studies in affected regions. DNA of ASFV genotype II has been detected in biting insects such as biting midges ( Culicoides spp.) [ 18 ], mosquitoes (Culicidae) [ 18 , 19 ] and Stomoxys calcitrans [ 18 , 20 ] near or on outbreak farms. In addition, ASFV DNA-positive hematophagous flies (i.e.: Tabanidae) have been identified on or near ASF free pig farms [ 20 , 8 ]. Still, to date, no studies have successfully isolated infectious ASFV from these biting insects. The transmission of ASFV to pigs by arthropods feeding on pigs has only been demonstrated under laboratory conditions. For instance, Mellor et al. [ 21 ] showed that S. calcitrans adults fed on ASFV infected blood were subsequently able to infect pigs during subsequent feedings on pigs. This study aimed to gather field evidence on the potential role of stable flies and biting midges in the mechanical transmission of ASFV. Conducted as part of a case-control study between August 4, 2021, and August 23, 2023, in Romania, Lithuania and Poland [ 22], the study hypothesized that if these insect groups contribute to ASFV transmission, the virus would at least be detectable in or on the insects. Such detection would indicate that the insects had fed on infectious blood or secretions, potentially serving as vehicles for introducing infectious virus onto ASF-free farms. Furthermore, a higher proportion of ASFV- positive insects or insect pools on affected farms compared to control farms would support their role in transmission within farms. Conversely, the detection of ASFV-positive insects on control farms could indicate the potential of these insects to transport the virus onto ASFV-free farms. Additionally, the collected data on biting midge species and S. calcitrans were analysed to identify species more likely to carry ASFV DNA and to investigate the presence of new Culicoides species in previously unreported areas. Methods Site selection This study was part of a prospective case control study that took place between August 4, 2021, and August 23, 2023, to investigate potential risk factors for the introduction of ASFV on commercial pig farms in Romania, Lithuania and Poland [ 22 ]. For each commercial pig farm where an ASF outbreak was confirmed, two control farms were selected, matched by herd size (e.g. 30–200 pigs; 201–1000 pigs; >1000 pigs) and by region, i.e. the Nomenclature of Territorial Units for Statistics (NUTS) 3 region in which the farm was located. If no control farm could be selected in the same NUTS 3 region, a control farm from the same herd size category was chosen from elsewhere in the country. Following the confirmation of the ASF outbreaks on commercial pig farms by the official Veterinary Services in Lithuania, Poland and Romania, the entomology teams were immediately informed about the locations of the affected farms to start vector collection. Simultaneously, two control farms were randomly assigned to each case farm by the team in EFSA. Case farms were to be visited within two days of ASF confirmation, and control farms within two weeks after the matching case farm’s outbreak confirmation. Vector traps Stable flies ( Stomoxys calcitrans ) Blue sticky traps were used to collect stable flies, based on the method described by Hogsette and Kline [ 23 ]. Four trap locations were selected on each farm, two inside and two outside the pig sheds. The traps were placed at least 15 meters apart in areas with high fly activity and direct sunlight to maximize visibility to stable flies. Outdoor traps were placed near the animals, at maximum 15 m from the shed at a height of 1.6 m. Traps were deployed for one day, covering the entire daylight period, as stable flies are diurnal and attracted to translucent and blue-colored objects. All stable flies were collected in dry containers and kept cool/frozen (+ 4 to -20) during transport. Upon arrival at the laboratory, samples were immediately stored at -80°C until morphological identification, ASFV DNA testing and blood meal source analysis. Biting midges ( Culicoides spp.) CDC miniature light traps equipped with UV light were used to collect biting midges following the methodology according to Medlock et al. [ 24 ]. Two traps were placed on each farm: one inside and one outside the pig shed. Traps were installed at a height of 1.5 to 2 m above the ground, as recommended by Diarra et al. [ 25 ]. Outdoor traps were placed in areas protected from wind and rain. Traps were situated in dark locations to minimize competition with other light sources. Based on the feeding activity pattern of biting midges, feeding mainly from dusk to dawn, [ 26 ], traps were activated one hour before sunset and deactivated one hour after sunrise, facilitating overnight collection of biting midges on each farm. Samples were collected early in the morning, placed in dry containers, and stored as quickly as possible at -80°C, allowing identification of Culicoides spp., virus isolation and blood meal source identification at a later stage. Vector species identification The identification of the arthropods was based on morphological characteristics. Identification of S. calcitrans was performed using descriptions and keys provided by Zumpt [ 27 , 28 ]. Culicoides spp. were identified using the Interactive Identification Key for Culicoides (IIKC), encompassing 110 taxonomic units (102 species and 8 morphological variants) as detailed by Mathieu et al. [ 29 ]. According to Augot et al. [ 30 ], Culicoides obsoletus , Culicoides scoticus , and Culicoides chiopterus were considered to be part of the Obsoletus complex. Identification of the vector species was carried out in the laboratories of each of the countries from where insects were collected, that is the Vilnius University, Life Sciences Centre in Lithuania; the University of Agricultural Sciences and Veterinary Medicine in Cluj-Napoca in Romania and the Veterinary Research Institute in Pulawy in Poland. The latter also performed the virus isolation for all three countries involved in the study. Detection of ASFV and its DNA and pig DNA in vector samples • Pooling of samples Prior to DNA extraction based on Cêtre-Sossah et al. [ 31 ] and Şevik and Oz [ 32 ], arthropod samples were pooled by species, collection site and date. Stable flies were pooled in pools of up to four maximum 4 specimens, while biting midges were pooled in pools of maximum 20 specimens. Before pooling, each individual stable fly was dissected longitudinally, creating two identical aliquots. These aliquots were then homogenized (with pools containing 1–4 halves of stable flies). Biting midges were pooled in groups of 1–20 individuals, homogenized, and subsequently divided into two equal portions. The first pool of aliquots of stable flies and the first portion of biting midges was used for detection of ASFV DNA by real-time PCR. Additionally, in Romania, the first aliquots also underwent PCR analysis to detect pig DNA. The second pool of aliquots was reserved for virus isolation, if the corresponding first pool tested positive by Real-Time PCR with a Ct value below 20. • ASFV DNA identification in vectors All pooled arthropod samples, including stable flies and biting midges, were tested for ASFV DNA by real-time PCR according to the standard operational procedures (SOPs) used by the ASF national laboratories in Poland (National Veterinary Research Institute, Pulawy), Romania (Institute for Diagnosis and Animal Health, Bucharest), and Lithuania (Lithuanian University of Health Sciences and the Life Sciences Centre of Vilnius University). These SOPs align with those of the European Union Reference Laboratory for ASF . The pooled arthropod samples were ground in MagNa Lyser Green Beads with 1.5 ml cold phosphate-buffered saline (PBS1x) containing 0.1% gentamicin sulphate. Suspensions were clarified by centrifugation at 5,000 g for 5 minutes, and 200 µl supernatant was used immediately for ASFV DNA detection or store at <-70ºC for future use. Ct values below 35 were considered positive for ASFV DNA. Validated extraction and amplification kits, routinely used for detection of ASFV in animal samples, were used, adhering to the SOPs implemented at the NRL’s for ASF. • Detection of Pig DNA Pig DNA analysis was conducted to evaluate whether the vectors had been in contact with pigs or pig secretions, to check if the ASFV DNA-positive vectors were exposed to pigs. Only pools positive for ASFV DNA were tested for the presence of pig DNA. All positive samples for ASFV genome were tested by conventional PCR using an in-house method developed by the ASF NRL of the IDAH based on the protocol described by the Dalmasso et. al. [ 33 ]. • Virus isolation in vectors Samples that tested real-time PCR positive for ASFV DNA with CT values lower than 20 were further analysed for virus isolation. Only strong positive pools were considered to be worth the resources to possible isolate ASFV, as described earlier [ 33 , 34 ]. The National Veterinary Research Institute in Pulawy, Department of Swine Diseases, performed the virus isolation for the three countries participating in the study. The potential presence of infectious virus in the insects’ homogenates was assessed using a hemadsorption assay in 96-well plates. An aliquot of 50 µL of each filtrated sample (0.45 µm Sartorius syringe filters, Göttingen, Germany) was added (in triplicate) to primary porcine alveolar macrophage (PPAM) cell culture or similar cultures supporting the growth of ASFV, such as porcine leukocytes or bone marrow. The cultures contained 200 µL of growth medium: RPMI (Gibco, Waltham, USA), 10% of foetal bovine serum (Gibco, Waltham, USA), 1% of Antibiotic/Antimitotic solution (Sigma Aldrich, St. Louis, USA) and 1:300 (v/v) swine red blood cells (RBC). Hemadsorption was monitored via microscopy over five days post-inoculation, as described by Walczak et al. [ 35 ]. Data collection, analysis and visualisation The results of the vector identification and laboratory tests conducted in the three countries were standardized, cleaned and processed using MS Access. Maps were created using R software 4.0. To study if there was a difference between the presence of ASFV DNA positive vector pools in the case and control farms three different analyses were conducted on the Culicoides results: Random Forest analysis, Classification Tree analysis and Logistic Regression analysis. Different variables were investigated for their potential influence on the ASFV DNA test results of the Culicoides spp. pools: the Sampling year, Sampling Month, Present of pigs on the farm at the moment of placing the vector traps, Numbers of insects in the pool, Country, Region, Farm category (case or control) and the Culicoides species. No statistical tests were performed on the S. calcitrans results, due to the small numbers of pools investigated. Results Geographic distribution of Culicoides species. Figure 1 displays the geographic distribution of the different Culicoides species that were observed on the 112 commercial pig farms included in the study, showing the predominance of C. punctatus , Obsoletus complex, C. pallidicornis and C. newsteadi on the pig farms. C. riethi and C. salinarius [36] were morphologically identified for the first time in Lithuania using Mathieu et al. [ 29 ]. The identification of the species was confirmed using PCR-based methods for analysis of Cytochrome c oxidase subunit I sequences (COI), which are used as barcoding for many insect species (Fig. 2 ). ASFV detection in insects collected on outbreak and control farms Vectors surveillance results were reported for 42 farms where ASF outbreaks were confirmed (case farms), and 70 control farms matched by farm size and location (Fig. 3 ). All case farms were visited within two days of ASF confirmation, and 85% of the control farms were visited within two weeks after the matching case farm’s outbreak confirmation, 8% between two and three weeks and 7% between three weeks and one month. In Lithuania, three case and six control farms were included in the study, with ASFV DNA-positive vectors detected on one case farm and one control farm (Fig. 3 ). In Poland, vector surveillance was conducted on 18 case farms and 36 control farms, with ASFV DNA-positive vectors identified on two case farms. In Romania, vector surveillance was carried out on 21 case farms and 28 control farms, with ASFV DNA-positive arthropods were found on seven case farms (Fig. 3 ). From the 1219 insect pools tested on the farms, 27 pools were positive for ASFV DNA. In 2021, 127 insect pools were tested for ASFV DNA of which 18 insect pools were positive. In 2022, 368 insect pools were tested, with one pool positive for ASFV DNA. In 2023, 697 pools were tested with eight pools positive for ASFV DNA. The detection of positive pools over tested pools in the three years together were spread over the months as following: two positives out of 8 tested pools in May, zero positive out of 331 tested in June, four positive out of 434 tested in July, 18 positive our of 285 tested in August, three positive out of 99 tested in September and zero positive out of 35 tested in October. Across the three countries, a total of 8,604 Culicoides spp. specimens were collected, of which 4,962 were caught on control farms and 3,642 were caught on case farms. Additionally, 742 S. calcitrans specimens were caught, of which 141 specimens on case farms and 616 on control farms (Table 1 ). Table 1. Counts of Culicoides spp. and S. calcitrans pools and specimens per species collected from pig farms and the positive detections (in red) of ASFV DNA in pools and individual specimens. Species/genus No. pools No. specimens Positive pools No. of specimens in positive pools No. pools No. specimens Positive pools No. of specimens in positive pools Farm Category Case farm Control farm Lithuania C. achrayi 5 19 0 0 0 0 0 0 C. circumscriptus 2 5 0 0 3 5 0 0 C. fascipennis 3 7 0 0 4 11 0 0 C. grisescens 1 1 0 0 0 0 0 0 C. impunctatus 1 1 0 0 0 0 0 0 C. kibunensis 1 2 0 0 3 17 1 4 C. nubeculosus 2 5 0 0 0 0 0 0 C. pallidicornis 1 2 0 0 12 120 0 0 C. pulicaris 1 1 0 0 0 0 0 0 C. punctatus 74 697 1 10 55 472 0 0 C. riethi 4 16 0 0 6 6 0 0 C. salinarius 3 5 0 0 0 0 0 0 Culicoides spp. 0 0 0 0 3 6 0 0 C. pictipennis 0 0 0 0 1 2 0 0 C. simulator 0 0 0 0 3 3 0 0 Obsoletus complex* 20 121 0 0 19 165 0 0 Total Lithuania 118 882 1 10 109 807 1 4 Poland C. achrayi 13 102 0 0 16 94 0 0 C. circumscriptus 4 4 0 0 6 33 0 0 C. fascipennis 3 5 0 0 4 4 0 0 C. pallidicornis 29 252 0 0 70 649 0 0 C. punctatus 49 498 0 0 110 987 0 0 C. riethi 6 28 0 0 6 20 0 0 C. furcillatus 0 0 0 0 1 4 0 0 C. grisescens 0 0 0 0 1 1 0 0 C. impunctatus 0 0 0 0 2 2 0 0 C. newsteadi 0 0 0 0 1 1 0 0 C. pulicaris 0 0 0 0 3 41 0 0 Obsoletus complex* 60 361 0 0 167 1659 0 0 Stomoxys calcitrans 30 104 2 4 133 516 0 0 Total Poland 194 1354 2 4 520 4011 0 0 Romania C. cataneii 2 20 0 0 0 0 0 0 C. circumscriptus 1 10 0 0 1 10 0 0 C. fagineus 1 10 0 0 1 10 0 0 C. fascipennis 1 10 0 0 0 0 0 0 C. longipennis 1 10 0 0 0 0 0 0 C. lupicaris 4 40 1 10 0 0 0 0 C. newsteadi 47 470 5 50 14 140 0 0 C. nubeculosus 12 120 0 0 2 20 0 0 C. punctatus 79 790 17 170 37 370 0 0 C. pulicaris 0 0 0 0 3 30 0 0 C. puncticollis 0 0 0 0 2 20 0 0 Obsoletus complex * 3 30 0 0 6 60 0 0 Stomoxys calcitrans 11 22 0 0 50 100 0 0 Total Romania 162 1532 23 230 116 760 0 0 Total 474 3768 26 244 745 5578 1 4 The most frequently caught Culicoides species across both farm categories were C. punctatus (n = 2,657), Obsoletus complex (n = 2,305), C. pallidicornis (n = 1,023) and C. newsteadi (n = 631). The majority of Culicoides species positive for ASFV DNA belonged to C. punctatus (18 positive pools out of 410 tested) followed by C. newsteadi (5 positive pools out of 64 tested) and Obsoletus complex (7 positive pools out of 275 tested). None of the real-time PCR- positive Culicoides pools had Ct values below 20 and were therefore not tested for virus isolation. In Romania, pig DNA was identified in three pools of C. punctatus , three pools of C. newsteadi and one pool of C. cataneii . Two of these pools tested negative for ASFV DNA but with doubtful results namely one of the C. cataneii pools with CT value of 37.3 and one of the C. newsteadi pools, with CT value of 36.2 but they were nonetheless tested for pig DNA, with negative results. All other pools that were negative for ASFV DNA were not tested for pig DNA. The other countries did not test for pig DNA in the arthropods. In total, two of the 224 S. calcitrans pools tested positive for ASFV DNA by real-time PCR. None of the PCR positive S. calcitrans pools yielded positive results in virus isolation. Statistical analysis None of the performed statistical analysis identified any significant association of the investigated variables (Sampling year, Sampling Month, Presence of pigs on the farm during vector survey, Numbers of insects in the pool, Country, Region, Farm category (case or control) and the Culicoides species) with the presence of ASFV DNA in the Culicoides pools. Discussion Surveillance of vectors was conducted on 42 outbreak farms and 70 control farms. ASFV DNA positive Culicoides pools were found on eight case farms and one control farm. Additionally, ASFV DNA positive S. calcitrans flies were found on two case farms. While there was a higher occurrence of DNA positive insect pools on case than control farms, statistical analysis (Random Forest, Classification Tree and Logistic Regression statistical analysis) showed no significant association between farm category and ASFV DNA test results. This lack of significance is probably due to the small sample size, because fewer outbreaks than anticipated occurred on commercial pig farms during the study period. ASFV could not be isolated from any of these DNA positive insect pools. The inability to detect viable ASFV was likely due to the very small quantities of blood available in the Culicoides pools tested by real-time PCR. Such pools comprised half the volume of homogenates from up to 20 Culicoides specimens, or fewer when fewer individuals of a given species were caught. In addition, ASFV’s short survival time at higher summer temperatures may have contributed to the inability to recover viable virus. ASFV survival has been studied in several matrices. At room temperature, it has been shown to survive up to 3 days in pig faeces and 5 days in urine [ 37 ]; less than 1 day on cereal grains; and up to 30 days on soy oil seeds and soybean meal [ 38 , 39 ]. Specific studies investigating the survival of ASF in insects, such as the study of Blome et al. [ 40 ] observed that ASFV could be isolated from Stomoxys calcitrans flies kept in an incubator at 20°C up to 2 days after feeding on viraemic blood. However, higher temperatures significantly reduced virus survival time. Although the traps in this study were placed in the shade, it is possible that high temperatures inside the traps were reached, negatively affecting the survival time of ASFV on mechanical vectors. Thus, while ASFV can be isolated from insects for short periods under laboratory conditions, environmental factors, particularly high summer temperatures, likely inhibit its long-term survival in insects during filed conditions. These findings align with previous studies that demonstrated the presence of ASFV DNA in Culicoides [ 18 ]and Stomoxys calcitrans [ 18 , 20 ], without isolating viable ASFV. They add to the growing body of evidence suggesting that Culicoides and S. calcitrans could serve as a mechanical vector of the virus. The repeated detection of ASFV DNA in these insects highlights the need for further studies to clarify their role in ASF transmission. It is possible that Culicoides merely landed on or fed from contaminated materials on the farm, without actively contributing to the transmission of ASFV. Additionally, the finding of the ASFV DNA positive biting midge on a control farm was not followed by infection of the pigs on the farm, as the farm remained ASF-free. However, this observation was limited to a single farm, and the biosecurity measures and insect control strategies in place may certainly have played a role in protecting the pigs from infection. Among the ASFV DNA-positive arthropods caught on the pig farms, the majority belonged to the species C. punctatus (18 positive pools), followed by Obsoletus complex ( 7 positive pools) and C. newsteadi (5 positive pools). These were also to the most frequently observed Culicoides species on the studied pig farms. In Romania, the only country where the insect pools were tested for host DNA, three C. punctatus pools, three C. newsteadi pools, and one C. catanei pool tested positive for pig DNA, which confirms these ASFV DNA positive insect must have been in direct or indirect contact with pigs. Tomazatos et al. [ 41 ] studied blood meals of common Culicoides species in the Danube Delta in Romania. The researchers identified Sus scrofa as the second preferred blood meal of C. punctatus , after cattle. The S. scrofa DNA in the blood meals identified in the biting midges caught in this study was from feeding on wild boar, as there were no pig farms in the vicinity of the trap sites. Few studies have specifically investigated Culicoides species feeding on pigs in Europe. This is likely because Culicoides research has primarily centred on their role as vectors of pathogens that affect ruminants (e.g. Schmallenberg virus, bluetongue virus) or horses (e.g. African horse sickness virus). A systematic review of Culicoides host-feeding patterns indicated that while most species predominantly feed on ruminants and horses, certain biting midges, such as Obsoletus ans Pulicaris complexes, exhibit opportunistic feeding behaviours. These species can feed on multiple host species, including pigs, depending on host availability and abundance [[ 41 , 42 ]. This study marks the first identification of C. riethi and C. salinarius in Lithuania. These vector species had not been previously recorded in the country, likely because earlier investigations were not performed near pig farms or habitats with similar suitable larval breeding sites. It was observed that C. riethi larvae develop in water bodies with extremely low pH and a high concentrations of sulphates, while larvae of C. salinarius can thrive in saline waters [ 43 ]. Our findings demonstrate the affinity of Culicoides to pig farms (8,604 Culicoides spp. specimens were detected on 112 farms over 1 night). While the actual number of midges captured in a single night can vary widely, depending on local conditions, trap type and trap configurations, comparing this with numbers of Culicoides caught on cattle or horse farms for instance, average catches of 1438 and 1161 midges per week, respectively, were reported in Poland [ 44 ]. Notably, species such as C. punctatus and C. newsteadi , were particularly prevalent on the pig farms in this study. The peak of positive pools was observed in August, with 6% of all the tested pools in August testing positive. The possible feeding of Culicoides on pigs increases the likelihood of virus acquisition and subsequent dissemination to other farms, provided the virus remains infective in the insect. Furthermore, environmental factors, including farm management practices, the density of pigs and other hosts, and proximity to natural habitats may influence the abundance and feeding behavior of Culicoides , further shaping their potential role in ASFV spread. Additional determinants related to husbandry practices that may have influenced the occurrence of ASF on commercial farms were evaluated in this case control study and reported by Dhollander et al.[ 22 ]. One notable finding was the significant difference in the use of insect nets between case and control farms, suggesting that insect nets could play a role in preventing the introduction of infected arthropods. Another interesting finding was that the application of manure from other farms near pig farms was identified as a risk factor for the occurrence of ASF. Manure may have served as an attractant for flies thereby increasing the risk of disease transmission. In addition, various types of dung have been shown to serve as suitable breeding sites substrates for the development of Culicoides vector species [ 45 , 46 ], highlighting the association between specific Culicoides species and manure as a breeding habitat. These findings emphasize the importance of implementing vector control strategies in ASF-affected areas. While the primary transmission routes of ASFV remain direct and indirect contact, such as through fomites, the potential role of incidental mechanic vectors such as Culicoides spp. or S. calcitrans should not be ignored. Future research should focus on elucidating the dynamics of ASFV infection in Culicoides , including the potential for virus survival in the mouth parts and gut of the biting midges, and the conditions that may facilitate vector-borne transmission. Such insights are crucial for refining biosecurity measures and mitigating the risk of ASFV transmission risk in pig populations. Declarations Conflicts of Interest The authors declare no conflict of interest. Funding statement The case control study in Lithuania, Poland and Romania was funded by the European Food Safety Authority through 3 monopoly grants GP/EFSA/ALPHA/2021/04, -05 and -06 respectively. The content of this article has been produced exclusively by the authors and it may not be considered as an output adopted by the European Food Safety Authority. Acknowledgements We acknowledge all the collaborators from the State Food and Veterinary Service and the Vilnius University, Life Sciences Centre in Lithuania; the University of Agricultural Sciences and Veterinary Medicine in Cluj-Napoca and the National Sanitary Veterinary and Food Safety Authority in Romania; the National Veterinary Research Institute in Pulawy and the General Veterinary Inspectorate in Warsaw in Poland. We sincerely appreciate the time and efforts provided by Romanian, Lithuanian and Polish farmers, the official veterinarians, laboratory workers and the entomologies in visiting the farms and filling out the questionnaires. We also like to thank all the colleagues in the European Food Safety Authority and the University of Copenhagen that helped with the study design and analysis of the data. This study could not have been done without all your efforts. Thank you!! Author Contributions Conceptualization, S.D. and A.E.B.; methodology, S.D., A.E.B., J.A.C; Execution S.D., A.S.B, A.D.M, M.F., A.G., M.K., W.R., A.M., J.T. statistical analysis, J.A.C..; investigation, S.D. J.A.C; E.C., data curation, S.D., E.C., writing—original draft preparation, S.D..; writing—review and editing, A.S.B, A.D.M, M.F., A.G.,M.K.,W.R., J.R., A.M., M.M., J.T. T.V., K.J., F.B.,M.A.M., J.A.S.; visualization, E.C.; supervision J.A.S. All authors have read and agreed to the published version of the manuscript. References Dixon, L. K., Sun, H., & Roberts, H. (2020). African swine fever. Antiviral Research, 174, 104725. https://doi.org/10.1016/j.antiviral.2019.104725 Penrith ML, Vosloo W. Review of African swine fever: Transmission, spread, and control. J S Afr Vet Assoc. 2009;80(2):58–62. https://doi.org/10.4102/jsava.v80i2.172. Berthe FCJ. African swine fever: An unprecedented global threat. 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Islands","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"Ángel","lastName":"Miranda","suffix":""},{"id":410498955,"identity":"c5f86bee-43a3-4768-80fd-b46cb06f9a45","order_by":20,"name":"Jan Arend Stegeman","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"Arend","lastName":"Stegeman","suffix":""}],"badges":[],"createdAt":"2025-02-01 15:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5942596/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5942596/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06816-w","type":"published","date":"2025-07-31T16:21:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75497433,"identity":"a37ab001-9cc8-47f0-8a51-05bc3d10f494","added_by":"auto","created_at":"2025-02-05 08:27:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":270430,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of \u003cem\u003eCulicoides\u003c/em\u003e species across farms participating in the vector surveillance in Lithuania (A), Poland (B) and Romania (C).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5942596/v1/6e3a350b00ab5f9fe717bb13.png"},{"id":75496295,"identity":"4bfa6564-7e60-400b-92cc-15a2a1a8d175","added_by":"auto","created_at":"2025-02-05 08:19:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33630,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of \u003cem\u003eC. riethi \u003c/em\u003eKieffer\u003cem\u003e \u003c/em\u003eand \u003cem\u003eC. salinarius\u003c/em\u003e Kieffer, newly reported in Lithuania.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5942596/v1/c4bceccf09ba0c002e12793b.png"},{"id":75497434,"identity":"f5e51a43-007e-42e9-b7c3-c1e13a20650d","added_by":"auto","created_at":"2025-02-05 08:27:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137945,"visible":true,"origin":"","legend":"\u003cp\u003eLocations of case and control farms in Lithuania, Poland and Romania where vector surveillance took place and ASFV DNA-positive vectors were found.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5942596/v1/52ca0689458d9a407e18b350.png"},{"id":88268307,"identity":"0b6dabd9-a97e-4dcc-a2d8-33427db92050","added_by":"auto","created_at":"2025-08-04 16:50:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1544659,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5942596/v1/0c4e68ac-b3ea-41f9-8f67-9154d60c2e9b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating the Role of Stable Flies (Stomoxys calcitrans) and Biting Midges (Culicoides spp.) as Potential Mechanical Vectors of African Swine Fever Virus in Lithuania, Poland and Romania.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAfrican swine fever (ASF) is a highly contagious and severe viral disease of domestic and wild suids, caused by a double-stranded DNA virus of the Asfarviridae family, genus \u003cem\u003eAsfivirus\u003c/em\u003e. The virus spreads through direct contact with infected animals or their secretions, and indirectly via contaminated feed or fomites [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During the last 10 years. ASF has caused devastating economic losses globally, affecting both backyard and commercial pig farmers. The disease has disrupted pork supply chains, led to the culling of millions of pigs, and significantly increased pork prices. Overall, the global pork industry has faced losses running into billions of dollars due to ASF outbreaks [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Europe, the epidemiological situation worsened in 2023, with ASF virus (ASFV) genotype II affecting 14 EU Member States, including new outbreaks in Croatia and a resurgence in Romania. The number of outbreaks among domestic pigs in 2023 was five times higher than in 2022, causing substantial disruptions to the EU\u0026rsquo;s pork production and trade. ASF outbreaks in domestic pigs were clearly seasonal in all countries, with 88% of outbreaks reported between July and October [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe seasonal nature of ASFV genotype II outbreaks in pigs in Europe has been evident since its introduction in Georgia in 2007. Summer-related husbandry practices and the transmission by arthropod vectors, abundant and active during the warmer months of the year, have been hypothesized to contribute to the increased risk of virus introduction on farms in the summer [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe soft tick from the \u003cem\u003eOrnithodoros erraticus\u003c/em\u003e complex (Ixodida; Argasidae) is currently the only confirmed biological vector of ASFV transmission in Europe. The biological transmission cycle with pigs has been demonstrated using field strains of ASFV collected during the epidemic in the Iberian Peninsula ([\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]). However, with the currently circulating Georgia 2007/1 ASFV strain, only oral transmission by feeding pigs with infected ticks has been demonstrated [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], despite infectious virus being detectable in ticks up to 8 months after exposure no transmission by feeding ticks was observed [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]). Thanks to its capacity to harbour infectious ASFV for several months, and its nidicolous behaviour (i.e. inhabiting burrows or cracks and feeding for short periods), \u003cem\u003eO. erraticus\u003c/em\u003e is considered a reservoir of ASFV in outdoor farms. In such environments, these ticks can thrive and remain close to pigs. However, they are not considered to play a relevant role in spreading the virus between farms [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eFurthermore, \u003cem\u003eO. erraticus\u003c/em\u003e and other potential soft tick vector species have not been observed in most areas currently affected by ASFV Georgia 2007/1 in Europe, making it highly unlikely that they are responsible for the peak in incidence during the summer months in these regions. However, mechanical transmission by arthropod vectors that are abundant during the warmer months has been the focus of several field studies in affected regions. DNA of ASFV genotype II has been detected in biting insects such as biting midges (\u003cem\u003eCulicoides\u003c/em\u003e spp.) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], mosquitoes (Culicidae) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and \u003cem\u003eStomoxys calcitrans\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] near or on outbreak farms. In addition, ASFV DNA-positive hematophagous flies (i.e.: Tabanidae) have been identified on or near ASF free pig farms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Still, to date, no studies have successfully isolated infectious ASFV from these biting insects. The transmission of ASFV to pigs by arthropods feeding on pigs has only been demonstrated under laboratory conditions. For instance, Mellor et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] showed that \u003cem\u003eS. calcitrans\u003c/em\u003e adults fed on ASFV infected blood were subsequently able to infect pigs during subsequent feedings on pigs.\u003c/p\u003e \u003cp\u003eThis study aimed to gather field evidence on the potential role of stable flies and biting midges in the mechanical transmission of ASFV. Conducted as part of a case-control study between August 4, 2021, and August 23, 2023, in Romania, Lithuania and Poland [ 22], the study hypothesized that if these insect groups contribute to ASFV transmission, the virus would at least be detectable in or on the insects. Such detection would indicate that the insects had fed on infectious blood or secretions, potentially serving as vehicles for introducing infectious virus onto ASF-free farms. Furthermore, a higher proportion of ASFV- positive insects or insect pools on affected farms compared to control farms would support their role in transmission within farms. Conversely, the detection of ASFV-positive insects on control farms could indicate the potential of these insects to transport the virus onto ASFV-free farms.\u003c/p\u003e \u003cp\u003eAdditionally, the collected data on biting midge species and \u003cem\u003eS. calcitrans\u003c/em\u003e were analysed to identify species more likely to carry ASFV DNA and to investigate the presence of new \u003cem\u003eCulicoides\u003c/em\u003e species in previously unreported areas.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSite selection\u003c/h2\u003e \u003cp\u003eThis study was part of a prospective case control study that took place between August 4, 2021, and August 23, 2023, to investigate potential risk factors for the introduction of ASFV on commercial pig farms in Romania, Lithuania and Poland [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For each commercial pig farm where an ASF outbreak was confirmed, two control farms were selected, matched by herd size (e.g. 30\u0026ndash;200 pigs; 201\u0026ndash;1000 pigs; \u0026gt;1000 pigs) and by region, i.e. the Nomenclature of Territorial Units for Statistics (NUTS) 3 region in which the farm was located. If no control farm could be selected in the same NUTS 3 region, a control farm from the same herd size category was chosen from elsewhere in the country. Following the confirmation of the ASF outbreaks on commercial pig farms by the official Veterinary Services in Lithuania, Poland and Romania, the entomology teams were immediately informed about the locations of the affected farms to start vector collection. Simultaneously, two control farms were randomly assigned to each case farm by the team in EFSA. Case farms were to be visited within two days of ASF confirmation, and control farms within two weeks after the matching case farm\u0026rsquo;s outbreak confirmation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVector traps\u003c/h3\u003e\n\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eStable flies (\u003c/b\u003e \u003cb\u003eStomoxys calcitrans\u003c/b\u003e \u003cb\u003e)\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eBlue sticky traps were used to collect stable flies, based on the method described by Hogsette and Kline [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Four trap locations were selected on each farm, two inside and two outside the pig sheds. The traps were placed at least 15 meters apart in areas with high fly activity and direct sunlight to maximize visibility to stable flies. Outdoor traps were placed near the animals, at maximum 15 m from the shed at a height of 1.6 m. Traps were deployed for one day, covering the entire daylight period, as stable flies are diurnal and attracted to translucent and blue-colored objects.\u003c/p\u003e \u003cp\u003eAll stable flies were collected in dry containers and kept cool/frozen (+\u0026thinsp;4 to -20) during transport. Upon arrival at the laboratory, samples were immediately stored at -80\u0026deg;C until morphological identification, ASFV DNA testing and blood meal source analysis.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eBiting midges (\u003c/b\u003e \u003cb\u003eCulicoides\u003c/b\u003e \u003cb\u003espp.)\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eCDC miniature light traps equipped with UV light were used to collect biting midges following the methodology according to Medlock et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Two traps were placed on each farm: one inside and one outside the pig shed. Traps were installed at a height of 1.5 to 2 m above the ground, as recommended by Diarra et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Outdoor traps were placed in areas protected from wind and rain. Traps were situated in dark locations to minimize competition with other light sources. Based on the feeding activity pattern of biting midges, feeding mainly from dusk to dawn, [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], traps were activated one hour before sunset and deactivated one hour after sunrise, facilitating overnight collection of biting midges on each farm. Samples were collected early in the morning, placed in dry containers, and stored as quickly as possible at -80\u0026deg;C, allowing identification of \u003cem\u003eCulicoides\u003c/em\u003e spp., virus isolation and blood meal source identification at a later stage.\u003c/p\u003e\n\u003ch3\u003eVector species identification\u003c/h3\u003e\n\u003cp\u003eThe identification of the arthropods was based on morphological characteristics. Identification of \u003cem\u003eS. calcitrans\u003c/em\u003e was performed using descriptions and keys provided by Zumpt [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. \u003cem\u003eCulicoides\u003c/em\u003e spp. were identified using the Interactive Identification Key for \u003cem\u003eCulicoides\u003c/em\u003e (IIKC), encompassing 110 taxonomic units (102 species and 8 morphological variants) as detailed by Mathieu et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. According to Augot et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], \u003cem\u003eCulicoides obsoletus\u003c/em\u003e, \u003cem\u003eCulicoides scoticus\u003c/em\u003e, and \u003cem\u003eCulicoides chiopterus\u003c/em\u003e were considered to be part of the Obsoletus complex. Identification of the vector species was carried out in the laboratories of each of the countries from where insects were collected, that is the Vilnius University, Life Sciences Centre in Lithuania; the University of Agricultural Sciences and Veterinary Medicine in Cluj-Napoca in Romania and the Veterinary Research Institute in Pulawy in Poland. The latter also performed the virus isolation for all three countries involved in the study.\u003c/p\u003e\n\u003ch3\u003eDetection of ASFV and its DNA and pig DNA in vector samples\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; Pooling of samples\u003c/h2\u003e \u003cp\u003ePrior to DNA extraction based on C\u0026ecirc;tre-Sossah et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Şevik and Oz [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], arthropod samples were pooled by species, collection site and date. Stable flies were pooled in pools of up to four maximum 4 specimens, while biting midges were pooled in pools of maximum 20 specimens. Before pooling, each individual stable fly was dissected longitudinally, creating two identical aliquots. These aliquots were then homogenized (with pools containing 1\u0026ndash;4 halves of stable flies). Biting midges were pooled in groups of 1\u0026ndash;20 individuals, homogenized, and subsequently divided into two equal portions. The first pool of aliquots of stable flies and the first portion of biting midges was used for detection of ASFV DNA by real-time PCR. Additionally, in Romania, the first aliquots also underwent PCR analysis to detect pig DNA. The second pool of aliquots was reserved for virus isolation, if the corresponding first pool tested positive by Real-Time PCR with a Ct value below 20.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; ASFV DNA identification in vectors\u003c/h2\u003e \u003cp\u003e All pooled arthropod samples, including stable flies and biting midges, were tested for ASFV DNA by real-time PCR according to the standard operational procedures (SOPs) used by the ASF national laboratories in Poland (National Veterinary Research Institute, Pulawy), Romania (Institute for Diagnosis and Animal Health, Bucharest), and Lithuania (Lithuanian University of Health Sciences and the Life Sciences Centre of Vilnius University). These SOPs align with those of the European Union Reference Laboratory for ASF .\u003c/p\u003e \u003cp\u003eThe pooled arthropod samples were ground in MagNa Lyser Green Beads with 1.5 ml cold phosphate-buffered saline (PBS1x) containing 0.1% gentamicin sulphate. Suspensions were clarified by centrifugation at 5,000 g for 5 minutes, and 200 \u0026micro;l supernatant was used immediately for ASFV DNA detection or store at \u0026lt;-70\u0026ordm;C for future use. Ct values below 35 were considered positive for ASFV DNA. Validated extraction and amplification kits, routinely used for detection of ASFV in animal samples, were used, adhering to the SOPs implemented at the NRL\u0026rsquo;s for ASF.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e• Detection of Pig DNA\u003c/h3\u003e\n\u003cp\u003ePig DNA analysis was conducted to evaluate whether the vectors had been in contact with pigs or pig secretions, to check if the ASFV DNA-positive vectors were exposed to pigs. Only pools positive for ASFV DNA were tested for the presence of pig DNA. All positive samples for ASFV genome were tested by conventional PCR using an in-house method developed by the ASF NRL of the IDAH based on the protocol described by the Dalmasso et. al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003e• Virus isolation in vectors\u003c/h3\u003e\n\u003cp\u003eSamples that tested real-time PCR positive for ASFV DNA with CT values lower than 20 were further analysed for virus isolation. Only strong positive pools were considered to be worth the resources to possible isolate ASFV, as described earlier [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The National Veterinary Research Institute in Pulawy, Department of Swine Diseases, performed the virus isolation for the three countries participating in the study. The potential presence of infectious virus in the insects\u0026rsquo; homogenates was assessed using a hemadsorption assay in 96-well plates. An aliquot of 50 \u0026micro;L of each filtrated sample (0.45 \u0026micro;m Sartorius syringe filters, G\u0026ouml;ttingen, Germany) was added (in triplicate) to primary porcine alveolar macrophage (PPAM) cell culture or similar cultures supporting the growth of ASFV, such as porcine leukocytes or bone marrow. The cultures contained 200 \u0026micro;L of growth medium: RPMI (Gibco, Waltham, USA), 10% of foetal bovine serum (Gibco, Waltham, USA), 1% of Antibiotic/Antimitotic solution (Sigma Aldrich, St. Louis, USA) and 1:300 (v/v) swine red blood cells (RBC). Hemadsorption was monitored via microscopy over five days post-inoculation, as described by Walczak et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData collection, analysis and visualisation\u003c/h2\u003e \u003cp\u003eThe results of the vector identification and laboratory tests conducted in the three countries were standardized, cleaned and processed using MS Access. Maps were created using R software 4.0. To study if there was a difference between the presence of ASFV DNA positive vector pools in the case and control farms three different analyses were conducted on the \u003cem\u003eCulicoides\u003c/em\u003e results: Random Forest analysis, Classification Tree analysis and Logistic Regression analysis. Different variables were investigated for their potential influence on the ASFV DNA test results of the \u003cem\u003eCulicoides\u003c/em\u003e spp. pools: the Sampling year, Sampling Month, Present of pigs on the farm at the moment of placing the vector traps, Numbers of insects in the pool, Country, Region, Farm category (case or control) and the Culicoides species. No statistical tests were performed on the \u003cem\u003eS. calcitrans\u003c/em\u003e results, due to the small numbers of pools investigated.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGeographic distribution of\u003c/b\u003e \u003cb\u003eCulicoides\u003c/b\u003e \u003cb\u003especies.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the geographic distribution of the different \u003cem\u003eCulicoides\u003c/em\u003e species that were observed on the 112 commercial pig farms included in the study, showing the predominance of \u003cem\u003eC. punctatus\u003c/em\u003e, Obsoletus complex, \u003cem\u003eC. pallidicornis\u003c/em\u003e and \u003cem\u003eC. newsteadi\u003c/em\u003e on the pig farms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eC. riethi\u003c/em\u003e and \u003cem\u003eC. salinarius\u003c/em\u003e [36] were morphologically identified for the first time in Lithuania using Mathieu et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The identification of the species was confirmed using PCR-based methods for analysis of Cytochrome c oxidase subunit I sequences (COI), which are used as barcoding for many insect species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eASFV detection in insects collected on outbreak and control farms\u003c/h2\u003e \u003cp\u003eVectors surveillance results were reported for 42 farms where ASF outbreaks were confirmed (case farms), and 70 control farms matched by farm size and location (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). All case farms were visited within two days of ASF confirmation, and 85% of the control farms were visited within two weeks after the matching case farm\u0026rsquo;s outbreak confirmation, 8% between two and three weeks and 7% between three weeks and one month.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Lithuania, three case and six control farms were included in the study, with ASFV DNA-positive vectors detected on one case farm and one control farm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In Poland, vector surveillance was conducted on 18 case farms and 36 control farms, with ASFV DNA-positive vectors identified on two case farms. In Romania, vector surveillance was carried out on 21 case farms and 28 control farms, with ASFV DNA-positive arthropods were found on seven case farms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom the 1219 insect pools tested on the farms, 27 pools were positive for ASFV DNA. In 2021, 127 insect pools were tested for ASFV DNA of which 18 insect pools were positive. In 2022, 368 insect pools were tested, with one pool positive for ASFV DNA. In 2023, 697 pools were tested with eight pools positive for ASFV DNA.\u003c/p\u003e \u003cp\u003eThe detection of positive pools over tested pools in the three years together were spread over the months as following: two positives out of 8 tested pools in May, zero positive out of 331 tested in June, four positive out of 434 tested in July, 18 positive our of 285 tested in August, three positive out of 99 tested in September and zero positive out of 35 tested in October.\u003c/p\u003e \u003cp\u003eAcross the three countries, a total of 8,604 \u003cem\u003eCulicoides\u003c/em\u003e spp. specimens were collected, of which 4,962 were caught on control farms and 3,642 were caught on case farms. Additionally, 742 \u003cem\u003eS. calcitrans\u003c/em\u003e specimens were caught, of which 141 specimens on case farms and 616 on control farms (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Counts of \u003cem\u003eCulicoides\u0026nbsp;\u003c/em\u003espp. and \u003cem\u003eS. calcitrans\u003c/em\u003e pools and specimens per species collected from pig farms and the positive detections (in red) of ASFV DNA in pools and individual specimens.\u003c/p\u003e\n\u003cdiv style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"border: none;border-collapse: collapse;width: 601px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eSpecies/genus\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNo. pools\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNo. specimens\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ePositive pools\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNo. of specimens in positive pools\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNo. pools\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNo. specimens\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ePositive pools\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 50.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNo. of specimens in positive pools\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eFarm Category\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 183.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eCase farm\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 183.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eControl farm\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eLithuania\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. achrayi\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. circumscriptus\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp 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solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;color:red;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;color:red;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. nubeculosus\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New 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style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. pallidicornis\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e120\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. pulicaris\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp 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0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. circumscriptus\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 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style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e649\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: 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windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e104\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt 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none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e133\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e516\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd 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1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;color:black;'\u003e2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New 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1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;color:black;'\u003e0\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan 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none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: 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none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n 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style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp 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windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. fascipennis\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n 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windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. lupicaris\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New 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0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. newsteadi\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e47\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e470\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e50\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e140\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. 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style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e120\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n 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top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eC. punctatus\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e79\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e790\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;color:red;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;color:red;'\u003e170\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e37\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e370\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 13.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eObsoletus 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top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:12px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003eStomoxys calcitrans\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: 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45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e162\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e1532\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e230\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e116\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e760\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84.7pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e474\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e3768\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e244\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e745\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e5578\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(166, 166, 166);padding: 0.75pt 0.75pt 0in;height: 14.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n \u003cp\u003eThe most frequently caught \u003cem\u003eCulicoides\u003c/em\u003e species across both farm categories were \u003cem\u003eC. punctatus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2,657), Obsoletus complex (n\u0026thinsp;=\u0026thinsp;2,305), \u003cem\u003eC. pallidicornis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1,023) and \u003cem\u003eC. newsteadi\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;631). The majority of \u003cem\u003eCulicoides\u003c/em\u003e species positive for ASFV DNA belonged to \u003cem\u003eC. punctatus\u003c/em\u003e (18 positive pools out of 410 tested) followed by \u003cem\u003eC. newsteadi\u003c/em\u003e (5 positive pools out of 64 tested) and \u003cem\u003eObsoletus\u003c/em\u003e complex (7 positive pools out of 275 tested). None of the real-time PCR- positive \u003cem\u003eCulicoides\u003c/em\u003e pools had Ct values below 20 and were therefore not tested for virus isolation.\u003c/p\u003e \u003cp\u003eIn Romania, pig DNA was identified in three pools of \u003cem\u003eC. punctatus\u003c/em\u003e, three pools of \u003cem\u003eC. newsteadi\u003c/em\u003e and one pool of \u003cem\u003eC. cataneii\u003c/em\u003e. Two of these pools tested negative for ASFV DNA but with doubtful results namely one of the \u003cem\u003eC. cataneii\u003c/em\u003e pools with CT value of 37.3 and one of the \u003cem\u003eC. newsteadi\u003c/em\u003e pools, with CT value of 36.2 but they were nonetheless tested for pig DNA, with negative results. All other pools that were negative for ASFV DNA were not tested for pig DNA. The other countries did not test for pig DNA in the arthropods.\u003c/p\u003e \u003cp\u003eIn total, two of the 224 \u003cem\u003eS. calcitrans\u003c/em\u003e pools tested positive for ASFV DNA by real-time PCR. None of the PCR positive \u003cem\u003eS. calcitrans\u003c/em\u003e pools yielded positive results in virus isolation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eNone of the performed statistical analysis identified any significant association of the investigated variables (Sampling year, Sampling Month, Presence of pigs on the farm during vector survey, Numbers of insects in the pool, Country, Region, Farm category (case or control) and the \u003cem\u003eCulicoides\u003c/em\u003e species) with the presence of ASFV DNA in the \u003cem\u003eCulicoides\u003c/em\u003e pools.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSurveillance of vectors was conducted on 42 outbreak farms and 70 control farms. ASFV DNA positive \u003cem\u003eCulicoides\u003c/em\u003e pools were found on eight case farms and one control farm. Additionally, ASFV DNA positive \u003cem\u003eS. calcitrans\u003c/em\u003e flies were found on two case farms. While there was a higher occurrence of DNA positive insect pools on case than control farms, statistical analysis (Random Forest, Classification Tree and Logistic Regression statistical analysis) showed no significant association between farm category and ASFV DNA test results. This lack of significance is probably due to the small sample size, because fewer outbreaks than anticipated occurred on commercial pig farms during the study period.\u003c/p\u003e \u003cp\u003eASFV could not be isolated from any of these DNA positive insect pools. The inability to detect viable ASFV was likely due to the very small quantities of blood available in the \u003cem\u003eCulicoides\u003c/em\u003e pools tested by real-time PCR. Such pools comprised half the volume of homogenates from up to 20 \u003cem\u003eCulicoides\u003c/em\u003e specimens, or fewer when fewer individuals of a given species were caught. In addition, ASFV\u0026rsquo;s short survival time at higher summer temperatures may have contributed to the inability to recover viable virus.\u003c/p\u003e \u003cp\u003eASFV survival has been studied in several matrices. At room temperature, it has been shown to survive up to 3 days in pig faeces and 5 days in urine [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]; less than 1 day on cereal grains; and up to 30 days on soy oil seeds and soybean meal [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Specific studies investigating the survival of ASF in insects, such as the study of Blome et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e] observed that ASFV could be isolated from \u003cem\u003eStomoxys calcitrans\u003c/em\u003e flies kept in an incubator at 20\u0026deg;C up to 2 days after feeding on viraemic blood. However, higher temperatures significantly reduced virus survival time. Although the traps in this study were placed in the shade, it is possible that high temperatures inside the traps were reached, negatively affecting the survival time of ASFV on mechanical vectors. Thus, while ASFV can be isolated from insects for short periods under laboratory conditions, environmental factors, particularly high summer temperatures, likely inhibit its long-term survival in insects during filed conditions.\u003c/p\u003e \u003cp\u003eThese findings align with previous studies that demonstrated the presence of ASFV DNA in \u003cem\u003eCulicoides\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]and \u003cem\u003eStomoxys calcitrans\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], without isolating viable ASFV. They add to the growing body of evidence suggesting that \u003cem\u003eCulicoides\u003c/em\u003e and \u003cem\u003eS. calcitrans\u003c/em\u003e could serve as a mechanical vector of the virus. The repeated detection of ASFV DNA in these insects highlights the need for further studies to clarify their role in ASF transmission.\u003c/p\u003e \u003cp\u003eIt is possible that \u003cem\u003eCulicoides\u003c/em\u003e merely landed on or fed from contaminated materials on the farm, without actively contributing to the transmission of ASFV. Additionally, the finding of the ASFV DNA positive biting midge on a control farm was not followed by infection of the pigs on the farm, as the farm remained ASF-free. However, this observation was limited to a single farm, and the biosecurity measures and insect control strategies in place may certainly have played a role in protecting the pigs from infection.\u003c/p\u003e \u003cp\u003eAmong the ASFV DNA-positive arthropods caught on the pig farms, the majority belonged to the species \u003cem\u003eC. punctatus\u003c/em\u003e (18 positive pools), followed by Obsoletus complex (\u003cem\u003e7\u003c/em\u003e positive pools) and \u003cem\u003eC. newsteadi\u003c/em\u003e (5 positive pools). These were also to the most frequently observed \u003cem\u003eCulicoides\u003c/em\u003e species on the studied pig farms. In Romania, the only country where the insect pools were tested for host DNA, three \u003cem\u003eC. punctatus\u003c/em\u003e pools, three \u003cem\u003eC. newsteadi\u003c/em\u003e pools, and one \u003cem\u003eC. catanei\u003c/em\u003e pool tested positive for pig DNA, which confirms these ASFV DNA positive insect must have been in direct or indirect contact with pigs. Tomazatos et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e] studied blood meals of common \u003cem\u003eCulicoides\u003c/em\u003e species in the Danube Delta in Romania. The researchers identified \u003cem\u003eSus scrofa\u003c/em\u003e as the second preferred blood meal of \u003cem\u003eC. punctatus\u003c/em\u003e, after cattle. The \u003cem\u003eS. scrofa\u003c/em\u003e DNA in the blood meals identified in the biting midges caught in this study was from feeding on wild boar, as there were no pig farms in the vicinity of the trap sites.\u003c/p\u003e \u003cp\u003eFew studies have specifically investigated \u003cem\u003eCulicoides\u003c/em\u003e species feeding on pigs in Europe. This is likely because \u003cem\u003eCulicoides\u003c/em\u003e research has primarily centred on their role as vectors of pathogens that affect ruminants (e.g. Schmallenberg virus, bluetongue virus) or horses (e.g. African horse sickness virus). A systematic review of \u003cem\u003eCulicoides\u003c/em\u003e host-feeding patterns indicated that while most species predominantly feed on ruminants and horses, certain biting midges, such as Obsoletus ans Pulicaris complexes, exhibit opportunistic feeding behaviours. These species can feed on multiple host species, including pigs, depending on host availability and abundance [[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study marks the first identification of \u003cem\u003eC. riethi\u003c/em\u003e and \u003cem\u003eC. salinarius\u003c/em\u003e in Lithuania. These vector species had not been previously recorded in the country, likely because earlier investigations were not performed near pig farms or habitats with similar suitable larval breeding sites. It was observed that \u003cem\u003eC. riethi\u003c/em\u003e larvae develop in water bodies with extremely low pH and a high concentrations of sulphates, while larvae of \u003cem\u003eC. salinarius\u003c/em\u003e can thrive in saline waters [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings demonstrate the affinity of \u003cem\u003eCulicoides\u003c/em\u003e to pig farms (8,604 \u003cem\u003eCulicoides\u003c/em\u003e spp. specimens were detected on 112 farms over 1 night). While the actual number of midges captured in a single night can vary widely, depending on local conditions, trap type and trap configurations, comparing this with numbers of \u003cem\u003eCulicoides\u003c/em\u003e caught on cattle or horse farms for instance, average catches of 1438 and 1161 midges per week, respectively, were reported in Poland [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, species such as \u003cem\u003eC. punctatus\u003c/em\u003e and \u003cem\u003eC. newsteadi\u003c/em\u003e, were particularly prevalent on the pig farms in this study. The peak of positive pools was observed in August, with 6% of all the tested pools in August testing positive. The possible feeding of \u003cem\u003eCulicoides\u003c/em\u003e on pigs increases the likelihood of virus acquisition and subsequent dissemination to other farms, provided the virus remains infective in the insect. Furthermore, environmental factors, including farm management practices, the density of pigs and other hosts, and proximity to natural habitats may influence the abundance and feeding behavior of \u003cem\u003eCulicoides\u003c/em\u003e, further shaping their potential role in ASFV spread.\u003c/p\u003e \u003cp\u003eAdditional determinants related to husbandry practices that may have influenced the occurrence of ASF on commercial farms were evaluated in this case control study and reported by Dhollander et al.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. One notable finding was the significant difference in the use of insect nets between case and control farms, suggesting that insect nets could play a role in preventing the introduction of infected arthropods. Another interesting finding was that the application of manure from other farms near pig farms was identified as a risk factor for the occurrence of ASF. Manure may have served as an attractant for flies thereby increasing the risk of disease transmission. In addition, various types of dung have been shown to serve as suitable breeding sites substrates for the development of \u003cem\u003eCulicoides\u003c/em\u003e vector species [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e46\u003c/span\u003e], highlighting the association between specific Culicoides species and manure as a breeding habitat.\u003c/p\u003e \u003cp\u003eThese findings emphasize the importance of implementing vector control strategies in ASF-affected areas. While the primary transmission routes of ASFV remain direct and indirect contact, such as through fomites, the potential role of incidental mechanic vectors such as \u003cem\u003eCulicoides\u003c/em\u003e spp. or \u003cem\u003eS. calcitrans\u003c/em\u003e should not be ignored. Future research should focus on elucidating the dynamics of ASFV infection in \u003cem\u003eCulicoides\u003c/em\u003e, including the potential for virus survival in the mouth parts and gut of the biting midges, and the conditions that may facilitate vector-borne transmission. Such insights are crucial for refining biosecurity measures and mitigating the risk of ASFV transmission risk in pig populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe case control study in Lithuania, Poland and Romania was funded by the European Food Safety Authority through 3 monopoly grants GP/EFSA/ALPHA/2021/04, -05 and -06 respectively. The content of this article has been produced exclusively by the authors and it may not be considered as an output adopted by the European Food Safety Authority.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge all the collaborators from the State Food and Veterinary Service and the Vilnius University, Life Sciences Centre in Lithuania; the University of Agricultural Sciences and Veterinary Medicine in Cluj-Napoca and the National Sanitary Veterinary and Food Safety Authority in Romania; the National Veterinary Research Institute in Pulawy and the General Veterinary Inspectorate in Warsaw in Poland. We sincerely appreciate the time and efforts provided by Romanian, Lithuanian and Polish farmers, the official veterinarians, laboratory workers and the entomologies in visiting the farms and filling out the questionnaires. We also like to thank all the colleagues in the European Food Safety Authority and the University of Copenhagen that helped with the study design and analysis of the data. \u0026nbsp;This study could not have been done without all your efforts. Thank you!!\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, S.D. and A.E.B.; methodology, S.D., A.E.B., J.A.C; Execution S.D., A.S.B, A.D.M, M.F., A.G., M.K., W.R., A.M., J.T. statistical analysis, J.A.C..; investigation, S.D. J.A.C; E.C., data curation, S.D., E.C., writing\u0026mdash;original draft preparation, S.D..; writing\u0026mdash;review and editing, A.S.B, A.D.M, M.F., A.G.,M.K.,W.R., J.R., A.M., M.M., J.T. T.V., K.J., F.B.,M.A.M., J.A.S.; visualization, E.C.; supervision J.A.S. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDixon, L. 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Identification of a new vector species of avian haemoproteids, with a description of methodology for the determination of natural vectors of haemosporidian parasites. \u003cem\u003eParasites \u0026amp; Vectors\u003c/em\u003e. 2019;12. https://doi.org/10.1186/s13071-019-3559-8\u003c/li\u003e\n\u003cli\u003eDavies, K., Goatley, L. C., Guinat, C., Netherton, C. L., Gubbins, S., Dixon, L. K., \u0026amp; Reis, A. L. (2017). Survival of African swine fever virus in excretions from pigs experimentally infected with the Georgia 2007/1 isolate. \u003cem\u003eTransboundary and Emerging Diseases, 64\u003c/em\u003e, 425\u0026ndash;431. https://doi.org/10.1111/tbed.12381\u003c/li\u003e\n\u003cli\u003eDee, S. A., Bauermann, F. V., Niederwerder, M. C., Singrey, A., \u0026amp; Clement, T. (2018). Survival of viral pathogens in animal feed ingredients under transboundary shipping models. \u003cem\u003ePLoS ONE, 13\u003c/em\u003e, e0194509. https://doi.org/10.1371/journal.pone.019\u003c/li\u003e\n\u003cli\u003eStoian AMM, Zimmerman J, Ji J, Hefley TJ, Dee S, Diel DG, et al. Half-life of African swine fever virus in shipped feed. Emerg Infect Dis. 2019;25:2261\u0026ndash;3. https://doi.org/10.3201/eid2512.191002.\u003c/li\u003e\n\u003cli\u003eBlome S, Sch\u0026auml;fer M, Ishchenko L, M\u0026uuml;ller C, Fischer M, Carrau T, et al. Survival of African swine fever virus in feed, bedding materials, and mechanical vectors and their potential role in virus transmission. \u003cem\u003eEFSA Supporting Publications\u003c/em\u003e. 2024;21(4):EN-8776. https://doi.org/10.2903/sp.efsa.2024.EN-8776\u003c/li\u003e\n\u003cli\u003eTomazatos A, J\u0026ouml;st H, Schulze J, Spinu M, Schmidt-Chanasit J, Cadar D, et al. Blood-meal analysis of \u003cem\u003eCulicoides\u003c/em\u003e (Diptera: Ceratopogonidae) reveals a broad host range and new species records for Romania. Parasites Vectors. 2020;13. https://doi.org/10.1186/s13071-020-3938-1.\u003c/li\u003e\n\u003cli\u003eViennet, E., Garros, C., Gardes, L., Rakotoarivony, I., All\u0026egrave;ne, X., et al. (2013). Host preferences of Palaearctic \u003cem\u003eCulicoides\u003c/em\u003e biting midges: implications for transmission of orbiviruses. \u003cem\u003eMedical and Veterinary Entomology, 27\u003c/em\u003e(3), 255\u0026ndash;266. https://doi.org/10.1111/j.1365-2915.2012.01042.x\u003c/li\u003e\n\u003cli\u003eGutsevich, A. V. (1973). Blood-sucking midges of the genera \u003cem\u003eCulicoides\u003c/em\u003e and \u003cem\u003eForcipomyia\u003c/em\u003e (\u003cem\u003eCeratopogonidae\u003c/em\u003e). \u003cem\u003eFauna USSR\u003c/em\u003e, 1st ed., 342. Leningrad: Nauka Press.\u003c/li\u003e\n\u003cli\u003eLarska M, Grochowska M, Lechowski L, Zmudzinski J. Abundance and species composition of Culicoides spp. biting midges near cattle and horse in South-Eastern Poland. Acta Parasitol. 2017;62:10.1515/ap-2017-0089.\u003c/li\u003e\n\u003cli\u003eWerner D, Groschupp S, Bauer C, Kampen H. Breeding habitat preferences of major \u003cem\u003eCulicoides\u003c/em\u003e species (Diptera: Ceratopogonidae) in Germany. Int J Environ Res Public Health. 2020 Jul 11;17(14):5000. doi: 10.3390/ijerph17145000. PMID: 32664561; PMCID: PMC7400431.\u003c/li\u003e\n\u003cli\u003eZimmer JY, Brostaux Y, Haubruge E, Francis F. Larval development sites of the main \u003cem\u003eCulicoides\u003c/em\u003e species (Diptera: Ceratopogonidae) in northern Europe and distribution of coprophilic species larvae in Belgian pastures. Vet Parasitol. 2014;205(3):676\u0026ndash;86. https://doi.org/10.1016/j.vetpar.2014.08.029.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Stomoxys calcitrans, Culicoides, Mechanical transmission, African Swine Fever Virus, pigs","lastPublishedDoi":"10.21203/rs.3.rs-5942596/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5942596/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSince its emergence in Georgia in 2007, the seasonal pattern of African swine fever virus (ASFV) genotype II outbreaks in European pig populations has been evident. It is hypothesized that summer-related farming practices, along with the increased activity and abundance of arthropod vectors during warmer months, contribute to the increased incidence of these outbreaks during this period. This study investigated the potential role of stable flies (\u003cem\u003eStomoxys calcitrans\u003c/em\u003e) and biting midges (\u003cem\u003eCulicoides\u003c/em\u003e spp.) as mechanical vectors of ASFV.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFrom August 2021 to August 2023, vector surveillance was conducted as part of a case-control study on 42 outbreak farms and 70 control farms across Romania, Lithuania, and Poland. Collected insect specimens were pooled and tested for ASFV DNA using real-time PCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 8,604 biting midges\u0026rsquo; specimens and 742 \u003cem\u003eS. calcitrans\u003c/em\u003e flies were collected, with ASF DNA detected in 27 out of 1,219 insect pools. Positive pools were predominantly observed in \u003cem\u003eCulicoides punctatus\u003c/em\u003e, \u003cem\u003eC. newsteadi\u003c/em\u003e, and the Obsoletus complex, with most detections occurring in August. However, ASFV isolation was unsuccessful. Statistical analyses revealed no significant association between farm status (outbreak vs. control) and ASFV DNA detection in biting midges\u0026rsquo; pools, likely due to limited sample size. Additionally, two ASFV-positive \u003cem\u003eS. calcitrans\u003c/em\u003e pools were identified, supporting their potential role as mechanical vehicles for ASFV. The findings highlight the affinity of \u003cem\u003eC. punctatus\u003c/em\u003e, \u003cem\u003eC. newsteadi\u003c/em\u003e, and the Obsoletus complex and \u003cem\u003eS. calcitrans\u003c/em\u003e for pig farms, which may increase the likelihood of ASFV acquisition and dissemination, assuming viral infectivity is maintained in these insects. Environmental factors, such as pig farm density and proximity to other hosts, likely influence this risk.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results emphasize the importance of vector control strategies, including insect netting, to mitigate ASFV transmission risks. Further research is needed to understand the dynamics of ASFV infection in arthropods. Notably, this study also reports the first identification of \u003cem\u003eCulicoides riethi\u003c/em\u003e and \u003cem\u003eCulicoides salinarius\u003c/em\u003e in Lithuania.\u003c/p\u003e","manuscriptTitle":"Investigating the Role of Stable Flies (Stomoxys calcitrans) and Biting Midges (Culicoides spp.) as Potential Mechanical Vectors of African Swine Fever Virus in Lithuania, Poland and Romania.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 08:19:28","doi":"10.21203/rs.3.rs-5942596/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-09T20:24:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-09T16:12:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-05T11:03:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-27T13:18:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51862498499696847276781828034370979598","date":"2025-02-15T19:40:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96516979250791377449082894962675148243","date":"2025-02-10T11:22:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331508946561988520822154901863820207045","date":"2025-02-10T09:51:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145804613247668424433583200834272728171","date":"2025-02-09T10:58:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227425089910380448373418418440083125465","date":"2025-02-08T21:46:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-08T18:39:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-03T11:30:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-03T11:21:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-02-01T15:40:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"95a1688d-5919-417f-a8e0-bd00edc91ae3","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:43:45+00:00","versionOfRecord":{"articleIdentity":"rs-5942596","link":"https://doi.org/10.1186/s13071-025-06816-w","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-07-31 16:21:26","publishedOnDateReadable":"July 31st, 2025"},"versionCreatedAt":"2025-02-05 08:19:28","video":"","vorDoi":"10.1186/s13071-025-06816-w","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06816-w","workflowStages":[]},"version":"v1","identity":"rs-5942596","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5942596","identity":"rs-5942596","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}