Epizootic hemorrhagic disease virus oral infection affects midge reproduction and is vertically transmitted to offspring in the biting midge, Culicoides sonorensis (Diptera: Ceratopogonidae)

Epizootic hemorrhagic disease virus oral infection affects midge reproduction and is vertically transmitted to offspring in the biting midge, Culicoides sonorensis (Diptera: Ceratopogonidae) | 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 Article Epizootic hemorrhagic disease virus oral infection affects midge reproduction and is vertically transmitted to offspring in the biting midge, Culicoides sonorensis (Diptera: Ceratopogonidae) Dinesh Erram, Bethany McGregor, Carolina Acevedo, Barry Alto, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4365421/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 May, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Epizootic hemorrhagic disease virus (EHDV: Reoviridae: Orbivirus ) is a Culicoides- borne pathogen that affects a variety of ruminants, causing significant economic losses and/or ecological impacts in animal agriculture/wildlife populations worldwide. In this study, we examined the effect of EHDV serotype-2 oral infection on the survival and reproduction of Culicoides sonorensis Wirth and Jones (a confirmed vector of EHDV in North America), and the potential vertical transmission of EHDV-2 (from infected female to its offspring) in this midge species. Culicoides sonorensis females were fed on defibrinated bovine blood with EHDV-2 (5.5 log 10 PFU/ml) or without EHDV-2 (control). Adult survival/longevity, oviposition rates, number of eggs deposited, egg hatch rates (fertility), larval survival, larval stage duration, eclosion rates, and sex-ratios of the progeny were recorded and compared between the two groups. In addition, the progeny (eggs and F 1 generation adults) of the EHDV-2 infected females were processed for viral detection through RT-qPCR and plaque assays. Survival/longevity of the blood-fed adults, oviposition rates, number of eggs deposited, larval stage duration, eclosion rates, and sex-ratios were not significantly different between the two groups. However, egg hatch rates were significantly lower in the EHDV-2 infected group (35.8 ± 5.2%) than the uninfected group (74.5 ± 6.8%), but larval survival rates were higher in the EHDV-2 infected group (59.8 ± 4.9%) compared to the control group (34.1 ± 6.5%). EHDV-2 was detected in the eggs (3.4%, 1/29 females tested, Cq value 22.1) and F 1 adult progeny (1.7%, 1/58 adults tested, Cq value 23.5) of the orally exposed females through RT-qPCR as well as plaque assays. Our findings suggest that EHDV-2 infection has no major impact on C. sonorensis survival/longevity or oviposition but has a significant negative effect on midge fecundity/fertility. Our study also provides evidence for the vertical transmission of EHDV-2 from an infected adult female to its offspring in C. sonorensis . However, salivary transmission of EHDV-2 from the vertically infected progeny and its significance in the epidemiology of hemorrhagic disease are currently unknown and remain to be examined in further studies. Overall, these findings collectively indicate that Orbivirus infection can negatively affect vector reproduction and that vertical transmission is a probable mechanism of overwintering of EHDV in North America. Biological sciences/Zoology/Entomology Biological sciences/Microbiology/Virology/Viral transmission Culicoides sonorensis biting midges survival longevity reproduction fecundity fertility EHDV BTV vertical transmission overwintering Figures Figure 1 Figure 2 1. Introduction Culicoides species (Diptera: Ceratopogonidae), commonly referred to as biting midges or no-see-ums, transmit numerous disease-causing agents to humans and animals worldwide 1 . In North America, Culicoides species are mainly important in the transmission of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) that cause hemorrhagic disease in susceptible hosts such as sheep, cattle, and white-tailed deer, and result in significant economic losses in animal agriculture. In the United States (US), Culicoides sonorensis Wirth & Jones and Culicoides insignis Lutz are the only confirmed vectors of EHDV/BTV 2 – 4 . However, other species such as Culicoides stellifer Coquillett, Culicoides venustus Hoffman, Culicoides debilipalpis Lutz, and others are likely involved in virus transmission in sylvatic cycles and in areas outside the geographic range of these vectors, particularly in the southeast 5 , 6 . Arboviral infections alter several life history traits of biting insects, influencing their overall vectorial capacity. For example, Aedes aegypti Linnaeus mosquitoes infected with dengue 7 or Chikungunya virus 8 show reduced survival and fecundity, A. aegypti infected with Zika virus show reduced lifespan but no change in fecundity 9 , while A. aegypti infected with Mayaro virus show reduced fecundity but no change in survival 10 . Similarly, Culex tarsalis Coquillett females infected with West Nile virus show reduced fecundity but no change in survival 11 , Cx. tarsalis infected with Western Equine Encephalitis virus show reduced survival and fecundity 12 , while Culiseta melanura Coquillett mosquitoes infected with Eastern Equine Encephalitis virus show reduced survival and fecundity 13 . Arboviruses are usually maintained in their natural transmission cycles through horizontal transmission between the arthropod vectors and the vertebrate hosts. However, during cold/dry seasons when the vector population densities are low or when herd immunity of the vertebrate hosts is high, alternate mechanisms such as vertical transmission (from infected arthropod vectors to their offspring) may drive the persistence of arboviruses in nature 14 . In the US, hemorrhagic disease is endemic with several serotypes of EHDV and BTV circulating in domestic and wild ruminants, particularly in the southeast 15 . However, the mechanisms by which these orbiviruses overwinter in the US remain uncertain to date. Previous studies concluded that vertical transmission of BTV is unlikely 16 – 18 , and suggested that long lived C. sonorensis females infected the previous year are more likely to contribute to the interseasonal maintenance of BTV in California 19 . However, the overwintering mechanisms of BTV outside California have received little attention to date 20 . In general, the vectorial capacity parameters of Culicoides species associated with EHDV/BTV transmission have not been studied comprehensively. In addition, the potential overwintering mechanisms of EHDV in North America have received little attention to date, collectively representing important knowledge gaps in our understanding of the overall transmission dynamics of orbiviruses in North America. In this study, we examined the survival and reproduction of C. sonorensis orally challenged with EHDV serotype-2 and examined the possibility of vertical transmission of EHDV-2 from an infected adult female to its offspring in this midge species. 2. Materials and Methods 2.1. Oral virus exposure The EHDV-2 strain used in this study was initially isolated from the spleen of a dead white-tailed deer from a commercial cervid facility in Quincy, Florida, US in 2016 (NCBI Accession #MF688816-MF688825). The virus was passaged twice on African green monkey kidney (Vero) cells maintained on medium 199 (HyClone Medium 199, GE Healthcare Life Sciences, Logan, UT, US) containing 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, US), 2% penicillin/streptomycin and 0.2% Amphotericin B (Thermo Fisher Scientific, Waltham, MA, US). Culicoides sonorensis females Ausman strain 21 (48 h post adult emergence) were allowed to feed for 1 h on defibrinated bovine blood (HemoStat Laboratories, Dixon, CA, US) inoculated with EHDV-2 in cell culture media (5.5 log 10 plaque forming unit equivalents [PFUe]/ml final concentration of blood + virus) or with clean media without EHDV-2 (control group) using a Hemotek artificial feeding system (Hemotek, Blackburn, United Kingdom) with a stretched Parafilm® M membrane (Amcor, Zurich, Switzerland). The viral titers of infectious blood fed to the midges are representative of viremia levels experienced by infected deer 22 . 2.2. Assessment of midge life history traits After feeding, blood-engorged females in each group were sorted using a CO 2 pad into individual 175 ml cardboard containers fitted (hot glued) with an oviposition substrate (Petri dish [60 × 15 mm] with moist cotton + filter paper) on the bottom. All females were provided with 10% sucrose solution on moistened cotton pads that were replaced daily, and individuals were monitored daily for survival and oviposition. The eggs deposited by females in each group were placed on 0.3% agar slants ( n = 10 eggs/dish) and the hatched larvae were reared on a diet of Panagrellus redivivus nematodes (Carolina Biological Supply Company, Burlington, North Carolina, US), according to previously established methods 23 . Developmental life history traits of the larvae were monitored and recorded every other day. Overall, several life history traits of C. sonorensis were recorded and compared between the two groups. These included: blood-fed adult survival/longevity, percentage of females that oviposited, number of eggs deposited, egg hatch rates, larval survival to pupal stage, larval stage duration, eclosion rates, and sex-ratios of the emerging adults. Environmental conditions throughout the experiments were 26°C, 60–80% RH, and 16:8 (L:D) h cycle. 2.3. Virus detection from the progeny of EHDV-2 fed females A randomly selected subset of the egg batch deposited by EHDV fed females ( n = 29) and F 1 generation adults reared from the other subset of eggs ( n = 58; 32 males and 26 females) were processed for the detection/presence of EHDV. Partial egg batches from each female and individual F 1 generation adults were placed in 200 µl of medium 199 (HyClone Medium 199, GE Healthcare Life Sciences, Logan, UT, US) in 1.5 ml microcentrifuge tubes containing 5–10 borosilicate glass beads (2 mm; Sigma-Aldrich, St. Louis, MO, US). This was followed by homogenization for five minutes using a Bullet Blender Storm homogenizer (Next Advance, Troy, NY, US). Viral RNA was then extracted from 150 µl of the homogenate using the QIAamp Viral RNA Mini Kit (Cat#52906, Qiagen, Hilden, Germany) using manufacturer’s protocol. The remainder of the homogenate (50 µl) was stored at − 80°C for later use as inoculum for culturing virus, if the sample was determined RT-qPCR positive. RT-qPCR was run on the extracted RNA using SuperScript III One-Step qRT-PCR kit (Thermo Fisher Scientific) using previously established protocols 24 employing primer and probe sequences from Wernicke et al. 25 and PCR conditions from Wilson et al. 26 , modified slightly (25 min at 55°C, 2 min at 95°C, and 45 cycles of 10 s at 95°C and 1 min at 57°C). All RT-qPCR assays were run with positive (EHDV-2 stock) and negative (water) controls. The RNA samples that were EHDV-2 positive through RT-qPCR were further processed using plaque assays to quantitate replication competent virions. Briefly, the remainder of homogenates were added to 6-well plates of confluent Vero cells, incubated for an hour at 37°C, and covered with an agarose gel overlay. One week post infection, overlays were removed, and crystal violet stain was used to view plaque formations. Each plaque was assumed to have originated from a single infecting virus. The samples showing plaques were again processed for EHDV-2 detection through RT-qPCR in the same manner as described above. The quantification cycle values (Cq) and standard curves for the EHDV-2 Florida strain used in the study were reported in our previous study and its detection limit was determined to be at Cq value 35 24 . 2.4. Statistical analysis Variation in the survival/longevity of adults between the two groups was analyzed using Kaplan-Meier survival curves and log rank (Mantel-Cox) tests. Variation in the percentage of females that oviposited, number of eggs deposited per female, egg hatch rates, larval survival to pupal stage, larval stage duration, eclosion rates, and sex-ratios of the emerging adults between the two groups were analyzed using generalized linear models (GLM) under Poisson or negative binomial distributions. All data were analyzed using R statistical software v.3.6.1 27 using packages Mass 28 , car 29 , and lsmeans 30 . 3. Results In total, there were 56 individual blood-fed females in the EHDV-2 infected group and 50 in the control group. Life history traits of adults : The longevity of females in the EHDV-2 infected group (13.7 ± 1.2 days post blood feeding [pbf]) (mean ± SE) was slightly, but not significantly longer than that in the control group (11.8 ± 1.2 days pbf) (χ 2 1 = 3.32, P = 0.0685) (Fig. 1 A). The percentage of females that oviposited in the EHDV-2 infected group was 57.1% (32/56; 95% CI: 43.2–70.3%), which was not significantly different from that in the uninfected (control) group (52.0%, 26/50; 95% CI: 37.4–66.3%) (χ 2 1 = 0.28, P < 0.5954) (Fig. 1 B). The number of eggs deposited per female in the EHDV-2 infected group (89.1 ± 10.4 eggs/female) was not significantly different from that in the control group (73.7 ± 9.1 eggs/female) (χ 2 1 = 0.72, P < 0.3964) (Fig. 1 C). The egg hatch rate in the EHDV-2 infected group was 35.8 ± 5.2%, which was significantly lower than that in the uninfected group (74.5 ± 6.8%) (χ 2 1 = 87.8, P < 0.0001) (Fig. 1 D). Life history traits of progeny : The rate of larval survival to pupal stage in the EHDV-2 infected group was 59.8 ± 4.9%, which was significantly higher than that in the control group (34.1 ± 6.5%) (χ 2 1 = 18.0, P < 0.0001) (Fig. 2 A). Larval development time to pupal stage in the EHDV-2 infected group (29 ± 0.9 days) was not significantly different from that in the uninfected group (27.3 ± 1.1 days) (χ 2 1 = 2.0, P = 0.1526) (Fig. 2 B). The eclosion rate of adults in the EHDV-2 infected group (44.6 ± 5.4%) was not significantly different from that in the control group (57.6 ± 8.8%) (χ 2 1 = 0.01, P = 0.9334) (Fig. 2 C). The sex-ratio of F 1 adults in the EHDV-2 infected group (1.1:1 [M:F]) was not significantly different from that in the uninfected group (0.9:1) (χ 2 1 = 0.21, P = 0.6490) (Fig. 2 D). Virus detection from the progeny of EHDV-2 fed females Partial egg batches from 29 different females were tested for EHDV-2. Out of these, 72.4% (21/29) were positive through RT-qPCR and 3.4% (1/29, Cq value 22.1, 4.9 log 10 PFUe/ml) were positive through plaque assays. Similarly, 58 F 1 adults (32 males and 26 females) were tested individually for EHDV-2. Out of these, 21.9% of the males (7/32) and 19.2% of the females (5/26) were positive through RT-qPCR. One F 1 adult male was positive (1.7%, 1/58 adults tested, Cq value 23.5, 4.5 log 10 PFUe/ml) through plaque assays. None of the F 1 adult females were positive through plaque assays (0.0%, 0/58 adults tested). All samples positive through plaque assays were subsequently re-confirmed as EHDV-2 through RT-qPCR (Table 1). 4. Discussion Overall, our findings demonstrate that EHDV-2 oral infection does not have a major impact on C. sonorensis survival/longevity or oviposition. However, EHDV-2 infection does have a significant negative effect on C. sonorensis fecundity (fertility) as it reduces the egg hatch rates in infected females. Interestingly, the larval survival rates to pupal stage were higher in the EHDV-2 infected group than in the uninfected group, which may possibly offset the lower egg hatch rates of EHDV-2 infected females and balance the overall reproductive output of the two groups. Nonetheless, the reasons behind this outcome are currently unknown, although they denote transgenerational virulence with consequences for offspring life history. Further studies will be needed to examine the impact of EHDV-2 on the overall reproductive output of C. sonorensis and to examine the physiological mechanisms by which EHDV-2 infection increases larval survival rates. The reasons behind the reduced egg hatch rates in EHDV-2 infected C. sonorensis females are currently unknown as well. However, it is possible that EHDV infection alters the expression of certain genes in the midge ovaries that are important for egg development/maturation. Previously, A. aegypti mosquitoes infected with Dengue-2 virus showed reduced fecundity compared to their uninfected counterparts 31 . Subsequent transcriptomic profiling of the ovaries of virus-infected mosquitoes showed the upregulation of several genes, which were suggested to interfere with egg production 31 . Similarly, A. aegypti infected with Chikungunya virus also showed reduced fecundity compared to uninfected mosquitoes 8 . Subsequent gene expression analyses on virus infected mosquitoes showed the downregulation of six transcripts in the egg laying pathway of A. aegypti 8 . A second possible reason is that the transfer of sperms from spermathecae into the micropyle of the eggs during oviposition (fertilization) is affected due to EHDV infection of certain tissues in the female reproductive tract (see further discussion below). Previously, EHDV-2 was detected in the epithelial cells of the spermathecae and the ovarian sheath in C. sonorensis 32 . Another possible reason is that EHDV infection causes unknown pathogenesis in the midges reducing their ability to produce fertile eggs or causes direct/indirect mortality in the eggs/embryos. Previously, Chikungunya virus infection caused reduced egg hatch rates in A. albopictus 33 and West Nile virus infection caused reduced egg hatch rates in Cx. tarsalis mosquitoes 11 . However, further studies will be needed to test these hypotheses. EHDV-2 was detected in the eggs and F 1 generation adults resulting from the virus infected C. sonorensis females through RT-qPCR as well as plaque assays, thus demonstrating that vertical transmission of EHDV may occur in nature. It was previously thought that EHDV and BTV are unlikely to be transmitted vertically in biting midges. This was because EHDV/BTV infections in the midge reproductive tissues in previous studies were detected essentially only along the outer layers, but not in the gametes. For example, BTV was detected only within the ovarian sheath, immature yolk bodies and vitelline membrane of the oocyte in C. sonorensis , but not in the oocytes or nurse cells 17 , 34 . Subsequent studies found no evidence for the vertical transmission of BTV under laboratory or field conditions 16 – 18 . Similarly, EHDV-2 was detected only within the spermathecal epithelium and the ovarian sheath in C. sonorensis , but not in the ovariolar sheath, ovarioles, follicular epithelium, oocytes or nurse cells 32 . Therefore, it was suggested that vertical transmission of EHDV is unlikely as well. However, our findings contradict previous notions and demonstrate clearly that EHDV-2 is vertically transmitted in C. sonorensis . It is possible that these discrepancies are due to differences in the EHDV-2 strains used in the studies. Mills et al. 32 used an EHDV-2 strain isolated from a white-tailed deer from Kansas, while we used an EHDV-2 strain isolated from a white-tailed deer from Florida in this study. It is possible that the EHDV-2 Florida strain infects different reproductive tissues/cells of C. sonorensis than the Kansas strain, which enable the virus to be vertically transmitted in the midge. Alternately, the virus may enter the fully developed egg later during oviposition. Currently, variations in the midge reproductive tissues/cells infected by the EHDV-2 Florida strain and other geographic strains (and serotypes) of EHDV/BTV are unknown. Further studies will be needed to examine the tissue specific infections caused by different serotypes/strains of orbiviruses in biting midges, which may provide clues towards the mode of vertical transmission of EHDV-2 found in C. sonorensis in this study. In mosquitoes, arboviruses are vertically transmitted through the transovarial or the transovum routes 35 , 36 . In a previous study, we found that the EHDV-2 Florida strain exhibited higher infection and dissemination rates than the EHDV-2 Can-Alberta strain in C. sonorensis , suggesting that the consequences of EHDV infection in biting midges vary with the geographic strain of the virus 24 . Therefore, it is likely that different serotypes/strains of EHDV and BTV influence the vector competence and other life history traits/vectorial capacity parameters of Culicoides species differently as well. The overall vertical transmission rates in this study were low (1.7%, 1/58 adults tested). Notably, our observations were made only during the first gonotrophic cycle of C. sonorensis . As such, it is possible that these vertical transmission rates persist or even increase during the subsequent gonotrophic cycles of C. sonorensis , as reported for other arboviruses in mosquitoes 37 . Interestingly, the F 1 generation adult from which EHDV-2 was detected in this study was a male (3.1%, 1/32) and EHDV-2 was not detected from any females examined (0.0%, 0/26). As such, it is possible that EHDV is also sexually transmitted from the infected male midges to females during mating (and possibly vice versa as well). Previously, sexual transmission of vesicular stomatitis virus was reported in C. sonorensis 38 , in addition to many other arboviruses in their insect vectors 39 – 42 . However, further studies will be needed to test these hypotheses. Hemorrhagic disease is known to be endemic in the US with several serotypes of EHDV and BTV frequently detected in a variety of domestic and wild ruminants, particularly in the southeast 15 . However, the potential mechanisms by which these orbiviruses overwinter in temperate regions such as North America remain enigmatic to date. In the past, several studies have concluded that vertical transmission of BTV is unlikely 16 – 18 , 34 . However, Mayo et al. 19 found BTV RNA in C. sonorensis parous females collected during the early part of the interseasonal period (Feb – Mar) of 2013 and 2014 in California, suggesting that in the absence of vertical transmission, long lived females infected the previous year can contribute to the interseasonal maintenance of BTV in temperate regions. Along similar lines, our current findings demonstrating the vertical transmission of EHDV-2 in C. sonorensis offer a strong explanation for one of the potential overwintering mechanisms of EHDV in North America and provide a plausible explanation for the endemic nature of hemorrhagic disease in the US. On a per capita basis, the vertically infected midges that are infectious are predicted to strongly contribute to EHDV transmission because, 1) they can transmit the virus to vertebrates upon their first bite, and 2) they will be infectious for a longer period as adults compared to midges that acquire EHDV by bite through horizontal transmission. It is possible that the low vertical transmission rates found in our study are adequate to sustain the virus between years while still requiring several months of amplification within host populations before outbreaks are observed, typically during late summer to fall. However, it is currently unknown whether the vertically infected progeny of orally infected midges can transmit EHDV-2 through their saliva/bite. Therefore, further studies will be needed to demonstrate the salivary transmission of EHDV-2 from the vertically infected progeny to susceptible hosts and to determine its significance in the epidemiology of hemorrhagic disease. Moreover, further studies will also be needed to gather evidence from field-collected C. sonorensis to ascertain whether these findings are biologically significant. However, it is to be noted that C. sonorensis is scattered/rare in the southeastern US 43 . Therefore, further studies will be needed to examine whether any of the endemic EHDV and BTV serotypes or strains in the US are vertically transmitted through other species associated with EHDV/BTV transmission in the southeast such as C. insignis, C. stellifer , C. venustus , C. debilipalpis , or others. However, the current lack of colonies of Culicoides vector species other than C. sonorensis may pose a challenge to such studies. 5. Conclusions In conclusion, our study demonstrates that EHDV does not have a major impact on the survival/longevity, or oviposition of C. sonorensis , but has a significant negative effect on midge reproduction as it reduces the egg hatch rates in infected females (but increases larval survival rates). In addition, our findings demonstrate that EHDV-2 can be vertically transmitted in C. sonorensis , a phenomenon that was previously thought unlikely. These findings collectively improve our understanding of the vectorial capacity parameters of C. sonorensis , provide valuable insight into the potential overwintering mechanisms of EHDV in North America, and offer a plausible explanation for the endemic nature of hemorrhagic disease in the US. Further studies will be needed to understand the physiological mechanisms by which EHDV-2 infection causes reduced fecundity/fertility and increased larval survival rates in biting midges and to determine the mode of vertical transmission of EHDV-2 in C. sonorensis . More importantly, further studies will be needed to examine the salivary transmission of EHDV-2 from the vertically infected progeny to susceptible hosts and to evaluate its significance in the epidemiology of hemorrhagic disease. In addition, further studies will also be needed to gather evidence from field-collected C. sonorensis to ascertain whether these findings are biologically significant and to determine whether any of the endemic (or novel) serotypes/strains of EHDV and BTV in North America are vertically transmitted through other species associated with Orbivirus transmission such as C. insignis , C. stellifer , C. venustus , C. debilipalpis or others. Declarations Author contributions: Dinesh Erram : Conceptualization, Methodology, Data Curation, Formal Analysis, Visualization, Validation, Investigation, Resources, Project Administration, Writing – Original Draft Preparation. Bethany McGregor : Methodology, Writing– Review & Editing. Carolina Acevedo : Methodology, Writing– Review & Editing. Barry W. Alto: Resources, Writing– Review & Editing. Nathan Burkett-Cadena : Funding Acquisition, Supervision, Writing– Review & Editing. All authors have reviewed and agreed to the final version of this manuscript. Funding: This study was funded by the Cervidae Health Research Initiative (CHeRI) sponsored by the State of Florida Legislature. NBC is partially supported by NIFA Project FLA-FME-006106. Acknowledgments: We thank Lee Cohnstaedt and William Yarnell (USDA, Manhattan, KS, US) for the maintenance and provision of colonized midges for this study. Agustin Quaglia provided help with statistical analyses of the data. Conflicts of Interest: The authors declare that they have no conflicts of interest. Data Availability: All data generated or analyzed during this study are included in this published article. Disclosures : Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. References Mellor, P. S., Boorman, J. & Baylis, M. Culicoides biting midges: their role as arbovirus vectors. Annu. Rev. Entomol. 45 , 307–340 (2000). Tabachnick, W. J. Culicoides variipennis and bluetongue-virus epidemiology in the United States. Annu. Rev. Entomol. 41 , 23–43 (1996). Tanya, V. N., Greiner, E. C. & Gibbs, E. P. Evaluation of Culicoides insignis (Diptera: Ceratopogonidae) as a vector of bluetongue virus. Vet. Microbiol. 32 , 1–14 (1992). McGregor, B. L. et al. 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Laboratory rearing of Culicoides stellifer (Diptera: Ceratopogonidae), a suspected vector of Orbiviruses in the United States. J. Med. Entomol. 57 , 25–32 (2020). McGregor, B. L., Erram, D., Acevedo, C., Alto, B. W. & Burkett-Cadena, N. D. Vector competence of Culicoides sonorensis (Diptera: Ceratopogonidae) for epizootic hemorrhagic disease virus serotype 2 strains from Canada and Florida. Viruses 11 , 367 (2019). Wernike, K., Hoffmann, B. & Beer, M. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR. J. Virol. Methods 217 , 28–35 (2015). Wilson, W. C. et al. Detection of all eight serotypes of Epizootic hemorrhagic disease virus by real-time reverse transcription polymerase chain reaction. J. Vet. Diagn. Invest. 21 , 220–225 (2009). R Core Team. R: A Language and Environment for Statistical Computing: R Foundation for Statistical Computing . (Vienna, Austria, 2019). Venables, W. N. & Ripley, B. D. Modern Applied Statistics with S . (Springer, Verlag, New York, NY, 2002). Fox, J. & Weisberg, S. An {R} Companion to Applied Regression . (Thousand Oaks, CA: Sage, 2011). Lenth, R. V. Least-squares means: the R package lsmeans. J. Stat. Softw. 69 , 1–33 (2016). Feitosa-Suntheimer, F. et al. Dengue virus-2 infection affects fecundity and elicits specific transcriptional changes in the ovaries of Aedes aegypti mosquitoes. Front. Microbiol. 13 , 886787 (2022). Mills, M. K., Ruder, M. G., Nayduch, D., Michel, K. & Drolet, B. S. Dynamics of epizootic hemorrhagic disease virus infection within the vector, Culicoides sonorensis (Diptera: Ceratopogonidae). PLoS One 12 , e0188865 (2017). Bellini, R. et al. Impact of Chikungunya virus on Aedes albopictus females and possibility of vertical transmission using the actors of the 2007 outbreak in Italy. PLoS One 7 , e28360 (2012). Fu, H., Leake, C. J., Mertens, P. P. C. & Mellor, P. S. The barriers to bluetongue virus infection, dissemination and transmission in the vector, Culicoides variipennis (Diptera: Ceratopogonidae). Arch. Virol. 144 , 747–761 (1999). Rosen, L. Further observations on the mechanism of vertical transmission of flaviviruses by Aedes mosquitoes. Am. J. Trop. Med. Hyg. 39 , 123–126 (1988). Tesh, R. B. & Cornet, M. The location of San Angelo virus in developing ovaries of transovarially infected Aedes albopictus mosquitoes as revealed by fluorescent antibody technique. Am. J. Trop. Med. Hyg. 30 , 212–218 (1981). Lequime, S., Paul, R. E. & Lambrechts, L. Determinants of arbovirus vertical transmission in mosquitoes. PLoS Pathog. 12 , e1005548 (2016). Rozo-Lopez, P., Londono-Renteria, B. & Drolet, B. S. Venereal transmission of vesicular stomatitis virus by Culicoides sonorensis midges. Pathogens 9 , 316 (2020). Pereira-Silva, J. W. et al. First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes. Mem. Inst. Oswaldo Cruz 113 , 56–61 (2018). Mavale, M. et al. Venereal transmission of chikungunya virus by Aedes aegypti mosquitoes (Diptera: Culicidae). Am. J. Trop. Med. Hyg. 83 , 1242–1244 (2010). Shroyer, D. A. Venereal transmission of St. Louis encephalitis virus by Culex quinquefasciatus males (Diptera: Culicidae). J. Med. Entomol. 27 , 334–337 (1990). Rosen, L. Sexual transmission of dengue viruses by Aedes albopictus . Am. J. Trop. Med. Hyg. 37 , 398–402 (1987). McGregor, B. L., Shults, P. T. & McDermott, E. G. A review of the vector status of North American Culicoides (Diptera: Ceratopogonidae) for bluetongue virus, epizootic hemorrhagic disease virus, and other arboviruses of concern. Curr. Trop. Med. Rep. 9 , 130–139 (2022). Tables Table 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Table1..pptx Table 1. Virus detection from the progeny of EHDV-2 infected females. Cite Share Download PDF Status: Published Journal Publication published 08 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Jun, 2024 Reviews received at journal 26 May, 2024 Reviews received at journal 16 May, 2024 Reviewers agreed at journal 15 May, 2024 Reviewers agreed at journal 15 May, 2024 Reviewers invited by journal 13 May, 2024 Editor assigned by journal 08 May, 2024 Editor invited by journal 08 May, 2024 Submission checks completed at journal 08 May, 2024 First submitted to journal 03 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4365421","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":302295471,"identity":"7a0c1e56-d666-4186-acbc-ea9311345e20","order_by":0,"name":"Dinesh Erram","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDCCA2DSBiHABybZCGpJQ3DYiNRymAQtfMd7n334mXNeXt798LHHHyruMbDxnzFg+FB2GKcWyTPHjWf2brttuPFMWrrBgTPFDGwSOQaMM87h1mJwI42ZgXfbbcaNM3jMJA62JQC18Bgw87bh0XL/GTPj323n7DfO4P8mcfBfAthhzH/xabnBxszMu+1A4nwJHjaJgw1ALQw5BsyMeLRInkljZpbdlpy8gSfNTOLMsQSgxrSCgz3n0nFq4Tt+jJnx7TY72/nth59JVNQkyPHzH9744EeZNU4tCBcegNA8IOIAYfVAIN9AlLJRMApGwSgYiQAAw/hTu1LW0gAAAAAASUVORK5CYII=","orcid":"","institution":"Louisiana State University","correspondingAuthor":true,"prefix":"","firstName":"Dinesh","middleName":"","lastName":"Erram","suffix":""},{"id":302295472,"identity":"c0a9a083-88e5-48d8-8906-203ba7731f66","order_by":1,"name":"Bethany McGregor","email":"","orcid":"","institution":"United States Department of Agriculture-ABADRU","correspondingAuthor":false,"prefix":"","firstName":"Bethany","middleName":"","lastName":"McGregor","suffix":""},{"id":302295473,"identity":"4da89a06-a635-4f4f-93c2-986f5eee1e8a","order_by":2,"name":"Carolina Acevedo","email":"","orcid":"","institution":"Minaris Regenerative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Acevedo","suffix":""},{"id":302295474,"identity":"6d043d4e-ee36-4219-9feb-f12aa9821420","order_by":3,"name":"Barry Alto","email":"","orcid":"","institution":"Department of Entomology and Nematology, Florida Medical Entomology Laboratory, University of Florida, Vero Beach, FL","correspondingAuthor":false,"prefix":"","firstName":"Barry","middleName":"","lastName":"Alto","suffix":""},{"id":302295475,"identity":"7e903a5d-7c19-409b-9590-6164c4495acd","order_by":4,"name":"Nathan Burkett-Cadena","email":"","orcid":"","institution":"Department of Entomology and Nematology, Florida Medical Entomology Laboratory, University of Florida, Vero Beach, FL","correspondingAuthor":false,"prefix":"","firstName":"Nathan","middleName":"","lastName":"Burkett-Cadena","suffix":""}],"badges":[],"createdAt":"2024-05-03 17:42:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4365421/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4365421/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-00849-y","type":"published","date":"2025-05-08T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56518523,"identity":"033bbb85-f883-4640-a8bc-a7b3bb2f77a5","added_by":"auto","created_at":"2024-05-15 08:20:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91876,"visible":true,"origin":"","legend":"\u003cp\u003eLife history traits of \u003cem\u003eC. sonorensis\u003c/em\u003e adults in the two groups, A) longevity, B) percentage of females that oviposited, C) number of eggs deposited, and D) egg hatch rates. Letters above bars represent significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4365421/v1/ebdf3c4014d80fa4c4b64f26.png"},{"id":56518521,"identity":"b5b75919-8e02-4c79-a615-9da31663335f","added_by":"auto","created_at":"2024-05-15 08:20:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130033,"visible":true,"origin":"","legend":"\u003cp\u003eLife history traits of \u003cem\u003eC. sonorensis\u003c/em\u003e progeny\u003cem\u003e \u003c/em\u003ein the two groups, A) larval survival rate to pupal stage, B) larval stage duration, C) eclosion rate, and D) sex-ratios. Letters above bars represent significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4365421/v1/f4e7561879003c63fca3d888.png"},{"id":82537554,"identity":"a8570e57-9164-4019-81dc-5d6137248dca","added_by":"auto","created_at":"2025-05-12 16:08:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":949309,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4365421/v1/c34772e1-e122-47f5-b792-8122093d040d.pdf"},{"id":56519246,"identity":"127ca5cb-954c-427e-b194-a84b306890ea","added_by":"auto","created_at":"2024-05-15 08:28:27","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. \u003c/strong\u003eVirus detection from the progeny of EHDV-2 infected females.\u003c/p\u003e","description":"","filename":"Table1..pptx","url":"https://assets-eu.researchsquare.com/files/rs-4365421/v1/88aacfd4d371282c6cc909f8.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epizootic hemorrhagic disease virus oral infection affects midge reproduction and is vertically transmitted to offspring in the biting midge, Culicoides sonorensis (Diptera: Ceratopogonidae)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eCulicoides\u003c/em\u003e species (Diptera: Ceratopogonidae), commonly referred to as biting midges or no-see-ums, transmit numerous disease-causing agents to humans and animals worldwide \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In North America, \u003cem\u003eCulicoides\u003c/em\u003e species are mainly important in the transmission of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) that cause hemorrhagic disease in susceptible hosts such as sheep, cattle, and white-tailed deer, and result in significant economic losses in animal agriculture. In the United States (US), \u003cem\u003eCulicoides sonorensis\u003c/em\u003e Wirth \u0026amp; Jones and \u003cem\u003eCulicoides insignis\u003c/em\u003e Lutz are the only confirmed vectors of EHDV/BTV \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, other species such as \u003cem\u003eCulicoides stellifer\u003c/em\u003e Coquillett, \u003cem\u003eCulicoides venustus\u003c/em\u003e Hoffman, \u003cem\u003eCulicoides debilipalpis\u003c/em\u003e Lutz, and others are likely involved in virus transmission in sylvatic cycles and in areas outside the geographic range of these vectors, particularly in the southeast \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eArboviral infections alter several life history traits of biting insects, influencing their overall vectorial capacity. For example, \u003cem\u003eAedes aegypti\u003c/em\u003e Linnaeus mosquitoes infected with dengue \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e or Chikungunya virus \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e show reduced survival and fecundity, \u003cem\u003eA. aegypti\u003c/em\u003e infected with Zika virus show reduced lifespan but no change in fecundity \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, while \u003cem\u003eA. aegypti\u003c/em\u003e infected with Mayaro virus show reduced fecundity but no change in survival \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eCulex tarsalis\u003c/em\u003e Coquillett females infected with West Nile virus show reduced fecundity but no change in survival \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eCx. tarsalis\u003c/em\u003e infected with Western Equine Encephalitis virus show reduced survival and fecundity \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, while \u003cem\u003eCuliseta melanura\u003c/em\u003e Coquillett mosquitoes infected with Eastern Equine Encephalitis virus show reduced survival and fecundity \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eArboviruses are usually maintained in their natural transmission cycles through horizontal transmission between the arthropod vectors and the vertebrate hosts. However, during cold/dry seasons when the vector population densities are low or when herd immunity of the vertebrate hosts is high, alternate mechanisms such as vertical transmission (from infected arthropod vectors to their offspring) may drive the persistence of arboviruses in nature \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In the US, hemorrhagic disease is endemic with several serotypes of EHDV and BTV circulating in domestic and wild ruminants, particularly in the southeast \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the mechanisms by which these orbiviruses overwinter in the US remain uncertain to date. Previous studies concluded that vertical transmission of BTV is unlikely \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and suggested that long lived \u003cem\u003eC. sonorensis\u003c/em\u003e females infected the previous year are more likely to contribute to the interseasonal maintenance of BTV in California \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the overwintering mechanisms of BTV outside California have received little attention to date \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn general, the vectorial capacity parameters of \u003cem\u003eCulicoides\u003c/em\u003e species associated with EHDV/BTV transmission have not been studied comprehensively. In addition, the potential overwintering mechanisms of EHDV in North America have received little attention to date, collectively representing important knowledge gaps in our understanding of the overall transmission dynamics of orbiviruses in North America. In this study, we examined the survival and reproduction of \u003cem\u003eC. sonorensis\u003c/em\u003e orally challenged with EHDV serotype-2 and examined the possibility of vertical transmission of EHDV-2 from an infected adult female to its offspring in this midge species.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Oral virus exposure\u003c/h2\u003e \u003cp\u003eThe EHDV-2 strain used in this study was initially isolated from the spleen of a dead white-tailed deer from a commercial cervid facility in Quincy, Florida, US in 2016 (NCBI Accession #MF688816-MF688825). The virus was passaged twice on African green monkey kidney (Vero) cells maintained on medium 199 (HyClone Medium 199, GE Healthcare Life Sciences, Logan, UT, US) containing 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, US), 2% penicillin/streptomycin and 0.2% Amphotericin B (Thermo Fisher Scientific, Waltham, MA, US). \u003cem\u003eCulicoides sonorensis\u003c/em\u003e females Ausman strain \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e (48 h post adult emergence) were allowed to feed for 1 h on defibrinated bovine blood (HemoStat Laboratories, Dixon, CA, US) inoculated with EHDV-2 in cell culture media (5.5 log\u003csub\u003e10\u003c/sub\u003e plaque forming unit equivalents [PFUe]/ml final concentration of blood\u0026thinsp;+\u0026thinsp;virus) or with clean media without EHDV-2 (control group) using a Hemotek artificial feeding system (Hemotek, Blackburn, United Kingdom) with a stretched Parafilm\u0026reg; M membrane (Amcor, Zurich, Switzerland). The viral titers of infectious blood fed to the midges are representative of viremia levels experienced by infected deer \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Assessment of midge life history traits\u003c/h2\u003e \u003cp\u003eAfter feeding, blood-engorged females in each group were sorted using a CO\u003csub\u003e2\u003c/sub\u003e pad into individual 175 ml cardboard containers fitted (hot glued) with an oviposition substrate (Petri dish [60 \u0026times; 15 mm] with moist cotton\u0026thinsp;+\u0026thinsp;filter paper) on the bottom. All females were provided with 10% sucrose solution on moistened cotton pads that were replaced daily, and individuals were monitored daily for survival and oviposition. The eggs deposited by females in each group were placed on 0.3% agar slants (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 eggs/dish) and the hatched larvae were reared on a diet of \u003cem\u003ePanagrellus redivivus\u003c/em\u003e nematodes (Carolina Biological Supply Company, Burlington, North Carolina, US), according to previously established methods \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Developmental life history traits of the larvae were monitored and recorded every other day. Overall, several life history traits of \u003cem\u003eC. sonorensis\u003c/em\u003e were recorded and compared between the two groups. These included: blood-fed adult survival/longevity, percentage of females that oviposited, number of eggs deposited, egg hatch rates, larval survival to pupal stage, larval stage duration, eclosion rates, and sex-ratios of the emerging adults. Environmental conditions throughout the experiments were 26\u0026deg;C, 60\u0026ndash;80% RH, and 16:8 (L:D) h cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Virus detection from the progeny of EHDV-2 fed females\u003c/h2\u003e \u003cp\u003eA randomly selected subset of the egg batch deposited by EHDV fed females (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;29) and F\u003csub\u003e1\u003c/sub\u003e generation adults reared from the other subset of eggs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;58; 32 males and 26 females) were processed for the detection/presence of EHDV. Partial egg batches from each female and individual F\u003csub\u003e1\u003c/sub\u003e generation adults were placed in 200 \u0026micro;l of medium 199 (HyClone Medium 199, GE Healthcare Life Sciences, Logan, UT, US) in 1.5 ml microcentrifuge tubes containing 5\u0026ndash;10 borosilicate glass beads (2 mm; Sigma-Aldrich, St. Louis, MO, US). This was followed by homogenization for five minutes using a Bullet Blender Storm homogenizer (Next Advance, Troy, NY, US). Viral RNA was then extracted from 150 \u0026micro;l of the homogenate using the QIAamp Viral RNA Mini Kit (Cat#52906, Qiagen, Hilden, Germany) using manufacturer\u0026rsquo;s protocol. The remainder of the homogenate (50 \u0026micro;l) was stored at \u0026minus;\u0026thinsp;80\u0026deg;C for later use as inoculum for culturing virus, if the sample was determined RT-qPCR positive. RT-qPCR was run on the extracted RNA using SuperScript III One-Step qRT-PCR kit (Thermo Fisher Scientific) using previously established protocols \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e employing primer and probe sequences from Wernicke et al. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and PCR conditions from Wilson et al. \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, modified slightly (25 min at 55\u0026deg;C, 2 min at 95\u0026deg;C, and 45 cycles of 10 s at 95\u0026deg;C and 1 min at 57\u0026deg;C). All RT-qPCR assays were run with positive (EHDV-2 stock) and negative (water) controls. The RNA samples that were EHDV-2 positive through RT-qPCR were further processed using plaque assays to quantitate replication competent virions. Briefly, the remainder of homogenates were added to 6-well plates of confluent Vero cells, incubated for an hour at 37\u0026deg;C, and covered with an agarose gel overlay. One week post infection, overlays were removed, and crystal violet stain was used to view plaque formations. Each plaque was assumed to have originated from a single infecting virus. The samples showing plaques were again processed for EHDV-2 detection through RT-qPCR in the same manner as described above. The quantification cycle values (Cq) and standard curves for the EHDV-2 Florida strain used in the study were reported in our previous study and its detection limit was determined to be at Cq value 35 \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. \u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eVariation in the survival/longevity of adults between the two groups was analyzed using Kaplan-Meier survival curves and log rank (Mantel-Cox) tests. Variation in the percentage of females that oviposited, number of eggs deposited per female, egg hatch rates, larval survival to pupal stage, larval stage duration, eclosion rates, and sex-ratios of the emerging adults between the two groups were analyzed using generalized linear models (GLM) under Poisson or negative binomial distributions. All data were analyzed using R statistical software v.3.6.1 \u003csup\u003e27\u003c/sup\u003e using packages \u003cem\u003eMass\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ecar\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003elsmeans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eIn total, there were 56 individual blood-fed females in the EHDV-2 infected group and 50 in the control group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLife history traits of adults\u003c/b\u003e: The longevity of females in the EHDV-2 infected group (13.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 days post blood feeding [pbf]) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) was slightly, but not significantly longer than that in the control group (11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 days pbf) (χ\u003csup\u003e2\u003c/sup\u003e \u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.32, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0685) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The percentage of females that oviposited in the EHDV-2 infected group was 57.1% (32/56; 95% CI: 43.2\u0026ndash;70.3%), which was not significantly different from that in the uninfected (control) group (52.0%, 26/50; 95% CI: 37.4\u0026ndash;66.3%) (χ\u003csup\u003e2\u003c/sup\u003e \u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.28, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.5954) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The number of eggs deposited per female in the EHDV-2 infected group (89.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4 eggs/female) was not significantly different from that in the control group (73.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1 eggs/female) (χ\u003csup\u003e2\u003c/sup\u003e \u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.72, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.3964) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The egg hatch rate in the EHDV-2 infected group was 35.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2%, which was significantly lower than that in the uninfected group (74.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8%) (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;87.8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLife history traits of progeny\u003c/b\u003e: The rate of larval survival to pupal stage in the EHDV-2 infected group was 59.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9%, which was significantly higher than that in the control group (34.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5%) (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;18.0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Larval development time to pupal stage in the EHDV-2 infected group (29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 days) was not significantly different from that in the uninfected group (27.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 days) (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1526) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The eclosion rate of adults in the EHDV-2 infected group (44.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4%) was not significantly different from that in the control group (57.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8%) (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9334) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The sex-ratio of F\u003csub\u003e1\u003c/sub\u003e adults in the EHDV-2 infected group (1.1:1 [M:F]) was not significantly different from that in the uninfected group (0.9:1) (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.6490) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eVirus detection from the progeny of EHDV-2 fed females\u003c/strong\u003e \u003cp\u003ePartial egg batches from 29 different females were tested for EHDV-2. Out of these, 72.4% (21/29) were positive through RT-qPCR and 3.4% (1/29, Cq value 22.1, 4.9 log\u003csub\u003e10\u003c/sub\u003e PFUe/ml) were positive through plaque assays. Similarly, 58 F\u003csub\u003e1\u003c/sub\u003e adults (32 males and 26 females) were tested individually for EHDV-2. Out of these, 21.9% of the males (7/32) and 19.2% of the females (5/26) were positive through RT-qPCR. One F\u003csub\u003e1\u003c/sub\u003e adult male was positive (1.7%, 1/58 adults tested, Cq value 23.5, 4.5 log\u003csub\u003e10\u003c/sub\u003e PFUe/ml) through plaque assays. None of the F\u003csub\u003e1\u003c/sub\u003e adult females were positive through plaque assays (0.0%, 0/58 adults tested). All samples positive through plaque assays were subsequently re-confirmed as EHDV-2 through RT-qPCR (Table\u0026nbsp;1).\u003c/p\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOverall, our findings demonstrate that EHDV-2 oral infection does not have a major impact on \u003cem\u003eC. sonorensis\u003c/em\u003e survival/longevity or oviposition. However, EHDV-2 infection does have a significant negative effect on \u003cem\u003eC. sonorensis\u003c/em\u003e fecundity (fertility) as it reduces the egg hatch rates in infected females. Interestingly, the larval survival rates to pupal stage were higher in the EHDV-2 infected group than in the uninfected group, which may possibly offset the lower egg hatch rates of EHDV-2 infected females and balance the overall reproductive output of the two groups. Nonetheless, the reasons behind this outcome are currently unknown, although they denote transgenerational virulence with consequences for offspring life history. Further studies will be needed to examine the impact of EHDV-2 on the overall reproductive output of \u003cem\u003eC. sonorensis\u003c/em\u003e and to examine the physiological mechanisms by which EHDV-2 infection increases larval survival rates. The reasons behind the reduced egg hatch rates in EHDV-2 infected \u003cem\u003eC. sonorensis\u003c/em\u003e females are currently unknown as well. However, it is possible that EHDV infection alters the expression of certain genes in the midge ovaries that are important for egg development/maturation. Previously, \u003cem\u003eA. aegypti\u003c/em\u003e mosquitoes infected with Dengue-2 virus showed reduced fecundity compared to their uninfected counterparts \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Subsequent transcriptomic profiling of the ovaries of virus-infected mosquitoes showed the upregulation of several genes, which were suggested to interfere with egg production \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eA. aegypti\u003c/em\u003e infected with Chikungunya virus also showed reduced fecundity compared to uninfected mosquitoes \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Subsequent gene expression analyses on virus infected mosquitoes showed the downregulation of six transcripts in the egg laying pathway of \u003cem\u003eA. aegypti\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A second possible reason is that the transfer of sperms from spermathecae into the micropyle of the eggs during oviposition (fertilization) is affected due to EHDV infection of certain tissues in the female reproductive tract (see further discussion below). Previously, EHDV-2 was detected in the epithelial cells of the spermathecae and the ovarian sheath in \u003cem\u003eC. sonorensis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Another possible reason is that EHDV infection causes unknown pathogenesis in the midges reducing their ability to produce fertile eggs or causes direct/indirect mortality in the eggs/embryos. Previously, Chikungunya virus infection caused reduced egg hatch rates in \u003cem\u003eA. albopictus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and West Nile virus infection caused reduced egg hatch rates in \u003cem\u003eCx. tarsalis\u003c/em\u003e mosquitoes \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, further studies will be needed to test these hypotheses.\u003c/p\u003e \u003cp\u003eEHDV-2 was detected in the eggs and F\u003csub\u003e1\u003c/sub\u003e generation adults resulting from the virus infected \u003cem\u003eC. sonorensis\u003c/em\u003e females through RT-qPCR as well as plaque assays, thus demonstrating that vertical transmission of EHDV may occur in nature. It was previously thought that EHDV and BTV are unlikely to be transmitted vertically in biting midges. This was because EHDV/BTV infections in the midge reproductive tissues in previous studies were detected essentially only along the outer layers, but not in the gametes. For example, BTV was detected only within the ovarian sheath, immature yolk bodies and vitelline membrane of the oocyte in \u003cem\u003eC. sonorensis\u003c/em\u003e, but not in the oocytes or nurse cells \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Subsequent studies found no evidence for the vertical transmission of BTV under laboratory or field conditions \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Similarly, EHDV-2 was detected only within the spermathecal epithelium and the ovarian sheath in \u003cem\u003eC. sonorensis\u003c/em\u003e, but not in the ovariolar sheath, ovarioles, follicular epithelium, oocytes or nurse cells \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, it was suggested that vertical transmission of EHDV is unlikely as well. However, our findings contradict previous notions and demonstrate clearly that EHDV-2 is vertically transmitted in \u003cem\u003eC. sonorensis\u003c/em\u003e. It is possible that these discrepancies are due to differences in the EHDV-2 strains used in the studies. Mills et al. \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e used an EHDV-2 strain isolated from a white-tailed deer from Kansas, while we used an EHDV-2 strain isolated from a white-tailed deer from Florida in this study. It is possible that the EHDV-2 Florida strain infects different reproductive tissues/cells of \u003cem\u003eC. sonorensis\u003c/em\u003e than the Kansas strain, which enable the virus to be vertically transmitted in the midge. Alternately, the virus may enter the fully developed egg later during oviposition. Currently, variations in the midge reproductive tissues/cells infected by the EHDV-2 Florida strain and other geographic strains (and serotypes) of EHDV/BTV are unknown. Further studies will be needed to examine the tissue specific infections caused by different serotypes/strains of orbiviruses in biting midges, which may provide clues towards the mode of vertical transmission of EHDV-2 found in \u003cem\u003eC. sonorensis\u003c/em\u003e in this study. In mosquitoes, arboviruses are vertically transmitted through the transovarial or the transovum routes \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In a previous study, we found that the EHDV-2 Florida strain exhibited higher infection and dissemination rates than the EHDV-2 Can-Alberta strain in \u003cem\u003eC. sonorensis\u003c/em\u003e, suggesting that the consequences of EHDV infection in biting midges vary with the geographic strain of the virus \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, it is likely that different serotypes/strains of EHDV and BTV influence the vector competence and other life history traits/vectorial capacity parameters of \u003cem\u003eCulicoides\u003c/em\u003e species differently as well. The overall vertical transmission rates in this study were low (1.7%, 1/58 adults tested). Notably, our observations were made only during the first gonotrophic cycle of \u003cem\u003eC. sonorensis\u003c/em\u003e. As such, it is possible that these vertical transmission rates persist or even increase during the subsequent gonotrophic cycles of \u003cem\u003eC. sonorensis\u003c/em\u003e, as reported for other arboviruses in mosquitoes \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Interestingly, the F\u003csub\u003e1\u003c/sub\u003e generation adult from which EHDV-2 was detected in this study was a male (3.1%, 1/32) and EHDV-2 was not detected from any females examined (0.0%, 0/26). As such, it is possible that EHDV is also sexually transmitted from the infected male midges to females during mating (and possibly vice versa as well). Previously, sexual transmission of vesicular stomatitis virus was reported in \u003cem\u003eC. sonorensis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, in addition to many other arboviruses in their insect vectors \u003csup\u003e\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. However, further studies will be needed to test these hypotheses.\u003c/p\u003e \u003cp\u003eHemorrhagic disease is known to be endemic in the US with several serotypes of EHDV and BTV frequently detected in a variety of domestic and wild ruminants, particularly in the southeast \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the potential mechanisms by which these orbiviruses overwinter in temperate regions such as North America remain enigmatic to date. In the past, several studies have concluded that vertical transmission of BTV is unlikely \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, Mayo et al. \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e found BTV RNA in \u003cem\u003eC. sonorensis\u003c/em\u003e parous females collected during the early part of the interseasonal period (Feb \u0026ndash; Mar) of 2013 and 2014 in California, suggesting that in the absence of vertical transmission, long lived females infected the previous year can contribute to the interseasonal maintenance of BTV in temperate regions. Along similar lines, our current findings demonstrating the vertical transmission of EHDV-2 in \u003cem\u003eC. sonorensis\u003c/em\u003e offer a strong explanation for one of the potential overwintering mechanisms of EHDV in North America and provide a plausible explanation for the endemic nature of hemorrhagic disease in the US. On a per capita basis, the vertically infected midges that are infectious are predicted to strongly contribute to EHDV transmission because, 1) they can transmit the virus to vertebrates upon their first bite, and 2) they will be infectious for a longer period as adults compared to midges that acquire EHDV by bite through horizontal transmission. It is possible that the low vertical transmission rates found in our study are adequate to sustain the virus between years while still requiring several months of amplification within host populations before outbreaks are observed, typically during late summer to fall. However, it is currently unknown whether the vertically infected progeny of orally infected midges can transmit EHDV-2 through their saliva/bite. Therefore, further studies will be needed to demonstrate the salivary transmission of EHDV-2 from the vertically infected progeny to susceptible hosts and to determine its significance in the epidemiology of hemorrhagic disease. Moreover, further studies will also be needed to gather evidence from field-collected \u003cem\u003eC. sonorensis\u003c/em\u003e to ascertain whether these findings are biologically significant. However, it is to be noted that \u003cem\u003eC. sonorensis\u003c/em\u003e is scattered/rare in the southeastern US \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Therefore, further studies will be needed to examine whether any of the endemic EHDV and BTV serotypes or strains in the US are vertically transmitted through other species associated with EHDV/BTV transmission in the southeast such as \u003cem\u003eC. insignis, C. stellifer\u003c/em\u003e, \u003cem\u003eC. venustus\u003c/em\u003e, \u003cem\u003eC. debilipalpis\u003c/em\u003e, or others. However, the current lack of colonies of \u003cem\u003eCulicoides\u003c/em\u003e vector species other than \u003cem\u003eC. sonorensis\u003c/em\u003e may pose a challenge to such studies.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, our study demonstrates that EHDV does not have a major impact on the survival/longevity, or oviposition of \u003cem\u003eC. sonorensis\u003c/em\u003e, but has a significant negative effect on midge reproduction as it reduces the egg hatch rates in infected females (but increases larval survival rates). In addition, our findings demonstrate that EHDV-2 can be vertically transmitted in \u003cem\u003eC. sonorensis\u003c/em\u003e, a phenomenon that was previously thought unlikely. These findings collectively improve our understanding of the vectorial capacity parameters of \u003cem\u003eC. sonorensis\u003c/em\u003e, provide valuable insight into the potential overwintering mechanisms of EHDV in North America, and offer a plausible explanation for the endemic nature of hemorrhagic disease in the US. Further studies will be needed to understand the physiological mechanisms by which EHDV-2 infection causes reduced fecundity/fertility and increased larval survival rates in biting midges and to determine the mode of vertical transmission of EHDV-2 in \u003cem\u003eC. sonorensis\u003c/em\u003e. More importantly, further studies will be needed to examine the salivary transmission of EHDV-2 from the vertically infected progeny to susceptible hosts and to evaluate its significance in the epidemiology of hemorrhagic disease. In addition, further studies will also be needed to gather evidence from field-collected \u003cem\u003eC. sonorensis\u003c/em\u003e to ascertain whether these findings are biologically significant and to determine whether any of the endemic (or novel) serotypes/strains of EHDV and BTV in North America are vertically transmitted through other species associated with \u003cem\u003eOrbivirus\u003c/em\u003e transmission such as \u003cem\u003eC. insignis\u003c/em\u003e, \u003cem\u003eC. stellifer\u003c/em\u003e, \u003cem\u003eC. venustus\u003c/em\u003e, \u003cem\u003eC. debilipalpis\u003c/em\u003e or others.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e \u003cstrong\u003eDinesh Erram\u003c/strong\u003e: Conceptualization, Methodology, Data Curation, Formal Analysis, Visualization, Validation, Investigation, Resources, Project Administration, Writing\u0026nbsp;–\u0026nbsp;Original Draft Preparation.\u0026nbsp;\u003cstrong\u003eBethany McGregor\u003c/strong\u003e: Methodology, Writing–\u0026nbsp;Review \u0026amp; Editing.\u0026nbsp;\u003cstrong\u003eCarolina Acevedo\u003c/strong\u003e: Methodology, Writing–\u0026nbsp;Review \u0026amp; Editing.\u0026nbsp;\u003cstrong\u003eBarry W. Alto:\u003c/strong\u003e Resources, Writing–\u0026nbsp;Review \u0026amp; Editing.\u003cstrong\u003e\u0026nbsp;Nathan Burkett-Cadena\u003c/strong\u003e: Funding Acquisition, Supervision, Writing–\u0026nbsp;Review \u0026amp; Editing. All authors have reviewed and agreed to the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was funded by the Cervidae Health Research Initiative (CHeRI) sponsored by the State of Florida Legislature. NBC is partially supported by NIFA Project FLA-FME-006106.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Lee Cohnstaedt and William Yarnell (USDA, Manhattan, KS, US) for the maintenance and provision of colonized midges for this study. Agustin Quaglia provided help with statistical analyses of the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e All data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures\u003c/strong\u003e: Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMellor, P. S., Boorman, J. \u0026amp; Baylis, M. \u003cem\u003eCulicoides\u003c/em\u003e biting midges: their role as arbovirus vectors. \u003cem\u003eAnnu. Rev. 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Rep.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 130\u0026ndash;139 (2022).\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Culicoides sonorensis, biting midges, survival, longevity, reproduction, fecundity, fertility, EHDV, BTV, vertical transmission, overwintering","lastPublishedDoi":"10.21203/rs.3.rs-4365421/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4365421/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpizootic hemorrhagic disease virus (EHDV: Reoviridae: \u003cem\u003eOrbivirus\u003c/em\u003e) is a \u003cem\u003eCulicoides-\u003c/em\u003eborne pathogen that affects a variety of ruminants, causing significant economic losses and/or ecological impacts in animal agriculture/wildlife populations worldwide. In this study, we examined the effect of EHDV serotype-2 oral infection on the survival and reproduction of \u003cem\u003eCulicoides sonorensis\u003c/em\u003e Wirth and Jones (a confirmed vector of EHDV in North America), and the potential vertical transmission of EHDV-2 (from infected female to its offspring) in this midge species. \u003cem\u003eCulicoides sonorensis\u003c/em\u003e females were fed on defibrinated bovine blood with EHDV-2 (5.5 log\u003csub\u003e10\u003c/sub\u003e PFU/ml) or without EHDV-2 (control). Adult survival/longevity, oviposition rates, number of eggs deposited, egg hatch rates (fertility), larval survival, larval stage duration, eclosion rates, and sex-ratios of the progeny were recorded and compared between the two groups. In addition, the progeny (eggs and F\u003csub\u003e1\u003c/sub\u003e generation adults) of the EHDV-2 infected females were processed for viral detection through RT-qPCR and plaque assays. Survival/longevity of the blood-fed adults, oviposition rates, number of eggs deposited, larval stage duration, eclosion rates, and sex-ratios were not significantly different between the two groups. However, egg hatch rates were significantly lower in the EHDV-2 infected group (35.8 ± 5.2%) than the uninfected group (74.5 ± 6.8%), but larval survival rates were higher in the EHDV-2 infected group (59.8 ± 4.9%) compared to the control group (34.1 ± 6.5%). EHDV-2 was detected in the eggs (3.4%, 1/29 females tested, Cq value 22.1) and F\u003csub\u003e1\u003c/sub\u003e adult progeny (1.7%, 1/58 adults tested, Cq value 23.5) of the orally exposed females through RT-qPCR as well as plaque assays. Our findings suggest that EHDV-2 infection has no major impact on \u003cem\u003eC. sonorensis\u003c/em\u003e survival/longevity or oviposition but has a significant negative effect on midge fecundity/fertility. Our study also provides evidence for the vertical transmission of EHDV-2 from an infected adult female to its offspring in \u003cem\u003eC. sonorensis\u003c/em\u003e. However, salivary transmission of EHDV-2 from the vertically infected progeny and its significance in the epidemiology of hemorrhagic disease are currently unknown and remain to be examined in further studies. Overall, these findings collectively indicate that \u003cem\u003eOrbivirus\u003c/em\u003e infection can negatively affect vector reproduction and that vertical transmission is a probable mechanism of overwintering of EHDV in North America.\u003c/p\u003e","manuscriptTitle":"Epizootic hemorrhagic disease virus oral infection affects midge reproduction and is vertically transmitted to offspring in the biting midge, Culicoides sonorensis (Diptera: Ceratopogonidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-15 08:20:22","doi":"10.21203/rs.3.rs-4365421/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-09T10:26:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-26T21:32:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-16T14:25:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237411482465593694473730506619312778398","date":"2024-05-15T17:17:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55214633303723643831803909870539448601","date":"2024-05-15T10:45:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-13T10:43:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-08T09:54:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-08T04:20:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-08T04:17:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-03T17:40:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46b86328-fdd1-4895-b0be-0cfcce41bc11","owner":[],"postedDate":"May 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31894871,"name":"Biological sciences/Zoology/Entomology"},{"id":31894872,"name":"Biological sciences/Microbiology/Virology/Viral transmission"}],"tags":[],"updatedAt":"2025-05-12T16:03:17+00:00","versionOfRecord":{"articleIdentity":"rs-4365421","link":"https://doi.org/10.1038/s41598-025-00849-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-08 15:57:42","publishedOnDateReadable":"May 8th, 2025"},"versionCreatedAt":"2024-05-15 08:20:22","video":"","vorDoi":"10.1038/s41598-025-00849-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-00849-y","workflowStages":[]},"version":"v1","identity":"rs-4365421","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4365421","identity":"rs-4365421","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}