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 F1 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 F1 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.