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Background: Usutu virus (USUV), which is closely related to West Nile virus (WNV), sharing a similar ecology and transmission cycle, was first reported in the UK in the southeast of England in 2020. Both USUV and WNV are emerging zoonotic viruses hosted by wild birds. The 2020 finding of USUV in England raised awareness of this virus and highlighted the importance of understanding the seasonality of Culex pipiens sensu lato (Cx. pipiens s.l.), the main enzootic vector of these viruses. Zoos are prime locations for trapping mosquitoes because of their infrastructure, security, and range of vertebrate hosts and aquatic habitats., Methods: Three independent zoo-based case studies at four locations that cover the seasonality of Cx. pipiens s.l. in England were undertaken: (i) London Zoo (Zoological Society London [ZSL]) and surrounding areas, London; (ii) Chester Zoo (Cheshire); (ii) Twycross Zoo (Leicestershire); and (iv) Flamingo Land (zoo; North Yorkshire). Various adult mosquito traps were used to catch adult Cx. pipiens s.l. across seasons., Results: High yields of Cx. pipiens s.l./Culex torrentium were observed in Biogents-Mosquitaire and Center for Disease Control and Prevention Gravid traps in all studies where these traps were used. Mosquito counts varied between sites and between years. Observations of adult Cx. pipiens s.l./Cx. torrentium abundance and modelling studies demonstrated peak adult abundance between late July and early August, with active adult female Cx. pipiens s.l./Cx. torrentium populations between May and September., Conclusions: The information collated in this study illustrates the value of multiple mosquito monitoring approaches in zoos to describe the seasonality of this UK vector across multiple sites in England and provides a framework that can be used for ongoing and future surveillance programmes and disease risk management strategies., (© 2024. The Author(s).)

Background: Zoological gardens contain unique configurations of exotic and endemic animals and plants that create a diverse range of developing sites and potential sources of blood meals for local mosquitoes. This may imply unusual interspecific pathogen transmission risks involving zoo vertebrates, like avian malaria to captive penguins. Understanding mosquito ecology and host feeding patterns is necessary to improve mosquito control and disease prevention measures in these environments., Methods: Mosquito sampling took place in Chester Zoo for 3 years (2017, 2018, and 2019) and for 1 year in Flamingo Land (2017) using different trapping methods. Blood-fed mosquitoes were identified and their blood meal was amplified by PCR, sequenced, and blasted for host species identification., Results: In total, 640 blood-fed mosquitoes were collected [Culex pipiens (n = 497), Culiseta annulata (n = 81), Anopheles maculipennis s.l. (n = 7), An. claviger (n = 1), and unidentifiable (n = 55)]. Successful identification of the host species was achieved from 159 blood-fed mosquitoes. Mosquitoes fed on birds (n = 74), non-human mammals (n = 20), and humans (n = 71). There were mixed blood meals from two hosts (n = 6). The proportions of blood-fed mosquitoes varied across sampling seasons and sites within the zoos. The use of resting traps and aspiration of vegetation were more efficient techniques for capturing blood-fed mosquitoes than traps for host-seeking or gravid mosquitoes. By relating the locations of zoo vertebrates to where fed mosquitoes were trapped, the minimum travelling distances were calculated (13.7 to 366.7 m). Temperature, precipitation, relative humidity, proximity to zoo vertebrate exhibits, and vegetation level were found to be significantly associated with the proportion of captured blood-fed mosquitoes by generalized linear modelling., Conclusions: Mosquito feeding behaviour in zoos is mainly influenced by time, location (sampling area), temperature, and host availability, which highlights the value of mosquito monitoring in complex settings to plan control strategies and potentially reduce inherent disease transmission risks for humans and threatened zoo vertebrates.

Background: Culicoides imicola (Diptera: Ceratopogonidae) is an important Afrotropical and Palearctic vector of disease, transmitting viruses of animal health and economic significance including African horse sickness and bluetongue viruses. Maternally inherited symbiotic bacteria (endosymbionts) of arthropods can alter the frequency of COI (cytochrome c oxidase subunit I) mitochondrial haplotypes (mitotypes) in a population, masking the true patterns of host movement and gene flow. Thus, this study aimed to assess the mtDNA structure of C. imicola in relation to infection with Candidatus Cardinum hertigii (Bacteroides), a common endosymbiont of Culicoides spp., Methods: Using haplotype network analysis, COI Sanger sequences from Cardinium-infected and -uninfected C. imicola individuals were first compared in a population from South Africa. The network was then extended to include mitotypes from a geographic range where Cardinium infection has previously been investigated., Results: The mitotype network of the South African population demonstrated the presence of two broad mitotype groups. All Cardinium-infected specimens fell into one group (Fisher's exact test, P = 0.00071) demonstrating a linkage disequilibrium between endosymbiont and mitochondria. Furthermore, by extending this haplotype network to include other C. imicola populations from the Mediterranean basin, we revealed mitotype variation between the Eastern and Western Mediterranean basins (EMB and WMB) mirrored Cardinium-infection heterogeneity., Conclusions: These observations suggest that the linkage disequilibrium of Cardinium and mitochondria reflects endosymbiont gene flow within the Mediterranean basin but may not assist in elucidating host gene flow. Subsequently, we urge caution on the single usage of the COI marker to determine population structure and movement in C. imicola and instead suggest the complementary utilisation of additional molecular markers.

Background: There has been no evidence of transmission of mosquito-borne arboviruses of equine or human health concern to date in the UK. However, in recent years there have been a number of outbreaks of viral diseases spread by vectors in Europe. These events, in conjunction with increasing rates of globalisation and climate change, have led to concern over the future risk of mosquito-borne viral disease outbreaks in northern Europe and have highlighted the importance of being prepared for potential disease outbreaks. Here we assess several UK mosquito species for their potential to transmit arboviruses important for both equine and human health, as measured by the presence of viral RNA in saliva at different time points after taking an infective blood meal., Results: The following wild-caught British mosquitoes were evaluated for their potential as vectors of zoonotic equine arboviruses: Ochlerotatus detritus for Venezuelan equine encephalitis virus (VEEV) and Ross River virus (RRV), and Culiseta annulata and Culex pipiens for Japanese encephalitis virus (JEV). Production of RNA in saliva was demonstrated at varying efficiencies for all mosquito-virus pairs. Ochlerotatus detritus was more permissive for production of RRV RNA in saliva than VEEV RNA. For RRV, 27.3% of mosquitoes expectorated viral RNA at 7 days post-infection when incubated at 21 °C and 50% at 24 °C. Strikingly, 72% of Cx. pipiens produced JEV RNA in saliva after 21 days at 18 °C. For some mosquito-virus pairs, infection and salivary RNA titres reduced over time, suggesting unstable infection dynamics., Conclusions: This study adds to the number of Palaearctic mosquito species that demonstrate expectoration of viral RNA, for arboviruses of importance to human and equine health. This work adds to evidence that native mosquito species should be investigated further for their potential to vector zoonotic mosquito-borne arboviral disease of equines in northern Europe. The evidence that Cx. pipiens is potentially an efficient laboratory vector of JEV at temperatures as low as 18 °C warrants further investigation, as this mosquito is abundant in cooler regions of Europe and is considered an important vector for West Nile Virus, which has a comparable transmission ecology.

Background: Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of arboviruses of international importance between ruminant livestock. These arboviruses include bluetongue virus (BTV) and Schmallenberg virus (SBV), which have emerged in unprecedented outbreaks in northern Europe. The impact of breed and shearing of sheep on Culicoides: host contact rates has not been investigated in detail and has the potential to influence arbovirus transmission and control measures employed to limit spread., Methods: Attraction of Culicoides to Hartline and Hartline/Suffolk cross-breed sheep was compared using 224 drop trap collections over 22 nights and 181 catches from sheared or unsheared Hartline/Suffolk ewes were made over 17 nights to compare Culicoides activity and rates of blood engorgement., Results: A total of 31,314 Culicoides was collected in the two trials and females of the subgenus Avaritia represented over 96.9% of individuals collected. Attraction to breed was dependent upon species of Culicoides and physiological status, with a significantly greater number of individuals collected on the cross-breed sheep. Shearing of sheep did not significantly increase or decrease the number of Culicoides attracted but increased the rate of successful engorgement., Conclusions: Both breed and shearing were shown to influence Culicoides biting rate on sheep. These data are useful in a direct context in understanding the likely impact of control measures against arboviruses including BTV and SBV and additionally in providing data from field-based studies to enable modelling exercises of arbovirus transmission and spread.

Background: To date there has been no evidence of mosquito-borne virus transmission of public health concern in the UK, despite the occurrence of more than 30 species of mosquito, including putative vectors of arboviruses. The saltmarsh mosquito Ochlerotatus detritus [syn. Aedes (Ochlerotatus) detritus] is locally common in parts of the UK where it can be a voracious feeder on people., Methods: Here, we assess the competence of O. detritus for three major arboviruses: dengue virus (DENV), chikungunya virus (CHIKV) and West Nile virus (WNV) using adult mosquitoes reared from wild, field-obtained immatures., Results: We demonstrate laboratory competence for WNV at 21 °C, with viral RNA detected in the mosquito's saliva 17 days after oral inoculation. By contrast, there was no evidence of laboratory competence of O. detritus for either DENV or CHIKV., Conclusions: To our knowledge, this is the first study to demonstrate competence of a UK mosquito for WNV and confirms that O. detritus may present a potential risk for arbovirus transmission in the UK and that further investigation of its vector role in the wild is required.

Background: Some Palaearctic biting midge species (subgenus Avaritia) have been implicated as vectors of bluetongue virus in northern Europe. Separation of two species (C. obsoletus and C. scoticus) is considered difficult morphologically and, often, these female specimens are grouped in entomological studies. However, species-specific identification is desirable to understand their life history characteristics, assess their roles in disease transmission or measure their abundance during arboviral outbreaks. This study aims to investigate whether morphometric identification techniques can be applied to female C. obsoletus and C. scoticus individuals trapped at different geographical regions and time periods during the vector season., Methods: C. obsoletus and C. scoticus were collected using light-suction traps from the UK, France and Spain, with two geographical locations sampled per country. A total of 759 C. obsoletus/C. scoticus individuals were identified using a molecular assay based on the cytochrome c oxidase subunit I gene. Fifteen morphometric measurements were taken from the head, wings and abdomen of slide-mounted specimens, and ratios calculated between these measurements. Multivariate analyses explored whether a combination of morphometric variables could lead to accurate species identification. Finally, Culicoides spp. collected in France at the start, middle and end of the adult vector season were compared, to determine whether seasonal variation exists in any of the morphometric measurements., Results: The principal component analyses revealed that abdominal characteristics: length and width of the smaller and larger spermathecae, and the length of the chitinous plates and width between them, are the most reliable morphometric characteristics to differentiate between the species. Seasonal variation in the size of each species was observed for head and wing measurements, but not abdominal measurements. Geographical variation in the size of Culicoides spp. was also observed and is likely to be related to temperature at the trapping sites, with smaller individuals trapped at more southern latitudes., Conclusions: Our results suggest that female C. obsoletus and C. scoticus individuals can be separated under a stereomicroscope using abdominal measurements. Although we show the length and width of the spermathecae can be used to differentiate between the species, this can be time-consuming, so we recommend undertaking this using standardized subsampling of catches.

Background: Plague, a zoonosis caused by Yersinia pestis, is found in Asia, the Americas but mainly in Africa, with the island of Madagascar reporting almost one third of human cases worldwide. In the highlands of Madagascar, plague is transmitted predominantly by two flea species which coexist on the island, but differ in their distribution. The endemic flea, Synopsyllus fonquerniei, dominates flea communities on rats caught outdoors, while the cosmopolitan flea, Xenopsylla cheopis, is found mostly on rats caught in houses. Additionally S. fonquerniei seems restricted to areas above 800 m. Climatic constraints on the development of the two main vectors of plague could explain the differences in their distribution and the seasonal changes in their abundance. Here we present the first study on effects of temperature and relative humidity on the immature stages of both vector species., Methods: We examined the two species' temperature and humidity requirements under experimental conditions at five different temperatures and two relative humidities. By employing multivariate and survival analysis we established the impact of temperature and relative humidity on development times and survival for both species. Using degree-day analysis we then predicted the average developmental threshold for larvae to reach pupation and for pupae to complete development under each treatment. This analysis was undertaken separately for the two relative humidities and for the two species., Results: Development times and time to death differed significantly, with the endemic S. fonquerniei taking on average 1.79 times longer to complete development and having a shorter time to death than X. cheopis under adverse conditions with high temperature and low humidity. Temperature had a significant effect on the development times of flea larvae and pupae. While humidity did not affect the development times of either species, it did influence the time of death of S. fonquerniei. Using degree-day analysis we estimated an average developmental threshold of 9 °C for S. fonquerniei, and 12.5 °C for X. cheopis., Conclusions: While many vector-borne diseases are limited to warm, low-lying regions, plague in Madagascar is unusual in being most prevalent in the cool, highland regions of the country. Our results point towards the possibility that this is because the endemic flea vector, S. fonquerniei, is better adapted to cool temperatures than the exotic flea vector, X. cheopis. Future warming caused by climate change might reduce the area suitable for S. fonquerniei and may thus reduce the incidence of plague in Madagascar.

Background: The response of Culicoides biting midges (Diptera: Ceratopogonidae) to artificial light sources has led to the use of light-suction traps in surveillance programmes. Recent integration of light emitting diodes (LED) in traps improves flexibility in trapping through reduced power requirements and also allows the wavelength of light used for trapping to be customized. This study investigates the responses of Culicoides to LED light-suction traps emitting different wavelengths of light to make recommendations for use in surveillance., Methods: The abundance and diversity of Culicoides collected using commercially available traps fitted with Light Emitting Diode (LED) platforms emitting ultraviolet (UV) (390 nm wavelength), blue (430 nm), green (570 nm), yellow (590 nm), red (660 nm) or white light (425 nm - 750 nm with peaks at 450 nm and 580 nm) were compared. A Centre for Disease Control (CDC) UV light-suction trap was also included within the experimental design which was fitted with a 4 watt UV tube (320-420 nm). Generalised linear models with negative binomial error structure and log-link function were used to compare trap abundance according to LED colour, meteorological conditions and seasonality., Results: The experiment was conducted over 49 nights with 42,766 Culicoides caught in 329 collections. Culicoides obsoletus Meigen and Culicoides scoticus Downes and Kettle responded indiscriminately to all wavelengths of LED used with the exception of red which was significantly less attractive. In contrast, Culicoides dewulfi Goetghebuer and Culicoides pulicaris Linnaeus were found in significantly greater numbers in the green LED trap than in the UV LED trap. The LED traps collected significantly fewer Culicoides than the standard CDC UV light-suction trap., Conclusions: Catches of Culicoides were reduced in LED traps when compared to the standard CDC UV trap, however, their reduced power requirement and small size fulfils a requirement for trapping in logistically challenging areas or where many traps are deployed at a single site. Future work should combine light wavelengths to improve trapping sensitivity and potentially enable direct comparisons with collections from hosts, although this may ultimately require different forms of baits to be developed.

Background: Farm to farm movement of Culicoides midges is believed to play a critical role in the spread of bluetongue (BT), Schmallenberg and other midge-borne diseases. To help understand and predict the spread of diseases carried by midges, there is a need to determine their dispersal patterns, and to identify factors contributing to the direction taken and distance travelled., Methods: The dispersal of Obsoletus Group members was studied on 19 farms around Bala, north Wales. Field-collected Culicoides were trapped in a black-light (OVI) trap and self-marked in the collecting vessel, using micronized fluorescent dust. Culicoides were released at a central farm and OVI traps set on 18 surrounding farms, at distances of 1 to 4 km. The study was repeated using six colours of fluorescent dust over an 18 day period., Results: An estimated 61,062 (95% CI = 56,298-65,830) marked Culicoides were released during the study and 12 (0.02%) Culicoides were recaptured. Of the females recaptured, six were C. obsoletus/scoticus, two C. dewulfi, two C. pulicaris and one C. festivipennis. The male was C. obsoletus. Recaptures occurred 1-2.5 km from the release site, with greatest numbers at 2.5 km. Most recaptures were 2 nights post-release; none were more than 3 nights post-release. Two females were recovered at 1.5 km on the night of release and one male at 1 km two nights post-release. The mean distance travelled (MDT) for males was 1 km, females was 2.21 km, and all recaptured Culicoides was 2.15 km. Recaptures were made both downwind and upwind of the prevailing wind direction during the trapping periods, highlighting possible passive and active dispersal of Culicoides between farms., Conclusions: This is the first study to demonstrate farm to farm movement of the main Palaearctic BT vector species, the Obsoletus Group. Such movement has disease control implications in terms of the vectoral movement of disease between farms. The results suggest that Culicoides control measures applied at an infected farm (trapping or killing Culicoides) will reduce risk of spread to neighbouring farms by lessening the number of Culicoides dispersing from that farm, as well as reducing transmission at the source farm itself.

Background: Investigation of insect flight patterns frequently involves the use of dispersal studies. A common method for studying insect dispersal is mark-release-recapture (MRR) techniques using wild-caught insects in their natural environment; however, this requires a suitable marker. At present, no studies have been performed to identify markers that are suitable for use in midges within the Obsoletus Group, and visible by eye or down a light microscope., Methods: A series of 11 experiments were undertaken to determine the effectiveness of three colours of Brilliant General Purpose (BGP) fluorescent dusts in marking Culicoides midges. Three areas were focused on: 1) dust properties, 2) the effect on Culicoides, and 3) dust application in the field., Results: All three dusts were insoluble in water, 10% washing-up liquid and 70% ethanol. They were visible down a microscope, with and without the use of a black light, and two were highly visible without the need for a microscope. The dusts remained adherent to the marked Culicoides for the duration of the experiments, did not transfer between marked and unmarked individuals or the environment, and remained adherent when the Culicoides were stored in an ethanol or water-based solution. The dusts had no effect on the mortality rate of the insects over the 48 hrs of the experiment. There were no significant differences between the recorded behaviours undertaken by undusted control Culicoides and the BGP fluorescent dusted Culicoides. Field-based marking of Culicoides can be achieved using a 'self-marking' technique, whereby the trapping vessel is pre-dusted with fluorescent dust prior to trapping the individuals to be marked., Conclusions: This is the first study to identify BGP fluorescent dusts as markers for use with Obsoletus Group Culicoides. BGP fluorescent dusts provide a quick and effective method of marking and identifying Culicoides for both field and laboratory studies. The self-marking technique minimises the time needed to handle specimens prior to release.