Mosquito bloodmeals can be used to determine vertebrate diversity, host preference, and pathogen exposure in humans and wildlife

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P. Vieira, Narayan Gyawali, Michael B. Onn, Martin A. Shivas, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3949962/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract The surveillance and detection of zoonotic pathogens in animals is essential for predicting disease transmission pathways and the risks of spillover, but challenges include the costs, ethics and technical expertise required for vertebrate trapping, serum sampling and antibody or virus screening. Surveillance using haematophagous arthropods as a sampling tool offers a unique opportunity to obtain blood samples from a wide range of vertebrate species, allowing the study of host-mosquito associations, and host exposure to pathogens. We explored vertebrate diversity and potential Ross River virus (RRV) transmission pathways by analysing blood-fed mosquitoes collected in Brisbane, Australia. Host origins were identified using barcode sequencing, and host exposure to RRV was assessed using a modified plaque reduction neutralisation test. In total, 480 blood-fed mosquitoes were collected between February 2021 and May 2022. The host origins of 346 (72%) bloodmeals were identified, with humans (73%) and cattle (9%) comprising the dominant hosts. RRV seroprevalence was high in both vertebrate species with evidence of RRV exposure in 70% (21/30) of cattle and 52% (132/253) of humans. This is a novel, non-invasive method of estimating seroprevalence in vertebrate host populations. Our results highlight the potential of blood-fed mosquitoes to provide species-specific insights into pathogen transmission dynamics. Biological sciences/Microbiology/Infectious disease diagnostics Earth and environmental sciences/Ecology/Ecological epidemiology Figures Figure 1 Introduction The emergence, and re-emergence of infectious diseases is difficult to predict and may have devastating economic and social consequences locally and globally. In recent decades we have witnessed the appearance of novel infectious diseases 1 a shifting distribution of vectors and pathogens 2 , and the resurgence of mosquito-borne diseases, such as malaria in the USA 3 , yellow fever in Brazil 4 and Japanese encephalitis in Australia 5 . This underscores an urgent and critical need for surveillance systems that can detect and monitor transmission across multiple vectors, pathogen reservoirs and environments. This challenge is complicated for mosquito-borne zoonoses, because extensive surveillance is required across time, space and multiple species. Understanding the transmission ecology of such multi-host pathogens is a major challenge to biomedical science in the 21st Century 6,7 . New, robust, scalable, and cost-effective approaches to surveillance are needed, for implementation across a variety of environments, vectors, reservoirs and pathogens 8 . Xeno-surveillance may offer a solution, using the blood-feeding behaviour of arthropods to determine vector-host relationships and to survey vertebrates for pathogen signals 9 without having to sample vertebrates directly. Haematophagous insects can yield information on local pathogen prevalence without being part of the transmission cycle. For example, collections of tsetse flies in Tanzania 10 and Culex mosquitoes in the Brazilian Amazon 11 yielded malaria parasites while urban Culex collections identified dengue, Zika and chikungunya viruses in Pernambuco, Brazil 12 .The presence of non-vector-borne pathogens, such as hepatitis C virus 8 and H5N1 virus 13 have also been noted in mosquito collections. Xeno-surveillance might also be used to detect vertebrate antibodies to pathogens. Proof-of-principle studies have demonstrated that pathogen-specific antibodies can be recovered from blood-engorged mosquitoes fed with vertebrate blood containing naturally-acquired antibodies in the laboratory (e.g. Toxoplasma gondii and SARS-CoV-2 antibodies 14 ) and from wild mosquitos collected from disease endemic areas (e.g. dengue and Japanese encephalitis virus antibodies 15 ). Antibodies to the pathogenic piroplasm Theileria sergenti have also been recovered from ticks 16 , However, these proofs detect host-specific conjugated antibodies, and so cannot be adapted to determine exposure in bloodmeals derived from multiple hosts. This makes them less suitable for investigating complex zoonoses, e.g. SARS-CoV-2 and RRV, where vertebrate blood sources are diverse or unknown. The analysis of the vertebrate host preferences of haematophagous arthropods, using molecular identification of bloodmeals is common practice and a key part of investigations on potential transmission pathways 17–20 , but only recently have bloodmeals also provided an opportunity to determine pathogen exposure in the host 21 . Screening for live virus in large mosquito collections is onerous and time consuming, and usually only a low detection rate is achievable 22 . Neither does it indicate the vertebrate source of the virus nor the transmission pathway. Simultaneous screening of pathogen-specific antibodies and host identification in mosquito bloodmeals can, however, yield information on vector-host interactions, vertebrate exposure to target pathogens and, therefore, potential virus transmission pathways. Plaque reduction neutralisation tests (PRNTs) are the gold standard for quantifying the presence of neutralising antibodies in serum samples. Unlike other assays (e.g. ELISAs or antigen assays) they are not reliant on host-specific conjugated antibodies and can therefore be applied to serum from any source. This is of particular relevance for the investigation of complex zoonoses like RRV where potential hosts are myriad or unknown. The PRNT has been adapted for screening RRV antibodies from the small volumes of blood present in mosquito abdomens 21 and can be used for all vertebrates 23,24 . In tandem with DNA barcoding of the vertebrate origin of the bloodmeal, the adapted PRNT can give a measure of vertebrate host exposure to RRV using mosquitoes. DNA barcoding is commonly applied to identify invertebrate bloodmeal source using species specific fragments of cytochrome c oxidase subunit 1 (CO1) and/or cytochrome b (cyt b ) genes for molecular identification 18,19,25,26 . Moreover, the use of haematophagous insects as “flying syringes” 10 represents a means of surveying vertebrate abundance and diversity by exploiting the presence of host-seeking insects that are abundant across temperate and tropical habitats and that, dependent on species, exhibit generalist, opportunistic feeding patterns. The objective of this study was to demonstrate that blood-fed mosquitoes, collected as a part of routine surveillance exercises, can yield crucial insights regarding vertebrate diversity, host-preference, and virus exposure in the vertebrate supplying the bloodmeal. This can identify potential disease transmission pathways. We focus on RRV as a case study, because it is endemic within our study area, a nationally notifiable disease of significant public health importance in Australia, and has the potential for global spread 27 . RRV is a complex mosquito-borne zoonosis, circulating between multiple vertebrate species and mosquito vectors, and regularly “spilling over” into humans 28 . We addressed three questions: ( 1 ) do existing trapping networks yield mosquitoes with bloodmeals of sufficient quality to yield data on host feeding patterns and antibody prevalence? ( 2 ) how do mosquito feeding patterns reflect vertebrate abundance? ( 3 ) how does host antibody prevalence revealed by bloodmeals compare with historical, conventional sero-surveys for RRV? Results A total of 54,670 mosquitoes were collected during the trapping period of this study (February 2021 to May 2022) as part of an established mosquito surveillance program implemented by Brisbane City Council (BCC), Queensland, Australia, and some additional traps deployed by Queensland Health, Australia. Of these, 480 mosquitoes (0.9% of the total catch) from 14 species were identified as being blood-fed ( Supplementary Table S1 ). Five species accounted for > 85% of blood-feds collected: Culex annulirostris (68.7%), Cx. orbostiensis (9.4%), Aedes procax (3.9%), Cx. sitiens (3.7%), and Verrallina Marks sp. No. 52 (2.71%). A single vertebrate origin was identified from 341 bloodmeals, while another five bloodmeals were of mixed origin (human and one other vertebrate), totalling 346 identified bloodmeals ( Table 1 and Supplementary Table S2 ). A total of 26 vertebrate species were identified from these bloodmeals, 10 birds, nine placental mammals, three marsupials, two reptiles, one amphibian, and one semi-aquatic fish. Humans were the dominant source of bloodmeals, accounting for 73.1% of bloodmeals (253/346), followed by cattle and marsupials (8.7% and 6.9% of bloodmeals, respectively). By comparison, conventional vertebrate surveys, conducted by human observers at the same sites, identified a much higher number of vertebrate species (85). Most of these species were birds (74), with 6 placental mammals, 2 marsupials, 2 amphibians and a reptile. The vertebrate survey did not observe cattle, deer, fox, sheep, or fish, all of which were fed on by mosquitoes. Human abundance was not quantified during conventional vertebrate surveys, but was estimated using population statistics from areas within the dispersal kernel of the key mosquito species sampled ( Fig. 1 ). All 480 bloodmeal samples collected in this study were screened for the presence of anti-RRV antibodies using an adapted micro-PRNT 21 and more than half (n=243, 51%) were seropositive ( Table 2 ). RRV seroprevalence was 52% (131/253) in humans, 70% in cattle and eastern grey kangaroos (21/30 and 14/20, respectively), and ranged from 25% to 100% in samples from other animals. Among the 14 blood-fed mosquito species, 12 were found to have fed on RRV seropositive hosts, with Ae. alternans (n=1) and Uranotaenia nivipes (n=1) being the exceptions. Discussion This is the first field demonstration that mosquito bloodmeals can be used a resource for estimating vertebrate seroprevalence. Our results indicate varying seroprevalence of RRV antibodies across different host species, highlighting potential transmission pathways. RRV is thought to be maintained in the environment by multiple host and vector species, with little understanding about how this community shifts across environments, seasons, and the degree of spillover to the human population. High seroprevalence demonstrated in this study and others ( Supplementary Table S3 ) among humans (ranging from 7 to 79%), horses (21 to 94%), cattle (6 to 100%), and kangaroos (36 to 100%), along with a very limited data-set on species presenting with RRV-induced viraemias 28–30 , suggest their susceptibility to RRV infection and their possible role as amplifying or diluting RRV hosts within the community needs to be re-examined; even for those species long thought of as ‘dead-end’ hosts (e.g. cattle). Humans are potential amplifying hosts of RRV 28,31,32 . Their high RRV seroprevalence rates indicate high rates of exposure. The human seropositivity in this study corroborates with previous sero-surveillance studies in QLD with antibody prevalence of 9–50% ( Supplementary Table S3 ). It is notable that in this study, we were sampling bloodmeals from mosquitoes caught in urban parklands. Although we do not know the movement patterns of those humans, the high seropositivity rates suggest that at least some of those humans are being infected in an urban transmission cycle rather than a rural one. Cattle are not considered important reservoir hosts for RRV, because under experimental conditions only one individual (of six) developed viraemia which was low and short-lived 29 . Yet, our results highlight that cattle are frequently exposed to RRV and may contribute to transmission dynamics. Notably, little corellas ( Cactua sanguinea ) and cattle develop near identical viraemic responses to infection in terms of peak and duration and the former was shown to infect 14% of susceptible Cx. annulirostris mosquitoes 29 . Additionally, varying seroprevalence in other placental mammals, including deer, red foxes and some birds, underscores the catholic feeding behaviour of RRV vectors and the potential complexity of RRV's host range. Our data emphasises the fact that, 60 years after the initial solation of RRV from a mosquito vector 33 we still have little understanding of the reservoirs or their role in maintain transmission cycles in different habitats. For example, we show that deer are clearly exposed to RRV, but there are no experimental studies exist to confirm transmission. Culex annulirostris , a major vector of RRV and other medically-important arboviruses in Australia 34,35 , is a freshwater mosquito that is abundant across the country. They are considered some of the most prominent vectors of RRV due to (i) being one of the most frequent sources of RRV isolates, with over 250 isolates 36–38 , (ii) their demonstrated ability to become infected with and transmit RRV under laboratory settings (e.g. from horses or little corellas 29 ), and (iii) spatio-temporal associations between infected Cx. annulirostris and RRV disease 36,39 . They are generalist-opportunistic feeders and feed readily on humans 18,40–42 . Cx. annulirostris was the most abundant species in this study, as also reported in other Queensland studies 36,39 . It also yielded the greatest number of bloodmeals for analysis and was found to have fed on six bird species and every mammal species identified in this study including humans (n = 12). This suggests a wide host range with potential for a variety of RRV transmission pathways. The species is likely a key means of spillover to humans Its eclectic feeding habits also makes this species a powerful sampling tool for identifying local vertebrate diversity and abundance. Further empirical research on viraemic response and transmission to mosquitoes is desirable for many of the potential hosts identified in this study. However, these experimental infection studies for complex zoonoses are ethically and logistically difficult. The micro-PRNT approach serves as a valuable, field-based but circumstantial alternative that can aid pathway incrimination and prioritise targets for further study. The micro-PRNT method estimates pathogen exposure by screening mosquito bloodmeals for antibodies produced in response to viral infection in the vertebrate host. These antibodies can persist in the bloodstream for extended periods. Since viraemia is typically short, antibodies provide several advantages for understanding population exposure and for inferring transmission dynamics. This approach also has advantages over virus screening of mosquito collections as means of investigating pathways. Mosquitoes are often extremely abundant, have short lifespans and only take bloodmeals at fixed intervals (the gonotrophic cycle). There are therefore limited opportunities for them to acquire virus, so “hit” rates during screening can be very low 22 . Moreover, the presence of virus in a population does not necessarily relate to transmission. Mosquitoes will often be incidental carriers of pathogens circulating in the environment and may not be responsible for transmission 10–12 . There is no “smoking gun” in terms of transmission proofs from field-collections of vertebrates or mosquitoes. The capacity to characterise host exposure to pathogens such as RRV, here demonstrated for the first time in the field, is an exciting development that can offer descriptive insights into potential virus reservoirs and transmission pathways. It is also a potentially powerful means of quantifying key parameters in deterministic compartmental models of disease transmission. All of these utilise some estimate of the size of the pathogen reservoir in order to propose values for the number of susceptible, infected and immune (recovered) individuals. For multi-host pathogens this can be highly challenging because estimates must be made for multiple populations 30 . This is traditionally done through vertebrate surveys and a great deal of guess work. Antibody screening in bloodmeals may allow us to estimate the S (susceptible) and R (immune) proportion of the reservoir population and therefore build SIR models of transmission. The “infected” parameter in these models could be derived through an additional screen of the mosquito bloodmeals for (rare) virus signals. Collection and analysis of blood-fed mosquitos offers an alternative to conventional vertebrate surveys. Excluding bird species, the number of vertebrate species quantified by screening mosquito bloodmeals was greater than that observed during conventional faunal surveys. This is probably because the observations made during traditional surveys are most likely to capture those species that are least cryptic, locally abundant and most active. Traditional surveys may record greater bird diversity because observers use additional cues such as bird calls. By using mosquitoes as the sampling tool, we have potentially greater access to cryptic species across all habitats and times of day. Moreover, trapped, blood-fed mosquitoes may have been foraging over much larger areas than those which are observable by a human operating along a fixed transect. It is notable that humans made up the majority of bloodmeals across all locations, although they only represent a small proportion of vertebrate diversity. Our estimates of accessible humans were based on residential populations within the effective dispersal kernel of mosquitoes. Thus, we undoubtedly overestimated the human population available to the mosquito. Nonetheless, the high proportion of human bloodmeals may reflect host availability in the study area. Humans were likely to represent a large proportion of the overall vertebrate biomass in the parks, particularly at some periods during the day (i.e., peak times for commuting, exercise or leisure may coincide with the crepuscular rhythms of many mosquito species). This would undoubtedly influence mosquito feeding patterns. In fact, there are over 2,180 parks across Brisbane 43 , and they may be major foci for transmission and human spillover, the specific patterns of which will depend on vertebrate diversity, virus amplification and virus dilution. The diversity of bloodmeal origins captured across a number of mosquito species, confirms that mosquitoes are a powerful, emerging tool for characterising vertebrate diversity. The technique has potential application to a range of ecological and conservation issues, such as monitoring rare or endangered species 44 , and measuring biodiversity 45,46 . It can also be used to understand host feeding patterns, which is a crucial component for assessing the risk of pathogen transmission pathways and human spillover. It is as important to identify non-reservoirs and the dilution of transmission as it is to identify potential virus sources. For example, some mosquito species will preferentially feed on reptiles, amphibians and even fish (particularly Uranotaenia spp.) 47 . An expansion to this study, involving greater numbers of bloodmeals, might explore whether some vertebrates are predictably “non-reactive” and whether some mosquito species showed strong preference for those vertebrates. Blood-fed mosquitoes can be used to characterise key aspects of disease ecology. However, it can be difficult to collect them in sufficient quantities to integrate them into surveillance campaigns 48 . The baited, fan-assisted traps commonly used for mosquito surveillance in the field (i.e., BioGents Sentinel traps, CDC light traps or CDC gravid traps) target mosquitoes that are seeking hosts or an oviposition site. Blood-fed mosquitoes are not optimally attracted by these traps but prefer to rest while digesting bloodmeals and developing eggs 49 . Nevertheless, in this study we demonstrate that existing networks of CO 2 baited traps, typically used for routine surveillance around the world are efficient and convenient tools for trapping blood-fed mosquitoes at least for some species and in some habitats. The BCC network of 9 traps, spread in the four urban parks and operating over a six-month period, collected a small percentage (0.9%, n = 480) of blood-fed mosquitoes (largely Cx. annulirostris ) but yielded sufficient numbers (346 bloodmeal IDs) to facilitate our investigations. The success of CO 2 baited light trap networks in collecting blood-feds is clearly dependent on locality, target species and the extent of the trap network. Flies et al. 19 assessed historic captures by a routine survey in South Australia and found just 280 (0.06%) blood-feds in a collection of 350,000 mosquitoes made over a 10-year period (mostly Ae. camptorhynchus and Cx. pipiens spp). Conversely, in Queensland, Kay et al. 40 caught 1,119 blood-fed mosquitoes over a seven month period representing 3.7% of the catch (mostly Cx. annulirostris ), showing its potential for efficiently trapping blood-fed mosquitoes. A notable limitation of our study is that, due to resource constraints we sampled vertebrates during the same months as the vectors, but in different years. However, comparisons of survey results between 2017/18 and 2020/21 does demonstrate that diversity and abundance are similar. Conclusion This study made a systematic comparison between vertebrate survey and xeno-surveillance techniques in relation to vertebrate diversity while also demonstrating the viability of xeno-surveillance as a tool to verify vertebrate species serostatus to a zoonotic virus. This is unique because no previous bloodmeal studies have included formal vertebrate surveys. This study validates the use of micro-PRNTs in the field for determine vertebrate exposure to pathogens from mosquito bloodmeals. Conducting seroprevalence studies, especially in non-human vertebrates, are challenging due to ethical and practical limitations. However, our method provides a novel, non-invasive approach to estimate pathogen exposure in vertebrates and linking that to vector-host networks. Overall, this can help determine which pathways are most likely to facilitate transmission of specific mosquito-borne pathogens to humans, as well as monitor any zoonotic disease. Methods Study area The study was undertaken in Brisbane (27°28'12'' S and 153°01'15'' E), the capital city of Queensland, which is the second largest state in Australia in terms of area. Brisbane is geographically the largest capital city in Australia and the third most populous city in the country, with approximately 2.5 million people. The city has a subtropical climate characterised by an average annual precipitation level of 1011.5 mm. The rainy season typically occurs from November to March, while the average monthly temperatures range from 10°C to 22°C in winter and 20°C to 29°C in summer 50 . Brisbane comprises diverse natural ecosystems, including freshwater and estuarine wetlands, mangroves, saltmarshes, bushlands, and rainforests 51 . These varied ecosystems provide a range of habitats suitable for both mosquito vector species and reservoir hosts. The presence of such ecological diversity, coupled with the persistently high rates of human notifications of RRV 34 , makes Brisbane an ideal location for investigating RRV dynamics. Mosquito collection and identification Mosquito collections were carried out by the BCC as part of their ongoing mosquito surveillance and control program from February to May 2021 and during the same period in 2022. Five sites were chosen based on historical detections of RRV in mosquito collections ( Supplementary Fig. S1 ). These were all urban recreational parks within established residential areas, with human population density ranging from 7.75 to 21.56 people per hectare. To increase the number of blood-fed individuals, particularly those that had potentially fed on a host that had previously been exposed to RRV, QH conducted additional surveillance at sites where RRV had recently been detected in sentinel traps during the trapping period. Traps were set once a week prior to dusk and then collected the following morning. We employed CDC-style light traps sourced from Pacific Biologics (Scarborough, Australia), with 1 kg of dry ice as a CO 2 source and supplemented by 1-octen-3-ol. After collection, mosquitoes were transported to the Mosquito Control Laboratory (MCL) for identification. Mosquitoes were identified to the species level using dichotomous keys 52–54 . Blood-engorged mosquitoes were stored at − 80°C until analysis by the respective assays. Sample preparation The abdomens of individual blood-fed mosquitoes were dissected under a stereo microscope. The dissected abdomens were subsequently homogenised in 70 µL of tissue culture media RPMI-1640, supplemented with 1% Penicillin, Streptomycin, and L-glutamine (Sigma-Aldrich, USA). To prevent fungal contamination 0.4% amphotericin B (Sigma-Aldrich, USA) was added to the homogenisation media. Following homogenisation, the samples were centrifuged at 10,000 x g for 10 minutes, and the resulting supernatant removed. A volume of 55 µL of the supernatant was transferred to a sterile 1.5 ml microtube for subsequent testing to determine the presence of neutralising antibodies against RRV. The remaining volume of the supernatant was retained in a separate tube for molecular identification of the host species from which the bloodmeal was obtained. Nucleic acid extraction and DNA barcoding The DNA barcoding was performed using different regions of the mitochondrial DNA: the subunit I of the cytochrome oxidase (COI) 55,56 or cytochrome b (cyt b ) 25,57 genes. The sets of primers chosen ( Supplementary Table S4 ) were tested and validated on known host species, including humans, sheep ( Ovis aries ), koalas ( Phascolarctos cinereus ), and birds (Australian magpie, Cracticus tibicen ), using whole blood or serum samples. DNA was extracted from up to 10 µL of the mosquito bloodmeal samples using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The extracted DNA was then amplified using a touchdown cycling sequence to minimize non-specific amplification. The PCR mixture consisted of 3 µL of template DNA, 2X Phusion High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs, Ipswich MA), 0.5% DMSO, and 0.5 µM of each of a forward and reverse primer in a 30 µL reaction mix. Each sample was submitted to the four combinations of primers ( Supplementary Table S4 ). Cycling conditions comprised a step at 98°C for 30s, followed by 5 touchdown cycles of 98°C 5s, 49.5°C 30s, 72°C 45s, 30 cycles of 98°C 5s, 54°C 30s, 72°C 45s, and a final elongation step of 72°C for 5min. The PCR products were visualised on a 2% agarose gel. Amplified target DNA bands were either purified directly from the PCR reaction or from the gel using Qiagen Gel kit or PCR purification kits. Samples were sequenced by the QIMRB DNA Sequencing Facility using the BigDye™ Terminator v3.1 Cycle Sequencing kit in an ABI-PRISM 3130 Genetic Analyser (Applied Biosystems, Foster City, CA). The obtained sequences were then compared to the NCBI nucleotide database using a Basic Local Alignment Search Tool (BLAST) search. Sequences showing a ≥ 95% sequence identity with those in the NCBI database were considered as matches. If the sequence identity was below 95%, the samples were considered inconclusive, and additional genus-specific PCR amplification and sequencing were performed. Micro-plaque reduction neutralisation test (micro-PRNT) The presence of neutralising antibodies against RRV in mosquito bloodmeals was assessed using a micro-PRNT, developed by Gyawali et al. 21 . Briefly, African green monkey kidney (Vero) cells (ATCC, CCL-81) were cultivated as monolayers in 96-well tissue culture plates (Nunclon, Thermo Scientific, Australia). The bloodmeals from mosquitoes were mixed with a quantity of RRV virus (T48 prototype strain 33 ), calibrated to form 30 plaques per well. The bloodmeal-virus mixture was added to wells in duplicate. Wells containing only Vero cells with and without RRV served as positive and negative controls, respectively. After incubation at 37°C for 2 hours, the mixture was removed and 0.75% w/v carboxymethyl cellulose (CMC, Sigma-Aldrich) in RPMI-1640 medium was added to each well. The plates were incubated for a further 40 hours at 37°C in an atmosphere of 5% v/v CO 2 /air. Following incubation, the media was decanted, and the wells stained with a solution of 0.05% w/v crystal violet in formaldehyde (1% v/v) and methanol (1% v/v). After 24 hours, the plates were rinsed, air-dried, and the number of plaques per well was counted. Plaques were counted in duplicate wells, and the average number was determined. Bloodmeals that resulted in a ≥ 50% reduction in the number of viral plaques compared to the positive control (RRV alone) were considered RRV seropositive. Host abundance surveys Methods for the vertebrate surveys were adapted from the Queensland Fauna Survey Guidelines 58 . In brief, we estimated the abundance of hosts using four-point surveys, six minutes in duration, performed at least 150m apart across Brisbane at the same sites used to collect mosquitoes monthly from November 2017 to March 2018 and again from March 2020 and April 2021. Vertebrate surveys were conducted during times of peak activity (within 30 minutes of dawn for birds, and after sunset for mammals). During each survey we recorded the species, number of individuals, whether the observation was seen or heard, and the microhabitat the species was in (i.e. on the ground, in the canopy, in the bottom third of a tree). Equipment used to identify vertebrates included binoculars, handheld torches, field guides and the Birds of Australis mobile phone application 59 . Human abundance in each area was calculated by multiplying the average number of people per household in each suburb (as per Census data 60 ) by the number of households within 2km of each vector trapping site. That effective radius is a modest estimate of the foraging kernel of the main mosquito species collected during this study. Declarations Author contributions G.J.D. devised the project. G.L.W., A.F.v.d.H., F.D.F. and G.J.D. supervised the project. C.J.S.P.V., M.B.O., M.A.S., D.S., J.M.D. and E.B.S. coordinated field activities. C.J.S.P.V. and N.G. analysed mosquito sera collections. C.J.S.P.V. and E.B.S. analysed vertebrate faunal surveys. C.J.S.P.V. collated and analysed the data, drafted the manuscript and designed the figures. J.M.D., N.G., G.L.W., A.F.v.d.H., F.D.F., E.B.S. and G.J.D. contributed to the editing of the final manuscript. All authors edited and approved the version submitted for publication. Data availability All data generated and analysed during this study are included in this published article and its Supplementary Information files. Competing interests The authors declare no competing interests. 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PLoS One 14 , e0215194 (2019). Gyawali, N., Murphy, A. K., Hugo, L. E. & Devine, G. J. A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: A useful tool for inferring transmission pathways. PLoS One 15 , e0229314 (2020). Ramírez, A. L., Van Den Hurk, A. F., Meyer, D. B. & Ritchie, S. A. Searching for the proverbial needle in a haystack: Advances in mosquito-borne arbovirus surveillance. Parasites and Vectors vol. 11 1–12 (2018). Togami, E. et al. First evidence of concurrent enzootic and endemic transmission of Ross River virus in the absence of marsupial reservoirs in Fiji. Int. J. Infect. Dis. 96 , 94–96 (2020). Johnson, B. J. et al. The environmental and ecological determinants of elevated Ross River Virus exposure in koalas residing in urban coastal landscapes. Sci. Rep. 11 , (2021). Townzen, J. S., Brower, A. V. Z. & Judd, D. D. Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences. Med. Vet. Entomol. 22 , 386–393 (2008). Gyawali, N. et al. Identification of the source of blood meals in mosquitoes collected from north-eastern Australia. Parasites Vectors 12 , 198 (2019). Shanks, G. D. Could Ross River Virus be the next Zika? Journal of Travel Medicine vol. 26 1–2 (2019). Kain, M. P., Skinner, E. B., van den Hurk, A. F., McCallum, H. & Mordecai, E. A. Physiology and ecology combine to determine host and vector importance for ross river virus. Elife 10 , 1–40 (2021). Kay, B. H. et al. Experimental infection of vertebrates with Murray Valley encephalitis and Ross River viruses. Arbovirus Res. Aust. 4 , 71–75 (1986). Stephenson, E. B., Peel, A. J., Reid, S. A., Jansen, C. C. & McCallum, H. The non-human reservoirs of Ross River virus: a systematic review of the evidence. Parasit. Vectors 11 , 188 (2018). Tesh, R. B., McLean, R. G., Shroyer, D. A., Calisher, C. H. & Rosen, L. Ross river virus (Togaviridae: Alphavirus) infection (epidemic polyarthritis) in American Samoa. Trans. R. Soc. Trop. Med. Hyg. 75 , 426–431 (1981). Lindsay, M. et al. The epidemiology of outbreaks of Ross River virus infection in Western Australia in 1991-1992. Arbovirus Res. Aust. 6 , 72–76 (1992). Doherty, R. L., Whitehead, R. H., Gorman, B. M. & O’Gower, A. K. The isolation of a third group A arbovirus in Australia, with preliminary observations on its relationship to epidemic polyarthritis. Aust. J. Sci. 26 , 183–184 (1963). Knope, K. et al. Arboviral diseases and malaria in Australia, 2014–15: Annual report of the National Arbovirus and Malaria Advisory Committee. Commun. Dis. Intell. 43 , (2019). Ong, O. T. W., Skinner, E. B., Johnson, B. J. & Old, J. M. Mosquito-Borne Viruses and Non-Human Vertebrates in Australia: A Review. Viruses 13 , 265 (2021). Webb, C. E. & Russell, P. R. C. Living with mosquitoes: In the Lower Hunter and Mid North Coast Region of NSW . (Department of Medical Entomology The University of Sydney & Westmead Hospital, 2009). Doggett, S. et al. The New South Wales Arbovirus Surveillance and Mosquito Monitoring Program. 2009 –2018 Annual Reports. (2018). Harley, D., Sleigh, A. & Ritchie, S. Ross river virus transmission, infection, and disease: A cross-disciplinary review. Clinical Microbiology Reviews vol. 14 909–932 (2001). Jansen, C. C. et al. Epidemiologic, entomologic, and virologic factors of the 2014-15 Ross River virus outbreak, Queensland, Australia. Emerg. Infect. Dis. 25 , 2243–2252 (2019). Kay, B. H., Boyd, A. M., Ryan, P. A. & Hall, R. A. Mosquito feeding patterns and natural infection of vertebrates with Ross River and Barmah Forest viruses in Brisbane, Australia. Am. J. Trop. Med. Hyg. 76 , 417–423 (2007). Van Den Hurk, A. F. et al. Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea. Med. Vet. Entomol. 17 , 403–411 (2003). Williams, C. R., Kokkinn, M. J. & Smith, B. P. Intraspecific variation in odor-mediated host preference of the mosquito Culex annulirostris. J. Chem. Ecol. 29 , 1889–1903 (2003). BCC, B. C. C. Brisbane Parks. Brisbane City Council https://www.brisbane.qld.gov.au/things-to-see-and-do/council-venues-and-precincts/parks#:~:text=Brisbane City Council maintains more,gardens and many bushland reserves. (2023). Abrams, J. F. et al. Shifting up a gear with iDNA : From mammal detection events to standardised surveys. J. Appl. Ecol. 56 , 1637–1648 (2019). Saranholi, B. et al. Comparing iDNA from mosquitoes and flies to survey mammals in a semi-controlled area. Mol. Ecol. Resour. 23 , 1790–1799 (2023). Cutajar, T. P. & Rowley, J. J. L. Surveying frogs from the bellies of their parasites : Invertebrate-derived DNA as a novel survey method for frogs. Glob. Ecol. Conserv. 22 , e00978 (2020). Hawkes, F. M. & Hopkins, R. J. The mosquito: An introduction. in Mosquitopia: The Place of Pests in a Healthy World (eds. Hall, M. & Tamïr, D.) 16–31 (Routledge, 2021). doi:https://doi.org/10.4324/9781003056034. Burkot, T. R. et al. Barrier screens: a method to sample blood-fed and host-seeking exophilic mosquitoes. Malar. J. 12 , 49 (2013). Williams, G. M. & Gingrich, J. B. Comparison of light traps, gravid traps, and resting boxes for West Nile virus surveillance. J. Vector Ecol. 32 , 285–291 (2007). BoM, B. of M. Climate statistics for Australian locations. Monthly climate statistics. Australian Government Bureau of Meteorology http://www.bom.gov.au/climate/averages/tables/cw_040913.shtml (2023). Queensland Government. Land zones within Southeast Queensland bioregion. https://apps.des.qld.gov.au/regional-ecosystems/landzones/?bioregion=12 (2023). Russell, R. C. & Debenham, M. L. A colour photo atlas of mosquitoes of southeastern Australia . (R.C. Russell, 1996). Marks, E. N. An atlas of common Queensland mosquitoes . (Queensland Institute of Medical Research, 1982). Russell, R. C. Mosquitoes and mosquito-borne disease in Southeastern Australia: A Guide to the Biology, Relation to Disease, Surveillance, Control and the Identification of Mosquitoes in Southeastern Australia . (Westmead Hospital, Department of Medical Entomology, 1993). Parodi, B. et al. Species identification and confirmation of human and animal cell lines: A PCR-based method. Biotechniques 32 , 432–440 (2002). Reeves, L. E., Gillett-Kaufman, J. L., Kawahara, A. Y. & Kaufman, P. E. Barcoding blood meals: New vertebrate-specific primer sets for assigning taxonomic identities to host DNA from mosquito blood meals. PLoS Negl. Trop. Dis. 12 , e0006767 (2018). Cicero, C. & Johnson, N. K. Higher-level phylogeny of new world vireos (aves: vireonidae) based on sequences of multiple mitochondrial DNA genes. Mol. Phylogenet. Evol. 20 , 27–40 (2001). Eyre, T. J. et al. Terrestrial vertebrate fauna survey assessment guidelines for Queensland . (Department of Environment and Science, Queensland Government, 2018). Morcombe, M. K. Field guide to Australian birds . (Steve Parish Publishing, 2003). Australian Bureau of Statistics. 2021 Census QuickStats. Australian Bureau of Statistics https://www.abs.gov.au/census/find-census-data/quickstats/2021/3GBRI (2022). Tables Tables 1 to 2 are available in the Supplementary Files section Additional Declarations No competing interests reported. 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As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3949962","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":272349079,"identity":"20fefd40-eb8f-4079-ab29-39bccea2a028","order_by":0,"name":"Carla Julia S. P. 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Devine","email":"","orcid":"","institution":"QIMR Berghofer Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Gregor","middleName":"J.","lastName":"Devine","suffix":""}],"badges":[],"createdAt":"2024-02-12 02:33:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3949962/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3949962/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-73820-y","type":"published","date":"2024-10-05T15:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51408612,"identity":"b1878e8c-22e0-4aa8-a4fc-a79a1c90076a","added_by":"auto","created_at":"2024-02-21 04:15:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59156,"visible":true,"origin":"","legend":"\u003cp\u003eRelative proportions of vertebrates across Brisbane collection sites.\u003c/p\u003e\n\u003cp\u003ea) Vertebrate diversity estimated by human observers November 2017 to March 2018 and March 2020 to April 2021; b) Vertebrate diversity from mosquito bloodmeals collected from February 2021 to May 2022.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3949962/v1/90d9269aca298da83d696218.png"},{"id":66097026,"identity":"0fa3ff06-abf7-4485-bd26-70bc036dadce","added_by":"auto","created_at":"2024-10-07 16:12:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":630860,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3949962/v1/3030ded8-eaa5-4e8a-8621-724da9cbd25a.pdf"},{"id":51408649,"identity":"51197a88-8f18-4adc-b225-2c23f4a5651b","added_by":"auto","created_at":"2024-02-21 04:15:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":327662,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDatabloodmealpaper.docx","url":"https://assets-eu.researchsquare.com/files/rs-3949962/v1/a9efa236a4544fa43cd34277.docx"},{"id":51408631,"identity":"32514b16-c1be-4455-ada3-ca20914f67e4","added_by":"auto","created_at":"2024-02-21 04:15:46","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":139264,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.doc","url":"https://assets-eu.researchsquare.com/files/rs-3949962/v1/74c724fe9bc7e6019452865e.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mosquito bloodmeals can be used to determine vertebrate diversity, host preference, and pathogen exposure in humans and wildlife","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe emergence, and re-emergence of infectious diseases is difficult to predict and may have devastating economic and social consequences locally and globally. In recent decades we have witnessed the appearance of novel infectious diseases\u003csup\u003e1\u003c/sup\u003e a shifting distribution of vectors and pathogens\u003csup\u003e2\u003c/sup\u003e, and the resurgence of mosquito-borne diseases, such as malaria in the USA\u003csup\u003e3\u003c/sup\u003e, yellow fever in Brazil\u003csup\u003e4\u003c/sup\u003e and Japanese encephalitis in Australia\u003csup\u003e5\u003c/sup\u003e. This underscores an urgent and critical need for surveillance systems that can detect and monitor transmission across multiple vectors, pathogen reservoirs and environments. This challenge is complicated for mosquito-borne zoonoses, because extensive surveillance is required across time, space and multiple species. Understanding the transmission ecology of such multi-host pathogens is a major challenge to biomedical science in the 21st Century\u003csup\u003e6,7\u003c/sup\u003e. New, robust, scalable, and cost-effective approaches to surveillance are needed, for implementation across a variety of environments, vectors, reservoirs and pathogens\u003csup\u003e8\u003c/sup\u003e. Xeno-surveillance may offer a solution, using the blood-feeding behaviour of arthropods to determine vector-host relationships and to survey vertebrates for pathogen signals\u003csup\u003e9\u003c/sup\u003e without having to sample vertebrates directly.\u003c/p\u003e \u003cp\u003eHaematophagous insects can yield information on local pathogen prevalence without being part of the transmission cycle. For example, collections of tsetse flies in Tanzania\u003csup\u003e10\u003c/sup\u003e and \u003cem\u003eCulex\u003c/em\u003e mosquitoes in the Brazilian Amazon\u003csup\u003e11\u003c/sup\u003e yielded malaria parasites while urban \u003cem\u003eCulex\u003c/em\u003e collections identified dengue, Zika and chikungunya viruses in Pernambuco, Brazil\u003csup\u003e12\u003c/sup\u003e.The presence of non-vector-borne pathogens, such as hepatitis C virus\u003csup\u003e8\u003c/sup\u003e and H5N1 virus\u003csup\u003e13\u003c/sup\u003e have also been noted in mosquito collections. Xeno-surveillance might also be used to detect vertebrate antibodies to pathogens. Proof-of-principle studies have demonstrated that pathogen-specific antibodies can be recovered from blood-engorged mosquitoes fed with vertebrate blood containing naturally-acquired antibodies in the laboratory (e.g. \u003cem\u003eToxoplasma gondii\u003c/em\u003e and SARS-CoV-2 antibodies\u003csup\u003e14\u003c/sup\u003e) and from wild mosquitos collected from disease endemic areas (e.g. dengue and Japanese encephalitis virus antibodies\u003csup\u003e15\u003c/sup\u003e). Antibodies to the pathogenic piroplasm \u003cem\u003eTheileria sergenti\u003c/em\u003e have also been recovered from ticks\u003csup\u003e16\u003c/sup\u003e, However, these proofs detect host-specific conjugated antibodies, and so cannot be adapted to determine exposure in bloodmeals derived from multiple hosts. This makes them less suitable for investigating complex zoonoses, e.g. SARS-CoV-2 and RRV, where vertebrate blood sources are diverse or unknown.\u003c/p\u003e \u003cp\u003eThe analysis of the vertebrate host preferences of haematophagous arthropods, using molecular identification of bloodmeals is common practice and a key part of investigations on potential transmission pathways\u003csup\u003e17\u0026ndash;20\u003c/sup\u003e, but only recently have bloodmeals also provided an opportunity to determine pathogen exposure in the host\u003csup\u003e21\u003c/sup\u003e. Screening for live virus in large mosquito collections is onerous and time consuming, and usually only a low detection rate is achievable\u003csup\u003e22\u003c/sup\u003e. Neither does it indicate the vertebrate source of the virus nor the transmission pathway. Simultaneous screening of pathogen-specific antibodies and host identification in mosquito bloodmeals can, however, yield information on vector-host interactions, vertebrate exposure to target pathogens and, therefore, potential virus transmission pathways.\u003c/p\u003e \u003cp\u003ePlaque reduction neutralisation tests (PRNTs) are the gold standard for quantifying the presence of neutralising antibodies in serum samples. Unlike other assays (e.g. ELISAs or antigen assays) they are not reliant on host-specific conjugated antibodies and can therefore be applied to serum from any source. This is of particular relevance for the investigation of complex zoonoses like RRV where potential hosts are myriad or unknown. The PRNT has been adapted for screening RRV antibodies from the small volumes of blood present in mosquito abdomens\u003csup\u003e21\u003c/sup\u003e and can be used for all vertebrates\u003csup\u003e23,24\u003c/sup\u003e. In tandem with DNA barcoding of the vertebrate origin of the bloodmeal, the adapted PRNT can give a measure of vertebrate host exposure to RRV using mosquitoes. DNA barcoding is commonly applied to identify invertebrate bloodmeal source using species specific fragments of cytochrome \u003cem\u003ec\u003c/em\u003e oxidase subunit 1 (CO1) and/or cytochrome \u003cem\u003eb\u003c/em\u003e (cyt \u003cem\u003eb\u003c/em\u003e) genes for molecular identification\u003csup\u003e18,19,25,26\u003c/sup\u003e. Moreover, the use of haematophagous insects as \u0026ldquo;flying syringes\u0026rdquo;\u003csup\u003e10\u003c/sup\u003e represents a means of surveying vertebrate abundance and diversity by exploiting the presence of host-seeking insects that are abundant across temperate and tropical habitats and that, dependent on species, exhibit generalist, opportunistic feeding patterns.\u003c/p\u003e \u003cp\u003eThe objective of this study was to demonstrate that blood-fed mosquitoes, collected as a part of routine surveillance exercises, can yield crucial insights regarding vertebrate diversity, host-preference, and virus exposure in the vertebrate supplying the bloodmeal. This can identify potential disease transmission pathways. We focus on RRV as a case study, because it is endemic within our study area, a nationally notifiable disease of significant public health importance in Australia, and has the potential for global spread\u003csup\u003e27\u003c/sup\u003e. RRV is a complex mosquito-borne zoonosis, circulating between multiple vertebrate species and mosquito vectors, and regularly \u0026ldquo;spilling over\u0026rdquo; into humans\u003csup\u003e28\u003c/sup\u003e. We addressed three questions: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) do existing trapping networks yield mosquitoes with bloodmeals of sufficient quality to yield data on host feeding patterns and antibody prevalence? (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) how do mosquito feeding patterns reflect vertebrate abundance? (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) how does host antibody prevalence revealed by bloodmeals compare with historical, conventional sero-surveys for RRV?\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 54,670 mosquitoes were collected during the trapping period of this study (February\u0026nbsp;2021 to May 2022) as part of an established mosquito surveillance program implemented by Brisbane City Council (BCC), Queensland, Australia, and some additional traps deployed by Queensland Health, Australia. Of these, 480 mosquitoes (0.9% of the total catch) from 14 species were identified as being blood-fed (\u003cstrong\u003eSupplementary\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e). Five species accounted for \u0026gt; 85% of blood-feds collected: \u003cem\u003eCulex annulirostris\u003c/em\u003e (68.7%), \u003cem\u003eCx. orbostiensis\u0026nbsp;\u003c/em\u003e(9.4%), \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eprocax\u003c/em\u003e (3.9%), \u003cem\u003eCx. sitiens\u0026nbsp;\u003c/em\u003e(3.7%), and\u003cem\u003e\u0026nbsp;Verrallina\u0026nbsp;\u003c/em\u003eMarks sp. No.\u003cem\u003e\u0026nbsp;\u003c/em\u003e52\u003cem\u003e\u0026nbsp;\u003c/em\u003e(2.71%).\u003c/p\u003e\n\u003cp\u003eA single vertebrate origin was identified from 341 bloodmeals, while another five bloodmeals were of mixed origin (human and one other vertebrate), totalling 346 identified bloodmeals\u0026nbsp;(\u003cstrong\u003eTable 1 and Supplementary Table S2\u003c/strong\u003e). A total of 26 vertebrate species were identified from these bloodmeals, 10 birds, nine placental mammals, three marsupials, two reptiles, one amphibian, and one semi-aquatic fish. Humans were the dominant source of bloodmeals, accounting for 73.1% of bloodmeals (253/346), followed by cattle and marsupials (8.7% and 6.9% of bloodmeals, respectively).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy comparison, conventional vertebrate surveys, conducted by human observers at the same sites, identified a much higher number of vertebrate species (85). Most of these species were birds (74), with 6 placental mammals, 2 marsupials, 2 amphibians and a reptile. The vertebrate survey did not observe cattle, deer, fox, sheep, or fish, all of which were fed on by mosquitoes. Human abundance was not quantified during conventional vertebrate surveys, but was estimated using population statistics from areas within the dispersal kernel of the key mosquito species sampled\u0026nbsp;(\u003cstrong\u003eFig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAll 480 bloodmeal samples collected in this study were screened for the presence of anti-RRV antibodies using an adapted micro-PRNT\u003csup\u003e21\u003c/sup\u003e and more than half (n=243, 51%) were seropositive (\u003cstrong\u003eTable 2\u003c/strong\u003e). RRV seroprevalence was 52% (131/253) in humans, 70% in cattle and eastern grey kangaroos (21/30 and 14/20, respectively), and ranged from 25% to 100% in samples from other animals. Among the 14 blood-fed mosquito species, 12 were found to have fed on RRV seropositive hosts, with \u003cem\u003eAe. alternans\u003c/em\u003e (n=1) and \u003cem\u003eUranotaenia nivipes\u003c/em\u003e (n=1) being the exceptions.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first field demonstration that mosquito bloodmeals can be used a resource for estimating vertebrate seroprevalence. Our results indicate varying seroprevalence of RRV antibodies across different host species, highlighting potential transmission pathways. RRV is thought to be maintained in the environment by multiple host and vector species, with little understanding about how this community shifts across environments, seasons, and the degree of spillover to the human population. High seroprevalence demonstrated in this study and others (\u003cb\u003eSupplementary Table S3\u003c/b\u003e) among humans (ranging from 7 to 79%), horses (21 to 94%), cattle (6 to 100%), and kangaroos (36 to 100%), along with a very limited data-set on species presenting with RRV-induced viraemias\u003csup\u003e28\u0026ndash;30\u003c/sup\u003e, suggest their susceptibility to RRV infection and their possible role as amplifying or diluting RRV hosts within the community needs to be re-examined; even for those species long thought of as \u0026lsquo;dead-end\u0026rsquo; hosts (e.g. cattle).\u003c/p\u003e \u003cp\u003eHumans are potential amplifying hosts of RRV\u003csup\u003e28,31,32\u003c/sup\u003e. Their high RRV seroprevalence rates indicate high rates of exposure. The human seropositivity in this study corroborates with previous sero-surveillance studies in QLD with antibody prevalence of 9\u0026ndash;50% (\u003cb\u003eSupplementary Table S3\u003c/b\u003e). It is notable that in this study, we were sampling bloodmeals from mosquitoes caught in urban parklands. Although we do not know the movement patterns of those humans, the high seropositivity rates suggest that at least some of those humans are being infected in an urban transmission cycle rather than a rural one. Cattle are not considered important reservoir hosts for RRV, because under experimental conditions only one individual (of six) developed viraemia which was low and short-lived\u003csup\u003e29\u003c/sup\u003e. Yet, our results highlight that cattle are frequently exposed to RRV and may contribute to transmission dynamics. Notably, little corellas (\u003cem\u003eCactua sanguinea\u003c/em\u003e) and cattle develop near identical viraemic responses to infection in terms of peak and duration and the former was shown to infect 14% of susceptible \u003cem\u003eCx. annulirostris\u003c/em\u003e mosquitoes\u003csup\u003e29\u003c/sup\u003e. Additionally, varying seroprevalence in other placental mammals, including deer, red foxes and some birds, underscores the catholic feeding behaviour of RRV vectors and the potential complexity of RRV's host range. Our data emphasises the fact that, 60 years after the initial solation of RRV from a mosquito vector\u003csup\u003e33\u003c/sup\u003e we still have little understanding of the reservoirs or their role in maintain transmission cycles in different habitats. For example, we show that deer are clearly exposed to RRV, but there are no experimental studies exist to confirm transmission.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCulex annulirostris\u003c/em\u003e, a major vector of RRV and other medically-important arboviruses in Australia\u003csup\u003e34,35\u003c/sup\u003e, is a freshwater mosquito that is abundant across the country. They are considered some of the most prominent vectors of RRV due to (i) being one of the most frequent sources of RRV isolates, with over 250 isolates\u003csup\u003e36\u0026ndash;38\u003c/sup\u003e, (ii) their demonstrated ability to become infected with and transmit RRV under laboratory settings (e.g. from horses or little corellas\u003csup\u003e29\u003c/sup\u003e), and (iii) spatio-temporal associations between infected \u003cem\u003eCx. annulirostris\u003c/em\u003e and RRV disease\u003csup\u003e36,39\u003c/sup\u003e. They are generalist-opportunistic feeders and feed readily on humans\u003csup\u003e18,40\u0026ndash;42\u003c/sup\u003e. \u003cem\u003eCx. annulirostris\u003c/em\u003e was the most abundant species in this study, as also reported in other Queensland studies\u003csup\u003e36,39\u003c/sup\u003e. It also yielded the greatest number of bloodmeals for analysis and was found to have fed on six bird species and every mammal species identified in this study including humans (n\u0026thinsp;=\u0026thinsp;12). This suggests a wide host range with potential for a variety of RRV transmission pathways. The species is likely a key means of spillover to humans Its eclectic feeding habits also makes this species a powerful sampling tool for identifying local vertebrate diversity and abundance.\u003c/p\u003e \u003cp\u003eFurther empirical research on viraemic response and transmission to mosquitoes is desirable for many of the potential hosts identified in this study. However, these experimental infection studies for complex zoonoses are ethically and logistically difficult. The micro-PRNT approach serves as a valuable, field-based but circumstantial alternative that can aid pathway incrimination and prioritise targets for further study.\u003c/p\u003e \u003cp\u003eThe micro-PRNT method estimates pathogen exposure by screening mosquito bloodmeals for antibodies produced in response to viral infection in the vertebrate host. These antibodies can persist in the bloodstream for extended periods. Since viraemia is typically short, antibodies provide several advantages for understanding population exposure and for inferring transmission dynamics. This approach also has advantages over virus screening of mosquito collections as means of investigating pathways. Mosquitoes are often extremely abundant, have short lifespans and only take bloodmeals at fixed intervals (the gonotrophic cycle). There are therefore limited opportunities for them to acquire virus, so \u0026ldquo;hit\u0026rdquo; rates during screening can be very low\u003csup\u003e22\u003c/sup\u003e. Moreover, the presence of virus in a population does not necessarily relate to transmission. Mosquitoes will often be incidental carriers of pathogens circulating in the environment and may not be responsible for transmission\u003csup\u003e10\u0026ndash;12\u003c/sup\u003e. There is no \u0026ldquo;smoking gun\u0026rdquo; in terms of transmission proofs from field-collections of vertebrates or mosquitoes.\u003c/p\u003e \u003cp\u003eThe capacity to characterise host exposure to pathogens such as RRV, here demonstrated for the first time in the field, is an exciting development that can offer descriptive insights into potential virus reservoirs and transmission pathways. It is also a potentially powerful means of quantifying key parameters in deterministic compartmental models of disease transmission. All of these utilise some estimate of the size of the pathogen reservoir in order to propose values for the number of susceptible, infected and immune (recovered) individuals. For multi-host pathogens this can be highly challenging because estimates must be made for multiple populations\u003csup\u003e30\u003c/sup\u003e. This is traditionally done through vertebrate surveys and a great deal of guess work. Antibody screening in bloodmeals may allow us to estimate the S (susceptible) and R (immune) proportion of the reservoir population and therefore build SIR models of transmission. The \u0026ldquo;infected\u0026rdquo; parameter in these models could be derived through an additional screen of the mosquito bloodmeals for (rare) virus signals.\u003c/p\u003e \u003cp\u003eCollection and analysis of blood-fed mosquitos offers an alternative to conventional vertebrate surveys. Excluding bird species, the number of vertebrate species quantified by screening mosquito bloodmeals was greater than that observed during conventional faunal surveys. This is probably because the observations made during traditional surveys are most likely to capture those species that are least cryptic, locally abundant and most active. Traditional surveys may record greater bird diversity because observers use additional cues such as bird calls. By using mosquitoes as the sampling tool, we have potentially greater access to cryptic species across all habitats and times of day. Moreover, trapped, blood-fed mosquitoes may have been foraging over much larger areas than those which are observable by a human operating along a fixed transect.\u003c/p\u003e \u003cp\u003eIt is notable that humans made up the majority of bloodmeals across all locations, although they only represent a small proportion of vertebrate diversity. Our estimates of accessible humans were based on residential populations within the effective dispersal kernel of mosquitoes. Thus, we undoubtedly overestimated the human population available to the mosquito. Nonetheless, the high proportion of human bloodmeals may reflect host availability in the study area. Humans were likely to represent a large proportion of the overall vertebrate biomass in the parks, particularly at some periods during the day (i.e., peak times for commuting, exercise or leisure may coincide with the crepuscular rhythms of many mosquito species). This would undoubtedly influence mosquito feeding patterns. In fact, there are over 2,180 parks across Brisbane\u003csup\u003e43\u003c/sup\u003e, and they may be major foci for transmission and human spillover, the specific patterns of which will depend on vertebrate diversity, virus amplification and virus dilution.\u003c/p\u003e \u003cp\u003eThe diversity of bloodmeal origins captured across a number of mosquito species, confirms that mosquitoes are a powerful, emerging tool for characterising vertebrate diversity. The technique has potential application to a range of ecological and conservation issues, such as monitoring rare or endangered species\u003csup\u003e44\u003c/sup\u003e, and measuring biodiversity\u003csup\u003e45,46\u003c/sup\u003e. It can also be used to understand host feeding patterns, which is a crucial component for assessing the risk of pathogen transmission pathways and human spillover. It is as important to identify non-reservoirs and the dilution of transmission as it is to identify potential virus sources. For example, some mosquito species will preferentially feed on reptiles, amphibians and even fish (particularly \u003cem\u003eUranotaenia\u003c/em\u003e spp.)\u003csup\u003e47\u003c/sup\u003e. An expansion to this study, involving greater numbers of bloodmeals, might explore whether some vertebrates are predictably \u0026ldquo;non-reactive\u0026rdquo; and whether some mosquito species showed strong preference for those vertebrates.\u003c/p\u003e \u003cp\u003eBlood-fed mosquitoes can be used to characterise key aspects of disease ecology. However, it can be difficult to collect them in sufficient quantities to integrate them into surveillance campaigns\u003csup\u003e48\u003c/sup\u003e. The baited, fan-assisted traps commonly used for mosquito surveillance in the field (i.e., BioGents Sentinel traps, CDC light traps or CDC gravid traps) target mosquitoes that are seeking hosts or an oviposition site. Blood-fed mosquitoes are not optimally attracted by these traps but prefer to rest while digesting bloodmeals and developing eggs\u003csup\u003e49\u003c/sup\u003e. Nevertheless, in this study we demonstrate that existing networks of CO\u003csub\u003e2\u003c/sub\u003e baited traps, typically used for routine surveillance around the world are efficient and convenient tools for trapping blood-fed mosquitoes at least for some species and in some habitats. The BCC network of 9 traps, spread in the four urban parks and operating over a six-month period, collected a small percentage (0.9%, n\u0026thinsp;=\u0026thinsp;480) of blood-fed mosquitoes (largely \u003cem\u003eCx. annulirostris\u003c/em\u003e) but yielded sufficient numbers (346 bloodmeal IDs) to facilitate our investigations. The success of CO\u003csub\u003e2\u003c/sub\u003e baited light trap networks in collecting blood-feds is clearly dependent on locality, target species and the extent of the trap network. Flies et al.\u003csup\u003e19\u003c/sup\u003e assessed historic captures by a routine survey in South Australia and found just 280 (0.06%) blood-feds in a collection of 350,000 mosquitoes made over a 10-year period (mostly \u003cem\u003eAe. camptorhynchus\u003c/em\u003e and \u003cem\u003eCx. pipiens\u003c/em\u003e spp). Conversely, in Queensland, Kay et al.\u003csup\u003e40\u003c/sup\u003e caught 1,119 blood-fed mosquitoes over a seven month period representing 3.7% of the catch (mostly \u003cem\u003eCx. annulirostris\u003c/em\u003e), showing its potential for efficiently trapping blood-fed mosquitoes.\u003c/p\u003e \u003cp\u003eA notable limitation of our study is that, due to resource constraints we sampled vertebrates during the same months as the vectors, but in different years. However, comparisons of survey results between 2017/18 and 2020/21 does demonstrate that diversity and abundance are similar.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study made a systematic comparison between vertebrate survey and xeno-surveillance techniques in relation to vertebrate diversity while also demonstrating the viability of xeno-surveillance as a tool to verify vertebrate species serostatus to a zoonotic virus. This is unique because no previous bloodmeal studies have included formal vertebrate surveys. This study validates the use of micro-PRNTs in the field for determine vertebrate exposure to pathogens from mosquito bloodmeals. Conducting seroprevalence studies, especially in non-human vertebrates, are challenging due to ethical and practical limitations. However, our method provides a novel, non-invasive approach to estimate pathogen exposure in vertebrates and linking that to vector-host networks. Overall, this can help determine which pathways are most likely to facilitate transmission of specific mosquito-borne pathogens to humans, as well as monitor any zoonotic disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eThe study was undertaken in Brisbane (27\u0026deg;28'12'' S and 153\u0026deg;01'15'' E), the capital city of Queensland, which is the second largest state in Australia in terms of area. Brisbane is geographically the largest capital city in Australia and the third most populous city in the country, with approximately 2.5\u0026nbsp;million people. The city has a subtropical climate characterised by an average annual precipitation level of 1011.5 mm. The rainy season typically occurs from November to March, while the average monthly temperatures range from 10\u0026deg;C to 22\u0026deg;C in winter and 20\u0026deg;C to 29\u0026deg;C in summer\u003csup\u003e50\u003c/sup\u003e. Brisbane comprises diverse natural ecosystems, including freshwater and estuarine wetlands, mangroves, saltmarshes, bushlands, and rainforests\u003csup\u003e51\u003c/sup\u003e. These varied ecosystems provide a range of habitats suitable for both mosquito vector species and reservoir hosts. The presence of such ecological diversity, coupled with the persistently high rates of human notifications of RRV\u003csup\u003e34\u003c/sup\u003e, makes Brisbane an ideal location for investigating RRV dynamics.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMosquito collection and identification\u003c/h3\u003e\n\u003cp\u003eMosquito collections were carried out by the BCC as part of their ongoing mosquito surveillance and control program from February to May 2021 and during the same period in 2022. Five sites were chosen based on historical detections of RRV in mosquito collections (\u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). These were all urban recreational parks within established residential areas, with human population density ranging from 7.75 to 21.56 people per hectare. To increase the number of blood-fed individuals, particularly those that had potentially fed on a host that had previously been exposed to RRV, QH conducted additional surveillance at sites where RRV had recently been detected in sentinel traps during the trapping period.\u003c/p\u003e \u003cp\u003eTraps were set once a week prior to dusk and then collected the following morning. We employed CDC-style light traps sourced from Pacific Biologics (Scarborough, Australia), with 1 kg of dry ice as a CO\u003csub\u003e2\u003c/sub\u003e source and supplemented by 1-octen-3-ol.\u003c/p\u003e \u003cp\u003eAfter collection, mosquitoes were transported to the Mosquito Control Laboratory (MCL) for identification. Mosquitoes were identified to the species level using dichotomous keys\u003csup\u003e52\u0026ndash;54\u003c/sup\u003e. Blood-engorged mosquitoes were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis by the respective assays.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation\u003c/h2\u003e \u003cp\u003eThe abdomens of individual blood-fed mosquitoes were dissected under a stereo microscope. The dissected abdomens were subsequently homogenised in 70 \u0026micro;L of tissue culture media RPMI-1640, supplemented with 1% Penicillin, Streptomycin, and L-glutamine (Sigma-Aldrich, USA). To prevent fungal contamination 0.4% amphotericin B (Sigma-Aldrich, USA) was added to the homogenisation media.\u003c/p\u003e \u003cp\u003eFollowing homogenisation, the samples were centrifuged at 10,000 \u003cem\u003ex\u003c/em\u003e g for 10 minutes, and the resulting supernatant removed. A volume of 55 \u0026micro;L of the supernatant was transferred to a sterile 1.5 ml microtube for subsequent testing to determine the presence of neutralising antibodies against RRV. The remaining volume of the supernatant was retained in a separate tube for molecular identification of the host species from which the bloodmeal was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eNucleic acid extraction and DNA barcoding\u003c/h2\u003e \u003cp\u003eThe DNA barcoding was performed using different regions of the mitochondrial DNA: the subunit I of the cytochrome oxidase (COI)\u003csup\u003e55,56\u003c/sup\u003e or cytochrome \u003cem\u003eb\u003c/em\u003e (cyt \u003cem\u003eb\u003c/em\u003e)\u003csup\u003e25,57\u003c/sup\u003e genes. The sets of primers chosen (\u003cb\u003eSupplementary Table S4\u003c/b\u003e) were tested and validated on known host species, including humans, sheep (\u003cem\u003eOvis aries\u003c/em\u003e), koalas (\u003cem\u003ePhascolarctos cinereus\u003c/em\u003e), and birds (Australian magpie, \u003cem\u003eCracticus tibicen\u003c/em\u003e), using whole blood or serum samples.\u003c/p\u003e \u003cp\u003eDNA was extracted from up to 10 \u0026micro;L of the mosquito bloodmeal samples using DNeasy Blood \u0026amp; Tissue Kit (Qiagen, Valencia, CA, USA), according to the manufacturer\u0026rsquo;s instructions. The extracted DNA was then amplified using a touchdown cycling sequence to minimize non-specific amplification. The PCR mixture consisted of 3 \u0026micro;L of template DNA, 2X Phusion High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs, Ipswich MA), 0.5% DMSO, and 0.5 \u0026micro;M of each of a forward and reverse primer in a 30 \u0026micro;L reaction mix. Each sample was submitted to the four combinations of primers (\u003cb\u003eSupplementary Table S4\u003c/b\u003e). Cycling conditions comprised a step at 98\u0026deg;C for 30s, followed by 5 touchdown cycles of 98\u0026deg;C 5s, 49.5\u0026deg;C 30s, 72\u0026deg;C 45s, 30 cycles of 98\u0026deg;C 5s, 54\u0026deg;C 30s, 72\u0026deg;C 45s, and a final elongation step of 72\u0026deg;C for 5min. The PCR products were visualised on a 2% agarose gel.\u003c/p\u003e \u003cp\u003eAmplified target DNA bands were either purified directly from the PCR reaction or from the gel using Qiagen Gel kit or PCR purification kits. Samples were sequenced by the QIMRB DNA Sequencing Facility using the BigDye\u0026trade; Terminator v3.1 Cycle Sequencing kit in an ABI-PRISM 3130 Genetic Analyser (Applied Biosystems, Foster City, CA). The obtained sequences were then compared to the NCBI nucleotide database using a Basic Local Alignment Search Tool (BLAST) search.\u003c/p\u003e \u003cp\u003eSequences showing a\u0026thinsp;\u0026ge;\u0026thinsp;95% sequence identity with those in the NCBI database were considered as matches. If the sequence identity was below 95%, the samples were considered inconclusive, and additional genus-specific PCR amplification and sequencing were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicro-plaque reduction neutralisation test (micro-PRNT)\u003c/h2\u003e \u003cp\u003eThe presence of neutralising antibodies against RRV in mosquito bloodmeals was assessed using a micro-PRNT, developed by Gyawali et al.\u003csup\u003e21\u003c/sup\u003e. Briefly, African green monkey kidney (Vero) cells (ATCC, CCL-81) were cultivated as monolayers in 96-well tissue culture plates (Nunclon, Thermo Scientific, Australia). The bloodmeals from mosquitoes were mixed with a quantity of RRV virus (T48 prototype strain\u003csup\u003e33\u003c/sup\u003e), calibrated to form 30 plaques per well.\u003c/p\u003e \u003cp\u003eThe bloodmeal-virus mixture was added to wells in duplicate. Wells containing only Vero cells with and without RRV served as positive and negative controls, respectively. After incubation at 37\u0026deg;C for 2 hours, the mixture was removed and 0.75% w/v carboxymethyl cellulose (CMC, Sigma-Aldrich) in RPMI-1640 medium was added to each well. The plates were incubated for a further 40 hours at 37\u0026deg;C in an atmosphere of 5% v/v CO\u003csub\u003e2\u003c/sub\u003e/air.\u003c/p\u003e \u003cp\u003eFollowing incubation, the media was decanted, and the wells stained with a solution of 0.05% w/v crystal violet in formaldehyde (1% v/v) and methanol (1% v/v). After 24 hours, the plates were rinsed, air-dried, and the number of plaques per well was counted. Plaques were counted in duplicate wells, and the average number was determined. Bloodmeals that resulted in a\u0026thinsp;\u0026ge;\u0026thinsp;50% reduction in the number of viral plaques compared to the positive control (RRV alone) were considered RRV seropositive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHost abundance surveys\u003c/h2\u003e \u003cp\u003eMethods for the vertebrate surveys were adapted from the Queensland Fauna Survey Guidelines\u003csup\u003e58\u003c/sup\u003e. In brief, we estimated the abundance of hosts using four-point surveys, six minutes in duration, performed at least 150m apart across Brisbane at the same sites used to collect mosquitoes monthly from November 2017 to March 2018 and again from March 2020 and April 2021. Vertebrate surveys were conducted during times of peak activity (within 30 minutes of dawn for birds, and after sunset for mammals). During each survey we recorded the species, number of individuals, whether the observation was seen or heard, and the microhabitat the species was in (i.e. on the ground, in the canopy, in the bottom third of a tree). Equipment used to identify vertebrates included binoculars, handheld torches, field guides and the Birds of Australis mobile phone application\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHuman abundance in each area was calculated by multiplying the average number of people per household in each suburb (as per Census data\u003csup\u003e60\u003c/sup\u003e) by the number of households within 2km of each vector trapping site. That effective radius is a modest estimate of the foraging kernel of the main mosquito species collected during this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.J.D. devised the project. G.L.W., A.F.v.d.H., F.D.F. and G.J.D.\u0026nbsp;supervised the project. C.J.S.P.V., M.B.O., M.A.S., D.S., J.M.D. and E.B.S. coordinated field activities. C.J.S.P.V. and N.G. analysed mosquito sera collections. C.J.S.P.V. and E.B.S. analysed vertebrate faunal surveys. C.J.S.P.V. collated and analysed the data, drafted the manuscript and designed the figures. J.M.D., N.G., G.L.W., A.F.v.d.H., F.D.F., E.B.S. and G.J.D.\u0026nbsp;contributed to the editing of the final manuscript. All authors edited and approved the version submitted for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analysed during this study are included in this published article and its Supplementary Information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.J.S.P.V. was supported by a QIMRB International PhD Scholarship and QUT Tuition Fee Sponsorship.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKhalil, A. M., Martinez-sobrido, L. \u0026amp; Mostafa, A. Zoonosis and zooanthroponosis of emerging respiratory viruses. \u003cem\u003eFront. Cell. Infect. 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(Steve Parish Publishing, 2003).\u003c/li\u003e\n\u003cli\u003eAustralian Bureau of Statistics. 2021 Census QuickStats. \u003cem\u003eAustralian Bureau of Statistics\u003c/em\u003e https://www.abs.gov.au/census/find-census-data/quickstats/2021/3GBRI (2022).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-3949962/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3949962/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe surveillance and detection of zoonotic pathogens in animals is essential for predicting disease transmission pathways and the risks of spillover, but challenges include the costs, ethics and technical expertise required for vertebrate trapping, serum sampling and antibody or virus screening. Surveillance using haematophagous arthropods as a sampling tool offers a unique opportunity to obtain blood samples from a wide range of vertebrate species, allowing the study of host-mosquito associations, and host exposure to pathogens. We explored vertebrate diversity and potential Ross River virus (RRV) transmission pathways by analysing blood-fed mosquitoes collected in Brisbane, Australia. Host origins were identified using barcode sequencing, and host exposure to RRV was assessed using a modified plaque reduction neutralisation test. In total, 480 blood-fed mosquitoes were collected between February 2021 and May 2022. The host origins of 346 (72%) bloodmeals were identified, with humans (73%) and cattle (9%) comprising the dominant hosts. RRV seroprevalence was high in both vertebrate species with evidence of RRV exposure in 70% (21/30) of cattle and 52% (132/253) of humans. This is a novel, non-invasive method of estimating seroprevalence in vertebrate host populations. Our results highlight the potential of blood-fed mosquitoes to provide species-specific insights into pathogen transmission dynamics.\u003c/p\u003e","manuscriptTitle":"Mosquito bloodmeals can be used to determine vertebrate diversity, host preference, and pathogen exposure in humans and wildlife","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 04:15:27","doi":"10.21203/rs.3.rs-3949962/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-16T05:49:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-15T15:31:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266640658392076108372855141126654631965","date":"2024-07-04T11:28:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-08T01:05:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44583aa4-0841-4f34-82b7-a90756023c93","date":"2024-04-18T00:19:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d4ca3af4-2527-4a32-b55e-15bcb7b8704c","date":"2024-04-16T18:57:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-05T09:33:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-05T09:32:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-05T05:32:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-05T03:21:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-12T02:15:52+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":"1f65c702-c215-4f38-8e1e-ea77c99582f2","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28703660,"name":"Biological sciences/Microbiology/Infectious disease diagnostics"},{"id":28703661,"name":"Earth and environmental sciences/Ecology/Ecological epidemiology"}],"tags":[],"updatedAt":"2024-10-07T16:05:21+00:00","versionOfRecord":{"articleIdentity":"rs-3949962","link":"https://doi.org/10.1038/s41598-024-73820-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-05 15:58:19","publishedOnDateReadable":"October 5th, 2024"},"versionCreatedAt":"2024-02-21 04:15:27","video":"","vorDoi":"10.1038/s41598-024-73820-y","vorDoiUrl":"https://doi.org/10.1038/s41598-024-73820-y","workflowStages":[]},"version":"v1","identity":"rs-3949962","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3949962","identity":"rs-3949962","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}