Mosquitoes in urban green spaces and cemeteries in northern Spain.

BACKGROUND: Mosquitoes inhabiting urban green spaces and cemeteries in Europe represent a crucial facet of public health concern and contribute to the ecological balance. As urbanization intensifies, these areas increasingly serve as vital habitats for various mosquito species, fostering breeding grounds and increasing the risk of disease transmission. METHODS: A study was conducted in the three main cities (inland, coastal, and estuarine) of the Basque Country, northern Spain, to investigate the species composition, abundance, dynamic populations, larval habitats, and host preferences of mosquitoes in urban green spaces and cemeteries. CDC traps and dipping were used to collect mosquitoes for 2 years (2019-2020). RESULTS: A total of 21 mosquito species were identified, with Culex pipiens s.l. being the most abundant and widespread. The three ecological forms of Cx. pipiens were found, and Cx. pipiens pipiens was the most common in both green areas and cemeteries. Morphological identification together with molecular tools identified 65 COI sequences with high homology. The highest species richness was found in the inland city, followed by the coastal city and the estuarine city. Mosquito abundance was significantly higher in green areas compared to cemeteries and in the coastal and estuarine cities compared to the inland city. The investigation of larval breeding sites highlighted the dominance of Cx. pipiens s.l., particularly in semi-artificial ponds, diverse water-holding containers (tyres and buckets) and drainage systems in green areas; in cemeteries, most of the larvae were found in flowerpots and funerary urns. Seasonal activity exhibited variable peaks in mosquito abundance in the different cities, with a notable increase in July or August. Additionally, blood meal analysis revealed that Cx. pipiens s.l. fed on several common urban avian species. CONCLUSIONS: Studies on mosquitoes are essential to understand their role in disease transmission and to design targeted and sustainable management strategies to mitigate the associated risks.

A fast and inexpensive genotyping system for the simultaneous analysis of human and Aedes albopictus short tandem repeats.

BACKGROUND: Determination of the interactions between hematophagous mosquitoes and their human hosts is of great importance for better understanding the transmission dynamics of mosquito-borne arboviruses and developing effective strategies to mitigate risk. Genetic analysis of human and mosquito DNA can play a key role in this, but commercial kits for human short tandem repeat (STR) genotyping are expensive and do not allow for the simultaneous STR analysis of host and vector DNA. Here, we present an inexpensive and straightforward STR-loci multiplex system capable of simultaneously amplifying Aedes albopictus and human STRs from blood-fed mosquitoes. Additionally, we examine the effect of storage methods and post-feeding time on the integrity of host DNA. METHODS: Thirty-five STRs (16 human and 19 Ae. albopictus STRs) subdivided in three multiplexes were tested for amplification and scoring reliability. Under laboratory conditions we compared the efficacy of two preservation methods (absolute ethanol vs lysis buffer) on the integrity of host DNA in Ae. albopictus blood meals. We also evaluated the effect of post-feeding time by sacrificing blood-fed mosquitoes at different time intervals after feeding, and we assessed our ability to detect multiple feedings. To determine if the system can be employed successfully under field conditions, we carried out a preliminary study using field-collected Ae. albopictus. RESULTS: All 35 STRs amplified consistently in the laboratory. Lysis buffer performed better than absolute ethanol in terms of allele peak height and clarity of electropherograms. Complete human DNA profiles could be obtained up to 48 h following the blood meal. Analysis of multiple feedings confirmed that peak heights can be used as a proxy to determine post-feeding time and thus derive the number of different people bitten by a mosquito. In the field trial, amplification was successful for 32 STRs. We found human DNA signal in 38 of the 61 field-collected mosquitoes (62%), of which 34 (89%) had ingested a single blood meal, while four (11%) contained double meals. CONCLUSIONS: Our new genotyping system allows fast and reliable screening of both host and vector species, and can be further adapted to other mosquito species living in close contact with humans.

Host Associations of Culicoides Biting Midges in Northeastern Kansas, USA.

Culicoides biting midges (Diptera: Ceratopogonidae) are hematophagous flies that transmit several viruses of veterinary concern to livestock. Understanding blood feeding behaviors is integral towards identification of putative vector species and preventing the transmission of these pathogens. PCR-based blood meal analysis was conducted on 440 blood-engorged Culicoides midges collected in northeastern Kansas, with 316 (71.8%) returning non-human vertebrate identifications at the ≥95% identity match level. Broadly, Culicoides sonorensis, Culicoides stellifer, and Culicoides variipennis were found to feed heavily on mammalian hosts, while Culicoides crepuscularis and Culicoides haematopotus fed on avian hosts. The blood meals in all specimens were graded prior to DNA extraction to determine whether blood meal size or digestion status significantly impacted the likelihood of a quality host match. Size had a significant impact on the likelihood of a quality match at grades 3-5, whereas digestion only significantly impacted outcomes at the most extreme grade. These vector-host dynamics have not previously been studied in Culicoides collected in Kansas, which represents a unique tallgrass prairie biome within the United States that is heavily interspersed with livestock operations. Based on these data, the highly abundant species C. crepuscularis and C. haematopotus are unlikely to be major vectors of mammalian viruses.

Molecular Identification of Culicoides Species and Host Preference Blood Meal in the African Horse Sickness Outbreak-Affected Area in Hua Hin District, Prachuap Khiri Khan Province, Thailand.

African horse sickness (AHS) was reported as an outbreak in Thailand in 2020. Hematophagous insects from the genus Culicoides are the suspected vector responsible for AHS transmission. Horses in Hua Hin district, Prachuab Khiri Khan province, Thailand, were affected and died from AHS in 2020. However, the potential Culicoides species and its host preference blood meal in the affected areas are unknown. To investigate the potential vectors of AHS, Culicoides were collected using ultraviolet light traps placed near horse stables. Six horse farms, including five farms with AHS history and one farm without AHS history, were included in this study. Morphological and molecular identification of the Culicoides species was performed. Polymerase chain reaction (PCR) targeting the cytochrome b oxidase I (COXI) gene for confirmation of the Culicoides species, identification of the prepronociceptin (PNOC) gene for host preference blood meal, and bidirectional sequencing were conducted. Consequently, 1008 female Culicoides were collected, consisting of 708 and 300 samples captured at positions A and B at a distance of <2 and >5 m from the horse, respectively. Twelve Culicoides species identified by morphology were noted, including C. oxystoma (71.92%), C. imicola (20.44%), C. actoni (2.28%), C. flavipunctatus (1.98%), C. asiana (0.99%), C. peregrinus (0.60%), C. huffi (0.60%), C. brevitarsis (0.40%), C. innoxius (0.30%), C. histrio (0.30%), C. minimus (0.10%), and C. geminus (0.10%). The PCR detection of the Culicoides COXI gene confirmed Culicoides species in 23 DNA samples. PCR targeting the PNOC gene revealed that the Culicoides collected in this study fed on Equus caballus (86.25%), Canis lupus familiaris (6.25%), Sus scrofa (3.75%), and Homo sapiens (3.75%) for their blood meal. Human blood was identified from two samples of C. oxystoma and a sample of C. imicola. Three dominant species including C. oxystoma, C. imicola, and C. actoni that were reported in the Hua Hin area prefer to feed on horse blood. Moreover, C. oxystoma, C. imicola, and C. bravatarsis also feed on canine blood. This study revealed the species of Culicoides in Hua Hin district, Thailand, after the AHS outbreak.

Blood feeding patterns of the Culex pipiens complex in equestrian land uses and their implications for arboviral encephalitis risk in temperate Argentina.

Blood feeding patterns of mosquitoes are a key component in the dynamics of arboviral encephalitides transmission. In temperate Argentina, the members of the Culex pipiens complex include Cx. pipiens molestus, Cx. quinquefasciatus and their hybrids. To characterize their blood feeding patterns, adult resting mosquitoes were collected monthly during the warm season in urban and rural equestrian fields. The availability of birds and domestic mammals per site was characterized. The blood source and the complex member were successfully identified for 89 specimens using PCR. Blood of 19 vertebrate species was isolated including four mammals (most common feeds from dog, Canus lupus 19% of the blood meals; and horse, Equus caballus 18%) and 15 birds (picazuro pigeon, Patagioenas picazuro 11%; eared dove, Zenaida auriculata 10%; chicken, Gallus gallus 9%). The Forage Ratio (FR), calculated as the proportion of feeds taken from a given host species with respect to that host availability in the environment, suggested preference for dog by all members of the complex (FR ≥4.5). On the contrary, FR values suggested avoidance for horse by Cx. quinquefasciatus and the hybrid (FR ≤0.8), and a use proportional to its abundance by Cx. pipiens molestus (FR = 1.1-1.2 in urban and rural sites, respectively). FR values suggesting preference were obtained for avian species of the orders Passeriformes (7 species in total) and Columbiformes (5) by all members of the complex (FR ≥ 3.3), whereas values for monk parakeet (Myiopsitta monachus, Psiitaciformes) suggested avoidance by Cx. quinquefasciatus in urban sites (FR = 0.4) and by Cx. pipiens molestus in rural sites (FR = 0.3) but not in urban sites (FR = 1.4). A mammal-bird index (MBI, from -1 all avian to +1 all mammalian blood meals) was calculated for each member of the complex and urbanization category. Values were negative for Cx. quinquefasciatus (MBIurban  = -0.60, MBIrural  = -0.33) and positive for Cx. pipiens molestus (MBIurban  = 0.20, MBIrural  = 0.60), indicating a higher proportion of feeds taken on birds and mammals, respectively, regardless of the urbanization category. In temperate Argentina, the members of the Cx. pipiens complex fed both on horses and on birds, thus representing a real risk of transmission of arboviral encephalitides from avian enzootic cycles to horse epizootics.

Blood feeding habits of mosquitoes: hardly a bite in South America.

Mosquito blood feeding plays a key role in epidemiology. Despite its importance and large number of studies worldwide, less attention has been paid in South America. We summarized some general concepts and methodological issues related to the study of mosquito blood feeding habits, and compiled and analyzed all published information regarding the subject in the continent until 2020. Available literature comprised 152 scientific studies, that pursued different approaches: human landing catches (102 studies), baited trap (19), and blood meal analyses of collected specimens (38). Among the latter, 23 used serological and 15 molecular techniques. Species most frequently studied were those incriminated in malaria transmission, whereas relevant vectors such as Aedes aegypti, Ae. albopictus, and Haemagogus janthinomys were surprisingly neglected. Brazil was the leading country both in number of works and species studied. For over 70% of the species and three out of 13 South American countries there is no single information on mosquito blood feeding habits. Data from baited traps included 143 mosquito species, 83.9% of which were attracted to humans, either exclusively (10.5%) or in combination with other vertebrates (73.4%). Host blood identification of field collected specimens provided data on 102 mosquito species, and 60.8% of these fed on humans (55.9% combined with other vertebrates). Only 17 of the 73 species assessed by both methods yielded similar feeding patterns. Finally, supplementary tables are provided in a comprehensive summary of all information available and information gaps are highlighted for future research in the continent.

The transmission ecology of Tahyna orthobunyavirus in Austria as revealed by longitudinal mosquito sampling and blood meal analysis in floodplain habitats.

BACKGROUND: Tahyna orthobunyavirus (TAHV) is a mosquito-borne virus that may cause mild flu-like symptoms or neurological symptoms in humans. It is historically associated with floodplain habitats in Central Europe, and the mammalophilic floodwater mosquito, Aedes vexans, is thought to be the principal vector. There are few contemporary reports of TAHV transmission ecology within mosquitoes or their vertebrate hosts, and virus infections are rarely reported (and probably seldom diagnosed). The objectives of this study were to survey the mosquito population for TAHV in three floodwater habitats and describe host usage by the predominant floodwater mosquito species to potentially define TAHV transmission at these foci. METHODS: We performed longitudinal mosquito sampling along three major rivers in eastern Austria to characterize the mosquito community in floodplain habitats, and tested for the presence of TAHV in pools of mosquitoes. We characterized TAHV rescued from mosquito pool homogenate by sequencing. We surveyed mosquito host selection by analyzing mosquito blood meals. RESULTS: We identified TAHV in two pools of Ae. vexans captured along the Leitha River. This mosquito, and other floodwater mosquitoes, used large mammals (red deer, roe deer, wild boar) as their hosts. The sequence of the rescued virus was remarkably similar to other TAHV isolates from the region, dating back to the first isolate of TAHV in 1958. CONCLUSIONS: In general, we confirmed that TAHV is most likely being transmitted by Ae. vexans, although the precise contribution of vertebrate-amplifying hosts to the ecological maintenance of the virus is unclear. The pattern of host selection matches the estimated exposure of the same large mammal species in the region to TAHV based on a recent serosurvey, but hares were also hosts at the site where TAHV was detected. We also confirm humans as hosts of two floodwater mosquito species, providing a potential mechanism for spillover of TAHV or other mosquito-borne viruses.

Understanding and interpreting mosquito blood feeding studies: the case of Aedes albopictus.

Blood feeding is a fundamental mosquito behavior with consequences for pathogen transmission and control. Feeding behavior can be studied through two lenses - patterns and preference. Feeding patterns are assessed via blood meal analyses, reflecting mosquito-host associations influenced by environmental and biological parameters. Bias can profoundly impact results, and we provide recommendations for mitigating these effects. We also outline design choices for host preference research, which can take many forms, and highlight their respective (dis)advantages for preference measurement. Finally, Aedes albopictus serves as a case study for how to apply these lessons to interpret data and understand feeding biology. We illustrate how assumptions and incomplete evidence can lead to inconsistent interpretations by reviewing Ae. albopictus feeding studies alongside prevalent narratives about perceived behavior.

Characterization and Host-Feeding Patterns of Culex pipiens s.l. Taxa in a West Nile Virus-Endemic Area in Southeastern Romania.

Culex pipiens sensu lato has been documented as West Nile virus (WNV) vector in southeastern Romania. Bucharest, the densely populated capital city of Romania, and the surrounding Ilfov county are WNV hotspots. In this area, the morphologically indistinguishable biotypes of Cx. pipiens, namely pipiens and molestus, are usually differentiated by their behavioral and physiological traits. Their involvement in WNV transmission, as suggested by entomological investigations, was not previously documented for each biotype. We used a Real-Time PCR assay based on CQ11 microsatellite to identify the Cx. pipiens biotypes and their hybrids collected in various habitats in the Bucharest metropolitan area. A sympatric distribution of both biotypes was observed, with a preference of green areas for pipiens, and human settings and animal farmlands for molestus. In the latter habitats, pipiens and molestus were found in mixed aboveground populations. A low number of hybrids was found suggesting existence of reproductive isolation. In subway tunnels molestus was dominant with a higher number of hybrids recorded than aboveground. Blood-engorged mosquitoes were identified to biotype and the blood meal source identified by DNA barcoding. Overall, Cx. pipiens s.l. fed mainly on birds, commonly on house sparrows, collared doves, and blackbirds, which are potential WNV-amplifying hosts. The preference for avian hosts was expressed strongest by pipiens biotype, while molestus was substantially less specific, feeding on avian and mammal hosts with similar frequency, with humans representing 20% of the hosts. Hybrids had a host choice closer to that of molestus. These findings highlight the role of pipiens biotype as enzootic/epizootic vector, and specifically show molestus as the bridge vector for WNV. The pipiens and molestus biotypes show important differences in habitat preferences, including oviposition; these findings demonstrate that targeted mosquito control to limit WNV transmission may be possible.

Retrotransposon-Based Blood Meal Analysis of Nymphal Deer Ticks Demonstrates Spatiotemporal Diversity of Borrelia burgdorferi and Babesia microti Reservoirs.

Deer tick-transmitted Borrelia burgdorferisensu stricto (Lyme disease) and Babesia microti (babesiosis) increasingly burden public health across eastern North America. The white-footed mouse is considered the primary host for subadult deer ticks and the most important reservoir host for these and other disease agents. Local transmission is thought to be modulated by less reservoir-competent hosts, such as deer, diverting ticks from feeding on mice. We measured the proportion of mouse-fed or deer-fed host-seeking nymphs from 4 sites during 2 transmission seasons by blood meal remnant analysis using a new retrotransposon-based quantitative PCR (qPCR) assay. We then determined the host that was associated with the infection status of the tick. During the first year, the proportion of mouse-fed ticks ranged from 17% on mainland sites to 100% on an island, while deer-fed ticks ranged from 4% to 24%. The proportion of ticks feeding on mice and deer was greater from island sites than mainland sites (on average, 92% versus 43%). Mouse-fed ticks decreased significantly during year 2 in 3 of 4 sites (most were <20%), while deer-fed ticks increased for all sites (75% at one site). Overall, ticks were more likely to be infected when they had fed on mice (odds ratio [OR] of 2.4 and 1.6 for Borrelia and Babesia, respectively) and were less likely to be infected if they had fed on deer (OR, 0.8 and 0.4). We conclude that host utilization by deer ticks is characterized by significant spatiotemporal diversity, which may confound efficacy tests of interventions targeting reservoir hosts.IMPORTANCE White-footed mice are thought to be the most important reservoir host for the deer tick-transmitted pathogens that cause Lyme disease and human babesiosis because they are the primary host for immature ticks. Transmission would be reduced, however, if ticks feed on deer, which are not capable of infecting ticks with either pathogen. By directly measuring whether ticks had fed on either mice or deer using a new quantitative PCR (qPCR) assay to detect remnants of host DNA leftover from the larval blood meal, we demonstrate that host utilization by ticks varies significantly over time and space and that mice often feed fewer ticks than expected. This finding has implications for our understanding of the ecology of these diseases and for the efficacy of control measures.

Culicoides (Diptera: Ceratopogonidae) Communities Differ Between a Game Preserve and Nearby Natural Areas in Northern Florida.

Culicoides Latreille biting midges are small hematophagous flies that feed on a variety of vertebrate animals. White-tailed deer (Odocoileus virginianus), a farmed species in the United States, can occur at high densities on farms. This elevated density of available hosts may result in greater abundance of midges and greater potential for disease transmission on farms than natural ecosystems. This research aimed to determine whether Culicoides abundance varied between a game preserve in Gadsden County, Florida, a site bordering the preserve ('adjacent'), a site 3.5 km away ('moderate'), and a site 13 km away ('distant'). CDC light traps were set one night per week at the preserve, adjacent site, and moderate site in 2016 and at all four sites in 2017. Total abundance was greatest at the preserve and second greatest at the adjacent site both years. Average abundance of female Culicoides stellifer (Coquillett) was an order of magnitude greater on the preserve (x¯=24.59 in 2016, 17.95 in 2017) than at any other site (x¯≤1.68 in 2016, x¯≤1.03 in 2017), whereas the greatest average abundance of Culicoides venustus Hoffman was found at the adjacent site (x¯=5.15 in 2016, x¯=1.92 in 2017). Distance from the preserve significantly affected overall average abundance for both species (P < 0.001), although pairwise significance varied. Species diversity was lowest on the preserve and highest at the moderate site both years. These data suggest that high densities of animals may increase transmission potential on high fence preserves and in adjacent areas by contributing to high densities of vector species.

Preliminary Characterization of Triatomine Bug Blood Meals on the Island of Trinidad Reveals Opportunistic Feeding Behavior on Both Human and Animal Hosts.

Chagas disease is a neglected tropical disease caused by infection with Trypanosoma cruzi. The parasite is endemic to the Americas, including the Caribbean, where it is vectored by triatomine bugs. Although Chagas disease is not considered a public health concern in the Caribbean islands, studies in Trinidad have found T. cruzi-seropositive humans and T. cruzi-infected triatomine bugs. However, little is known about triatomine bug host preferences in Trinidad, making it difficult to evaluate local risk of vector-borne T. cruzi transmission to humans. To investigate this question, we collected triatomine bugs in Trinidad and diagnosed each one for T. cruzi infection (microscopy and PCR). We then carried out a blood meal analysis using DNA extracted from each bug (PCR and sequencing). Fifty-five adult bugs (54 Panstrongylus geniculatus and one Rhodnius pictipes) were collected from five of 21 sample sites. All successful collection sites were residential. Forty-six out of the 55 bugs (83.6%) were infected with T. cruzi. Fifty-three blood meal hosts were successfully analyzed (one per bug), which consisted of wild birds (7% of all blood meals), wild mammals (17%), chickens (19%), and humans (57%). Of the 30 bugs with human blood meals, 26 (87%) were from bugs infected with T. cruzi. Although preliminary, our results align with previous work in which P. geniculatus in Trinidad had high levels of T. cruzi infection. Furthermore, our findings suggest that P. geniculatus moves between human and animal environments in Trinidad, feeding opportunistically on a wide range of species. Our findings highlight a critical need for further studies of Chagas disease in Trinidad in order to estimate the public health risk and implement necessary preventative and control measures.

No evidence for sylvatic cycles of chikungunya, dengue and Zika viruses in African green monkeys (Chlorocebus aethiops sabaeus) on St. Kitts, West Indies.

BACKGROUND: Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS: We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS: All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION: Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.

An overview of the sandfly fauna (Diptera: Psychodidae) followed by the detection of Leishmania DNA and blood meal identification in the state of Acre, Amazonian Brazil

BACKGROUND In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved. OBJECTIVES Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.

Host Associations Of Biting Midges (Diptera: Ceratopogonidae: Culicoides) Near Sentinel Chicken Surveillance Locations In Florida, USA.

Quantifying host use is important for understanding transmission of vector-borne pathogens. Despite the importance of biting midges (Diptera: Ceratopogonidae) in pathogen transmission, the vector-host relationships of most Culicoides species are poorly documented, even in locations where active arbovirus surveillance is conducted. Polymerase chain reaction-based blood-meal analysis was performed on 663 blood-engorged Culicoides females collected by 7 Florida mosquito control districts at 24 sentinel chicken arbovirus surveillance sites in 2017. A total of 638 blood meals were successfully analyzed to determine host species source, representing 11 Culicoides species. The most commonly bitten host was domestic chicken (Gallus gallus) (presumably sentinel chickens), constituting 565 of 638 (88.6%) the total blood meals. Other common hosts included humans (5.8%), white-tailed deer (Odoocoileus virginianus) (2.5%), and brown basilisk (Basiliscus vittatus) (1.6%). Significant differences in distribution of mammal and avian blood meals were found for a number of Culicoides species, and these patterns did not vary across locations. These results indicate that sentinel chickens are exposed to bites by Culicoides, potentially exposing them to Culicoides-borne pathogens. The findings that vertebrate host use was consistent across locations suggests that each Culicoides species has affinity for one or more specific animal groups, and does not feed randomly upon available animals.

Host Associations of Culex (Melanoconion) atratus (Diptera: Culicidae) and Culex (Melanoconion) pilosus from Florida, USA.

Characterizing the host-use patterns of mosquitoes is an essential component of understanding the transmission dynamics of mosquito-vectored pathogens. The host associations of two species of the medically important Culex subgenus Melanoconion, Culex atratus, and Culex pilosus are unknown or unclear, respectively. Both species have wide neotropical distributions. In the United States of America (USA), Culex pilosus occurs throughout the southeastern coastal plain, while Culex atratus is restricted to the southern Florida Peninsula. Using PCR-based blood meal analysis, we investigated the host associations of Culex atratus and Culex pilosus that were collected from Everglades National Park, Florida, USA We identified the host species of 208 Culex atratus and 168 Culex pilosus. Both species were narrowly associated with reptilian host species, particularly native and non-native lizards of the genus Anolis. Sampled Culex atratus exclusively fed on reptilian hosts, with >99% of blood meals derived from Anolis lizards. Culex pilosus fed predominantly from reptiles, but avian and mammalian blood meals were also detected. Of these, 92% of blood meals were derived from Anolis species. For both species, Anolis sagrei, an invasive exotic lizard in Florida, was the most frequently detected host species. These data indicate that Culex atratus and Culex pilosus are specialists of reptilian hosts, particularly Anolis lizards.

Host DNA integrity within blood meals of hematophagous larval gnathiid isopods (Crustacea, Isopoda, Gnathiidae).

BACKGROUND: Juvenile gnathiid isopods are common ectoparasites of marine fishes. Each of the three juvenile stages briefly attach to a host to obtain a blood meal but spend most of their time living in the substrate, thus making it difficult to determine patterns of host exploitation. Sequencing of host blood meals from wild-caught specimens is a promising tool to determine host identity. Although established protocols for this approach exist, certain challenges must be overcome when samples are subjected to typical field conditions that may contribute to DNA degradation. The goal of this study was to address a key methodological issue associated with molecular-based host identification from free-living, blood-engorged gnathiid isopods-the degradation of host DNA within blood meals. Here we have assessed the length of time host DNA within gnathiid blood meals can remain viable for positive host identification. METHODS: Juvenile gnathiids were allowed to feed on fish of known species and subsets were preserved at 4-h intervals over 24 h and then every 24 h up to 5 days post-feeding. Host DNA extracted from gnathiid blood meals was sequenced to validate the integrity of host DNA at each time interval. DNA was also extracted from blood meals of wild-fed gnathiids for comparison. Attempts were also made to extract host DNA from metamorphosed juveniles. RESULTS: Using a cox1 universal fish primer set, known fish host DNA sequences were successfully identified for nearly 100% of third-stage juvenile gnathiid blood meals, digested for up to 5 days post-feeding. For second-stage juveniles, host identification was 100% successful when gnathiids were preserved within 24 h of collection. Fish hosts were positively identified for 69% of sequences from wild-fed gnathiid isopods. Of the 31% of sequences not receiving a ≥ 98 % match to a sequence in GenBank, 25 sequences were of possible invertebrate origin. CONCLUSIONS: To our knowledge, this is the first study to examine the degradation rate of gnathiid isopod blood meals. Determining the rate at which gnathiids digest their blood meal is an important step in ensuring the successful host identification by DNA-based methods in large field studies.

Generalist host species drive Trypanosoma cruzi vector infection in oil palm plantations in the Orinoco region, Colombia.

BACKGROUND: Oil palm plantation establishment in Colombia has the potential to impact Chagas disease transmission by increasing the distribution range of Rhodnius prolixus. In fact, previous studies have reported Trypanosoma cruzi natural infection in R. prolixus captured in oil palms (Elaeis guineensis) in the Orinoco region, Colombia. The aim of this study is to understand T. cruzi infection in vectors in oil palm plantations relative to community composition and host dietary specialization by analyzing vector blood meals and comparing these results to vectors captured in a native palm tree species, Attalea butyracea. METHODS: Rhodnius prolixus nymphs (n = 316) were collected from A. butyracea and E. guineensis palms in Tauramena, Casanare, Colombia. Vector blood meals from these nymphs were determined by amplifying and sequencing a vertebrate-specific 12S rRNA gene fragment. RESULTS: Eighteen vertebrate species were identified and pigs (Sus scrofa) made up the highest proportion of blood meals in both habitats, followed by house mouse (Mus musculus) and opossum (Didelphis marsupialis). Individual bugs feeding only from generalist mammal species had the highest predicted vector infection rate, suggesting that generalist mammalian species are more competent hosts for T. cruzi infection . CONCLUSIONS: Oil palm plantations and A. butyracea palms found in altered areas provide a similar quality habitat for R. prolixus populations in terms of blood meal availability. Both habitats showed similarities in vector infection rate and potential host species, representing a single T. cruzi transmission scenario at the introduced oil palm plantation and native Attalea palm interface.

Investigating the blood-host plasticity and dispersal of Anopheles coluzzii using a novel field-based methodology.

BACKGROUND: The biting behaviour and dispersal of insect vectors in the field underlies the transmission of many diseases. Here, a novel collection methodology coupled with the molecular analysis of blood-meal sources and digestion rates is introduced with the aim of aiding the understanding of two critical and relatively understudied mosquito behaviours: plasticity in blood-host choice and vector dispersal. RESULTS: A collection strategy utilising a transect of mosquito traps placed at 50 m intervals allowed the collection of blood-fed Anopheles coluzzii from a malaria-endemic village of southern Ghana where human host availability ranged from zero (a cattle pen), increasing until humans were the dominant host choice (the middle of the village). Blood-meal analysis using PCR showed statistically significant variation in blood-meal origins for mosquitoes collected across the 250 m transect: with decreasing trend in Bovine Blood Index (OR = 0.60 95% CI: 0.49-0.73, P < 0.01) and correspondingly, an increasing trend in Human Blood Index (OR = 1.50 95% CI: 1.05-2.16, P = 0.028) as the transect approached the village. Using qPCR, the host DNA remaining in the blood meal was quantified for field-caught mosquitoes and calibrated according to timed blood digestion in colony mosquitoes. Time since blood meal was consumed and the corresponding distance the vector was caught from its blood-host allowed the estimation of An. coluzzii dispersal rates. Within 7 hours of feeding, mosquitoes typically remained within 50 m of their blood-host but at 60 hours they had dispersed up to 250 m. CONCLUSIONS: Using this methodology the remarkably small spatial scale at which An. coluzzii blood-host choice can change was demonstrated. In addition, conducting qPCR on host blood from field-caught mosquitoes and calibrating with timed experiments with colonised mosquitoes presents a novel methodology for investigating the dispersal behaviour of vectors. Future adaptations to this novel method to make it broadly applicable to other types of setting are also discussed.

Using the human blood index to investigate host biting plasticity: a systematic review and meta-regression of the three major African malaria vectors.

BACKGROUND: The proportion of mosquito blood-meals that are of human origin, referred to as the 'human blood index' or HBI, is a key determinant of malaria transmission. METHODS: A systematic review was conducted followed by meta-regression of the HBI for the major African malaria vectors. RESULTS: Evidence is presented for higher HBI among Anopheles gambiae (M/S forms and Anopheles coluzzii/An. gambiae sensu stricto are not distinguished for most studies and, therefore, combined) as well as Anopheles funestus when compared with Anopheles arabiensis (prevalence odds ratio adjusted for collection location [i.e. indoor or outdoor]: 1.62; 95% CI 1.09-2.42; 1.84; 95% CI 1.35-2.52, respectively). This finding is in keeping with the entomological literature which describes An. arabiensis to be more zoophagic than the other major African vectors. However, analysis also revealed that HBI was more associated with location of mosquito captures (R2 = 0.29) than with mosquito (sibling) species (R2 = 0.11). CONCLUSIONS: These findings call into question the appropriateness of current methods of assessing host preferences among disease vectors and have important implications for strategizing vector control.