A metagenomic analysis of the phase 2 Anopheles gambiae 1000 genomes dataset reveals a wide diversity of cobionts associated with field collected mosquitoes.

The Anopheles gambiae 1000 Genomes (Ag1000G) Consortium previously utilized deep sequencing methods to catalogue genetic diversity across African An. gambiae populations. We analyzed the complete datasets of 1142 individually sequenced mosquitoes through Microsoft Premonition's Bayesian mixture model based (BMM) metagenomics pipeline. All specimens were confirmed as either An. gambiae sensu stricto (s.s.) or An. coluzzii with a high degree of confidence ( > 98% identity to reference). Homo sapiens DNA was identified in all specimens indicating contamination may have occurred either at the time of specimen collection, preparation and/or sequencing. We found evidence of vertebrate hosts in 162 specimens. 59 specimens contained validated Plasmodium falciparum reads. Human hepatitis B and primate erythroparvovirus-1 viral sequences were identified in fifteen and three mosquito specimens, respectively. 478 of the 1,142 specimens were found to contain bacterial reads and bacteriophage-related contigs were detected in 27 specimens. This analysis demonstrates the capacity of metagenomic approaches to elucidate important vector-host-pathogen interactions of epidemiological significance.

Stability and detection of nucleic acid from viruses and hosts in controlled mosquito blood feeds.

Monitoring the presence and spread of pathogens in the environment is of critical importance. Rapid detection of infectious disease outbreaks and prediction of their spread can facilitate early responses of health agencies and reduce the severity of outbreaks. Current sampling methods are sorely limited by available personnel and throughput. For instance, xenosurveillance utilizes captured arthropod vectors, such as mosquitoes, as sampling tools to access blood from a wide variety of vertebrate hosts. Next generation sequencing (NGS) of nucleic acid from individual blooded mosquitoes can be used to identify mosquito and host species, and microorganisms including pathogens circulating within either host. However, there are practical challenges to collecting and processing mosquitoes for xenosurveillance, such as the rapid metabolization or decay of microorganisms within the mosquito midgut. This particularly affects pathogens that do not replicate in mosquitoes, preventing their detection by NGS or other methods. Accordingly, we performed a series of experiments to establish the windows of detection for DNA or RNA from human blood and/or viruses present in mosquito blood meals. Our results will contribute to the development of xenosurveillance techniques with respect to optimal timing of sample collection and NGS processing and will also aid trap design by demonstrating the stabilizing effect of temperature control on viral genome recovery from blood-fed mosquitoes.

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.

Patterns of Abundance, Host Use, and Everglades Virus Infection in Culex (Melanoconion) cedecei Mosquitoes, Florida, USA.

Everglades virus (EVEV), subtype II within the Venezuelan equine encephalitis (VEE) virus complex, is a mosquitoborne zoonotic pathogen endemic to south Florida, USA. EVEV infection in humans is considered rare, probably because of the sylvatic nature of the vector, the Culex (Melanoconion) cedecei mosquito. The introduction of Cx. panocossa, a tropical vector mosquito of VEE virus subtypes that inhabits urban areas, may increase human EVEV exposure. Field studies investigating spatial and temporal patterns of abundance, host use, and EVEV infection of Cx. cedecei mosquitoes in Everglades National Park found that vector abundance was dynamic across season and region. Rodents, particularly Sigmodon hispidus rats, were primary vertebrate hosts, constituting 77%-100% of Cx. cedecei blood meals. Humans were fed upon at several locations. We detected EVEV infection in Cx. cedecei mosquitoes in lower and upper regions of Everglades National Park only during the wet season, despite an abundance of Cx. cedecei mosquitoes at other sampling times.

Human-powered pop-up resting shelter for sampling cavity-resting mosquitoes.

Methods for collecting mosquitoes without the use of electricity are advantageous in remote field locations where electricity is not present or is unreliable. Here we report on the development and evaluation of a human-powered system for sampling cavity-resting mosquitoes. The system is composed of a spring-steel framed resting shelter and pneumatic lid that "pumps" mosquitoes resting within the shelter into a capture chamber when the resting shelter is manually compressed. Field evaluation of the system in the Everglades of Florida, USA indicates that the human-powered system is not statistically different than resting shelter vacuum aspiration with regard to the number or physiological status of the adults sampled. Culex (Melanoconion) females dominated collections, indicating that this system would have greatest utility in sampling vectors of zoonotic Alphaviruses in the American tropics.

Mammal decline, linked to invasive Burmese python, shifts host use of vector mosquito towards reservoir hosts of a zoonotic disease.

Invasive apex predators have profound impacts on natural communities, yet the consequences of these impacts on the transmission of zoonotic pathogens are unexplored. Collapse of large- and medium-sized mammal populations in the Florida Everglades has been linked to the invasive Burmese python, Python bivittatus Kuhl. We used historic and current data to investigate potential impacts of these community effects on contact between the reservoir hosts (certain rodents) and vectors of Everglades virus, a zoonotic mosquito-borne pathogen that circulates in southern Florida. The percentage of blood meals taken from the primary reservoir host, the hispid cotton rat, Sigmodon hispidus Say and Ord, increased dramatically (422.2%) from 1979 (14.7%) to 2016 (76.8%), while blood meals from deer, raccoons and opossums decreased by 98.2%, reflecting precipitous declines in relative abundance of these larger mammals, attributed to python predation. Overall species diversity of hosts detected in Culex cedecei blood meals from the Everglades declined by 40.2% over the same period (H(1979) = 1.68, H(2016) = 1.01). Predictions based upon the dilution effect theory suggest that increased relative feedings upon reservoir hosts translate into increased abundance of infectious vectors, and a corresponding upsurge of Everglades virus occurrence and risk of human exposure, although this was not tested in the current study. This work constitutes the first indication that an invasive predator can increase contact between vectors and reservoirs of a human pathogen and highlights unrecognized indirect impacts of invasive predators.