A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

University of Florida

Mosquito community composition and population dynamics were compared to weather variables and land use/cover data during 2008 to determine which variables affected population dynamics at the J.W. Jones Ecological Research Center in southwestern Georgia. Models relating adult mosquito distributions to weather variables and time of year were compared using Akaike's Information Criterion (AIC) model selection. Precipitation, temperature, humidity, and Keetch-Byram Drought Index were important factors correlated with mosquito abundance or presence/absence for the species considered. A cluster analysis, which grouped eight sites based on the percentages of land use/cover and hydric soils located in a 1-km radius surrounding collection sites, and an indicator species analysis were used to investigate the associations among 11 mosquito species and sites with similar land use/cover. Aedes albopictus (Skuse), Culex coronator Dyar & Knab, Culex quinquefasciatus Say, and Culex salinarius Coquillett were associated with sites that had the most anthropogenic influence, while Coquillettidia perturbans (Walker) and Psorophora ferox (von Humboldt) were associated with natural land cover such as wetlands and forested land. This study demonstrates that regional climate and land use/cover data can be predictive of the population dynamics of certain mosquito populations and is the first to examine how the distribution of Cx. coronator adults relate to land use/cover in the southeastern United States.

Weather and landscape factors associated with adult mosquito abundance in southwestern Georgia, U.S.A.

Following the 2009 and 2010 dengue-1 (DENV-1) outbreaks in Key West, FL, we used Florida Aedes aegypti (L.) mosquitoes and DENV-1 isolated from Key West in 2010 to test the hypothesis that if the 2009 and 2010 DENV-1 genome sequences are similar, then vertical transmission of DENV-1 from infected Ae. aegypti female mosquitoes to their eggs could have served as an interepidemic reservoir between outbreaks. We also investigated the ability of Florida Aedes albopictus (Skuse) mosquitoes to vertically transmit DENV-1. In addition, we determined the rates of infection and dissemination of these Florida mosquito species for DENV-1 and the effect of DENV-1 infection on oviposition success and number of mosquito eggs laid by females. Vertical transmission of DENV-1 was documented, with rates of 11.11% (2 out of 18) for Ae. albopictus and 8.33% (3 out of 36) for Ae. aegypti. Approximately 93% (111 out of 119) of Ae. aegypti that fed on DENV-1 in blood became infected, and 80% (89 out of 111) of infections were disseminated. Similarly, 93% of Ae. albopictus became infected (53 out of 57), and 85% (45 out of 53) of infections were disseminated. No significant differences were detected in numbers of eggs laid by either species after imbibing DENV-1 in blood, suggesting little cost of infection on number of eggs laid. Our results demonstrate that Florida Ae. aegypti and Ae. albopictus mosquitoes are competent vectors for DENV-1, whose maintenance between the 2009 and 2010 Key West outbreaks may have been facilitated by vertical transmission.

Vertical transmission of Key West dengue-1 virus by Aedes aegypti and Aedes albopictus (Diptera: Culicidae) mosquitoes from Florida.

Temperature-food interactions in the larval environment can affect life history and population growth of container mosquitoes Aedes aegypti (L.) and Aedes albopictus Skuse, the primary vectors of chikungunya and dengue viruses. We used Ae. aegypti, Ae. albopictus, and dengue-1 virus (DENV-1) from Florida to investigate whether larval rearing temperature can alter the effects of larval food levels on Ae. aegypti and Ae. albopictus life history and DENV-1 infection and vertical transmission. Although we found no effect of larval treatments on survivorship to adulthood, DENV-1 titer, or DENV-1 vertical transmission, rates of vertical transmission up to 16-24% were observed in Ae. albopictus and Ae. aegypti, which may contribute to maintenance of this virus in nature. Larval treatments had no effect on number of progeny and DENV-1 infection in Ae. aegypti, but the interaction between temperature and food affected number of progeny and DENV-1 infection of the female Ae. albopictus parent. The cooler temperature (24°C) yielded the most progeny and this effect was accentuated by high food relative to the other conditions. Low and high food led to the highest (∼90%) and lowest (∼65%) parental infection at the cooler temperature, respectively, whereas intermediate infection rates (∼75-80%) were observed for all food conditions at the elevated temperature. These results suggest that temperature and food availability have minimal influence on rate of vertical transmission and a stronger influence on adults of Ae. albopictus than of Ae. aegypti, which could have consequences for dengue virus epidemiology.

Larval Temperature-Food Effects on Adult Mosquito Infection and Vertical Transmission of Dengue-1 Virus.

The incidence of locally acquired dengue infections increased during the last decade in the United States, compelling a sustained research effort concerning the dengue mosquito vector, , and its microbiome, which has been shown to influence virus transmission success. We examined the "metavirome" of four populations of  mosquitoes collected in 2016 to 2017 in Manatee County, FL. Unexpectedly, we discovered that dengue virus serotype 4 (DENV4) was circulating in these mosquito populations, representing the first documented case of such a phenomenon in the absence of a local DENV4 human case in this county over a 2-year period. We confirmed that all of the mosquito populations carried the same DENV4 strain, assembled its full genome, validated infection orthogonally by reverse transcriptase PCR, traced the virus origin, estimated the time period of its introduction to the Caribbean region, and explored the viral genetic signatures and mosquito-specific virome associations that potentially mediated DENV4 persistence in mosquitoes. We discuss the significance of prolonged maintenance of the DENV4 infections in  that occurred in the absence of a DENV4 human index case in Manatee County with respect to the inability of current surveillance paradigms to detect mosquito vector infections prior to a potential local outbreak. Since 1999, dengue outbreaks in the continental United States involving local transmission have occurred only episodically and only in Florida and Texas. In Florida, these episodes appear to be coincident with increased introductions of dengue virus into the region through human travel and migration from countries where the disease is endemic. To date, the U.S. public health response to dengue outbreaks has been largely reactive, and implementation of comprehensive arbovirus surveillance in advance of predictable transmission seasons, which would enable proactive preventative efforts, remains unsupported. The significance of our finding is that it is the first documented report of DENV4 transmission to and maintenance within a local mosquito vector population in the continental United States in the absence of a human case during two consecutive years. Our data suggest that molecular surveillance of mosquito populations in high-risk, high-tourism areas of the United States may enable proactive, targeted vector control before potential arbovirus outbreaks.

Eva A. Buckner

Florida Medical Entomology Laboratory,
Department of Entomology and Nematology,
Institute of Food and Agricultural Sciences,
Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences

University of Florida

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing