Invasion, establishment, and spread of invasive mosquitoes from the Culex coronator complex in urban areas of Miami-Dade County, Florida

Species from the Culex coronator complex are Neotropical species and potential vectors of Saint Louis and West Nile viruses. Culex coronator was first described in Trinidad and Tobago in the early twentieth century and since then it has invaded and has been reported established in most countries of the Americas. Species from the Culex coronator complex were first detected in the United States in the state of Louisiana in 2004 and were subsequently detected in Florida in 2005, reaching Miami-Dade County in 2008. We hypothesize that species from the Cx. coronator complex are adapting to urban environments in Miami-Dade County, Florida, and are becoming more present and abundant in these areas. Therefore, our objective was to investigate the patterns of the presence and abundance of species from the Cx. coronator complex in the urban areas of Miami-Dade County. Here we used weekly data comprised of 32 CDC traps from 2012 to 2020 and 150 BG-Sentinel traps from 2016 to 2020. A total of 34,146 female mosquitoes from the Cx. coronator complex were collected, 26,138 by CDC traps and 8008 by BG-Sentinel traps. While the number of CDC traps that were positive was relatively constant at 26–30 positive traps per year, the number of positive BG-Sentinel traps varied substantially from 50 to 87 positive traps per year. Furthermore, the heat map and logistic general linear model for repeated measures analyses showed a significant increase in both the distribution and abundance of mosquitoes from the Cx. coronator complex, indicating that these species are becoming more common in anthropized habitats being able to thrive in highly urbanized areas. The increase in the distribution and abundance of species from the Cx. coronator complex is a major public health concern. The ability of species from the Cx. coronator complex to benefit from urbanization highlights the need to better understand the mechanisms of how invasive vector mosquito species are adapting and exploiting urban habitats.

Culex coronator Dyar and Knab is a highly invasive Neotropical species 16 that was first observed in Trinidad and Tobago in 1906 25 . It belongs to the Culex coronator complex with another four sibling species Culex camposi Dyar, Culex ousqua Dyar, Culex usquatissimus Dyar, and Culex usquatus Dyar 26 . These species can occur in sympatry and are only distinguishable by a few morphological features present at the larval stage and in the male genitalia 26,27 .
Species from the Culex coronator complex have an unknown epidemiological role in the transmission of arboviruses to humans. However, they have been found to be competent vectors of WNV 28,29 and are considered potential vectors of SLEV 30,31 . They have greatly expanded their range and are now present in most countries of the Americas. Since its first detection in Louisiana in 2004, species from the Cx. coronator complex are now considered established in most of the southern states in the United States 16 . Even though the invasion of species from the Cx. coronator complex is well documented in the United States, the mechanisms (e.g., changes in their ecology and behavior, adaptation to local artificial aquatic habitats and resources, etc.) employed on their invasion, establishment, and colonization of new areas remain largely unknown.
Several factors could have contributed to the dispersion of species from the Cx. coronator complex over large geographic areas. However, their ability to thrive and establish themselves in urban areas that have undergone abrupt and substantial anthropogenic land use and land cover transformations indicates that these species are able to exploit the resources present in urban areas at the microgeographic scale (i.e., neighborhood level). Furthermore, many mosquito vector species have the potential to be passively or actively introduced into urban areas [32][33][34] , but only a selected few species are able to reach high abundances. It is clear that being able to exploit the resources available in urban areas at the microgeographic scale is crucial for their establishment and proliferation in urban areas 14 . Determining to what extent species from the Cx. coronator complex are expanding their range in urban areas at the microgeographic scale and whether they are increasing their contact with human hosts in the process is essential for the development of effective and targeted mosquito control strategies.
Species from the Culex coronator complex have only recently been detected in Florida with the first reports dating from 2005, reaching Miami-Dade County in 2008 35 . Since then they have been found breeding in artificial aquatic habitats such as pools, garbage cans, and fountains 36 and have become abundant in the urban areas of the county 13 . We hypothesize that species from the Cx. coronator complex are adapting to urban environments in Miami-Dade County, Florida, and are becoming gradually more present and abundant in those areas. Therefore, our objective was to investigate the patterns of the presence and abundance of species from the Cx. coronator complex in the urban areas of Miami-Dade County.

Results
A total of 34,146 female specimens from the Cx. coronator complex were collected by the Miami-Dade Mosquito Control surveillance system, 26,138 by CDC traps from July 2012 to June 2020, and 8,008 by BG-Sentinel traps from July 2016 to June 2020. From the 32 CDC traps in the surveillance system, species from the Cx. coronator complex were collected by a minimum of 26 traps and a maximum of 30 traps in a given year. On the other hand, from the 150 BG-Sentinel traps in the surveillance system, species from the Cx. coronator complex were collected by a minimum of 50 and a maximum of 87 traps in a given year (Table 1). Our results also indicate that despite having only recently been detect in Miami-Dade, species from the Cx. coronator complex are increasing their range and abundance in urban areas of the county, and even though they are not as abundant and widespread as Ae. aegypti or Cx. quinquefasciatus, they should be considered in future mosquito control interventions ( Table 2).
The heat map based on the Kernel density estimator of the relative abundance of species from the Cx. coronator complex collected by the CDC traps indicates that since their first detection in 2008, they have become abundant in the peri-urban areas of Miami-Dade County. The presence and abundance of specimens from the Cx. coronator complex remained constant from 2012 to 2020 indicating that these species are adapted to the conditions and resource availability found in those areas. Furthermore, specific locations with optimum conditions continually yielded high abundances of species from the Cx. coronator complex throughout all 8 years of data obtained by the CDC traps (Fig. 1). www.nature.com/scientificreports/ The heat map based on the Kernel density estimator of the relative abundance of species from the Cx. coronator complex collected by the BG-Sentinel traps showed a clear increase in their presence and abundance in urban areas. Since 2016, when the surveillance system results showed that species from the Cx. coronator complex had limited presence and abundance relegated to specific and well-defined locations in the county, they have greatly increased their presence and abundance over the years. Our results showed a gradual increase in presence and abundance of species from the Cx. coronator complex in urban areas. The increase of the kernels of the heat maps indicates that more traps collected more species from the Cx. coronator complex in a more consistent way over the years culminating in the colonization of most of Miami-Dade urban areas by Cx. coronator in 2020. Furthermore, our results indicate that even though fewer mosquitoes from the Cx. coronator complex were collected from July 2019 to June 2020 than from July 2016 to June 2017 they were more homogeneously distributed throughout Miami-Dade. As a result, the kernel density heatmap shows larger and more widespread dots ranging from 1 to 20 mosquitoes per trap (Fig. 2).
The logistic general linear repeated measures model of the mean of positive BG-Sentinel traps for species from the Culex coronator complex collected from July 2016 to June 2020 resulted in a significant quadratic effect over the 4 years of collections yielding a P value of < 0.001. The estimated 95% confidence interval standard errors did not show a linear increase but a quadratic curve, in which more BG-Sentinel traps collected significantly more mosquitoes from the Cx. coronator complex from July 2017 to June 2018 and July 2018 to June 2019 when compared to the number of positive traps from July 2016 to June 2017. On the other hand, even though more traps were positive for species from the Cx. coronator complex from July 2019 to June 2020 when compared to July 2016 to June 2017 the difference was not significant (Fig. 3).

Discussion
Anthropogenic changes in the environment are major drivers of biotic homogenization of species, greatly benefiting species that can exploit the resources available in urban areas. Our results show that species from the Cx. coronator complex successfully invaded and colonized Miami-Dade County becoming one of the most abundant mosquito species with epidemiological relevance. Since its introduction in Miami-Dade County in 2008, species from the Cx. coronator complex gradually increased their presence and abundance. First, they increased their presence and abundance in the peri-urban areas, then they subsequently invaded and colonized the urban areas of Miami-Dade, ranging from residential areas with low population densities to highly urbanized areas comprised mostly of skyscrapers. Regardless of the availability of several species occurrence record studies, to our knowledge, this is the first study to assess the microgeographic demographic expansion of species from the Cx. coronator complex in urban areas in the United States. The increase in presence and abundance of species from the Cx. coronator complex in Miami-Dade represents a major public health concern. One of the side effects of urbanization is the health disparities due to monetary inequities resulting in underserved populations that are more exposed to infectious diseases, including vectorborne. People living in lower-income neighborhoods are more exposed to vector mosquitoes 37 . Some houses in these under-served neighborhoods do not have air conditioning or screens on windows, and for these reasons, people tend to spend more time outdoors and are more exposed to mosquito bites during the night when species from the Cx. coronator complex are more active 38 . Furthermore, these areas have higher concentrations of homeless people that are constantly exposed to vector mosquito bites. The size of the homeless population fluctuates in Miami-Dade during the year, increasing substantially during the colder months of the year due to homeless people seeking warmer environments 39 . The homeless migration to Miami-Dade coincides with bird  www.nature.com/scientificreports/ migration passing through Miami migrating south. The introduction of another mosquito vector species such as the species from the Cx. coronator complex that are adapted to urban environments and are recognized as an efficient vector of WNV can drastically increase the likelihood of WNV spillover from birds to humans 13,28 . The mechanisms used by invasive species to adapt their ecology and behavior to urban environments are not fully understood. It is still unknown what allows a given species to successfully invade and colonize a new area. For example, the invasive species Culex panocossa Dyar has been detected in great numbers in natural areas in Miami-Dade County 40 but has not been detected in peri-urban and urban areas 13 . The most widely accepted hypothesis is that Cx. panocossa close association with water lettuce plants, commonly found in natural areas in Miami-Dade but not in peri-urban and urban areas, is a limiting factor for its expansion into urban areas 41 . Furthermore, regional variation has also to be taken into consideration. For example, even though species from the Cx. coronator complex are considered to be invasive vector species well adapted to urban environments in the United States 16 , in Brazil they are not commonly found in urban areas [42][43][44] .
Even though invasion, establishment, and colonization of new areas by vector mosquito species have been well documented 16,35 , there are currently no contingency plans or specific guidelines to deter and mitigate both mosquito geographic and demographic expansions. Our results showed that since they were abundantly found in the outskirts of the urbanized areas of Miami-Dade and commonly collected by the CDC traps, species from the Cx. coronator complex were able to gradually colonize most of Miami-Dade incorporated areas in less than a decade, as uncovered by the results obtained by the BG-Sentinel traps, highlighting the need for the development of integrated and effective mosquito surveillance and control strategies at the national level rather than a more localized approach at the county level. Those measures together with the Integrated Vector Management (IVM) in the form of source reduction of potential artificial and natural aquatic habitats, improvement of environmental ordinance guidelines and policies, and adequate sanitary infrastructure, especially for underserved populations to attenuate social inequities are crucial for thwarting the invasion and expansion of vector mosquito species and prevent non-endemic areas from becoming endemic for arboviruses 45,46 .
The results of this study can be used to increase awareness of the danger of the introduction of invasive species. The implementation of mosquito surveillance systems capable of detecting invasive species at an early stage is paramount for the development of effective contingency plans and targeted mosquito control strategies. In this context, Miami-Dade is a gateway city and consequently has the potential to export invasive mosquito species to other parts of the contiguous United States that have suitable resources and conducive conditions to support mosquito populations.
Recently, the invasive species Aedes vittatus (Bigot, 1861), a primary vector of arbovirus in Europe and Asia, has been detected in Cuba and the Dominican Republic. Subsequent phylogenetic analysis indicated that Ae. vittatus had been introduced multiple times into the Caribbean region 47 . Due to the increased connectivity between the Caribbean region and Miami-Dade, the introduction of Ae. vittatus to Miami-Dade is considered unavoidable and should be monitored closely.
The continuous environmental degradation due to anthropogenic changes in the environment is a major driver for the introduction, colonization, and spread of vector mosquito species. A new and improved mosquito control framework is urgently needed to prevent both the introduction of mosquito vector species to nonendemic regions (i.e., the introduction of Ae. vittatus in the United States) and to manage the spread of invasive species that have already been introduced and are spreading to new areas (i.e., range expansion of species from the Cx. coronator complex in the United States and Ae. albopictus in Europe 16 ).
Essential information about species from the Cx. coronator complex biology, behavior, and physiology is currently lacking. Most of the information currently available has been acquired through adult mosquito surveillance systems that focus on female mosquitoes. Due to caveats in the morphological identification, the correct identification of adult specimens from the Cx. coronator complex is only possible by observing differences in the male genitalia. For this reason, it is so far unknown what species from the Cx. coronator complex are present in the United States, and if one or more species from the Cx. coronator complex are more prone to adapt to urban environments than others and should be targeted in control strategies. The correct species identification is critical for the precise assessment of behavioral traits such as blood-and sugar-feeding, mating, resting, etc. Without that information, it is impossible to determine what environmental resources are driving the population dynamics of Cx. coronator and the other species in the Cx. coronator complex.
This study can serve as a stepping stone to future studies to determine what invasive mosquito vector species represent a risk to public health and should be targeted by mosquito control operations. Furthermore, there is no coordinated mosquito surveillance at the international and national level, which is essential to prevent the introduction and spread of invasive mosquito vector species to new areas. The introduction of invasive mosquito vector species can have an unpredictable impact on public health and represent one of the most important challenges in mosquito control and vector-borne disease transmission.

Methods
Study design. This study used a longitudinal design to assess the presence and relative abundance of species from the Cx. coronator complex in peri-urban and urban areas in Miami-Dade County, Florida. Miami-Dade is the most populous county in Florida, with approximately 3 million people. Current predictions indicate that Miami-Dade will gain 700,000 more residents by 2030 48 causing a substantial increase in urbanization processes. Miami-Dade has a year-round wet sub-tropical climate that is conducive for the year-round proliferation of mosquitoes 13 . Miami-Dade is also a destination for many bird species that are migrating south seeking warmer weather conditions 49 . 4) 13 . All traps were deployed weekly for 24 h and baited with CO 2 using a container filled with 1 kg of dry ice pellets 13,51 . All collected mosquitoes were transported to the Miami-Dade County Mosquito Control Laboratory and morphologically identified to species using taxonomic keys 52 . Since both the BG-Sentinel and the CDC trap mainly attract females seeking hosts male mosquitoes were not included in the analysis.
Geospatial analysis. We used a Kernel density estimator to calculate the magnitude-per-unit area (number of input per point (i.e., species from the Cx. coronator complex relative abundance)) from both the BG-Sentinel and CDC traps using ArcGIS 10.2 (Esri, Redlands, CA) 53 . We used the geodesic method for densities in square kilometers with an output cell size of 0.001. We used freely available layers from the Miami-Dade County's Open Data Hub-https:// gis-mdc. opend ata. arcgis. com/. We used a logistic general linear model (GLM) for repeated measures for the number of BG-Sentinel traps that collected mosquitoes from the Cx. coronator complex on each year (positive traps) as the dependent variable, with time as the independent variable. Since this study posed less than minimal risk to participants and did not involve endangered or protected species the Institutional Review Board at the University of Miami determined that the study be exempt from institutional review board assessment (IRB Protocol Number: 20161212).