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Interactions between the invasive Burmese python, Python bivittatus Kuhl, and the local mosquito community in Florida, USA

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Title:
Interactions between the invasive Burmese python, Python bivittatus Kuhl, and the local mosquito community in Florida, USA
Series Title:
Reeves LE, Krysko KL, Avery ML, Gillett- Kaufman JL, Kawahara AY, Connelly CR, et al. (2018) Interactions between the invasive Burmese python, Python bivittatus Kuhl, and the local mosquito community in Florida, USA. PLoS ONE 13(1): e0190633.
Creator:
Reeves, Lawrence
Kenneth L. Krysko
Michael L. Avery
Jennifer L. Gillett-Kaufman
Akito Y. Kawahara
C. Roxanne Connelly
Phillip E. Kaufman
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PLOS ONE
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English
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The Burmese python, Python bivittatus Kuhl, is a well-established invasive species in the greater Everglades ecosystem of southern Florida, USA. Most research on its ecological impacts focuses on its role as a predator and its trophic interactions with native vertebrate species, particularly mammals. Beyond predation, there is little known about the ecological interactions between P. bivittatus and native faunal communities. It is likely that established populations of P. bivittatus in southern Florida serve as hosts for native mosquito communities. To test this concept, we used mitochondrial cytochrome c oxidase subunit I DNA barcoding to determine the hosts of blood fed mosquitoes collected at a research facility in northern Florida where captive P. bivittatus and Argentine black and white tegu, Salvator merianae (Dume ril and Bibron), are maintained in outdoor enclosures, accessible to local mosquitoes. We recovered python DNA from the blood meals of three species of Culex mosquitoes: Culex erraticus (Dyar and Knab), Culex quinquefasciatus Say, and Culex pilosus (Dyar and Knab). Culex erraticus conclusively (P = 0.001; Fisher's Exact Test) took more blood meals from P. bivittatus than from any other available host. While the majority of mosquito blood meals in our sample were derived from P. bivittatus, only one was derived from S. merianae. These results demonstrate that local mosquitoes will feed on invasive P. bivittatus, a recently introduced host. If these interactions also occur in southern Florida, P. bivittatus may be involved in the transmission networks of mosquito-vectored pathogens. Our results also illustrate the potential of detecting the presence of P. bivittatus in the field through screening mosquito blood meals for their DNA.
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Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Lawrence Reeves.

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RESEA RCH ARTICL E Interactions between the invasive Burmese python, Python bivittatus Kuhl, and the local mosquito community in Florida, USA Lawrence E. Reeves 1 r, Kenneth L. Krysko 2 Michael L. Avery 3n Jennifer L. GillettKaufman 1 Akito Y. Kawahara 4 C. Roxanne Connelly 5 Phillip E. Kaufman 1n 1 Entomolo gy and Nema tology Department, Institute of Food and Agricultural Sciences, Univers ity of Florida, Gainesvi lle, Florida, United States of America, 2 Division of Herpetology Florida Museum of Natural History, University of Florida, Gainesvi lle, Florida, United States of America, 3 National Wildlife Researc h Center, United States Department of Agriculture, Gaines ville, Florida, United States of America, 4 McGuire Center for Lepidopt era and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesvi lle, Florida, United States of America, 5 Florid a Medical Entomolo gy Laborat ory, Institute of Food and Agricu ltural Sciences, Univers ity of Florida, Vero Beach, Florida, United States of America n These authors contributed equally to this work. These authors also contributed equally to this work. lereeve s@ufl.edu Abstract The Burmese python, Python bivittatus Kuhl, is a well-established invasive species in the greater Everglades ecosystem of southern Florida, USA. Most research on its ecological impacts focuses on its role as a predator and its trophic interactions with native vertebrate species, particularly mammals. Beyond predation, there is little known about the ecological interactions between P. bivittatus and native faunal communities. It is likely that established populations of P. bivittatus in southern Florida serve as hosts for native mosquito communities. To test this concept, we used mitochondrial cytochrome c oxidase subunit I DNA barcoding to determine the hosts of blood fed mosquitoes collected at a research facility in northern Florida where captive P. bivittatus and Argentine black and white tegu, Salvator merianae (Dume ril and Bibron), are maintained in outdoor enclosures, accessible to local mosquitoes. We recovered python DNA from the blood meals of three species of Culex mosquitoes: Culex erraticus (Dyar and Knab), Culex quinquefasciatus Say, and Culex pilosus (Dyar and Knab). Culex erraticus conclusively (P = 0.001; Fisher's Exact Test) took more blood meals from P. bivittatus than from any other available host. While the majority of mosquito blood meals in our sample were derived from P. bivittatus, only one was derived from S. merianae. These results demonstrate that local mosquitoes will feed on invasive P. bivittatus, a recently introduced host. If these interactions also occur in southern Florida, P. bivittatus may be involved in the transmission networks of mosquito-vec tored pathogens. Our results also illustrate the potential of detecting the presence of P. bivittatus in the field through screening mosquito blood meals for their DNA. PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 1 / 15 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 23(1 $&&(66 Citation: Reeves LE, Krysko KL, Avery ML, GillettKaufman JL, Kawahara AY, Connelly CR, et al. (2018) Interactions between the invasive Burmese python, Python bivittatus Kuhl, and the local mosquito community in Florida, USA. PLoS ONE 13(1): e0190633. https:// doi.org/10.1371 /journal. pone.019063 3 Editor: Richard Paul, Institut Pasteur, FRANCE Received: October 4, 2017 Accepted: December 18, 2017 Published: January 17, 2018 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmi tted, modifie d, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availabilit y Statement: All relevant data are within the paper and its Support ing Information files. Funding: This material is based upon work supported by the National Science Foundation (http://ww w.nsf.gov) Gradua te Research Fellowship under Grant No. 00107251 to LER. Additional support for this study was provided by the United States Department of Agriculture, National Wildlife Research Center (http://ww w. usda.gov). Publication of this article was funded in

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Introduction Introductions of plant and animal species to areas outside their native ranges can destabilize ecosystems [1, 2], contribute to biotic homogenization [3], impose public health risks [4], and impose large economic costs through direct damage or mitigation efforts [5]. Introduced animal species interact with various ecological components within ecosystems. Direct competitive and trophic interactions are traditionally considered the primary drivers of invasive speciesassociated impacts. However, direct or indirect interactions with native pathogens or parasites similarly may lead to damaging impacts and may go undetected. Species introductions have the potential to dramatically alter the prevalence or transmission systems of parasites and pathogens through the introduction of novel vectors [6], spillover of co-introduced pathogens from introduced hosts to native hosts [7], or spillback of endemic pathogens to native hosts through competent introduced reservoir hosts [8]. In the United States, Florida is particularly susceptible to species introductions [9, 10]. The state has both tropical and subtropical climates, major international ports of entry for tourists and commercial goods from the Caribbean, South America and elsewhere, active horticultural and captive wildlife industries, and empty niches created by human altered environments [11]. These factors facilitate the introduction and establishment of many non-native species, particularly, amphibian and reptile taxa. Currently, at least 180 non-native herpetofaunal taxa have been introduced to the state and 63 are established, more than in any other U.S. state or global region [12]. Some of these species, such as the Burmese python, n, and Argentine black and white tegu, rnn nn, are high profile invasive species that have attracted public attention and scientific interest [13]. n is presumed to have been introduced to the Flamingo area of Everglades National Park prior to 1985 and became established thereafter [1416]. n is an opportunistic predator of mammals, birds and reptiles. In Florida, its diet consists of both small and large animals including protected and managed species, such as wading birds, alligators and deer, and federally protected species [1720]. Research on the ecological impacts of n are largely limited to its trophic interactions and direct effects on prey populations. The establishment and expansion of python populations is correlated with precipitous declines in relative abundance of several mammal species [2123]. Through the diminished abundance or extirpation of mammalian mesopredators, a cascade of python-mediated indirect effects is possible [24]. The integration of a novel, large-bodied predator into the ecosystems of southern Florida has the potential to alter the transmission dynamics of endemic parasites and pathogens. In Florida, mosquitoes are the vectors of numerous zoonotic parasites and pathogens, many of which are poorly known. Species diversity and composition of vertebrate host communities are among the factors that structure transmission networks and affect the prevalence of mosquito-vectored pathogens [25]. In much of southern Florida, n is an available host for a diverse mosquito fauna (Fig 1) that includes more than 50 native and introduced mosquito species, some of which are potential pathogen vectors [26]. Females of many North American mosquito species take blood meals from reptilian hosts, and some specialize entirely on ectothermic hosts [27, 28]. Through mosquito feeding, there is potential that pythons are exposed to mosquito-vectored parasites and pathogens and it is possible that n is now directly involved in the transmission of endemic parasites and pathogens. Further, by dramatically restructuring the vertebrate host community and eliminating some host taxa through predation, the establishment of n may have broader implications for mosquitoborne pathogen transmission in Florida. Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 2 / 15 part by the University of Florida Open Access Publishing Fund. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests : The authors have declared that no competing interests exist.

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The diversity, host range, vectors, and transmission dynamics of many mosquito-vectored reptile pathogens are poorly known. While no known data are available on the pathogens of n in its native or introduced range, squamates (lizards and snakes) in Florida are the hosts of many mosquito-borne parasites including species of the taxonomically diverse genera n (Apicomplexa: Adeleorina) and n (Apicomplexa: Haemosporida) [29 31]. Similarly, the role of snakes in the transmission systems of medically-import ant mosquito-borne viruses is not well understood. However, snakes are competent hosts for the medically-important zoonoses Eastern equine encephalitis virus, Western equine encephalitis virus and West Nile virus, and may promote the persistence of these viruses within an ecosystem [3235]. The intent of this study is to determine if local mosquito communities interact with n, an introduced host. Native mosquito species have been documented feeding from other exotic reptile species in Florida [36], but interactions between n and mosquitoes are not known in the state. Determining whether native mosquitoes feed upon n and identifying those species is the first step towards understanding any potential pathogen transmission implications of python establishment in Florida. Using traditional survey methods Fig 1. Burmes e python (Python bivittatus) and Aedes taeniorh ynchus mosqui toes in Everglades National Park, October 2015. In the Greater Everglades Ecosystem of southern Florida, introduced populations of P. bivittatus are sympatric with a local mosquito fauna that includes more than 50 native and introduced mosquito species. https://do i.org/10.1371/j ournal.pone .0190633.g00 1 Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 3 / 15

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(i.e., transect walks, road surveys), n is difficult to detect in the habitats it occupies. Environmental DNA approaches using water sampling have been proposed to improve the ability of detecting n at field sites in southern Florida [37, 38]. If mosquitoes take blood meals from n, mosquito blood meals could be used as an alternative source of environmental DNA in monitoring python populations. Materials and methods Mosquito sampling Mosquitoes were collected at the United States Department of Agriculture (USDA), National Wildlife Research Center (NWRC) (29 39' 14.6", N 82 17' 17.1" W; datum WGS84, elev. 45 m), in Gainesville, Alachua County, Florida, USA. At this facility, eight adult n and 12 adult r. nn were housed individually in outdoor pens arranged in three parallel rows (Fig 2) where they were accessible to the local mosquito community. One row of eight adjacent python pens (1.5 m wide 3.0 m deep 1.8 m high) was located approximately 20 m from two rows of six adjacent tegu pens (1.5 m wide 3.0 m deep 1.8 m high). Rows of tegu pens were approximately 4 m apart. Pen walls and ceilings consisted of polyvinyl chloride (PVC) coated steel wire mesh with square openings 2.54 2.54 cm. Each reptile pen included an animal shelter. Python pens each contained one 1 1 0.5 m insulated plastic box with a 15 30 cm opening. Tegu pens each had one artificial burrow consisting of a 1.5 m tunnel made from 10.2 cm diameter corrugated plastic tubing leading to an underground chamber (48 66 33 cm). All python and tegu pens were positioned within a larger, aviary-like enclosure (60 30 4 m) of fiber netting (mesh size ~2.5 cm). Vegetation within this enclosure and surrounding reptile pens was limited to grasses consistently maintained by mowing, a few small thickets of saw palmetto, rn and young hardwood trees. The vegetation immediately surrounding the larger enclosure consisted of a pine flatwoods community with a canopy of n, and interspersed with hardwoods, and an understory dominated by saw palmetto. Mosquitoes were collected from July-October 2015 and April-December 2016. Mosquitoes were not collected from November 2015March 2016 when snakes were relocated to indoor enclosures to avoid potentially cold temperatures. Five resting shelter traps were placed inside the larger enclosure. Two traps were located alongside the python pens, two alongside the tegu pens, and one in a saw palmetto thicket (Fig 2). Resting shelter traps were constructed after Burkett-Cadena [39], and consisted of a 1 2 m section of 5.1 10.2 cm steel fencing, rolled into a cylindrical frame, set inside a 159 L black heavy-duty trash bag to make a cylinder, open at one end. The trash bag was secured tightly to the frame with duct tape. To remove mosquitoes from the traps, the open end of the resting shelter was covered by a 76 102 cm piece of cardboard that had a 9 cm circular hole cut in the center. The hole was positioned over the opening of the trap and a battery-powered aspirator, made from a modified 12V vacuum (BioQuip Products, Rancho Domiguez, CA), was turned on and held parallel to the hole, immediately outside the trap. The trap was then shaken vigorously to flush resting mosquitoes. The aspirator collected mosquitoes attempting to escape the shelter through the hole in the cardboard cover. After shaking, the cardboard cover was removed and the aspirator, still running, was moved to the interior of trap to collect any mosquitoes that had not attempted to exit through the hole in the cover. We collected mosquitoes from one to three times per week, between 0800 and 0900 and immediately transported them 10.8 km to the Entomology and Nematology Department, University of Florida, Gainesville, Florida, USA. Collected mosquitoes were killed within 30 minutes of collection by exposure to ethyl acetate for approximately 10 minutes. Mosquitoes were Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 4 / 15

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visually inspected under a stereoscope for the presence of a blood meal and identified to species using a morphological taxonomic key to adult females [26]. Gut contents of blood fed mosquitoes were preserved on Whatman 1 four-sample Flinders Technology Associates (FTA) cards [40]. Each blood fed mosquito was assigned a unique number and transferred to the sampling area of an FTA card. For blood fed females, a sterile pipette tip was used to apply pressure to the abdomen, releasing the blood meal onto the card. The blood meal was spread over the sampling area of the FTA card with the pipette tip until all viscous droplets were absorbed. The extent of blood meal digestion was visually estimated by scoring blood meals on a rank of 13; an approach modified from Detinova [41]. The following scores were based on the stage of digestion after feeding: (1) fresh blood meal, bright red in color, large size in proportion to mosquito abdomen and absence of visible developing ovaries; (2) blood meal that was still red in color, but developing ovaries were apparent; and (3) smaller blood meal, characterized by a brownish or blackish color, and ovaries that occupied more than 50% of the abdomen. A subset of the collected male and unfed female mosquitoes (n = 23, representing all species collected) were processed identically and served as negative controls, with the contents of the Fig 2. Location of mosquito resting shelter traps in relation to reptile pens. Arrangemen t (from above) of pens (light gray shaded boxes) housing Burmese (Python bivittatus) or Argentine black and white tegu (Salvator merianae) where blood fed mosquitoes were collected in resting shelter traps (stars) at the USDA National Wildlife Research Center in Gainesville, Florida, USA. Pens were set within the perimete r of a 60 30 4 m aviary-l ike enclosure (dark outer line). Two shelter traps were placed alongsid e python pens, two alongside tegu pens, and one in a saw palmetto thicket between the two pens. Areas that are shaded darker gray indicate areas of natural, unmainta ined vegetation. Unshaded areas indicate mowed grassy areas. https://do i.org/10.1371/j ournal.pone .0190633.g00 2 Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 5 / 15

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abdomen spread over the sampling area of the FTA card with a sterile pipette tip. FTA cards were then labeled and stored at room temperature until DNA extraction. Blood meal analysis DNA was extracted following the HotSHOT method [42] using hot sodium hydroxide to lyse cells and a Tris-hydrogen chloride neutralization buffer solution. For each FTA card-preserved blood meal, a hole punch was used to remove two circular 1 mm diameter sections of the FTA card sampling area. The two FTA-card sections were transferred to a 0.2 mL tube with flamesterilized forceps. Initially, 50 L of lysis solution (25 mM NaOH and 0.2 mM EDTA) was added to each tube. Tubes were then incubated in a BioRad 1 DNA Engine thermocycler at 95C for 30 minutes followed by 4C for five minutes. Thereafter, 50 L of neutralization solution (40 mM Tris-HCl) was added to each tube and vortexed for ~10 s. Extracted DNA was stored at -20C until PCR. A degenerate primer set [43] was used to amplify a 664 base pair (bp) fragment of the vertebrate host cytochrome oxidase subunit I gene (COI). Three nucleotide mismatches at the forward priming site of mosquito templates inhibited co-amplification of mosquito DNA. The primers RepCOI-F (717 707 &$$ &1$ $&& $&$ $$* $) and RepCOI-R ( $&7 7&7 **5 7*. &&$ $$5 $$7 &$) were used in 20 L reactions each consisting of 4 L 10X PCR buffer, 0.6 L 50 mM MgCl 2 0.4 L of 10 mM dNTPs, 0.48 L of each 10 mM primer, 0.2 L of Taq polymerase (5 U/L), 1 L of extracted DNA, and 12.84 L of sterile, double distilled water. Reaction conditions followed a standard profile of 94C for 3 min, followed by 40 cycles of 94C for 40 s, 48.5C for 30 s, and 72C for 60 s, and a final extension step of 72C for 7 min. Reaction products were stained with ethidium bromide and visualized under ultra-violet light on a 1.5% agarose gel. A 50 bp DNA ladder (Invitrogen TM ) was used to determine the approximate fragment size of PCR products. Negative controls in which sterile, double-distilled water was used in place of extracted DNA were included in all reactions to monitor for contamination. All PCR products a visible band at the expected fragment size were sent to the University of Florida Interdisciplinary Center for Biotechnology Research, Gainesville, Florida, for Sanger sequencing on an ABI 3130 1 automated sequencer. Resulting sequencing chromatograms were examined for quality and edited to truncate ambiguous terminal stretches from the sequence using the software Geneious 1 Version R10 [44]. Edited sequences were searched on the National Center for Biotechnology Information (NCBI) GenBank database (https://www.ncbi.nlm.nih.gov/ genbank/) using the Basic Local Alignment Search Tool (BLAST). Species-level taxonomic identities were assigned to blood meals when a host sequence was 98% homologous to a sequence referenced in the database [45] or sequences obtained from museum specimens. Statistical analyses were performed in the statistical program R 1 Version 3.2.0 using the stats package [46]. For each mosquito species that was determined to feed from n, we used Fisher's Exact Test to compare the proportion of blood meals derived from n to the proportion derived from all other identified host species. Results of these tests were considered significant if p < 0.05. No permits were required for the described study, which complied with all relevant regulations. Results We collected a total of 511 adult female mosquitoes using resting shelter traps (325 traps nights) from the USDA NWRC. This sample represented seven species in four genera (Table 1). n was the most abundant species collected and represented 72% of all adult females collected. nn, and to a lesser extent, and Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 6 / 15

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n also were common, together representing 24% of the total sample. n, n, and nnn were collected in small numbers (< 10 individuals, each species). We observed other mosquito species (including nn sp., n n, nn, n and an ( n) species) searching for hosts or feeding from humans at the site, but these species were absent from our resting shelter samples. Sixty-one (12.3%) of the 511 adult female mosquitoes in our sample had visible blood meals. The majority of blood-fed specimens were n (n = 51; 84% of blood fed specimens), while five or fewer (< 8% of blood fed specimens) blood-fed n, and nn were collected. n, n, and were not represented in our sample of blood fed mosquitoes. Polymerase chain reactions successfully amplified a DNA template of the expected target size (664 bp) from 57 of 61 (93.4%) mosquito blood meal samples, but failed to produce an amplicon for DNA templates from four blood meals. The likely cause of unsuccessful reactions was an advanced extent of digestion as all four blood meals received a digestion score of 3. The taxonomic origin of host DNA from all other blood meals was identified through Sanger sequencing. Edited chromatograms of host sequences had distinct and well-defined peaks with little background noise, indicating that the sequences were of good quality. No evidence of multiple host feedings was detected in the sequencing chromatograms. The majority of sequences and all sequences derived from n, were 99100% similar to referenced sequences in the NCBI database. Ten vertebrate host species representing the classes Reptilia, Aves, and Mammalia were identified (Table 2). n was identified as the host in 78.4% of blood meal samples. Only one blood meal was attributed to r. nn. Twelve blood meals had been acquired from other hosts, including humans. Three mosquito species (. n, nn) had obtained blood meals from n, while no python blood meal was detected in either n specimen sampled. Three blood meal sequences from n and specimens were ~95% homologous to NCBI-referenced n sequences. To confirm that these blood meals were derived from n, their sequences were subsequently aligned to corresponding sequences obtained from n museum specimens (UF-Herpetology 170869, 170871), resulting in 99% homology. In n, the number of blood meals derived from n was greater than those from all other hosts combined. Forty n had fed on n, compared to Table 1. Taxonomic composit ion of female mosquitoe s collecte d in resting shelters at the USDA National Wildlife Research Center, Gainesvi lle, Florida, USA over 325 trap nights between July 2015 and December 2016. Mosquito Specie s # Collect ed Mean per day (S.D.) # Blood fed (%) Mean # blood fed per day (S.D.) Aedes albopictu s 23 0.35 (0.65) 0 (0) Aedes infirmatu s 9 0.14 (0.39) 0 (0) Anopheles crucians 8 0.12 (0.38) 2 (25) 0.03 (0.17) Culex erraticus 369 5.67 (2.71) 51 (14) 0.69 (1.13) Culex pilosus 26 0.4 (0.66) 5 (19) 0.077 (0.27) Culex quinquefas ciatus 75 1.15 (1.62) 3 (4) 0.046 (0.21) Uranotaeni a lowii 1 0.015 (0.12) 0 (0) Total 511 61 57 For each mosquito species, the total number of blood fed individuals overall mean number of adult females collected per day, total number of blood fed individuals and overall daily mean of blood fed individuals are indicated. https://do i.org/10.1371/j ournal.pone .0190633.t001 Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 7 / 15

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eight that fed from other hosts. At this site, where n was an available host, n was significantly (P = 0.0002) more likely to feed on n than any other identified host. and nn each had taken blood meals from n, but neither was more likely to feed on pythons over other identified host species, although the sample size for each species was small (n < 5). Discussion Mosquito species vary in their host-use patterns and vectorial competency to pathogens [47]. The transmission networks of mosquito-vectored pathogens are, in large part, structured by these factors and the diversity of vertebrate communities within an ecosystem [48]. The presence of introduced vertebrate species has direct and indirect implications for mosquito-vectored pathogens [8]. An introduced species that is a competent host for a pathogen may serve as a reservoir of infection, facilitating its transmission to native hosts. Alternatively, introduced species that are not competent can serve as dead-end hosts resulting in reduced pathogen circulation. Introduced vertebrates also can have indirect effects on pathogen transmission networks if they influence or restructure host communities. In southern Florida, where there are >50 species of native and introduced mosquitoes, interactions between mosquitoes and the introduced n have not been previously documented. A first step towards characterizing these interactions is a determination of the mosquito species that are likely to feed on n. We identified n DNA in the blood meals of three species (. n, and nn) collected at a facility in northern Florida that housed n in outdoor enclosures, suggesting that when pythons are available hosts, they are likely to be fed upon by local Florida mosquitoes. All three mosquitoes are widely distributed across the southeastern coastal plain and occur throughout Florida. In southern Florida, they are sympatric with established populations of n [26]. mosquitoes take blood meals from mammals, birds, reptiles and amphibians in Florida [49]. There is variation among species in their degree of host specialization and preference for certain host classes. Some species feed from a narrow range of vertebrate hosts, while others are relative generalists. We also collected blood-fed n, but the sample size was small, and only one blood Table 2. Host use of blood fed female mosquitoe s collecte d at the USDA National Wildlife Research Center, Gainesvi lle, Florida, USA between July 2015 and December 2016. Host specie s Host class Cx. erraticus Cx. pilosus Cx. quinquefa sciatus An. crucians Anolis carolinensis Reptilia 1 2 0 0 Coluber constric tor Reptilia 1 0 0 0 Python bivittatus Reptilia 40 2 2 0 Salvator meriana e Reptilia 1 0 0 0 Cathartes aura Aves 1 1 0 0 Toxostoma rufum Aves 0 0 1 0 Didelphis virginiana Mammal ia 2 0 0 0 Homo sapiens Mammal ia 1 0 0 0 Felis catus Mammal ia 1 0 0 0 Sylvilagus florida nus Mammal ia 0 0 0 1 Unidentif ied 3 0 0 1 Total 51 5 3 2 Values represent the number of individual blood meals for each mosquito species derived from a host species. https://do i.org/10.1371/j ournal.pone .0190633.t002 Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 8 / 15

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meal was identified, as rn! n. n, like other studied mosquitoes, feeds predominantly from mammalian hosts. Only one blood meal specimen, from n, contained r. nn DNA. rnn nn is diurnal, and retreats to underground burrows at night or during unfavorable environmental conditions. Edman et al. [50] suggested that the cavity-roosting habits of woodpeckers make them less susceptible to mosquito feeding. n and nn search for and feed from hosts nocturnally [51]. Therefore, by spending nights underground, tegus may similarly avoid hostseeking mosquitoes. Alternatively, the artificial burrows inside the enclosures may provide suitable resting sites for mosquitoes that had fed from sleeping tegus, making them disinclined to leave the burrows before blood meals are digested. n conclusively took more blood meals from n than from any other identified host. This mosquito feeds opportunistically; previous studies have reported either birds or mammals as frequent hosts, with smaller proportions of reptilian, and to a lesser extent, amphibian hosts recorded [27, 5256]. Combined with our results, this suggests that n is a likely host for n in southern Florida, where the species co-occur. Sample size was small for both and nn, but n DNA was recovered from two of five, and two of three blood meals, respectively. nn is ornithophilic, and feeds predominantly from birds, especially passerines [49, 57], although reports of frequent mammalian, and occasionally reptilian host use exist [58]. Host associations for are not well known, but the available data suggest it feeds primarily on reptiles, particularly lizards [52]. In the southeastern U.S., n and nn are medically-import ant vectors of arboviruses and parasites. n is suspected to be an important bridge vector for the Eastern equine encephalitis virus [59]. In North America, Eastern equine encephalitis virus is circulated and amplified among bird populations by the primary vector, n nn, which is ornithophilic and largely specializes on passerines. The virus can escape bird-mosquito transmission to infect mammals and reptiles through competent, more generalist mosquito species. Recent evidence suggests that snakes may be important to the transmission dynamics and overwintering of Eastern equine encephalitis virus [27, 28, 35, 60] and West Nile virus [61]. The overwintering mechanisms of these viruses are unknown. Because these viruses are not transmitted directly from mosquito to mosquito, they may persist through the winter, in the absence of mosquitoes, in infected host animals that remain viremic for extended time periods, such as reptiles [34]. nn is important in the transmission of a wide range of human and wildlife pathogens including viruses (West Nile virus, St. Louis encephalitis virus), avian malaria parasites, and filarial nematodes. A recent competency study suggested that nn may be a vector for Zika virus [62], but others have found contradictory results [63, 64]. Our results suggest that n, as a host for medically-important mosquito vectors, is likely to be exposed to arboviruses and other endemic pathogens and may influence pathogen transmission within its introduced range. While little is known about wildlife pathogens transmitted by mosquitoes, there are records of nn transmitting n blood parasites between boid snakes and lizards in Brazil [65]. In the Pantanal of South America, various mosquito species in the subgenus n, which includes n are the vectors of another reptile-specific n species, nn [66]. n is also a vector of the lizard malaria parasite n [67]. Introduced mosquito species may affect vertebrate hosts and the transmission of local pathogens [68]. In southern Florida, >10 species of nonnative mosquitoes are present and their host-use patterns, in many cases, are not well characterized. Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 9 / 15

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In Florida, n may indirectly influence the transmission dynamics of mosquitovectored pathogens through predation of mammalian hosts. There is evidence that predation of mammals by n increases contact between the vectors and reservoir hosts of Everglades virus, a strain of the Venezuelan equine encephalitis virus [69]. Everglades virus circulates in an enzootic transmission cycle between reservoir rodent hosts via the mosquito [70]. Reservoir hosts, specifically the cricetid rodents r! and !, become infected with the virus, and effectively transmit it to the mosquitoes that feed from them. This virus is currently not considered a substantial public health threat, although human populations living near native habitats that support virus circulation show high levels of exposure [71]. In humans, Everglades virus infection causes febrile symptoms that occasionally progress to neurologic disease [72]. Although feeds largely on rodents, it also feeds on other mammals including raccoons, opossums, deer, and rabbits [52]. Populations of some mammal species have declined precipitously since the introduction of the Burmese python [22, 23], correlating with a shift in the host use of towards the reservoir hosts of Everglades virus, and potentially leading to an increase in the prevalence of Everglades virus [69]. Currently, there are limited data on abundance trends of the rodent reservoir hosts of Everglades virus. We speculate that python-mediated declines in or extirpations of mammal species could affect the transmission and prevalence of Everglades virus in southern Florida by increasing or decreasing contact between vector mosquitoes and reservoir hosts. For example, predation of rodents by pythons could reduce the abundance of reservoir hosts (leading to a decrease in virus prevalence); predation of mammalian mesopredators by pythons could release reservoir rodents from mesopredator predation pressure (leading to an increase in virus prevalence); or mammal declines could push mammalophilic mosquito species that may serve as bridge-vectors, toward the rodent reservoir hosts through the decreased availability of non-reservoir mammals (leading to an increase in virus prevalence). In addition to Everglades virus, many other poorly known arboviruses (e.g., Mahogany Hammock virus, Pahayokee virus, Shark River virus, Gumbo Limbo virus) occur in mosquitonative mammal transmission cycles in the Everglades [7375], and their transmission dynamics may be influenced by python predation of mammalian hosts. Field surveys for n in difficult-to-access habitats are challenging and resourceintensive. In the Everglades, n is elusive, cryptic and semi-aquatic, making it difficult to detect through direct observation or traditional methods. For these reasons, molecular approaches through environmental DNA analyses have been developed that are designed to detect the presence of small fragments of python DNA in water [37, 38]. Our results demonstrate that local mosquito communities use n as a host. Assuming these interactions are occurring in the Everglades, mosquito blood meals could be used as an alternate source of DNA for detecting the presence of n. Although blood fed mosquitoes cannot be obtained as easily as water samples, one advantage of screening mosquito blood meals for python DNA is that host DNA is more concentrated and less degraded, potentially providing the ability to attribute DNA sequences to individual snakes [76], which may enable estimations of population size. Further, such an approach to n detection would generate datasets that are simultaneously valuable to both biodiversity monitoring and to the epidemiology of mosquito-vectored pathogens in southern Florida. The feasibility of n detection through mosquito blood meals depends on the extent to which mosquitoes and pythons interact in the Greater Everglades Ecosystem of southern Florida. The habitats, mosquito and vertebrate host communities, and climatic conditions of southern Florida differ from those in northern Florida, where this research was performed. Southern Florida supports a similar mosquito assemblage to northern Florida with many species in common, including n, and nn [26]. Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 10 / 15

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These mosquitoes are likely to co-occur with n in the hardwood hammocks and coniferous forests with substantial canopy cover that are preferred by pythons [77]. Mosquito populations may be substantially denser in southern Florida, which may be beneficial to n detection. n, in particular, reaches high abundances at sites where n is common. This species was not collected in our sample from northern Florida, but may feed from n in the Everglades, as it is known to feed from reptiles elsewhere [78]. Unlike northern Florida, the climate of southern Florida is also conducive to year-round mosquito activity, which would be expected to benefit the use of mosquito blood meals as a source of environmental DNA for n detection. Additional work, particularly field study, is needed to examine mosquito-python interactions and implications in nature in the Greater Everglades Ecosystem. Research examining the impacts of n on the ecology of southern Florida has focused primarily on trophic interactions between pythons and native vertebrates, particularly mammals. Beyond predation, little is known about ecological interactions between n and other elements of Florida's ecosystems. In addition to the potential of using mosquitoes as a source of environmental DNA for monitoring python populations, understanding interactions between introduced n populations and Florida's mosquito fauna is necessary to determine any potential role n may play in the transmission systems of locallyoccurring parasites and pathogens. The integration of a novel, large-bodied vertebrate into Florida's ecosystems may directly or indirectly affect the transmission dynamics of local mosquito-borne pathogens. Future research should work towards understanding these impacts, particularly on Everglades virus, as environmental change has been hypothesized to promote the epizootic emergence of other Venezuelan equine encephalitis strains [79]. Supporting information S1 Table. Collection details and results of blood meal analysis for mosquitoes collected at the USDA National Wildlife Research Center, Gainesville, Alachua Co., Florida, USA. Each collected mosquito was given a unique identifying number (ID). The taxonomic identification (Species), date of collection (Date), sex (Sex), and the estimated extent of digestion are indicated for each mosquito specimen. A small sample of unfed female and male mosquitoes were collected to serve as negative controls to monitor for contamination and co-amplification of non-host templates. For each specimen, amplification success (Amp) and host identity (Host) are indicated. (DOCX) Acknowledgmen ts We thank Kandy L. Keacher and the staff of the USDA National Wildlife Research Center for assistance with collecting mosquito specimens, Lois Wood of the University of Florida Veterinary Entomology Lab, and Leroy Nuez of the Florida Museum of Natural History Herpetology Division. Author Contributions Conceptualization: Lawrence E. Reeves, Kenneth L. Krysko, Jennifer L. Gillett-Kaufman, Akito Y. Kawahara, C. Roxanne Connelly. Data curation: Lawrence E. Reeves. Formal analysis: Lawrence E. Reeves. Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 11 / 15

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Funding acquisition: Lawrence E. Reeves. Investigation: Lawrence E. Reeves, Michael L. Avery. Methodology: Lawrence E. Reeves, Kenneth L. Krysko, Michael L. Avery, Jennifer L. GillettKaufman, Akito Y. Kawahara, C. Roxanne Connelly. Project administration: Lawrence E. Reeves, Kenneth L. Krysko, Michael L. Avery, Jennifer L. Gillett-Kaufman, Akito Y. Kawahara. Resources: Kenneth L. Krysko, Michael L. Avery, Phillip E. Kaufman. Software: Lawrence E. Reeves. Supervision: Jennifer L. Gillett-Kaufman, Akito Y. Kawahara, C. Roxanne Connelly, Phillip E. Kaufman. Writing original draft: Lawrence E. Reeves. Writing review & editing: Lawrence E. Reeves, Kenneth L. Krysko, Michael L. Avery, Jennifer L. Gillett-Kaufman, Akito Y. Kawahara, C. Roxanne Connelly, Phillip E. Kaufman. References 1. Pysek P, Jarosik V, Hulme PE, Pergl J, Hejda M, Schaffner U, et al. A global assessmen t of invasive plant impacts on resident species, commu nities and ecosystems : The interacti on of impact measures, invading species traits and environm ent. Glob Chang Biol. 2012; 18:17251737 2. Cronk QCB, Fuller CL. Plant Invaders: The Threat to Natural Ecosyste ms. London: Chapm an & Hall; 1995. 3. McKinney ML, Lockwood JL. Biotic homogen ization: A few winners replacing many losers in the next mass extinction. Trends Ecol Evol. 1999; 14:450453. PMID: 10511724 4. Benedict MQ, Levine RS, Hawley WA, Lounibos LP. Spread of the tiger: Global risk of invasion by the mosquito Aedes albopi ctus. Vector Borne Zoonotic Dis. 2007; 7:7685. https:// doi.org/10.10 89/vbz. 2006.0562 PMID: 17417960 5. Pimental D, Zuniga R, Morrison D. Update on the environment al and economic costs associat ed with alien-invas ive species in the United States. Ecol Econ. 2005; 52:273288. 6. Fonseca DM, Lapoint e DA, Fleischer RC. Bottlen ecks and multiple introduc tions: Popula tion genetics of the vector of avian malaria in Hawaii. Mol Ecol. 2000; 9:1803 1814. PMID: 11091316 7. Prenter J, MacNei l C, Dick JTA, Dunn AM. Roles of parasites in animal invasions. Trends Ecol Evol. 2004; 19:385 390. https://doi.or g/10.101 6/j.tree.2004.0 5.002 PMID: 16701290 8. Kelly DW, Paterso n RA, Townse nd CR, Poulin R, Tompkins DM. Parasit e spillback: A neglected concept in invasion ecology? Ecology. 2009; 90:2047 2056. PMID: 197393 67 9. Simberloff D, Schmitz DC, Brown TC. Strangers in Paradise: Impact and Managemen t of Nonindig enous Species in Florida. Washingto n D.C.: Island Press; 1997. 10. Hardin S. Managin g non-nativ e wildlife in Florida: State perspective policy and practice. In: Witmer G, Pitt W, Fagerstone K, editors. Managin g Vertebrate Invasive Species. Fort Collins: USDA/APH IS/WS, National Wildlife Research Center; 2007. p 4352. 11. Krysko KL, Burgess JP, Rochford MR, Gillette CR, Cueva D, Enge KM, et al. Verified non-indige nous amphibi ans and reptiles in Florida from 1863 through 2010: Outlining the invasion process and identifying invasion pathways and stages. Zootaxa. 2011; 3028:164. 12. Krysko KL, Somma LA, Smith DC, Gillette CR, Cueva D, Wasilewsk i JA. New verified nonindigeno us amphibi ans and reptiles in Florida through 2015, with a summa ry of over 152 years of introduc tions. Reptiles Amphib 2016; 23:110143. 13. Engema n R, Jacobs on E, Avery ML, Meshaka ME. The aggressive invasion of exotic reptiles in Florida with a focus on prominent species: A review. Curr Zool. 2011; 57:599 612. 14. Meshaka WE, Loftus WF, Steiner T. The herpetofauna of Evergl ades National Park. Fla Sci. 2000; 63:84103. 15. Snow RW, Krysko KL, Enge KM, Oberhofer L, Warren-B radley A, Wilkins L. Introdu ced populations of Boa constrictor (Boidae) and Python molurus bivittatus (Python idae) in southern Florida. In: Henderson Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 12 / 15

PAGE 13

RW, Powell R, editors. The Biology of Boas and Pythons. Eagle Mountain : Eagle Mountain Publish ing; 2007. p. 41743 8. 16. Willson JD, Dorcas ME, Snow RW. Identifying plausible scenarios for the establishmen t of invasive Burmese pythons (Python molurus) in Southern Florida. Biol Invasions. 2011; 13:14931504 17. Greene DU, Potts JM, Duquesnel JG, Snow RW. Python molurus bivittatus (Burmes e python). Herpetol Rev. 2007; 38:355. 18. Snow RW, Brien ML, Cherkiss MS, Wilkins L, Mazzotti FJ. Dietary habits of the Burmese python, Python molurus bivittatus, in Evergl ades National Park, Florida. Herpetol Bull. 2007; 101:57 19. Dove C, Snow RW, Rochfo rd MR, Mazzotti FJ. Birds preyed upon by the introduced Burmese python (Python molurus bivittatus) in Everglades National Park, FL. Wilson J Ornithol. 2011; 123:126 131. 20. Boback SM, Snow RM, Hsu T, Peurac h SC, Dove CJ, Reed RN. Supers ize me: Remains of three white-tail ed deer (Odocoileu s virginianus ) in an invasive Burmese python (Python molurus bivittatus) in Florida. Bioinvasi ons Rec. 2016; 5:197203. 21. Holbroo k J, Chesnes T. An effect of Burmese pythons (Python molorus bivittatus) on mammal populations in southern Florida. Fla Sci. 2011; 74:1724. 22. Dorcas ME, Willson JD, Reed RN, Snow RW, Rochford MR, Miller MA, et al. Severe mamma l declines coincide with proliferatio n of invasive Burmese pythons in Everglade s National Park. Proc Natl Acad Sci U S A. 2012; 109:2418242 2. https://doi.or g/10.1073/ pnas.11152261 09 PMID: 22308381 23. McCleery RA, Sovie A, Reed RN, Cunningham MW, Hunter ME, Hart KM. Marsh rabbit mortalities tie pythons to the precipi tous declines of mamma ls in the Everglades Proc R Soc Lond B Biol Sci. 2015; 282:2015. 0120. 24. Willson JD. Indirect effects of invasive Burmese pythons on ecosystem s in southern Florida. J Appl Ecol. 2017; https://doi.or g/10.1111/ 1365-2664.1 2844 25. Keesing F, Holt RD, Ostfeld RS. Effect of species diversity on disease risk. Ecol Lett. 2006; 9:4854 98. https://doi.or g/10.111 1/j.1461-024 8.2006.0 0885.x PMID: 16623733 26. Darsie RF, Morris CD. Keys to the Adult Females and Fourth Instar Larvae of the Mosquitoes of Florida (Diptera: Culicidae). Fort Myers: Florida Mosquito Control Assoc iation; 2000. 27. Cupp EW, Zhang D, Yue X, Cupp MS, Guyer C, Sprenger TR, et al. Identificati on of reptilian and amphibi an blood meals from mosquitoes in an Eastern equine encepha lomyelitis virus focus in central Alabama Am J Trop Med Hyg. 2004; 71:272 276. PMID: 153818 05 28. Burkett-Ca dena ND, Graham SP, Hassan HK, Guyer C, Eubank s MD, Kathol i CR, et al. Blood feeding patterns of potential arbovirus vectors of the genus Culex targeti ng ectothermic hosts. Am J Trop Med Hyg. 2008; 79:809 815. PMID: 189815 28 29. Smith TG. The genus Hepatoz oon (Apicomp lexa: Adeleina) J Parasit ol. 1996; 82:565585 PMID: 8691364 30. Telford SR, Wozniak EJ, Butler JF. Haemogreg arine specificity in two commu nities of Florida snakes with descriptions of six new species of Hepatozoon (Apicomp lexa: Hepatozoida e) and a possible species of Haemogreg arina (Apicomp lexa: Haemogreg arinidae). J Parsito l. 2001; 87:890905. 31. Perkins SL, Rothschild A. Walter E. Infections of the malaria parasite Plasmodi um floridense in the invasive lizard, Anolis sagrei, in Florida. J Herpetol. 2007; 41:750 754. 32. Gebhardt LP, Stanton JG, Hill DW, Collett GC. Natural overwinter ing hosts of the virus of western equine encepha litis. N Engl J Med. 1964; 271:171177 33. Steinman A, Banet-Noac h C, Simano v L, Grinfeld N, Aizenberg Z, Levi O, et al. Experime ntal infection of commo n garter snakes (Thamnophis sirtalis) with West Nile virus. Vector Borne Zoonotic Dis. 2006; 6:361368. https://doi.or g/10.1089/ vbz.2006.6.3 61 PMID: 171875 70 34. White G, Ottend orfer C, Graham S, Unnas ch TR. Competenc y of reptiles and amphibians for Eastern equine encepha litis virus. Am J Trop Med Hyg. 2011; 85:421 425. https://doi.or g/10.426 9/ajtmh.201 1. 11-0006 PMID: 218967 98 35. Bingham AM, Graham SP, Burkett-Ca dena ND, White GS, Hassan HK, Unnasc h TR. Detection of Eastern equine encepha lomyelitis virus RNA in North American snakes. J Am Trop Med Hyg. 2012; 87:114011 44. 36. Blosser EM, Stenn T, Acevedo C, Burkett-Ca dena ND. Host use and seasonali ty of Culex (Melano conion) iolambdis (Diptera: Culicidae) from eastern Florida, USA. Acta Trop. 2016; 164:352359. https:// doi.org/10.10 16/j.actatrop ica.2016.10. 001 PMID: 27712940 37. Piaggio AJ, Engema n R, Hopken MW, Humphrey JS, Keacher KL, Bruce WE et al. Detecting an elusive invasive species: A diagnos tic PCR to detect Burmese python in Florida waters and an assessmen t of persistence of environ mental DNA. Mol Ecol Resour. 2014; 14:374 380. https://doi. org/10.1111/1 7550998.12180 PMID: 24119154 Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 13 / 15

PAGE 14

38. Hunter ME, Oyler-Mc Cance SJ, Dorazio RM, Fike JA, Smith BJ, Hunter CT, et al. Environm ental DNA (eDNA) sampling improves occurrenc e and detection estima tes of invasive Burmese pythons. PLOS ONE 2015; 10:e0121655. https://d oi.org/10.137 1/journal.po ne.0121 655 PMID: 25874630 39. Burkett-Ca dena ND. A wire-fram e shelter for collecting resting mosquit oes. J Am Mosq Control Assoc. 2011; 27:153 155. https://doi.or g/10.298 7/10-6076.1 PMID: 218058 49 40. Reeves LE, Holderman CJ, Gillett-Kauf man JL, Kawahara AY, Kaufman PE. Maintena nce of host DNA integrity in field-pres erved mosquito (Diptera: Culicidae) blood meals for identifica tion by DNA barcoding. Parasit Vectors. 2016; 9:503. https://doi.or g/10.118 6/s13071-0161791-z PMID: 27629021 41. Detinova TS. Age-grou ping methods in Diptera of medical importanc e: With special reference to some vectors of malaria Bull World Health Organ. 1962; 47:4868. 42. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. Preparatio n of PCR quality mouse genomic DNA with sodium hydroxide and Tris (HotSHOT) Biotechniques 2000; 29:525 4. PMID: 10907076 43. Nagy ZT, Sonet G, Glaw F, Vences M. First large-sc ale DNA barcoding assessmen t of reptiles in the biodivers ity hotspot of Madagascar based on newly designed COI primers. PLOS One. 2012; 7: e34506. https:// doi.org/10.13 71/journal.p one.003 4506 PMID: 2247963 6 44. Kearse M, Moir R, Wilson A, StonesHavas S, Cheung M, Sturrock S, et al. Geneious Basic: An integrated and extendab le desktop software platform for the organization and analysis of sequence data. Bioinformat ics. 2012; 28:16471649 https:// doi.org/10.10 93/bioinfo rmatics/bts199 PMID: 22543367 45. Hebert PDN, Cywinska A, Ball SL, DeWaard JR. Biological identifica tions throug h DNA barcodes. Proc R Soc Lond B Biol Sci. 2003; 270:313 321. 46. R Development Core Team. R: A Langua ge and Environment for Statistical Computing. Vienna, Austria: The R Foundation for Statistical Comp uting. 2012. ISBN: 3-90005107-0. 47. Templis CH. Host-fee ding patterns of mosquitoes with a review of advances in analysis of blood meals by serolog y. J Med Entomol. 1975; 11:63565 3. PMID: 235647 48. Dobson A, Cattadori I, Holt RD, Ostfeld RS, Keesing F, Krichbaum K, et al. Sacred cows and sympathetic squirrels: The importan ce of biological diversity to human health. PLOS Med. 2006; 3:e231. https://doi.or g/10.137 1/journal.pme d.0030231 PMID: 167298 46 49. Edman JD. Host-feedin g patterns of Florida mosquitoes: III. Culex (Culex) and Culex (Neoculex ). J Med Entomol. 1974; 11:95104 PMID: 4828351 50. Edman JD, Webber LA, Kale HW II. Host-feedin g pattern s of Florida mosquitoes II. Culiseta. J Med Entomol. 1972; 9:429434 PMID: 4404140 51. Savage HM, Aggarwal D, Apperson CS, Katholi CR, Gordon E, Hassan HK, et al. Host choice and West Nile virus infection rates in blood-fed mosquitoes, including members of the Culex pipiens complex, from Memphis and Shelby County, Tennessee, 20022 003. Vector Borne Zoonotic Dis. 2007; 7:365386. https://doi.or g/10.1089/ vbz.2006.06 02 PMID: 1776741 3 52. Edman JD. Host-feedin g patterns of Florida mosquitoes VI. Culex (Melano conion). J Med Entomol. 1979; 15:521 525. PMID: 544824 53. Rodrigu es SCG, Marunia k JE. Blood meal identifica tion from mosquitoes collected at a commercial alligator farm. J Am Mosq Control Assoc. 2006; 22:557 560. https://doi.or g/10.298 7/8756-971X (2006)22 [557:BMIFM C]2.0.CO;2 PMID: 17067063 54. Savage HM, Anderson M, Gordon E, McMillen L, Colton L, Delorey M, et al. Host-se eking heights, hostseeking activity patterns, and West Nile virus infection rates for members of the Culex pipiens complex at different habitat types within the hybrid zone, Shelby County, TN, 2002 (Diptera : Culicidae) J Med Entomol. 2007; 45:27628 8. 55. Mendenh all IH, Tello SA, Neira LA, Castillo LF, Ocampo CB, Wesson DM. Host preferenc e of the arbovirus vector Culex erraticus (Diptera: Culicidae) at Sonso Lake, Cauca Valley Departm ent, Colombia. J Med Entomol. 2012; 49:10921102 PMID: 23025191 56. Bingham AM, Burkett-Ca dena ND, Hassan HK, McClur e CJW, Unnasc h TR. Field investigatio ns of winter transmiss ion of Eastern equine encepha litis virus in Florid a. Am J Trop Med Hyg. 2014; 91:685 693. https://doi.or g/10.426 9/ajtmh.140081 PMID: 25070997 57. Molaei G, Andreadis TG, Armstrong PM, Bueno R, Dennett JA, Real SV, et al. Host feeding pattern of Culex quinquefas ciatus (Diptera: Culicidae) and its role in transmiss ion of West Nile virus in Harris County, Texas. Am J Trop Med Hyg. 2007; 77:738 1. PMID: 17620633 58. Unlu I, Kramer WL, Roy AF, Foil LD. Detection of West Nile virus RNA in mosquitoes and identification of mosquito blood meals collected at alligator farms in Louisiana. J Med Entomol. 2010; 47:625 633. PMID: 206952 78 Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 14 / 15

PAGE 15

59. Bingham AM, Burkett-Ca dena ND, Hassan HK, Unnas ch TR. Vector competenc e and capacity of Culex erraticus (Diptera : Culicidae) for Eastern equine encepha litis virus in the southeaster n United States. J Med Entomol. 2016; 53:473476. https://d oi.org/10.109 3/jme/tjv195 PMID: 26659606 60. Graham SP, Hassan HK, Chapman T, White G, Guyer C, Unnasch TR. Serosu rveillance of Eastern equine encepha litis virus in amphibi ans and reptiles from Alabam a, USA. Am J Trop Med Hyg. 2012; 86:540544 https://doi.or g/10.4269/ ajtmh.201 2.11-0283 PMID: 22403333 61. Dahlin CR, Hughes DF, Meshaka WE Jr., Coleman C, Henning JD. Wild snakes harbor West Nile virus. One Health. 2016; 2:13613 8. https://doi.or g/10.1016/ j.onehlt.2016.0 9.003 PMID: 28616487 62. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasc iatus in Brazil. bioRxiv org. 2016;073197. https:// doi.org/10. 1101/07319 7 63. Fernandes RS, Campo s SS, Ferreira-de-B rito A, de Miranda RM, da Silva KAB, de Castro MG, et al. Culex quinquefas ciatus from Rio de Janeiro is not compete nt to transmit the local Zika virus. PLOS Negl Trop Dis. 2016; 10:e000499 3. https://doi.or g/10.1371 /journal.pntd .0004993 PMID: 27598421 64. Dodson BL, Rasgon JL. Vector competenc e of Anopheles and Culex mosquitoe s for Zika virus. PeerJ. 2017; 5:e3096 https://doi.or g/10.771 7/peerj.3096 PMID: 2831689 6 65. Paperna I, Lainson R. Hepatoz oon c.f. terzii (Sambon & Seligman, 1907) infection in the snake Boa constrictor from north Brazil: Transmission to the mosquito Culex quinquefas ciatus and the lizard Tropidurus torquat u. Parasite. 2004; 11:175 181. https://doi.or g/10.105 1/parasite/ 2004112175 PMID: 15224579 66. Viana LA, Soares P, Paiva F, Lourenc o-De-Oliv eira R. Caiman-bi ting mosquitoes and the natural vectors of Hepatoz oon caimani in Brazil. J Med Entomol. 2010; 47:670676. PMID: 20695284 67. Klein TA, Young DG, Telford SR. Vector incrimina tion and experime ntal transmission of Plasmodi um floridense by bite of infected Culex (Melanoc onion) erratic us (Diptera: Culicidae) J Am Mosq Control Assoc. 1987; 3:1651 75. PMID: 350490 6 68. Juliano SA, Lounibo s LP. Ecology of invasive mosquitoes: Effects on resident species and on human health. Ecol Lett. 2005; 8:5585 74. https://doi.or g/10.111 1/j.1461-024 8.2005.0 0755 PMID: 17637849 69. Hoyer IJ, Blosser EM, Acevedo C, Thompson AC, Reeves LE, BurkettCadena ND. Mammal decline, linked to invasiv e Burmese python, shifts host use of vector mosquito towards reservoir hosts of a zoonotic disease. Biol Lett. 2017;2017035 3. 70. Coffey LL, Carrer a A-S, Paessler S, Haynie ML, Bradley RD, Tesh RB, et al. Experimental Evergl ades virus infection of cotton rats (Sigmo don hispidus). Emerg Infect Dis. 2004; 10:21822188 https://doi. org/10.3201/ eid1012.0404 42 PMID: 156638 57 71. Work TH. Serological evidence of arbovirus infection in the Seminole Indians of southern Florida. Science. 1964; 145:270272. PMID: 14171866 72. Coffey LL, Crawfo rd C, Dee J, Miller R, Freier J, Weaver SC. Serologic evidence of widesprea d Everglades virus activity in dogs, Florida. Emerg Infect Dis. 2006; 12:1873 1879. https://doi.o rg/10.3201/ eid1212.0604 46 PMID: 17326938 73. Coleman PH, Ryder S, Work TH. Mahogan y Hammock virus, a new Guama group arbovirus from the Florida Everglades Am J Epidem iol. 1969; 89:217 221. PMID: 5765959 74. Fields BN, Henderson BE, Coleman PH, Work TH. Pahayo kee and Shark River, two new arboviruses related to Patois and Zegla from the Florida Everglades. Am J Epidem iol. 1969; 89:222226. PMID: 5765960 75. Henderson BE, Calisher CH, Coleman PH, Fields BN, Work TH. Gumbo Limbo, A new group C arbovirus from the Florida Everglades Am J Epidem iol. 1969; 89:227 231. PMID: 4387912 76. Martinezde la Puente J, Mendez M, Ruiz S, Godoy JA, Soriguer RC, Figuerola J. Individua l identification of endang ered species using mosquito blood meals: A proof-of-con cept study in Iberian lynx. Parasitol Res. 2015; 114:1607161 0. https://doi.or g/10.1007/ s00436-0154343-0 PMID: 25656463 77. Walters TM, Mazzotti FJ, Fitz HC. Habitat selection by the invasive species Burmese python in southern Florida. J Herpetol. 2016; 50:5056. 78. Bataille A, Fournie G, Cruz M, Cedexo V, Parker PG, Cunningh am AA, Goodm an SJ. Host selection and parasite infection in Aedes taenior hynchus, endemic disease vector in the Galapagos Islands. Infect Genet Evol. 2012; 12:18311841 https://d oi.org/10.101 6/j.meegid .2012.07 .019 PMID: 22921730 79. Brault AC, Powers AM, Ortiz D, Estrada-Fra nco JG, Navarro-Lopez R, Weaver SC. Venezuelan equine encepha litis emergenc e: Enhanced vector infection from a single amino acid substitution in the envelope glycoprote in. Proc Natl Acad Sci U S A. 2004; 101:1134 411349. https://doi.or g/10.107 3/pnas. 0402905101 PMID: 15277679 Invasive Burmese python and local mosquito communities in Florida PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01906 33 January 17, 2018 15 / 15