DOI: 10.5061/dryad.jm63xsjh8
title: Spatiotemporal organization of cryptic North American Culex species along an urbanization gradient

description: Landscape heterogeneity creates diverse habitat and resources for mosquito
 vectors of disease. A consequence may be varied distribution and abundance
 of vector species over space and time dependent on niche requirements. We
 tested the hypothesis that landscape heterogeneity driven by urbanization
 influences the distribution and relative abundance of Culex pipiens , Cx.
 restuans, and Cx. quinquefasciatus, three vectors of West Nile virus (WNv)
 in the eastern North American landscape. We collected 9,803 cryptic Culex
 from urban, suburban, and rural sites in metropolitan Washington, District
 of Columbia, during the months of June-October, 2019-2021. In 2021, we
 also collected mosquitoes in April and May to measure early-season
 abundance and distribution. Molecular techniques were used to identify a
 subset of collected Culex to species (n = 2,461). Ecological correlates of
 the spatiotemporal distribution of these cryptic Culex were examined using
 constrained and unconstrained ordination. Seasonality was not associated
 with Culex community composition in June-October over three years but
 introducing April and May data revealed seasonal shifts in community
 composition in the final year of our study. Culex pipiens were dominant
 across site types, while Cx. quinquefasciatus were associated with urban
 environments, and Cx. restuans were associated with rural and suburban
 sites. All three species rarely coexisted. Synthesis and applications: Our
 work demonstrates that human-mediated land-use changes influence the
 distribution and relative abundance of Culex vectors of WNv, even on fine
 geospatial scales. Site classification, percent impervious surface,
 distance to city center, and longitude predicted Culex community
 composition. We documented active Culex months before vector surveillance
 typically commences in this region, with Culex restuans being most
 abundant during April and May. Active suppression of Cx. restuans in April
 and May could reduce early enzootic transmission, delay the seasonal
 spread of WNv, and thereby reduce overall WNv burden. By June, the highest
 risk of epizootic spillover of WNv to human hosts may be in suburban areas
 with high human population density and mixed Culex  assemblages
 that can transmit WNv between birds and humans. Focusing management
 efforts there may further reduce human disease burden.
description: Mosquito collection was completed by authors over three years.
 Specimens were morphologically identified to the lowest taxon possible and
 cryptic Culex were identified with molecular methods (PCR). Once
 individuals were identified, species distribution patterns were analyzed
 by site, by site class, and by season using R and associated R
 packages. 
description: # Spatiotemporal organization of cryptic North American Culex species
 along an urbanization gradient; Arsenault-Benoit and Fritz 2023 *** From
 2019-2021, the authors collected mosquitoes via gravid traps from 15 sites
 in Washington D.C. and Maryland. Cryptic Culex specimens were molecularly
 identified to assess patterns of community composition, habitat use, and
 seasonality among these species. ## Description of the Data and file
 structure All data files in this dataset are in .xlsx format. The contents
 of each dataset are as follows: #### File Name: MD\_3yr\_totalcatch.xlsx
 **Description of file:** MD\_3yr\_totalcatch.xlsx: The number of unfed,
 cryptic Culex collected in each trap. This is the total number of Culex
 collected across sites and seasons, from which a subset were molecularly
 identified. Culex collected are individual counts/integers. This dataset
 includes individual trap identification codes, site (nominal variable),
 site classificaitons (nominal variable), and collection dates.
 **Variables:** Trap\_ID: Identification code for each trap, including
 year, a two letter abbeviation for site, and collection event. Nominal
 variable CollNum: A unique identifier for each trap set- corresponds to
 the site and date of collection; each individual collected in the same
 trap has the same trap ID. Nominal variable Date: Calendar date that
 mosquitoes were collected on. Site: Site name where collection occurred
 (Factor) CollEvent:Number of the trapping instance at each site throughout
 the year from 1-11 (Apr-Oct) in 2021 and 3-11 (Jun-Oct) in 2019 and 2020.
 Orderd numerical variable Prim\_Class: nominal factor variable determined
 by visual classification in combination with the quantifiable urbanization
 measures used. Sites are classified as either urban, suburban, or rural.
 Site classifications were assigned at the beginning of the study and
 remained consistant. Sec\_Class:nominal factor variable determined by
 visual classification for land use in combination with the quantifiable
 urbanization measures used. Sites are classified as either commercial,
 residential, agricultural, or natural. Site classifications were assigned
 at the beginning of the study and remained consistant. Season:an ordinal
 factor variable separating collections into seasonal bins. (very early
 (April and May), early (Jun 1-Jul 8, mid (Jul 9-Aug 20, late (Aug 20-Oct
 1)) Unfed\_PerTrap:Integer variable indicating the number of unfed cryptic
 Culex (target taxa for this study)collected in each trap on the indicated
 site and date Year:Ordered numerical variable indiciating whether a
 collection was done in 2019, 2020, or 2021. WOY:week of the year, and is
 ordinal with week 1 corresponding to the week containing Jan. 1. \*This
 file contains NAs. These are due to either trap failures (traps
 malfunctioned or were tampered with) or instances where we could not
 access sites in 2020 due to COV19 restrictions. #### File Name:
 summary.matrix.xlsx **Description of file:** A summary of total unfed
 Culex collected across site classification and season, generated from the
 "aggregate" function in R. **Variables:** Class:Primary site
 classification assigned to the site. Classes are either urban, suburban,
 or rural; this is a factor variable Season:An ordinal factor variable
 describing whether collection was in the early, mid, or late season,
 Unfed\_PerTrap:Mean number of unfed cryptic Culex collected in each class
 x season combination. (Continuous variable) SE:standard error of the mean
 of unfed cryptic Culex collected in traps of the same site class x season
 combination. Continuous variable #### File Name: summatrix21.xlsx
 **Description of file:** A summary of unfed Culex per trap similar to
 summary.matrix.xlsx, but inclusive of the "very early season",
 from which collections were only done in 2021. **Variables:**
 Class:Primary site classification assigned to the site. Classes are either
 urban, suburban, or rural; this is a factor variable Season:An ordinal
 factor variable describing whether collection was in the very early,
 early, mid, or late season, Unfed\_PerTrap:Mean number of unfed cryptic
 Culex collected in each class x season combination. (Continuous variable)
 SE:standard error of the mean of unfed cryptic Culex collected in traps of
 the same site class x season combination. Continuous variable N:Total
 number of individuals collected in traps of the same site class x season
 combination. (Integer) #### File Name: MD\_3yr\_finals.xlsx **Description
 of file:** Each individual included in the analysis, with a unique
 identifier, molecular identification and collection information.
 **Variables:** TotID\_Yr:Identification code for each trap, including
 year, a two letter abbeviation for site, and collection event. Nominal
 variable TotID:Identification code for each trap, including a two letter
 abbeviation for site, and collection event. Nominal variable
 CollectionNum:The collection number is a unique identified for a trap, so
 individuals collected in a single trap share a collection number
 (nominal). UniqueID: An identifier given to each individual specimen
 processed. ID begins with year, then collection number corresponding to
 site and date, and an alphanumeric code for the individual ID. Location:
 Collection site name (factor) Final\_ID:Final ID is the species
 identification (pipiens, restuans, or quinquefasciatus)for each individual
 using PCR (factor) Prim\_Class: nominal factor variable determined by
 visual classification in combination with the quantifiable urbanization
 measures used. Sites are classified as either urban, suburban, or rural.
 Site classifications were assigned at the beginning of the study and
 remained consistant. Sec\_Class:nominal factor variable determined by
 visual classification for land use in combination with the quantifiable
 urbanization measures used. Sites are classified as either commercial,
 residential, agricultural, or natural. Site classifications were assigned
 at the beginning of the study and remained consistant. Season:an ordinal
 factor variable separating collections into seasonal bins. (very early
 (April and May), early (Jun 1-Jul 8, mid (Jul 9-Aug 20, late (Aug 20-Oct
 1) CollDate:Calendar date when collection occurred (month/day/year)
 Coll\_Event:Number of the trapping instance at each site throughout the
 year from 1-11 (Apr-Oct) in 2021 and 3-11 (Jun-Oct) in 2019 and 2020.
 Orderd numerical variable WOY:week of the year, and is ordinal with week 1
 corresponding to the week containing Jan. 1. Year:Year specimens were
 collected, from 2019-2021 #### File Name: pq\_HWE.xlsx **Description of
 file:** Used for Hardy Weinberg Equilibirum analysis for specimens
 molecularly identifed as Culex pipiens or Culex restuans at a single
 locus. These data represent the number of alleles for pipiens homozygotes
 (pp), quinquefasciatus homozygotes (qq) and heterozygotes by year, site,
 and season. **Variables:** Year: Year of collection from 2019-2021
 (ordered factor) Site: Name of collection site (factor) Season: Season
 (very early, early, mid, late) when collection occurred pp: number of
 individuals in each year, site, and season combination homozygous for the
 pipiens allele (integer) pq:number of heterozygous individuals in each
 year, site, and season combination (integer) pp: number of individuals in
 each year, site, and season combination homozygous for the
 quinquefascaitus allele (integer) #### File Name: env\_phen.xslx
 **Description of file:** Environmental data, primarily generated using
 ArcGIS tools, associated with each collection. **Variables:**
 Tot\_ID\_Yr:is a combination of the two-letter abbreviation for site and
 collection event for each trap, with year as well (factor) TotID: is a
 combination of the two-letter abbreviation for site and collection event
 for each trap (factor) CollectionNum: The collection number is associated
 with each trap that was set (values are numeric but the variable is meant
 to be nominal).. Location: Collection site name (factor) Prim\_Class:
 nominal factor variable determined by visual classification in combination
 with the quantifiable urbanization measures used. Sites are classified as
 either urban, suburban, or rural. Site classifications were assigned at
 the beginning of the study and remained consistant. Sec\_Class:nominal
 factor variable determined by visual classification for land use in
 combination with the quantifiable urbanization measures used. Sites are
 classified as either commercial, residential, agricultural, or natural.
 Site classifications were assigned at the beginning of the study and
 remained consistant. Season:an ordinal factor variable separating
 collections into seasonal bins. (very early (April and May), early (Jun
 1-Jul 8, mid (Jul 9-Aug 20, late (Aug 20-Oct 1) CollDate:Calendar date
 when collection occurred (month/day/year) Coll\_Event:is the number in a
 sequence of traps annually (1-11) that correspond with the trap date and
 is ordinal. WOY:is week of the year, and is ordinal with week 1
 corresponding to the week containing Jan. 1. Year: calendar year from
 2019-2021 (ordered factor). X: site latitude Y: site longitude
 LandCover\_Maj: the land cover classification code number, based on the
 NLCD 2016 datalayer and legend, that makes up the majority or greatest
 number of pixels within a 0.5km buffer of each trap site (nominal).
 LandCov\_Name: the land cover classifcation that corresponds to the codes
 in LandCov\_Maj variable. PerentImp\_Mean: mean percent impervious
 surface, within a 0.5km buffer of the trap site, based on NLCD percent
 impervious surface data layer (continuous from 0-100%) PerentImp\_Med:
 median percent impervious surface, within a 0.5km buffer of the trap site,
 based on NLCD percent impervious surface data layer (integer from 0-100%)
 Percent\_tree: the percent tree cover, based on NLCD 2016 data, in a 0.5km
 buffer radius around each trap site (continous from 0-100%). Pop: human
 popululation counts in a 0.5km radius buffer around trap sites from the
 2010 US Cenusus (integer) House: Number of housing units within a 0.5km
 radius buffer around trap sites based on the 2010 US Census DCC: Distance
 to city center: linear distance to Washington, DC from each trap site in
 km (continuous) WaterTbl: the depth required to reach surface water at
 each trap site based on USGS data (cm, continuous) Elev: Site elevation
 above sea level (m, continuous) NDVI:Normalized differential vegetation
 index: a measure of vegetation density at each site based on light
 refraction. These are mean values within a 0.5km radius buffer of each
 site (continuous on a 0-200 scale). #### File Name: env\_21.xslx
 **Description of file:** Environmental data for each collection, similar
 to env\_phen.xslx, but only including collections from 2021, which were
 analyzed separately. **Variables:** TotID: is a combination of the
 two-letter abbreviation for site and collection event for each trap
 (factor) CollectionNum: The collection number is associated with each trap
 that was set (values are numeric but the variable is meant to be nominal).
 Location: Collection site name (factor) Prim\_Class: nominal factor
 variable determined by visual classification in combination with the
 quantifiable urbanization measures used. Sites are classified as either
 urban, suburban, or rural. Site classifications were assigned at the
 beginning of the study and remained consistant. Sec\_Class:nominal factor
 variable determined by visual classification for land use in combination
 with the quantifiable urbanization measures used. Sites are classified as
 either commercial, residential, agricultural, or natural. Site
 classifications were assigned at the beginning of the study and remained
 consistant. Season:an ordinal factor variable separating collections into
 seasonal bins. (very early (April and May), early (Jun 1-Jul 8, mid (Jul
 9-Aug 20, late (Aug 20-Oct 1) CollDate:Calendar date when collection
 occurred (month/day/year) Coll\_Event:is the number in a sequence of traps
 annually (1-11) that correspond with the trap date and is ordinal. WOY:is
 week of the year, and is ordinal with week 1 corresponding to the week
 containing Jan. 1. Year: calendar year from 2019-2021 (ordered factor). X:
 site latitude Y: site longitude LandCover\_Maj: the land cover
 classification code number, based on the NLCD 2016 datalayer and legend,
 that makes up the majority or greatest number of pixels within a 0.5km
 buffer of each trap site (nominal). LandCov\_Name: the land cover
 classifcation that corresponds to the codes in LandCov\_Maj variable.
 PerentImp\_Mean: mean percent impervious surface, within a 0.5km buffer of
 the trap site, based on NLCD percent impervious surface data layer
 (continuous from 0-100%) PerentImp\_Med: median percent impervious
 surface, within a 0.5km buffer of the trap site, based on NLCD percent
 impervious surface data layer (integer from 0-100%) <br>
 Percent\_tree: the percent tree cover, based on NLCD 2016 data, in a 0.5km
 buffer radius around each trap site (continous from 0-100%). Pop: human
 popululation counts in a 0.5km radius buffer around trap sites from the
 2010 US Cenusus (integer) House: Number of housing units within a 0.5km
 radius buffer around trap sites based on the 2010 US Census DCC: Distance
 to city center: linear distance to Washington, DC from each trap site in
 km (continuous) WaterTbl: the depth required to reach surface water at
 each trap site based on USGS data (cm, continuous) Elev: Site elevation
 above sea level (m, continuous) NDVI:Normalized differential vegetation
 index: a measure of vegetation density at each site based on light
 refraction. These are mean values within a 0.5km radius buffer of each
 site (continuous on a 0-200 scale). ### File Name: dat\_phen.csv
 **Description of file:** Molecular identification of each individual, as
 well as collection information for each individual. **Variables:**
 Tot\_ID\_Yr:is a combination of the two-letter abbreviation for site and
 collection event for each trap, with year as well (factor) TotID: is a
 combination of the two-letter abbreviation for site and collection event
 for each trap (factor) CollectionNum: The collection number is associated
 with each trap that was set (values are numeric but the variable is meant
 to be nominal). UniqueID: An identifier given to each individual specimen
 processed. ID begins with year, then collection number corresponding to
 site and date, and an alphanumeric code for the individual ID. Location:
 Collection site name (factor) Final\_ID:Final ID is the species
 identification (pipiens, restuans, or quinquefasciatus)for each individual
 using PCR (factor) Prim\_Class: nominal factor variable determined by
 visual classification in combination with the quantifiable urbanization
 measures used. Sites are classified as either urban, suburban, or rural.
 Site classifications were assigned at the beginning of the study and
 remained consistant. Sec\_Class:nominal factor variable determined by
 visual classification for land use in combination with the quantifiable
 urbanization measures used. Sites are classified as either commercial,
 residential, agricultural, or natural. Site classifications were assigned
 at the beginning of the study and remained consistant. Season:an ordinal
 factor variable separating collections into seasonal bins. (very early
 (April and May), early (Jun 1-Jul 8, mid (Jul 9-Aug 20, late (Aug 20-Oct
 1) CollDate:Calendar date when collection occurred (month/day/year)
 Coll\_Event:is the number in a sequence of traps annually (1-11) that
 correspond with the trap date and is ordinal. WOY:is week of the year, and
 is ordinal with week 1 corresponding to the week containing Jan. 1. Year:
 calendar year from 2019-2021 (ordered factor). #### CSV File Name:
 dat\_reg.csv **Description of file:** Counts of each species at each site
 and collection event, but summed across years. **Variables:** TotID: is a
 combination of the two-letter abbreviation for site and collection event
 for each trap (factor) pip: Count of Cx. pipiens for each TotID (integer)
 quinq: Count of individuals with Cx. quinquefasciatus ancestry for each
 TotID (integer) rest: Count of Cx. restuans for each TotID (integer) ####
 CSV File Name: dat\_21.csv **Description of file:** Same as dat\_reg.xslx
 but including collection events 1-3, corresponding to the "very early
 season" that were only collected in 2021. This data was analyzed
 separately. **Variables:** TotID: is a combination of the two-letter
 abbreviation for site and collection event for each trap (factor) pip:
 Count of Cx. pipiens for each TotID (integer) quinq: Count of individuals
 with Cx. quinquefasciatus ancestry for each TotID (integer) rest: Count of
 Cx. restuans for each TotID (integer) #### File Name: TotID\_m.csv
 **Description of file:** A matrix generated in long form to represent the
 full species counts per site per collection event in the dataset.
 **Variables:** TotID: is a combination of the two-letter abbreviation for
 site and collection event for each trap (factor) pip: Count of Cx. pipiens
 for each TotID (integer) quinq: Count of individuals with Cx.
 quinquefasciatus ancestry for each TotID (integer) rest: Count of Cx.
 restuans for each TotID (integer) ### Codes and abbreviations: Fifteen
 sites were used in this study, and each has a two-letter abbreviation
 commonly used throughout the dataset. Sites and site codes are described
 in env\_phen.xlsx. See the description of env\_phen.xlsx for variable
 abbreviations, format, and scale for environmental data. We completed
 eight collection events per season in 2019-2020, and eleven in 2021. Those
 collection event (CE) numbers correspond to the season of collection. CE
 1-3 are "very early season" (April and May), CE 4-6 are
 "early season" (June -mid-July), CE 7-9 are "mid
 season" (mid July-late August) and CE 10&11 are "late
 season" (late August- early October).WOY= week of the year, with Jan
 1 as week one. Abbreviations for species identifications are as follows:
 pip: Culex pipiens quinq: Culex quinquefasciatus (or having Cx.
 quinquefasciatus ancestry at the CQ11 locus) rest: Culex restuans Some
 cells throughout the dataset are blank or have NA values. These are due to
 either trap malfunctions or instances where traps could not be set due to
 COV19 restrictions. Each instance is described in Arsenault-Benoit and
 Fritz 2023. ## Sharing/access Information Data was generated by the
 authors. Description of methodology and analysis, including a link to the
 script used, can be found in Arsenault-Benoit and Fritz, 2023.