Data from: Haemosporidian infection risk across an urban gradient in a songbird

Description

Notes

Methods

Field Sampling

Bird capture was conducted during three consecutive breeding seasons (January–August) from 2021–2023. The study sites spanned four major metropolitan areas (Los Angeles, San Diego, Santa Barbara, and San Francisco, CA) and included two non-urban sites (Angeles Nation Forest, ANF; Santa Monica Mountains, SMM) and six urban sites (University of California, Los Angeles campus, UCLA; University of California, San Diego campus, UCSD; University of California, Santa Barbara, UCSB; Occidental College campus, Occ; San Francisco State University campus, SFSU; and parks throughout Los Angeles, LA).

At each site, wild juncos were captured by targeted mist netting. Trapping efforts included audio lures recorded from juncos in Los Angeles. All captured individuals, including males, females, and juveniles, were used in this analysis. Upon capture, individuals were banded with metal federal aluminum bands and three plastic color bands to create unique band combinations for behavioral studies performed in tandem with this study. Body morphometrics, including weight, wing cord length, tail length, tarsus length, bill width, depth, and length, were recorded. Age is determined based on plumage characteristics, and sex is determined by a combination of plumage and brood patches or cloacal protuberances (Pyle et al. 1997). Blood samples, >10% of the total mass with an average volume of 50 µL, were collected from individuals via brachial venipuncture and a 30G needle following sterilization of the puncture site with alcohol pads. Blood was collected via heparinized capillary tubes. Parasitemia was not assessed for this study, but two blood smears were prepared per individual, and whole blood was then stored in lysis buffer (Valkiūnas 2005). Samples were collected from recaptures if the capture date exceeded two weeks.

Abiotic Environmental Assessment

Habitats were characterized on the basis of local vegetation surveys and remote sensing data. For both the local and remote datasets, we focused on the 50 m radius surrounding a capture site. This space presents a reasonable approximation of the space used by small territorial passerines, including juncos (Chandler et al. 1994, Blair 2001).

Local vegetation was assessed between 2022 and 2023 via a modified version of the combined Rapid Assessment Protocol (CNPS-RAP) developed by the California Native Plant Society and California Department of Fish and Wildlife (California Native Plant Society (CNPS), 2022). These modifications included conducting surveys at a junco capture location, regardless of whether they fell within the CNPS-RAP definition of a vegetation stand. Rapid assessments were conducted in circular plots with a 50-meter radius around each individual's capture location. Finally, we counted the number of trash cans and tables within the assessment plot as a proxy for anthropogenic waste availability (Mazué et al., 2023).

Broader habitat conditions included Built-Up Index (BU) as a metric for urbanization and monthly precipitation as a metric for water availability. To calculate the BU values, the difference between the normalized difference built-up index (NDBI) and the normalized difference vegetation index (NDVI) was calculated (He et al. 2010). The raster files for both the NDBI and the NDVI were from the U.S. Geological Survey Earth Resources Observation and Science (EROS) Science Processing Architecture (ESPA) Collection 2 Level 2 Landsat Surface Reflectance-Derived Spectral Indices and had a resolution of 30 m. The BU values corresponded to the average within a 50 m radius of a banding site. The monthly precipitation values were obtained from the PRISM Climate Group via the prism 0.2.0 R package. The total rainfall for the month of the capture date was obtained.

Laboratory analysis

DNA was extracted from whole blood lysis solution via a Qiagen DNeasy Blood and Tissue Extraction Kit (San Diego, CA, USA) or a Wizard® SV Blood and Tissue Extraction Kit (Madison, WI, USA) following the manufacturer's instructions. A total of 10 µL of DNA was then used to screen for the presence of Haemoproteus/Plasmodium via the previously described nested PCR protocol (Waldenström et al. 2004). In brief, an initial 25-µL reaction was performed with the primers HaemNF and HaemNR2, followed by HaemF and HaemR2 for nested PCR (Waldenström et al. 2004). All reactions were conducted via ThermoFisher DreamTaq MasterMix (Hanover Park, IL, USA). Positive samples were submitted for Sanger sequencing (Azenta US Inc., La Jolla, CA). No coinfections were detected via chromatography; however, this is likely due to PCR bias, which preferentially amplifies Haemoproteus over Plasmodium (Ciloglu et al. 2019).

Sanger sequences were used to identify parasite genera and lineages. All sequences were able to be identified to the genus level; however, only sequences with >80% high-quality reads were used to assign lineages. All sequences were initially aligned via the MUSCLE alignment feature available in Geneious Prime 2023.2.1, with all sequences that exceeded 1% (4 bp) dissimilarity classified as a unique lineage (Bensch et al. 2009). Each unique lineage was compared via BLAST with sequences available in the GenBank and MalAvi databases to determine if it was previously observed.

Host-parasite specificity was reported as S_TD and calculated via TAXOBIODIV2 http://www.otago.ac.nz/parasitegroup/downloads.html.While other metrics of host-parasite specificity exist (Svensson-Coelho et al. 2013), S_TD is a comparable method and is broadly used (Poulin and Mouillot 2005). The data on hosts were based on reported observations available from the MalAvi data for each parasite lineage. Host-parasite specificity was reported via S_TD values ranging from 1--4, with higher values suggesting more generalist parasites. Lineages that were unique from those previously published were given a default S_TD value of 1.