This Perryman_2022__DATA_README.txt file was generated on 2022-04-21 by Danielle Perryman GENERAL INFORMATION 1. Title of Dataset: Data from: Effects of supplemental feeding on nesting success and physiological metrics in Eastern Bluebirds (Sialia sialis). 2. Author Information Corresponding Investigator Name: Dr Jennifer L. Grindstaff Institution: Oklahoma State University, Stillwater, Oklahoma USA Email: jen.grindstaff@okstate.edu Co-investigator 1 Name: Danielle C. Perryman Institution: Oklahoma State University, Stillwater, Oklahoma USA Co-investigator 2 Name: Meeyln M. Pandit Institution: Oklahoma State University, Stillwater, Oklahoma USA Co-investigator 3 Name: Dr. Eric A. Riddell Institution: Museum of Vertebrate Zoology, University of California, Berkeley, Berkeley, California USA Co-investigator 4 Name: Tiana L. Sanders Institution: Oklahoma State University, Stillwater, Oklahoma USA Co-investigator 5 Name: Ian A. Kanda Institution: Oklahoma State University, Stillwater, Oklahoma USA Co-investigator 6 Name: Dr. Jennifer L. Grindstaff (Corresponding) Institution: Oklahoma State University, Stillwater, Oklahoma USA 3. Date of data collection: 2015-2017 4. Geographic location of data collection: Stillwater, Payne County, Oklahoma, USA 5. Funding sources that supported the collection of the data: Oklahoma State University, Oklahoma Ornithological Society, Southwestern Association of Naturalists, North American Bluebird Society, New York State Bluebird Society 6. Recommended citation for this dataset: Perryman, Danielle C. et al. (2022), Data from: Effects of supplemental feeding on nesting success and physiological metrics in Eastern Bluebirds (Sialia sialis). DATA & FILE OVERVIEW 1. Description of dataset Data was gathered for the purpose of studying physiological responses of eastern bluedbirds to supplemental feeding. Each metric (BKA, H:L, and CORT) help describe the responses of the birds in this population. 2. File List: File 1 Name: Perryman_2022_MassDatawithNestID.csv File 1 Description: Datasheet including both NestIDs and mass data to make mass comparisons while accounting for non-independent nestlings. 'Male' or 'Female' columns indicate the parents captured for the nest if applicable. NAs indicate we could not capture the associated male/female. File 2 Name: Perryman_2022_HLFilewithNestID.csv File 2 Description: Datasheet including both NestIDs and H:L data to make mass comparisons while accounting for non-independent nestlings. 'Male' or 'Female' columns indicate the parents captured for the nest if applicable. NAs indicate we could not capture the associated male/female. File 3 Name: Perryman_2022_CORTDatawithNestID.csv File 3 Description: Datasheet including both NestIDs and CORT data to make mass comparisons while accounting for non-independent nestlings. 'Male' or 'Female' columns indicate the parents captured for the nest if applicable. NAs indicate we could not capture the associated male/female. File 4 Name: Perryman_2022_FullBKADatawithNestID.csv File 4 Description: Datasheet including both NestIDs and CORT data to make mass comparisons while accounting for non-independent nestlings. File 5 Name: Perryman_2022_cort_reduced.csv File 5 Description: Adult only data file for nesting success comparisons with CORT. Morphometric NAs are data excluded due to being outside a plausible range for the species. File 6 Name: Perryman_2022_bka_reduced.csv File 6: Description: Adult only data file for nesting success comparisons with BKA. File 7 Name: Perryman_2022_hl_reduced.csv File 7 Description: Adult only data file for nesting success comparisons with HL. CORT and BKA NAs are missing values as not every adult could be tested for H:L, BKA, AND CORT. Morphometric NAs are removed due to being outside a plausible range for the species File 8 Name: Perryman_2022_Supplemental_Feeding_Rscript.Rmd File 8 Description: Annotated R script which was used to generate all out put, except the figures, seen in the manuscript METHODOLOGICAL INFORMATION Material and methods Field methods The range of eastern bluebirds extends from the central United States to the Atlantic Coast (Sibley et al. 2014). Bluebirds prefer to nest in edge habitats, including edges created by human development, which brings them into close contact with humans (Jones et al. 2014). Eastern bluebirds are secondary cavity nesters, and breed in artificial nest cavities. For this study, nest boxes (2015: n=187, 2016: n=176) located along walking trails and roads in Stillwater, Oklahoma (36°7′18″N 97°4′7″W) were utilized to manipulate the food available to breeding eastern bluebirds. Nest boxes were checked 2–3 times weekly throughout the breeding season during March–August in 2015 and 2016. Individual boxes were monitored daily when complete nests were detected to determine first egg dates. Boxes were again monitored daily when clutches were within two days of their expected hatch date (clutch completion date +13 d). Then nest boxes were monitored during regular nest checks to determine if fledging occurred. This frequency of monitoring generally follows methods used previously in this population (Grindstaff et al. 2012). Experimental manipulation of food availability Small feeding cups (2 oz, plastic) were mounted in all nest boxes prior to the breeding season. In these cups, supplemental food was provided by adding fifteen mealworms (1.909 ± 0.177 g) per individual bluebird in the nest box, including both adults and nestlings, three times per week (~11.4–40 g weekly) (Smith 2020). Each time the nest boxes were visited, it was noted whether the cups were empty or contained mealworms to track if the birds had consumed them. Any nest boxes with consistently full cups, likely neophobic individuals or recently failed nests, were removed from all analyses (9.53% of 63 boxes total for both years which received PART/FULL treatments and survived to day 5). All boxes included in the analyses consumed greater than half of all mealworms provided to them. The experimental groups were randomly allocated to FULL (2015: n=35, 2016: n=22), PART (2015: n=26, 2016: n=20) or CONT (2015: n=36, 2016: n=35) treatment groups. An additional subset of boxes was included as a control for potential effects of additional foot traffic at the boxes. The boxes in this second control group did not receive supplemental food and were visited only 2–3 times per week during regular nest monitoring. All other boxes, regardless of experimental treatment, were visited and opened the same number of times to control for any effects of disturbance. Because the two control groups did not differ significantly in any of the main effects (all p > 0.058), these groups were combined in the analyses described below. Morphology All adult birds in the study were captured for banding and weighing using a nest box trap no sooner than two days after nestlings hatched (Friedman et al. 2008). Mass for each individual was determined using either a digital balance (accuracy=0.01 g) or Pesola (accuracy=0.1 g). Nestlings were weighed on day 14 post-hatch (day 0=hatch day). Nestlings were banded with an aluminum USFWS band on day 11 post-hatch, and were examined to determine sex based on plumage coloration on day 14 (Pyle 1997). Nest success A clutch was considered complete when the number of eggs did not increase for more than one day. Hatching success was quantified as the proportion of eggs in the clutch that hatched. Fledging success was determined as the proportion of nestlings that left the nest after the day 14 measurement. The typical time to fledging in eastern bluebirds is 17–19 d (Gowaty and Plissner 2015) with nestlings capable of weak flight at day 14. Nestlings absent from the nest after day 14 were considered successfully fledged, unless there was evidence of death or predation. Blood sampling and laboratory methods Blood samples (50–100 µl) were collected from all nestlings and adults within three minutes of removal from the nest box to determine CORT levels (Owen 2011). Compared to other studies on this population in which CORT has been measured, CORT levels in this study were elevated (Grindstaff et al. 2012). In this study, we collected blood samples within three minutes of removal from the nest box, rather than within three minutes of initial disturbance, as in previous studies. All blood samples were taken between 07:00 and 11:00 h to minimize variation due to circadian rhythms in hormone levels (Remage-Healey and Romero 2000). Approximately 5–10 µl of each whole blood sample was used to prepare blood smears in the field. The remainder of each whole blood sample was kept cool on ice until it was brought to the laboratory for plasma separation with a centrifuge at 1846×g for seven minutes, which took place within 4h of collection. Plasma was stored at −20°C until it was used in BKA assays, or to measure plasma CORT. Average storage length prior to BKA analysis was 158 ± 3.37 d. Storage length was not significantly related to BKA; therefore, we did not include this as a covariate in our analyses (t(76)=2.28, p=0.99). Storage length also was not significantly related to CORT levels; therefore, we did not include this as a covariate in our analyses (t(276)=0.055, p=0.956). Slides were stained with differential quik stain following kit instructions (Davis et al. 2008). A complete blood cell count was performed following the techniques described in 'Avian and exotic animal hematology and cytology’ (Clark et al. 2009, Campbell and Ellis 2013) by an expert in white blood cell identification (IK) who was blind to the identity of the treatment groups. Heterophil to lymphocyte (H:L) ratios were calculated by dividing the total number of heterophils by the total number of lymphocytes. Enzyme immunoassays (Corticosterone ELISA Kit ADI901-097, Enzo Life Sciences) were used to quantify circulating plasma CORT levels. Based on previous optimization of the assay for eastern bluebirds (unpubl.), plasma samples were diluted 1:40 in 1.5% steroid displacement reagent (SDR). All samples were run in duplicate and the corticosterone standards (20 000, 4000, 800, 500, 160 and 32 pg ml−1) were run in triplicate. The optical density of the plates was then read at 405 nm on a BioTek ELx808 microplate reader. Samples with intra-assay coefficients of variation (CVs) higher than 15% were re-run (average intra-assay CV: 6.25%). The average inter-assay CV was 14.57%. Tryptic soy agar plates were made under sterile conditions and stored at 4°C the day prior to conducting BKA assays. Escherichia coli (ATCC no. 8739, Microbiologics) stocks and dilutions were prepared the day before assays were conducted. E. coli stock solutions were prepared by adding one lyophilized bacteria pellet (5.6 × 107 CFU) to 40 ml of phosphate-buffered saline, which was then incubated at 37°C for 30 min and stored at 4°C. Plasma and bacteria were incubated in carbon dioxide independent media. Additionally, 200 µl of 200 mM L-glutamine was added to the solution prior to incubation. To perform the assay, 5 µl of plasma was combined with 100 µl of the media solution and 10 µl of bacterial working solution. The bacteria and plasma were incubated together for 30 min at 41°C. The samples were then plated on agar plates and incubated for a minimum of 12 h at 37°C. Control plates were prepared in the same manner as experimental plates; however, control plates did not contain plasma. The number of colonies on each plate was counted after the 12 h incubation. Finally, the BKA of each plasma sample was quantified as the percent difference in the number of colonies on plasma treated plates compared to the number of colonies on control plates. DATA-SPECIFIC INFORMATION FOR: Perryman_2022_cort_reduced.csv 1. Number of variables: 31 2. Number of cases/rows: 46 3. Variable List: band_number: USFWS Band attributed to the bird mass: Mass of bird Age: Age of bird, Nestling/juvenile OR Adult BoxNumber: Nest box number SEX: Male/Female Year: Year of Collection NestIDNumber: Concatenate variable of nestbox and date (grouped by the date of adult association with the box) to account for multiple nestlings in a brood. CollectionDate: Date of collection month: Month of collection Supplement_Group: Assigned experimental group supplement: Did this box receive mealworms, yes/no Brood_Size_Managed_or_Part_of: The size of the individuals affiliated brood Conc: CORT concentration pg.ml: CORT concentration in pg/mL ng.ml: CORT concentration in ng/mL log10ng.mL: Log10 of ng/mL Std_Dev: Standard deviation og Log 10 ng/mL CoeOfVariance: Coefficient of variance og Log 10 ng/mL date: Date of collection supplement_group: Numeric assignments of experimental groups, 1 = control 2 = partial 3 = full Wing.Measure.Median: Wing measure, median of three measures taken Tail.Measure.R.Median: Tail measure, median of three measures taken Tail.L.Measure.Median: Tail measure, median of three measures taken Tarsus.Measure.median: Tarsus measure, median of three measures taken Mass.measure.median: Mass measure, median of three measures taken brood_size: The size of the individuals affiliated brood number_fledged: Of the brood size, this is the number which clearly fledged fledging_success: Proportion fledged original_clutch_size: The size of the individuals affiliated clutch number_hatched: Of the clutch size, this is the number which clearly hatched hatching_success: Proportion hatched 4. Missing data codes: NA 5. Abbreviations used: NA; not applicable XX-XX (nest boxes); unique identifier of each nest box 6. Other relevant information: CORT values do not represent a baseline as noted in the manuscript. DATA-SPECIFIC INFORMATION FOR: Perryman_2022_MassDatawithNestID.csv 1. Number of variables: 25 2. Number of cases/rows: 280 3. Variable List: AssayDate: Date of Assay SampleShortID: Short name used for labeling only Band_Number: USFWS Band attributed to the bird Mass: Mass of bird Age: Age of bird, Nestling/juvenile OR Adult Male: Adult male associated with the box; Band number or NA Female: Adult female associated with the box; Band number or NA BoxNumber: Nest box number SEX: Male/Female Year: Year of collection Nestbox: Nest box number NestIDNumber: Concatenate variable of nestbox and date (grouped by the date of adult association with the box) to account for multiple nestlings in a brood. Julian: Julian Collection date CollectionDate: Collection date month: Month collected Supplement_Group: Numeric assignments of experimental groups, 1 = control 2 = paartial 3 = full supplement: Did this box receive mealworms, yes/no Brood_Size_Managed_or_Part_of: The size of the individuals affiliated brood Conc: CORT concentration Dilution: Dilution prior to assay pg/ml: CORT Concentration in pg/mL ng/ml: CORT Concentration in ng/mL log10ng.mL: Log10 of ng/mL Std_Dev: Standard deviation of Log 10 ng/mL CoeOfVariance: Coefficient of variance of Log 10 ng/mL 4. Missing data codes: NA; Not Applicable 5. Abbreviations used: NA; not applicable XX-XX (nest boxes); unique identifier of each nest box 6. Other relevant information: Where "Male" or "Female" equals NA, this indicates we did not capture this adult with this nesting attempt. DATA-SPECIFIC INFORMATION FOR: Perryman_2022_HLFilewithNestID.csv 1. Number of variables: 36 2. Number of cases/rows: 92 3. Variable List: spot: Organizational variable only year: Year of Collection bandnumber: USFWS Band attributed to the bird age: Age of bird, Nestling/juvenile OR Adult supplementgroup: Numeric assignments of experimental groups, 1 = control 2 = partial 3 = full mass: Mass of bird CORT: CORT, Log10 of ng/mL bka: Percent bacterial killing nestsize: Brood Size sex: Male/Female boxnumber: Nest box number NestID: Concatenate variable of nestbox and date (grouped by the date of adult association with the box) to account for multiple nestlings in a brood. collectiondate: collection date month: Month of collection wcper10hpf: White cells per 10 high power fields het_cnt: Heterophil count lym_cnt: Lymphocyte count mon_cnt: Monocyte count eos_cnt: Eosinophil count bas_cnt: Basophil count total_cnt: Total Count wceperuL: Wc per uL het_perc: Heterophil percentage of count lym_perc: Lyphocyte percentage of count mon_perc: Monocyte percentage of count eos_perc: Eosinophil percentage of count bas_perc: Basophil percentage of count hetperuL: Heterophils per uL lymperuL: Lymphocytes per uL monperuL: Monocytes per uL eosperuL: Eosinophils per uL basperuL: Basophils per uL hlratio: Literal H:L ratio log10hl: Log 10 transformed h:l ratio hemopar: Hemoparasites notes: Specific notes on slide presentation 4. Missing data codes: NA 5. Abbreviations used: NA; not applicable XX-XX (nest boxes); unique identifier of each nest box 6. Other relevant information: Where NA information is missing DATA-SPECIFIC INFORMATION FOR: Perryman_2022_CORTDatawithNestID.csv 1. Number of variables: 26 2. Number of cases/rows: 276 3. Variable List: AssayDate: Assay date Storage Length: Length of storage in the freezer; Assay date - collection date SampleShortID: Short label name for labeling Band_Number: USFWS Band attributed to the bird Mass: Mass of bird Age: Age of bird, Nestling/juvenile OR Adult Male: Adult male associated with the box; Band number or NA Female: Adult female associated with the box; Band number or NA BoxNumber: Nestbox number SEX: Male/Female Year: Year of collection Nestbox: Nest box number NestIDNumber: Concatenate variable of nestbox and date (grouped by the date of adult association with the box) to account for multiple nestlings in a brood. Julian: Julian date CollectionDate: Date of collection month: Month of collection Supplement_Group: Numeric assignement of supplement group, 1 = control 2 = partial 3 = full supplement: Did this box revceive supplement, yes/no Brood_Size_Managed_or_Part_of: The size of the individuals affiliated brood Conc: CORT concentration Dilution: Dilution prior to assay pg/ml: CORT concentration in pg/mL ng/ml: CORT concentration in ng/mL log10ng.mL: Log10 of ng/mL Std_Dev: Standard deviation og Log 10 ng/mL CoeOfVariance: Coefficient of variance og Log 10 ng/mL 4. Missing data codes: NA 5. Abbreviations used: NA; not applicable XX-XX (nest boxes); unique identifier of each nest box 6. Other relevant information: CORT values do not represent a baseline as noted in the manuscript. DATA-SPECIFIC INFORMATION FOR: Perryman_2022_FullBKADatawithNestID.csv 1. Number of variables: 17 2. Number of cases/rows: 118 3. Variable List: year: Year of collection Nestbox: Nest box number NestIDNumber: Concatenate variable of nestbox and date (grouped by the date of adult association with the box) to account for multiple nestlings in a brood. band_number: USFWS Band attributed to the bird age: Adult/Nestling age_code: 0 = Adult, 1 = Nestling sex: F = Female, M = Male gender_code: 1= Female, 0 = Male treatment: Experiment group numeric code. 0 = Et Cont, 1 = Cont, 2 = Part, 3 = Full Plate_A: Colonies on plate A Plate_B: Colonies on Plate B average: Average number of colonies on the plates perc_killing: Percent difference of experimental plates to the control collection_date: Date of collection month: Month of collection nest_size: brood size mass: Mass of bird 4. Missing data codes: NA 5. Abbreviations used: NA; not applicable XX-XX (nest boxes); unique identifier of each nest box 6. Other relevant information: None DATA-SPECIFIC INFORMATION FOR: Perryman_2022_bka_reduced.csv 1. Number of variables: 30 2. Number of cases/rows: 39 3. Variable List: band_number: USFWS Band attributed to the bird year.x: Year of collection date: Date of collection box_number: Nestbox number Supplement_Group: Supplement group assigned sex: Sex of the bird Wing.Measure.Median: Median measure of wing of three measures taken Tail.Measure.R.Median: (Right) Median measure of tai of three measures taken Tail.L.Measure.Median: (Left) Median measure of wing of three measures taken Tarsus.Measure.median: Median measure of tarsus of three measures taken Mass.measure.median: Median measure of mass of three measures taken brood_size: Brood size number_fledged: Of those which hatched, this is the number that fledged fledging_success: Proportion fledged original_clutch_size: Original clutch size number_hatched: Number successfully hatched from a clutch hatching_success: Proportion successfully hatched nestbox: Next box number NestIDNumber: Concatenate variable of nestbox and date (grouped by the date of adult association with the box) to account for multiple nestlings in a brood. age.y: Age of the bird sex.y: Sex of the bird treatment: Numeric supplement group assignment, 1 = control, 2 = partial, 3 = full Plate_A: Plate A colony count Plate_B: Plate B colony count average: Average colonies on each plate perc_killing: Percent difference bacterial killing ability collection_date: Date of collection month: Month of collection nest_size: Brood size mass: Mass of bird 4. Missing data codes: NA 5. Abbreviations used: NA; not applicable XX-XX (nest boxes); unique identifier of each nest box 6. Other relevant information: None DATA-SPECIFIC INFORMATION FOR: Perryman_2022_hl_reduced.csv 1. Number of variables: 49 2. Number of cases/rows: 40 3. Variable List: band_number: USFWS Band attributed to the bird age.x: Age of the bird, all adults in this dataset supplementgroup: Numeric supplement group assignment where 1 = control, 2 = Partial, 3 = Full mass: Mass of the bird CORT: Respective CORT value bka: Respective percent killing nestsize: Brood size sex.x: Sex of the bird (male or female) boxnumber: Nest box number NestID: Concatenate variable of nestbox and date (grouped by the date of adult association with the box) to account for multiple nestlings in a brood. collectiondate: Date of collection month: month of collection wcper10hpf: White cells per 10 high power fields het_cnt: Heterophil count lym_cnt: Lymphocyte count mon_cnt: Monocyte count eos_cnt: Eosinophil count bas_cnt: Basophil count total_cnt: Total Count wceperuL: Wc per uL het_perc: Heterophil percentage of count lym_perc: Lyphocyte percentage of count mon_perc: Monocyte percentage of count eos_perc: Eosinophil percentage of count bas_perc: Basophil percentage of count hetperuL: Heterophils per uL lymperuL: Lymphocytes per uL monperuL: Monocytes per uL eosperuL: Eosinophils per uL basperuL: Basophils per uL hlratio: Literal H:L ratio log10hl: Log 10 transformed h:l ratio hemopar: Hemoparasites year.y: Year of collection date: Date of collection box_number: Nest box number supplement_group_: Numeric supplement group where 0 = extra traffic control, 1 = control, 2 = partial, 3 = full sex.y: Sex of the bird Wing.Measure.Median: Median measure of wing of three measures taken Tail.Measure.R.Median: (Right) Median measure of tai of three measures taken Tail.L.Measure.Median: (Left) Median measure of wing of three measures taken Tarsus.Measure.median: Median measure of tarsus of three measures taken Mass.measure.median: Median measure of mass of three measures taken brood_size: Brood size number_fledged: Of those which hatched, this is the number that fledged fledging_success: Propotion fledged original_clutch_size: Original clutch size number_hatched: Number successfully hatched from a clutch hatching_success: Proportion successfully hatched 4. Missing data codes: NA 5. Abbreviations used: NA; not applicable XX-XX (nest boxes); unique identifier of each nest box 6. Other relevant information: Where NA in BKA and CORT columns, data was not collected