DOI: 10.5061/dryad.7d7wm3888
title: Data from: Does nocturnal light pollution impair immune function in a wild-living amphibian?

description: Light pollution is among the most rapidly growing anthropogenic stressors
 on Earth. Experimental studies suggest that light pollution have
 deleterious effects on metabolism and immunity (among others) of animals.
 The aim of this study was to assess, for the first time, whether the
 effects of light pollution on immunity previously reported in experimental
 studies, are also observable in a wild species. To do so, we measured a
 large set of body condition and immune traits, to obtain a comprehensive
 picture of individuals’ health, in 15 natural populations of common toads
 (Bufo bufo) distributed along a gradient of exposure to light pollution.
 We measured these sites' exposure to light pollution and because we
 expected that other landscape features would covary with light pollution,
 additional landscape variables associated with urbanisation and climate
 were extracted: altitude (as a proxy of ambient temperature); the
 percentage of forest, urban, agricultural and wetlands areas in a buffer
 of 500 meters around the pond of toads’ capture were calculated. Ethics.
 The protocols of capture and blood sampling of toads were approved by the
 French Government via prefectural decrees n° 69-2023-01-20-00005 for the
 Rhône department; n° DDPP01-23-044 for the Ain department; n°
 38-2023-02-21-00003 for the Isère department. All experiments were
 performed in accordance with guidelines and regulations of the Ethical
 Committee of Lyon 1 University (project APAFIS #41025-2023021713471183,
 20th February, 2023).
description: # Data from: Does nocturnal light pollution impair immune function in a
 wild-living amphibian? Dataset DOI:
 [10.5061/dryad.7d7wm3888](https://doi.org/10.5061/dryad.7d7wm3888) ##
 Description of the data and file structure **Ethics** The protocols of
 capture and blood sampling of toads were approved by the French Government
 *via* prefectural decrees n° 69-2023-01-20-00005 for the Rhône department;
 n° DDPP01-23-044 for the Ain department; n° 38-2023-02-21-00003 for the
 Isère department. All experiments were performed in accordance with
 guidelines and regulations of the Ethical Committee of Lyon 1 University
 (project APAFIS #41025-2023021713471183, 20^th^ February, 2023). **Sites**
 We selected 15 populations of toads in sites around a 50-km radius from
 the Lyon Metropole in France. This area characterized by its variety of
 environments, from dense urban areas (Lyon metropolis, 1.4 million
 inhabitants), residential areas/villages, agricultural zones to isolated
 natural areas. The sites have been chosen in this area to be located on a
 gradient of exposure to light pollution. Exposure of the sites to light
 pollution has been measured with a sensitive lightmeter SQM (Sky Quality
 Meter, Unihedron – following the methodology of Spoelstra 2014; Kyba *et
 al*. 2015): the highest the value obtained is, the darkest is the site. As
 an example, an urban night sky will display a value around 16-17 and a
 dark site with a starry sky will display a value around 21-22. For this,
 we used a tripod with a fixed structure to attach the SQM at similar
 height (1 m above the ground) and position (at 90° or zenith, 45° and 0°
 or horizon; at North/West/Est/South directions) in each site – giving us a
 total of 12 standardized measures in each site. These measures have been
 recorded on moonless sky to ensure that only the illuminance from light
 pollution was captured. Recordings were also conducted during periods of
 dense cloud cover to obtain the maximal value of light pollution exposure
 in a site, as cloud cover amplifies light pollution (Kyba *et al.* 2011).
 Because we expected that other landscape features would covary with light
 pollution, additional landscape variables associated with urbanisation and
 climate were extracted: altitude (as a proxy of ambient temperature); the
 percentage of forest, urban, agricultural and wetlands areas in a buffer
 of 500 meters around the pond of toads’ capture were calculated using QGIS
 software. **Captures and sampling** Toads were captured at spring during
 the short period of time when they are migrating from their hibernation
 sites in the woods to ponds to breed. In this study, they were captured
 between the 8^th^ and the 16^th^ of March, 2023, but on the same night for
 all individuals of a given site. As the sex ratio is strongly biased
 towards males in this species, only male toads were captured here. Ten
 toads per sites were captured, *i.e.,* 150 toads in total. After capture,
 toads were weighed to the nearest g using a digital scale and a blood
 sample was taken by cardiocentesis (25G heparinized and sterile needles)
 after local anesthesia with a lidocaine-prilocaine cream (EMLA 5%). The
 volume of blood samples taken represented a maximum of 1% of the
 individual body mass. After sampling, toads were immediately released on
 site. A drop of fresh blood was immediately used for a blood smear of each
 individual; while a small amount of blood was collected in an heparinized
 microcapillary tube for hematocrit determination (centrifuged 5 minutes at
 10,000 g). Hematocrit (%) is an hematological parameter that reflect
 changes in an animal's oxygen-carrying capacity. The remaining blood
 sample was centrifuged for 5 minutes at 1,000 g, after which the plasma
 was transferred into an eppendorf and stored at -80°C. **Immune
 phenotype** We measured a large set of immune traits in order to depict
 both the innate and the adaptive responses of toads, encompassing humoral
 and cell-mediated components. First, we assessed the cellular part of
 innate and adaptive immunity, by determining the composition of the white
 blood cells (WBC) populations, *i.e*., five different cell types, based on
 the identification of the first hundred WBC in Wright-Giemsa-stained blood
 smears. Among these, neutrophils and monocytes are phagocytes involved in
 the innate response (Claver & Quaglia 2009). Eosinophil and basophil
 function remain poorly understood in amphibians. Eosinophil seem to be
 associated with defense against internal parasites such as trematode
 infections but also in response to pollutants (Kiesecker 2002; Claver
 & Quaglia 2009). Basophils play a role in early inflammation and in
 innate immune responses by releasing proteins such as toxins (Claver &
 Quaglia 2009). Cell-mediated adaptive immunity was assessed by lymphocyte
 counts including both T and B cells (see blood smears above), B cells
 being particularly involved in the production of antibodies. We also
 determined the total proportion of white blood cells relative to
 erythrocytes, based on the identification and count of the first hundred
 cells in randomly selected areas of the blood smears. This metrics was
 used as a proxy of investment in cellular immunity. The humoral part of
 innate and adaptive immunity was then determined by measuring levels of
 the following plasma proteins: alpha1-globulins, alpha2-globulins,
 beta1-globulins, beta2-globulins and gamma-globulins. Alpha- and
 beta-globulins fractions include acute phase proteins (APPs) of the
 inflammatory response. Gamma-globulins, or immunoglobulins, represent the
 majority of circulating antibodies and thus their concentration reflects
 adaptive immunity. We also calculated the albumin to globulins ratio
 (A:G), as an altered A:G ratio could indicate changes in physiological
 status. In particular, a decrease in albumin synthesis and/or an increase
 in the synthesis of one of the APPs can reflect inflammation (Zaias &
 Cray 2002). The total protein content (in mg/mL) was first assessed using
 Pierce BCA Protein Assay Kit (ThermoFisher Scientific, reference 23225),
 followed by automatic agarose gel electrophoresis (HYDRASYS, Sebia, Evry,
 France) that separates albumin and the 5 fractions of globulins (α1, α2,
 β1, β2, and γ). Finally, albumin levels obtained (mg/mL) also reflects the
 blood level of protein resources of an individual. In addition, we
 measured the level of haptoglobin as a proxy of inflammation. These
 proteins bind free hemoglobin in order to mitigate damages caused by
 reactive oxygen components produced during inflammation (Quaye *et al.*
 2008). We measured haptoglobin using 7.5 µL of plasma with a
 commercially-available assay (TP801, Phase Haptoglobin Assay, Tri-Delta
 Diagnotics, United States) and following recommendations from Matson *et
 al.* (2012, in birds) and Lorrain-Solignon *et al.* (2022, in frogs). This
 colorimetric assay measures the preservation of the peroxidase activity of
 bounded hemoglobin, which should be proportional to the concentration of
 the hemoglobin-binding proteins and thus inflammation in the plasma
 sample. All samples were run in duplicates. ### Files and variables ####
 File: Dataset_DRYAD_FE202500618_bufodata.csv **Description:**  #####
 Variables * Bufo_id: unique identifier for each individual * Site_number:
 number of the site (1 to 15), similar in the second dataset available on
 sites characteristics. * Size_cm: size of individuals in centimeters *
 Bodymass_g: body mass of individuals in grams * Hematocrit_percent_plasma:
 percentage of the plasma fraction of the hematocrit *
 Hematocrit_percent_redbloodcells: percentage of the red blood cells
 fraction of the hematocrit * Proteins_plasma_mgml: dosage of total plasma
 proteins in mg/ml used for the calculation of the concentration value of
 the different plasma proteins (measured first in percentage) *
 Albumin_percent: Percentage of the different forms of plasma proteins
 assessed by electrophoresis, here albumin * Alpha1_percent: Percentage of
 the different forms of plasma proteins assessed by electrophoresis, here
 alpha-1-globulins * Alpha2_percent: Percentage of the different forms of
 plasma proteins assessed by electrophoresis, here alpha-2-globulins *
 Alpha_total_percent: Percentage of the different forms of plasma proteins
 assessed by electrophoresis, here total alpha-globulins (alpha1 + alpha2)
 * Beta1_percent: Percentage of the different forms of plasma proteins
 assessed by electrophoresis, here beta-1-globulins * Beta2_percent:
 Percentage of the different forms of plasma proteins assessed by
 electrophoresis, here beta-2-globulins * Beta3_percent: Percentage of the
 different forms of plasma proteins assessed by electrophoresis, here
 beta-3-globulins * Beta_total_percent: Percentage of the different forms
 of plasma proteins assessed by electrophoresis, here tota beta-globulins
 (beta1 + beta2 + beta3) * Gamma_percent: Percentage of the different forms
 of plasma proteins assessed by electrophoresis, here gamma-globulins *
 Globulines_total_percent: Percentage of the different forms of plasma
 proteins assessed by electrophoresis, here total globulins (alpha + beta +
 gamma) * Albumin_mgml: Concentrations values (in mg/ml) of the different
 forms of plasma proteins calculated from the percentages (columns H to Q)
 and the total protein concentration assay (column G) : here albumin *
 Alpha1_mgml: Concentrations values (in mg/ml) of the different forms of
 plasma proteins calculated from the percentages (columns H to Q) and the
 total protein concentration assay (column G) : here, alpha-1-globulins *
 Alpha2_mgml: Concentrations values (in mg/ml) of the different forms of
 plasma proteins calculated from the percentages (columns H to Q) and the
 total protein concentration assay (column G) : here, alpha-2-globulins *
 Alpha_total_mgml: Concentrations values (in mg/ml) of the different forms
 of plasma proteins calculated from the percentages (columns H to Q) and
 the total protein concentration assay (column G) : here, total
 alpha-globulins (alpha1 + alpha2) * Beta1_mgml: Concentrations values (in
 mg/ml) of the different forms of plasma proteins calculated from the
 percentages (columns H to Q) and the total protein concentration assay
 (column G) : here, beta1-globulins * Beta2_mgml: Concentrations values (in
 mg/ml) of the different forms of plasma proteins calculated from the
 percentages (columns H to Q) and the total protein concentration assay
 (column G) : here, beta2-globulins * Beta3_mgml: Concentrations values (in
 mg/ml) of the different forms of plasma proteins calculated from the
 percentages (columns H to Q) and the total protein concentration assay
 (column G) : here, beta3-globulins * Beta_total_mgml: Concentrations
 values (in mg/ml) of the different forms of plasma proteins calculated
 from the percentages (columns H to Q) and the total protein concentration
 assay (column G) : here, total beta-globulins (beta1 + beta2 + beta3) *
 Gamma_mgml: Concentrations values (in mg/ml) of the different forms of
 plasma proteins calculated from the percentages (columns H to Q) and the
 total protein concentration assay (column G) : here, gamma-globulins *
 Globulines_total_mgml: Concentrations values (in mg/ml) of the different
 forms of plasma proteins calculated from the percentages (columns H to Q)
 and the total protein concentration assay (column G) : here, total
 globulins (alpha + beta + gamma) * AlbuminGlobulins_ratio: ratio albumin /
 globulins * Neutrophils_percent: Percentage of the 5 forms of white blood
 cells assessed from blood smears reading, here neutrophils *
 Eosinophils_percent: Percentage of the 5 forms of white blood cells
 assessed from blood smears reading, here eosinophils * Basophils_percent:
 Percentage of the 5 forms of white blood cells assessed from blood smears
 reading, here basophils * Lymphocytes_percent: Percentage of the 5 forms
 of white blood cells assessed from blood smears reading, here lymphocytes
 * Monocytes_percent: Percentage of the 5 forms of white blood cells
 assessed from blood smears reading, here monocytes *
 Whitebloodcells_percent: Percentage of white blood cells assessed from
 blood smears reading * Redbloodcells_percent: Percentage of red blood
 cells assessed from blood smears reading * Haptoglobin_mgml: Haptoglobin
 concentration (in mg/ml) assayed in plasma #### File:
 Dataset_DRYAD_FE202500618_sitesdata.csv **Description:**  ##### Variables
 * Site_number: number of the site (1 to 15), similar in the dataset
 available on Bufo bufo physiological data. * Site_name: name of the city
 of the site * Hamlet: precise name of the pond/hamlet * Altitude_meters:
 Altitude of the site in meters * Latitude: latitude of the site *
 Longitude: longitude of the site * Mean_SQMvalue: mean SQM value at night
 for each site (Sky Quality Meters, i.e. for nocturnal light pollution
 assessment). Values obtained on a cloudy night, no moon, and mean value
 calculated from columns H to S. * SE_SQMvalue: SE of mean SQM value at
 night, calculated from columns H to S * SQMvalue_north_180: unique SQM
 value measured in the site in the North direction at 180° (using a tripod
 for standardization). This value is used for the calulation of the mean
 SQM value of each site (column G). * SQMvalue_north_45: unique SQM value
 measured in the site in the North direction at 45° (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_north_azimuth: unique SQM value
 measured in the site in the North direction at azimuth (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_south_180: unique SQM value
 measured in the site in the South direction at 180° (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_south_45: unique SQM value
 measured in the site in the South direction at 45° (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_south_azimuth: unique SQM value
 measured in the site in the South direction at azimuth (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_east_180: unique SQM value
 measured in the site in the East direction at 180° (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_east_45: unique SQM value
 measured in the site in the East direction at 45° (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_east_azimuth: unique SQM value
 measured in the site in the East direction at azimuth (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_west_180: unique SQM value
 measured in the site in the West direction at 180° (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_west_45: unique SQM value
 measured in the site in the West direction at 45° (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * SQMvalue_west_azimuth: unique SQM value
 measured in the site in the West direction at azimuth (using a tripod for
 standardization). This value is used for the calulation of the mean SQM
 value of each site (column G). * Forest_percent_500maroundpond: Landscape
 variables in the 500 meters area around the site (in %, calculated using
 QGIS), here percentage of forest areas. * Urban_percent_500maroundpond:
 Landscape variables in the 500 meters area around the site (in %,
 calculated using QGIS), here percentage of urbanized areas (buildings,
 houses, main roads). * Agriculture_percent_500maroundpond: Landscape
 variables in the 500 meters area around the site (in %, calculated using
 QGIS), here percentage of agricultural lands. *
 Wetlands_percent_500maroundpond: Landscape variables in the 500 meters
 area around the site (in %, calculated using QGIS), here percentage of
 wetlands. ## Access information Other publicly accessible locations of the
 data: * NA Data was derived from the following sources: * NA