Dataset DOI: 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