BioRodDis: Managing BIOdiversity in forests and urban green spaces - Dilution and amplification effects on RODent microbiomes and rodent-borne DISeases

Coordinator: Charbonnel Nathalie

BioRodDis includes occurrence of small terrestrial mammals from forests and urban parks in five countries: Belgium, France, Germany, Ireland and Poland.

The BioRodDis project aims at elucidating the interlinkages between small mammal biodiversity and diseases at local and European scales using standardised assessments of biodiversity and disease risks. In particular, the dilution/amplification effect is assessed by integrating new key research directions, i.e. host microbiome and multiple pathogen diversity levels on one hand, seasonal and multi-annual dynamics on the other hand, including climate change scenarios and interactions with socioeconomic contexts. More information is provided in the website: https://www6.inrae.fr/biodiversa-bioroddis.

This project is funded through the 2018-2019 BiodivERsA joint call for research proposals, under the BiodivERsA3 ERA-Net COFUND programme, and with the funding organisations ANR (France), DFG (Germany), EPA (Ireland), FWO (Belgium) and NCN (Poland).

This study includes six sampling sites (2 forest reserves, 2 public forests, 2 urban parks) in eastern France (see Figs 1, 2). The current dataset extends from February 2020 to June 2022.

Figure 1.  

General map of the studied areas and their position in France. Localities are indicated by circles, red circles = urban parks; green circles = managed forests; light green circles = protected forests. FRPLTO: Lyon, Parc de la Tête d’Or; FRPDLL: Marcy l'étoile, Domaine Lacroix Laval; FRFCOR: Cormaranche en Bugey; FRFGRI: Arvière, La Griffe au diable; FRFMIG: Mignovillard; FRFGLA: Esserval-Tartre, La Glacière.

Figure 2.  

Pictures of some of the trapping lines within each locality. FRPLTO: Lyon, Parc de la Tête d’Or; FRPDLL: Marcy l'étoile, Domaine Lacroix Laval; FRFCOR: Cormaranche en Bugey; FRFGRI: Arvière, La Griffe au diable; FRFMIG: Mignovillard; FRFGLA: Esserval-Tartre, La Glacière

Figure 3.  

Maps of the trapping lines (red points) represented at a local scale for each of the following localities: A- FRPLTO: Lyon, Parc de la Tête d’Or; B- FRPDLL: Marcy l'étoile, Domaine Lacroix Laval; C- FRFCOR: Cormaranche en Bugey; D- FRFGRI: Arvière, La Griffe au diable; E- FRFMIG: Mignovillard; F- FRFGLA: Esserval-Tartre, La Glacière. The landscape around the trapping lines is represented by different colours corresponding to water bodies (blue; corine land cover 5), Forest and semi-natural areas (green, Forest database), artificial areas (grey; corine land cover 1) or other land-cover classes (white).

Small mammals were live-trapped using INRA traps for all rodents and shrews, except rats (an INRA trap is composed of a 160 × 50 × 50-mm aluminium tunnel, coupled with a 150 × 70 × 70-mm plastic rest box). Rats were trapped using meshed traps of 500 x 175 x 175 cm (Fig. 4).

Figure 4.  

Pictures illustrating the different steps leading to trapping and individual information. A- Preparation of traps (bait, cotton, trap number); B- An INRA trap sets in a forest; C- Meshed trap sets in a urban park; D- Trap checking in the morning (masks and gloves are important to protect animals and humans from zoonotic agents); E- Capture information recorded on a digital tablet (this picture was taken during the lockdown, which explains the mask); F- An INRA trap used to capture small mammals; G- A meshed trap containing a rat (Rattus norvegicus); H- A plastic rest box containing a woodmouse (Apodemus sylvaticus) that is released; I- Disinfection of traps (masks and gloves are important to protect humans from zoonotic agents).

Six to ten lines of 20 INRA live-traps and one meshed trap, with about 3 m interval, were set up so that each sampling locality consisted of a few km² area (see Table 1, Fig. 3). The rest box was filled with hydrophobic cotton to provide good conditions for trapped animals. All traps were baited with sunflower seeds, carrots and sardine. Each trap was geolocated. The traps were checked daily, early in the morning. Trapping session per locality lasted at least three nights, except when abundances were too low and new trap lines had to be set up. Moreover, because we encountered difficulties in trapping rats and mice in urban parks, traps were set in particular places where animals had been detected and they were removed after 10 to 11 nights (see details in Suppl. material 1). For ethical reasons, we could not trap more than 35 animals per species, locality and session. As such, these were released individuals when we reached 35 individuals of a species for a given locality and session. Relative abundance (e.g. trapping success) might nevertheless be estimated using all capture information gathered from the three first trapping nights (see details in Suppl. material 1). In addition, note that rare shrews were released for all sessions, except autumn 2020 and spring 2021, to limit our impact on these populations. Altogether, this dataset gathered information on 1593 small mammals and 21,494 trap-nights. The details of the number of animals dissected per locality and session are provided in Table 1.

All captured animals were determined to species level using morphological criteria in the field or using molecular methods when necessary (CO1 sequencing for Microtus species and shrews, Pagès et al. 2010 or DNA fingerprinting for Apodemus species, Bugarski-Stanojević et al. 2013, Suppl. material 2). Animal dissections and measurements were performed according to the protocols described in Herbreteau et al. (2011). Biological samples (organs, blood, parasites) were deposited in a public biobank at the Centre de Biologie pour la Gestion des Populations (CBGP) in Montpellier, France. Metadata associated with the samples and occurrence set were deposited in the CBGP small mammal database (BPM, Base Petits Mammifères, https://doi.org/10.15454/WWNUPO).

Fieldwork: The different steps of the fieldwork are detailed in Fig. 4 and in the ‘sampling description’ section above. All information related to traps and captures is recorded on paper sheets and/or on digital tablets using the KoBo software. These results are described in Suppl. material 1.

Animal dissection: On the day of capture, animals are anaesthetised using isofluorane and euthanised by cervical dislocation, as recommended by Mills et al. (1995). Morphological measures are taken, amongst which were mass and head-body, tail and hind-foot length. Species identification is recorded, based on this morphological information. In case of doubt, only the genus is noted. Sex is recorded and sexual maturity is inferred considering testes length and position (abdominal or descended into the scrotal sac) and seminal vesicle development (visible or not) for males or vaginal opening, nipples (visible or not), pregnancy and uterus size (very thin or thick) for females. Ectoparasites (ticks and fleas) are collected and stored in ethanol at 96°C. Macroparasites that are detected in the cavity are recorded (e.g. cestodes on the liver). This protocol is described in Herbreteau et al. (2011). All information related to individual dissections is recorded on paper sheets and on an MS Еxcel file. Several samples are collected from each animal for further mammal genetics and parasitological (bacteria, viruses, macroparasites, protozoa) analyses. The heart is removed and is stored in PBS at -20°C. The lungs, rectum and a piece of liver are collected and are stored in RNA shield solution (one day at 4° then -20°C). The spleen, the digestive tract, the ears, the left hind-foot and a piece of the tail are collected and are stored in 96% ethanol. Faeces and a piece of lungs are collected and are stored at -20°C. All these samples are given a unique identifier and datamatrix. Metadata associated with the samples and occurrence set were deposited in the CBGP small mammal database (BPM, https://doi.org/10.15454/WWNUPO).

Lastly, all waste products were eliminated using the official incineration process, that is a safe way of destroying hazardous potentially infectious waste, protecting both human and the environment.

The different steps of the dissection are detailed in Fig. 5.

Figure 5.  

Pictures illustrating the different steps during small mammal dissection. Masks (FFP2), gloves and glasses protect the experimenter who manipulates and dissects the animals. A- All tubes are prepared in advance with unique identifier and datamatrix. One colour is dedicated to each type of sample (e.g. red tip for heart in PBS, purple tip for liver in RNA later). Morphological information was recorded on a paper sheet. Dissection instruments are disinfected between each animal. B- The experimenter is preparing the animal (here a brown rat Rattus norvegicus) for the dissection. C- The experimenter is weighing the animal (here a woodmouse Apodemus sylvaticus) for the dissection. D- A male common vole (Microtus arvalis). E- Tubes corresponding to liver samples, with unique identifiers and datamatrices, stored in a clearly identified box (unique identifier and datamatrix).

Ethical statements: Animal capture and handling have been conducted according to the French and European regulations on care and protection of laboratory animals (French Law 2001-486 issued on 6 June 2001 and Directive 2010/63/EU issued on 22 September 2010). The CBGP laboratory has approval (D-34-169-003) from the Departmental Direction of Population Protection (DDPP, Hérault, France) and from the regional ethical committee (Comite d'Ethique pour l'Expérimentation Animale Languedoc Roussillon), for the sampling of rodents and the storage and use of their tissues.

Molecular analyses: DNA was extracted from kidney using Qiagen DNeasy® Blood & Tissue Kit. The specific identification of Microtus species and shrews was next performed using CO1 sequencing (BatL5310 and R6036R), following Pagès et al. (2010). The specific identification of Apodemus species was determined using DNA fingerprinting (AP-PCR) and the E8S primer, following Bugarski-Stanojević et al. (2013).

The data were collected in six forested areas in eastern France, within three administrative Departments (Rhône, Ain, Jura).

44.84 and 48.633 Latitude; 2.021 and 7.734 Longitude.