DOI: 10.5061/dryad.5tb2rbpf0
title: Fecal corticosterone metabolite levels in two closely related rodent species in a sub-Mediterranean environment

description: Glucocorticoids regulate many physiological functions and play an
 important role in coping with challenging stimuli. The non-invasive
 assessment of glucocorticoids is increasingly used as a tool to evaluate
 individual and population health status in wild animals. Given the crucial
 role of rodents in forest ecosystems, it may be useful to study the
 glucocorticoid profile of these species to find possible links with the
 environmental characteristics in which they live and facilitate the
 development of biodiversity management plans. We studied two closely
 related species that are the main representatives of the ground-dwelling
 rodent communities in sub-Mediterranean forested areas: Apodemus
 sylvaticus and A. flavicollis. Fecal corticosterone metabolite (FCM)
 levels of animals captured in a Mediterranean agroforestry system were
 assessed. We found that A. sylvaticus males excreted lower FCM levels than
 females, while A. flavicollis males showed higher FCM levels than females.
 Males of the two species excreted similar FCM levels, while higher FCM
 levels were recorded in A. sylvaticus females than in their A. flavicollis
 counterparts. The FCM levels in both species were similar between breeding
 conditions, seasons, and habitat types. The results of our exploratory
 investigation suggest that traditional silviculture may not trigger the
 activity of the hypothalamus-pituitary-adrenal axis. Further studies are
 required for a more detailed examination of how environmental factors
 affect FCM levels. Long-term studies may disclose possible effects of
 interannual environmental factor variability in ground-dwelling rodents.
description: To evaluate the effects induced by different habitat types on
 mouse FCM levels we selected fifteen sampling sites within the three main
 habitat types: coppiced deciduous woodlands, conifer plantations and
 long-fallow fields. Trapping sessions were conducted
 every other month from September 2011 to September 2012. In each sampling
 site, handmade LOT (Locasciulli Osvaldo Trap) live traps (9 x 8 x 23 cm)
 (Locasciulli et al. 2015), were placed on a 60 x 60 m grid. The grids were
 placed at least 100 m away from the habitat border. The minimum distance
 between two grids was around 500 m. The traps were equipped with nesting
 material (hemp) and baited with sunflower seeds, apples, and peanut
 butter. In each session, the traps were checked every morning for three
 consecutive days. To evaluate the effects induced by
 trap confinement on mouse FCM levels and post-excretion FCM stability,
 opportunistic trapping events were carried out using food-baited L.O.T.
 traps placed in transects (20 traps, 10 m spacing). In particular, to test
 if the time mice spent in the traps was longer than their gut transit
 time, we added shredded colored waxes (Giotto be-bè®, Fila, Spain) to the
 bait as indigestible nontoxic markers (Serres-Corral et al. 2021). Traps
 remained active for three consecutive nights and were checked every
 morning. The use of shredded colored waxes made it
 possible to distinguish the fecal boluses excreted in the trap before the
 digestion of the bait (i.e. boluses excreted in the 8-10 hours following
 capture) from those excreted afterwards. Almost all of the animals
 captured (around 90%) excreted both uncolored and colored fecal boluses
 inside the traps. The FCM levels of uncolored and colored fecal samples
 were assessed separately to compare their values (<em>A.
 sylvaticus</em>: 10 males and 10 females; <em>A.
 flavicollis</em>: 10 males and 9 females).  A
 possible influence of factors such as temperature, humidity, and bacterial
 enzymes on the types and concentrations of fecal glucocorticoid
 metabolites after defecation has been described in some mammal species
 (Touma & Palme 2005). For this reason, certain studies assessing
 FCM levels in wild rodent species (e.g. Navarro-Castilla et al. 2014;
 Navarro-Castilla & Barja 2014) chose to collect only fresh fecal
 boluses (i.e. only feces with a soft texture) to avoid any issues
 regarding hormone metabolite stability. However, no data are available on
 the post-excretion stability of hormone metabolites in mice. To evaluate
 post-excretion FCM level stability, we collected fresh boluses excreted by
 the mice during handling for identification. The boluses of different
 animals from both species and sexes were mixed to obtain several sample
 pools (n = 6) for statistical analyses. Each pool was mixed to be
 homogeneous and five aliquots of 100 mg were taken from each pool for
 sampling. One aliquot from each set was immediately frozen at -20 °C while
 the others were left at ambient temperature for different time periods (6,
 12, 18, 24 h) before being stored at −20 °C until hormone extraction was
 carried out.
description: # Fecal corticosterone metabolite levels of *Apodemus sylvaticus* and *A.
 flavicollis*
 [https://doi.org/10.5061/dryad.5tb2rbpf0](https://doi.org/10.5061/dryad.5tb2rbpf0) ## Description of the data and file structure The datasets as_f_color.xls, as_m_color.xls, af_f_color.xls, af_m_color.xls, and stability.xls were used to evaluate fecal corticosterone metabolites levels time excretion and stability differences postexcretion. The datasets dataset.xls, dataset_flavicollis.xls and dataset_sylvaticus.xls were used to investigate the effects of individual (i.e. sex and breeding condition) and environmental (i.e. season and habitat type) variables on the fecal corticosterone metabolite levels in *Apodemus sylvaticus* and *Apodemus flavicollis* ### Files and variables #### File: af\_f\_color.xls **Description:** Female *Apodemus flavicollis* uncolored and colored fecal boluses collected from the live-trap  ##### Variables * IDanimal: number of the trap and date of animal capture * type: type of fecal boluses, uncolored = fecal boluses that did not have any trace of colored wax marker; colored =  boluses with marked evidence or wax colored traces * species: species of the animals; AF = *Apodemus flavicollis* * sex: sex of the animals * FCM: fecal corticosterone metabolite concentration (ng/g) * logFCM: log-transformed fecal corticosterone metabolite concentration (ng/g) #### File: af\_m\_color.xls **Description:** Male *Apodemus flavicollis* uncolored and colored fecal boluses collected from the live-trap  ##### Variables * IDanimal: number of the trap and date of animal capture * type: type of fecal boluses, uncolored = fecal boluses that did not have any trace of colored wax marker; colored =  boluses with marked evidence or wax colored traces uncolored or colored boluses * species: species of the animals; AF = *Apodemus flavicollis* * sex: sex of the animals * FCM: fecal corticosterone metabolite concentration (ng/g) * logFCM: log-transformed fecal corticosterone metabolite concentration (ng/g) #### File: as\_f\_color.xls **Description:** Female *Apodemus sylvaticus* uncolored and colored fecal boluses collected from the live-trap  ##### Variables * IDanimal: number of the trap and date of animal capture * type : type of fecal boluses, uncolored = fecal boluses that did not have any trace of colored wax marker; colored =  boluses with marked evidence or wax colored traces * species: species of the animals; AS = *Apodemus sylvaticus* * sex: sex of the animals * FCM: fecal corticosterone metabolite concentration (ng/g) * logFCM: log-transformed fecal corticosterone metabolite concentration (ng/g) #### File: as\_m\_color.xls **Description:** Male Apodemus sylvaticus uncolored and colored fecal boluses collected from the live-trap ##### Variables * IDanimal: number of the trap and date of animal capture * type: type of fecal boluses, uncolored = fecal boluses that did not have any trace of colored wax marker; colored =  boluses with marked evidence or wax colored traces * species: Species of the animals; AS = *Apodemus sylvaticus* * sex: sex of the animals * FCM: fecal corticosterone metabolite concentration (ng/g) * logFCM: log-transformed fecal corticosterone metabolite concentration (ng/g) #### File: stability.xls **Description:** Pool fecal samples left at ambient temperature before to be stored at -20°C to evaluate the stability of fecal corticosterone metabolite levels during the time windows of the test ##### Variables * time_h: Number of hours the samples remained after their excretion at ambient temperature before to be stored at -20°C  * id: Id number of the pool used to prepare the aliquots  * FCM: fecal corticosterone metabolite concentration (ng/g) * logFCM: log-transformed fecal corticosterone metabolite concentration (ng/g) #### File: dataset\_sylvaticus.xls **Description:** Dataset of the *Apodemus sylvaticus* capture in the Selva forest between September 2011 and September 2012 **Variables** * Session: Session of capture * Season: Season of capture, hot : spring-summer period (May, July, and September); cold: autumn-winter (January, March, and November)  * Date: date of capture (YYMMDD) * Grid: Grid of capture * Habitat: Habitat type of capture, woodland: coppiced deciduous woodland, fallow: fallow field, conifer plantation:  conifer plantation  * Species: Species of the animal * Mark: Id of the captured animal * Weight_g: Weight of the animal at capture (g) * Sex: Sex of the animal * Testes: visible size of the test for male animals (A = abdominal; S = small; L = large) ; NA for female animals * Pregnant: No or Yes for female animals; NA for male animals, * Nipples: Visible size of the nipples for female animals (N= non visible; S= small; B = big); NA for male animals * Vagina: Opening of vagina (C= close; O = open); NA for male animals * Breeding: Breeding condition of the animals (Yes = females with opened vagina and/or small or big nipples, males with small or large testes; No = females with closed vagina and no visible nipples, males with abdominal testes) * FCM_ng: Fecal corticosterone metabolite levels (ng/g) * log_FCM: log-transformed fecal corticosterone metabolite concentration (ng/g) #### File: dataset\_flavicollis.xls **Description:** Dataset of the *Apodemus flavicollis* capture in Selva forest between September 2011 and September 2012 ##### Variables * Session: Session of capture * Season: Season of capture, hot : spring-summer period (May, July and September); cold: autumn-winter (January, March, and November) * Date: date of capture (YYMMDD) * Grid: Grid of capture * Habitat: Habitat type of capture, woodland: coppiced deciduous woodland, fallow: fallow field, conifer plantation:  conifer plantation * Species: Species of the animal * Mark: Id of the captured animal * Weight_g: Weight of the animal at capture (g) * Sex: Sex of the animal * Testes: visible size of the test for male animals (A = abdominal; S = small; L = large) ; NA for female animals * Pregnant: No or Yes for female animals; NA for male animals, * Nipples: Visible size of the nipples for female animals (N= non visible; S= small; B = big); NA for male animals * Vagina: Opening of vagina (C= close; O = open); NA for male animals * Breeding: Breeding condition of the animals (Yes = females with opened vagina and/or small or big nipples, males with small or large testes; No = females with closed vagina and no visible nipples, males with abdominal testes) * FCM_ng: Fecal corticosterone metabolite levels (ng/g) * log_FCM: log-transformed fecal corticosterone metabolite concentration (ng/g) #### File: dataset.xls **Description:** Dataset of the *Apodemus sylvaticus and A. flavicollis* captured in Selva forest between September 2011 and September 2012 ##### Variables * Session: Session of capture * Season: Season of capture, hot : spring-summer period (May, July, and September); cold: autumn-winter (January, March, and November)  * Date: date of capture (YYMMDD) * Grid: Grid of capture * Habitat: Habitat type of capture, woodland: coppiced deciduous woodland, fallow: fallow field, conifer plantation:  conifer plantation  * Species: Species of the animal * Mark: Id of the capture animal * Weight_g: Weight of the animal at capture (g) * Sex: Sex of the animal * Testes: visible size of the test for male animals (A = abdominal; S = small; L = large) ; NA for female animals * Pregnant: No or Yes for female animals; NA for male animals, * Nipples: Visible size of the nipples for female animals (N= non visible; S= small; B = big); NA for male animals * Vagina: Opening of vagina (C= close; O = open); NA for male animals * Breeding: Breeding condition of the animals (Yes = females with opened vagina and/or small or big nipples, males with small or large testes; No = females with closed vagina and no visible nipples, males with abdominal testes) * FCM_ng: Fecal corticosterone metabolite levels (ng/g) * log_FCM: log-transformed fecal corticosterone metabolite concentration (ng/g) ## Code/software Programm used R version 4.4 or Rstudio version 2024.12.0 Script: Apodemus_spp.R