Instability of fecal glucocorticoid metabolites at 4°C: Time to freeze matters.

The use of fecal corticosteroid metabolites (FCMs) has proven to be well suited to evaluate adrenocortical activity, a major component of the stress response, particularly in wildlife. As with any tools, confounding factors and drawbacks must be carefully considered. Among them, sample preservation and storage are of particular importance, as they can affect stability of FCMs and lead to biased results and interpretations. Arguably, immediate freezing of fecal samples upon collection is the best practice to preserve FCM integrity, however, for logistical reasons, this condition is rarely feasible in the field. It is generally argued that temporary storage of samples at low above-zero temperature is an acceptable way of preserving samples in the field before freezing them for long-term storage. However, to our knowledge, there is no empirical study that demonstrates the stability of fecal metabolites in samples stored at +4°C. In this study, we collected a fresh fecal sample from 20 captive roe deer, each of which was homogenized and split into three subsamples (60 subsamples in total) to investigate the effects on FCMs levels of temporary storage at +4°C for 24 h and 48 h before freezing versus immediate freezing at -20°C after feces collection. Compared to immediate freezing, mean FCMs levels decreased by 25% every 24 h when feces were stored at +4°C before freezing. The variance of FCMs levels followed the same pattern, leading to a clear reduction in the ability to detect biological effects. Minimizing the storage time at +4°C before freezing should therefore be seriously considered when establishing sampling and storage protocols for feces in the field for adequate hormonal profiling.

Meteorological and climatic variables predict the phenology of Ixodes ricinus nymph activity in France, accounting for habitat heterogeneity.

Ixodes ricinus ticks (Acari: Ixodidae) are the most important vector for Lyme borreliosis in Europe. As climate change might affect their distributions and activities, this study aimed to determine the effects of environmental factors, i.e., meteorological, bioclimatic, and habitat characteristics on host-seeking (questing) activity of I. ricinus nymphs, an important stage in disease transmissions, across diverse climatic types in France over 8 years. Questing activity was observed using a repeated removal sampling with a cloth-dragging technique in 11 sampling sites from 7 tick observatories from 2014 to 2021 at approximately 1-month intervals, involving 631 sampling campaigns. Three phenological patterns were observed, potentially following a climatic gradient. The mixed-effects negative binomial regression revealed that observed nymph counts were driven by different interval-average meteorological variables, including 1-month moving average temperature, previous 3-to-6-month moving average temperature, and 6-month moving average minimum relative humidity. The interaction effects indicated that the phenology in colder climates peaked differently from that of warmer climates. Also, land cover characteristics that support the highest baseline abundance were moderate forest fragmentation with transition borders with agricultural areas. Finally, our model could potentially be used to predict seasonal human-tick exposure risks in France that could contribute to mitigating Lyme borreliosis risk.

Covariation between glucocorticoids, behaviour and immunity supports the pace-of-life syndrome hypothesis: an experimental approach.

The biomedical literature has consistently highlighted that long-term elevation of glucocorticoids might impair immune functions. However, patterns are less clear in wild animals. Here, we re-explored the stress-immunity relationship considering the potential effects of behavioural profiles. Thirteen captive roe deer (Capreolus capreolus) were monitored over an eight-week period encompassing two capture events. We assessed how changes in baseline faecal cortisol metabolite (FCM) concentrations following a standardized capture protocol and an immune challenge using anti-rabies vaccination affected changes in 13 immune parameters of innate and adaptive immunity, and whether these changes in baseline FCM levels and immune parameters related to behavioural profiles. We found that individuals with increased baseline FCM levels also exhibited increased immunity and were characterized by more reactive behavioural profiles (low activity levels, docility to manipulation and neophilia). Our results suggest that the immunity of large mammals may be influenced by glucocorticoids, but also behavioural profiles, as it is predicted by the pace-of-life syndrome hypothesis. Our results highlight the need to consider covariations between behaviour, immunity and glucocorticoids in order to improve our understanding of the among-individual variability in the stress-immunity relationships observed in wildlife, as they may be underpinned by different life-history strategies.

Where to find questing Ixodes frontalis ticks? Under bamboo bushes!

Tick-borne diseases have a complex epidemiology that depends on different ecological communities, associating several species of vertebrate hosts, vectors and pathogens. While most studies in Europe are focused on Ixodes ricinus, other Ixodes species may also be involved in the transmission or maintenance of pathogens. This is the case of Ixodes frontalis, a poorly known species associated with different bird species such as blackbirds, thrushes and robins, with a wide distribution covering most European countries. In a previous study, high densities of questing I. frontalis larvae were found during autumn-winter at a site close to Nantes (western France) where a long-term survey focused on I. ricinus was conducted. These I. frontalis were mostly observed under bamboo bushes. In the present study, we investigated the presence of I. frontalis under bamboo bushes at various locations. With that aim in mind, a systematic search for questing I. frontalis was undertaken by the flagging method in public urban parks and private gardens presenting bamboo bushes (32 sites). This survey was carried out during autumn-winter to maximize the probability of finding the most abundant stage, i.e. larvae. We searched for I. frontalis first in the area of Nantes (10 sites), then in other regions of France (21 sites) and at one site in northern Italy. A single visit to each site revealed the presence of I. frontalis at 29 out of 32 sites: larvae were always present, nymphs were frequent (59 % of the positive sites), while adults were found at only 14 % of the sites. Questing stages of this understudied species are thus easy to find, by dragging or flagging under bamboo bushes in autumn or winter. We make the assumption that bamboo offers a favourable place for birds to roost overnight outside their breeding period (i.e. spring), sheltered from both predators and wind. This would explain higher densities of I. frontalis under bamboo, relative to other biotopes. As I. frontalis is known to harbour zoonotic pathogens, the consequences of this discovery on the epidemiology of tick-borne diseases are discussed.

Distribution of ticks, tick-borne pathogens and the associated local environmental factors including small mammals and livestock, in two French agricultural sites: the OSCAR database.

BACKGROUND: In Europe, ticks are major vectors of both human and livestock pathogens (e.g. Lyme disease, granulocytic anaplasmosis, bovine babesiosis). Agricultural landscapes, where animal breeding is a major activity, constitute a mosaic of habitat types of various quality for tick survival and are used at different frequencies by wild and domestic hosts across seasons. This habitat heterogeneity, in time and space, conditions the dynamics of these host-vector-pathogen systems and thus drives acarological risk (defined as the density of infected ticks). The principal objective of the OSCAR project (2011-2016) was to examine the links between this heterogeneity and acarological risk for humans and their domestic animals. Here, we present the data associated with this project. NEW INFORMATION: This paper reports a database on the distribution and densities of I. ricinus ticks - the most common tick species in French agricultural landscapes - and the prevalence of three tick-borne pathogens (Anaplasma phagocytophilum, Borrelia spp. and Babesia spp.) in two sites in north-western ("Zone Atelier Armorique": ZA site) and south-western ("Vallées et Coteaux de Gascogne": VG site) France. The distribution and density of ticks along a gradient of wooded habitats, as well as biotic variables, such as the presence and abundance of their principal domestic (livestock) and wild hosts (small mammals), were measured from forest cores and edges to more or less isolated hedges, all bordering meadows. Ticks, small mammals and information on local environmental conditions were collected along 90 transects in each of the two sites in spring and autumn 2012 and 2013 and in spring 2014, corresponding to the main periods of tick activity. Local environmental conditions were recorded along each tick and small mammal transect: habitat type, vegetation type and characteristics, slope and traces of livestock presence. Samples consisted of questing ticks collected on the vegetation (mainly I. ricinus nymphs), biopsies of captured small mammals and ticks fixed on small mammals. In the VG site, livestock occurrence and abundance were recorded each week along each tick transect.A total of 29004 questing ticks and 1230 small mammals were captured during the study across the two sites and over the five field campaigns. All questing nymphs (N = 12287) and questing adults (N = 646) were identified to species. Ticks from small mammals (N = 1359) were also identified to life stage. Questing nymphs (N = 4518 I. ricinus) and trapped small mammals (N = 908) were analysed for three pathogenic agents: A. phagocytophilum, Borrelia spp. and Babesia spp.In the VG site, the average prevalence in I. ricinus nymphs for A. phagocytophilum, Borrelia spp. and Babesia spp. were, respectively 1.9% [95% CI: 1.2-2.5], 2.5% [95% CI: 1.8-3.2] and 2.7% [95% CI: 2.0-3.4]. In small mammals, no A. phagocytophilum was detected, but the prevalence for Borrelia spp. was 4.2% [95% CI: 0.9-7.5]. On this site, there was no screening of small mammals for Babesia spp. In ZA site, the average prevalence in nymphs for A. phagocytophilum, Borrelia spp. and Babesia were, respectively 2.2% [95% CI: 1.6-2.7], 3.0% [95% CI: 2.3-3.6] and 3.1% [95% CI: 2.5-3.8]. In small mammals, the prevalence of A. phagocytophilum and Borrelia spp. were, respectively 6.9% [95% CI: 4.9-8.9] and 4.1% [95% CI: 2.7-5.9]. A single animal was found positive for Babesia microti at this site amongst the 597 tested.

Individual variation in an acute stress response reflects divergent coping strategies in a large herbivore.

Individuals differ in the manner that they cope with risk. When these behavioral differences are manifested in risky or challenging environments (i.e. stressful situations), they are generally interpreted within the "coping style" framework. As studying inter-individual variability in behavior is particularly challenging in the wild, we used a captive facility to explore consistency in the individual behavioral response to an acute stress in roe deer (Capreolus capreolus). Using behavioral and physiological parameters measured six times across a calendar year, we first quantified individual repeatability and, second, explored the correlations among these parameters that might indicate a coherent stress response. Finally, we analyzed the link between the stress response and individual body mass, a reliable indicator of phenotypic quality in roe deer. We found that the measured parameters were highly repeatable across seasons, indicating that the individual stress response is consistent over time. Furthermore, there was considerable covariation among the stress response parameters, describing a proactivity-reactivity gradient at the individual level. Finally, proactive individuals had higher body mass than reactive individuals. We suggest that consistent individual differences in energy metabolism and physiology may promote consistent individual differences in behavioral traits, providing a mechanistic link between food acquisition tactics and demographic performance.

Ranging behaviour of roe deer in an experimental high-density population: are females territorial?

We studied the ranging behaviour and spatial relationships between seven roe deer during more than 4 years in a partly wooded 14.2-ha enclosure. The animals (three young males, four adult females) were monitored with GPS telemetry collars. As expected, the surface area and overlap of the males' bimonthly ranges decreased, and the distance between their arithmetic centres increased, as they became adult and, for two of them, territorial. Unexpectedly, females also tended to space out, the surface area and overlap of their bimonthly ranges being minimal in May to June, i.e. during the birth period. The distance between their arithmetic centres reached its maximum at the same time. Overlap between females' ranges was consistently lower than those between males and females' ranges, or between 1-year old males' ranges. Our results raise the questions of female seasonal territoriality and of independence of the spacing systems of the two sexes in roe deer.