Physiological stress response to urbanisation differs between native and invasive squirrel species.

Novel pressures derived from urbanisation can alter native habitats and ultimately impact wildlife. Coping with such human-driven changes might induce shifts in species phenotypic traits, such as physiological responses to anthropogenic stressors. Preadaptation to face those challenges has been suggested to favour settlement and spread of invasive alien species in urbanised areas which, consequently, might respond differently than ecologically similar native species to stressors posed by urbanisation. The activation of the hypothalamic-pituitary-adrenal (HPA) axis and the subsequent release of glucocorticoids (GCs) has been suggested to mediate responses to anthropogenic disturbance in vertebrates. Furthermore, intraspecific competition, in conjunction with stressors related to urbanisation, might affect invasive and native species physiological stress responses differently. Using a parallel pseudo-experimental study system we measured faecal glucocorticoid metabolite (FGM) concentrations of the native Eurasian red squirrel and the invasive alien Eastern grey squirrel along a rural-urban gradient and in relation to conspecific density. The two species responded differently to challenges posed by the synergic effect of urbanisation and intraspecific competition. Association of FGMs and conspecific density in native red squirrels varied between rural and suburban sites, potentially depending on differential HPA axis responses. In urban sites, this relationship did not differ significantly from that in rural and suburban ones. Conversely, invasive grey squirrels' FGMs did not vary in relation to conspecific density, nor differed along the rural-urban gradient. Improving knowledge about native and competing invasive species' physiological responses to anthropogenic stressors can support conservation strategies in habitats altered by man. Our findings suggested that the invasive squirrels might be preadapted to cope with these challenges in urbanised areas, potentially increasing their success under the future global change scenario.

Shifts in immune responses of an invasive alien species: A test of the evolution of increased competitive ability hypothesis using American Eastern gray squirrels in Italy.

Based on the Evolution of Increased Competitive Ability (EICA) hypothesis, a reduced investment in immunity, consequent to parasite loss, could partly explain the success of invasive alien species. We investigated variation in parasite load and immune responses of alien Eastern gray squirrels (Sciurus carolinensis) along the invasion wave of an expanding population. We first verified by fecal analyses that 1) parasite abundance decreased moving from the core towards the invasion front. Next, we used multiple measures of immunity to investigate whether, in response to the lower parasite pressure, individuals at the invasion front 2) dampened their costly inflammatory response, and 3) increased their investment in less expensive acquired immunity. We first explored variation in hematological variables related either to the inflammatory or the acquired response. On a subset of individuals, we carried out ex vivo cell cultures to analyse the basal expression of MHC class II genes and the expression of TNF-α genes in response to an immune challenge. Platelet counts and TNF-α expression suggested higher inflammation in individuals living at the invasion core, whereas parameters associated with an acquired response (lymphocyte counts and MHC II expression by spleen cells), conversely, were higher in squirrels at the front. Overall, our results suggest a shift between different immune strategies along the invasion wave, supporting a reduced investment in costly inflammatory responses and an increased investment in acquired immunity in individuals at the expanding edge of the range, which are subjected to high selective pressures for dispersal and reproduction.

Exotic Pet Trade as a Cause of Biological Invasions: The Case of Tree Squirrels of the Genus Callosciurus.

The trade of non-native pets, especially of non-domesticated and exotic animals, and their subsequent release and establishment of populations is one of the major pathways of introduction for invasive alien reptiles, amphibia, birds and mammals. Here, we use a group of arboreal mammals, tree squirrels of the genus Callosciurus, as a well-documented case study, reviewing the pathways of introduction, the current areas of non-native distribution, the rate of establishment success and the challenge and legal importance of species identification. We further illustrate the importance of early detection and effective monitoring methods and plans. Next, we document how they interfere with native species, their risk of acting as vectors for emerging infectious diseases and their potential role in maintaining parasitic infections that can affect human health. We conclude by reviewing the current management, or the lack of it, and highlight the diverse biological, social, political and economic reasons that make control/eradication of these charismatic species difficult or even impractical in most countries. However, reviewing the only two successful eradications of the IAS, we highlight the need to acknowledge the public opinion and the importance of communication, transparency and the engagement of a diversity of stakeholders to create a consensus about the actions to undertake.

Camera Trapping to Assess Status and Composition of Mammal Communities in a Biodiversity Hotspot in Myanmar.

Tropical forests comprise a critically impacted habitat, and it is known that altered forests host a lower diversity of mammal communities. In this study, we investigated the mammal communities of two areas in Myanmar with similar environmental conditions but with great differences in habitat degradation and human disturbance. The main goal was to understand the status and composition of these communities in an understudied area like Myanmar at a broad scale. Using camera trap data from a three-year-long campaign and hierarchical occupancy models with a Bayesian formulation, we evaluated the biodiversity level (species richness) and different ecosystem functions (diet and body mass), as well as the occupancy values of single species as a proxy for population density. We found a lower mammal diversity in the disturbed area, with a significantly lower number of carnivores and herbivores species. Interestingly, the area did not show alteration in its functional composition. Almost all the specific roles in the community were present except for apex predators, thus suggesting that the effects of human disturbance are mainly effecting the communities highest levels. Furthermore, two species showed significantly lower occupancies in the disturbed area during all the monitoring campaigns: one with a strong pressure for bushmeat consumption and a vulnerable carnivore threatened by illegal wildlife trade.

Personality traits, sex and food abundance shape space use in an arboreal mammal.

Animal space use is affected by spatio-temporal variation in food availability and/or population density and varies among individuals. This inter-individual variation in spacing behaviour can be further influenced by sex, body condition, social dominance, and by the animal's personality. We used capture-mark-recapture and radio-tracking to examine the relationship between space use and personality in Eurasian red squirrels (Sciurus vulgaris) in three conifer forests in the Italian Alps. We further explored to what extent this was influenced by changes in food abundance and/or population density. Measures of an individual's trappability and trap diversity had high repeatability and were used in a Principal Component Analysis to obtain a single personality score representing a boldness-exploration tendency. Males increased home-range size with low food abundance and low female density, independent of their personality. However, bolder males used larger core-areas that overlapped less with other males than shy ones, suggesting different resource (food, partners) utilization strategies among personality types. For females, space use-personality relationships varied with food abundance, and bolder females used larger home ranges than shy ones at low female density, but the trend was opposite at high female density. Females' intrasexual core-area overlap was negatively related to body mass, with no effect of personality. We conclude that relationships between personality traits and space use in free-ranging squirrels varied with sex, and were further influenced by spatio-temporal fluctuations in food availability. Moreover, different personality types (bold-explorative vs. shy) seemed to adopt different space-use strategies to increase access to food and/or partners.

Complex relationships between physiological stress and endoparasite infections in natural populations.

Short-term elevation of glucocorticoids (GCs) is one of the major physiological mechanisms by which vertebrates cope with challenging environmental or social factors (stressors). However, when exposure to stressors occurs repeatedly or over a prolonged period of time, animals may experience chronic elevation of GCs, which reduces the immune response efficiency and can lead to higher intensity of parasitic infection. Here, we used invasive gray squirrels Sciurus carolinensis introduced in Northern Italy and their 2 most prevalent gastrointestinal parasites, the nematode Strongyloides robustus and coccidia of the genus Eimeria, as a model to investigate relationships among macroparasite infection and concentrations of fecal glucocorticoid metabolites (FGMs), an integrated measure of circulating GCs. Our results revealed an association of FGMs with infection by St. robustus, but not with coccidia. Individuals with higher FGMs appear to be responsible for the greatest St. robustus egg shedding within gray squirrel populations, thus possibly acting as superspreaders. However, FGMs were negatively associated with adult St. robustus, suggesting that the abundance of adults of this nematode species does not induce elevation in FGMs, but is only affected by it through immune-mediated effects on its fecundity. Finally, the relationship between St. robustus (both eggs and adult parasites) and FGMs was not linear, suggesting that only high levels of physiological stress influence parasite infection. Our findings highlight that the direction and magnitude of the stress-infection relationship may depend not only on the specific host-parasite system, but also on the different life stages of the same parasite.

Relationships between personality traits and the physiological stress response in a wild mammal.

Glucocorticoids (GCs) are involved in the regulation of an animal's energetic state. Under stressful situations, they are part of the neuroendocrine response to cope with environmental challenges. Animals react to aversive stimuli also through behavioral responses, defined as coping styles. Both in captive and wild populations, individuals differ in their behavior along a proactive-reactive continuum. Proactive animals exhibit a bold, active-explorative and social personality, whereas reactive ones are shy, less active-explorative and less social. Here, we test the hypothesis that personality traits and physiological responses to stressors covary, with more proactive individuals having a less pronounced GC stress response. In wild populations of invasive gray squirrels Sciurus carolinensis, we measured fecal glucocorticoid metabolites (FGMs), an integrated measure of circulating GCs, and 3 personality traits (activity, sociability, and exploration) derived from open field test (OFT) and mirror image stimulation (MIS) test. Gray squirrels had higher FGMs in Autumn than in Winter and males with scrotal testes had higher FGMs than nonbreeding males. Personality varied with body mass and population density. Squirrels expressed more activity-exploration at higher than at lower density and heavier squirrels had higher scores for activity-exploration than animals that weighed less. Variation in FGM concentrations was not correlated with the expression of the 3 personality traits. Hence, our results do not support a strong association between the behavioral and physiological stress responses but show that in wild populations, where animals experience varying environmental conditions, the GC endocrine response and the expression of personality are uncorrelated traits among individuals.

Spillover of an alien parasite reduces expression of costly behaviour in native host species.

Understanding the effects of invasive alien species (IAS) on native host-parasite relationships is of importance for enhancing ecological theory and IAS management. When IAS and their parasite(s) invade a guild, the effects of interspecific resource competition and/or parasite-mediated competition can alter existing native host-parasite relationships and the dependent biological traits such as native species' behaviour. We used a natural experiment of populations of native red squirrels Sciurus vulgaris that were colonized by the alien grey squirrel Sciurus carolinensis, comparing repeated measurements of red squirrel parasite infection and personality with those taken in sites where only the native species occurred. We explored two alternative hypotheses: (a) individual differences in personality traits (activity and/or sociability) of native red squirrel positively affect the probability of macroparasite spillover and thus the likelihood to acquire the alien's parasitic helminth Strongyloides robustus; (b) the combined effects of grey squirrel presence and parasite infection result in a reduction of costly personality traits (activity and/or exploration). Using data from 323 arena tests across three experimental (native species and IAS) and three control sites (only native species), we found negative correlations between native species' activity and infection with S. robustus in the sites invaded by the alien species. Activity was also negatively correlated with infection by its native helminth Trypanoxyuris sciuri but only when grey squirrels were present, while in the red-only sites there was no relationship of T. sciuri infection with any of the personality traits. Moreover, individuals that acquired S. robustus during the study reduced their activity after infection, while this was not the case for animals that remained uninfected. Our results show that parasite-mediated competition is costly, reducing activity in individuals of the native species, and altering the native host-native parasite relationships.

Interspecific competition affects the expression of personality-traits in natural populations.

Competition between animal species can cause niche partitioning and shape an individual's phenotype, including its behaviour. However, little is known about effects of interspecific competition on personality, the among-individual variation in behaviour that is consistent across different spatial and temporal contexts. We investigated whether alien grey squirrels (Sciurus carolinensis) influenced the expression of personality traits in native red squirrels (Sciurus vulgaris). In Italy, alien grey squirrels replaced native reds through competition for food resources and space, reducing breeding and recruitment in the native species. We compared personality of red squirrels in red-only (no interspecific competition) and red-grey (with interspecific competition) sites, using arena-tests. The trait activity was measured by Open Field Test while sociability and avoidance were quantified by Mirror Image Stimulation test. Red squirrels co-occurring with the alien species had higher sociability scores and higher between-individual variation in sociability than in red-only sites. Differences in activity and avoidance were not significant. Personality - fitness relationships were not affected by presence or absence of grey squirrels, suggesting that the expression of sociability in red squirrels was not due to short-term selection, but was likely the result of context-related advantages when co-occurring with the competing species.

Native and introduced squirrels in Italy host different Cryptosporidium spp.

The present study was undertaken to describe Cryptosporidium spp. infection in tree squirrels from 17 locations in Northern Italy. A total of 357 squirrels were examined, including species native to Europe (Sciurus vulgaris; n=123), and species introduced from North America (Sciurus carolinensis; n=162) and Southeast Asia (Callosciurus erythraeus; n=72). Faecal samples of all squirrels were examined for the presence of Cryptosporidium infection by microscopy (flotation method) and PCR/sequence analysis of the Cryptosporidium 18S rRNA, actin, and gp60 genes. Despite the overlapping ranges of native and introduced tree squirrel species in the study area, they host different Cryptosporidium spp. Sciurus vulgaris were exclusively infected with Cryptosporidium ferret genotype (n=13) belonging to three novel gp60 subtypes, VIIIb-VIIId. Sciurus carolinensis hosted C. ubiquitum subtype XIIb (n=2), Cryptosporidium skunk genotype subtype XVIa (n=3), and chipmunk genotype I subtype XIVa (n=1). Cryptosporidium chipmunk genotype I subtype XIVa was also found in two C. erythraeus. Comparing data from this and previous studies, we propose that Cryptosporidium skunk genotype and possibly C. ubiquitum subtype XIIb were introduced to Europe with eastern grey squirrels. Cryptosporidium chipmunk genotype I and ferret genotype were associated with high intensity infections, but there was no association with diarrhoea.

Preventing species invasion: A role for integrative taxonomy?

Integrative taxonomy, a multi-disciplinary approach adding modern techniques to traditional morphology-based methods (e.g. molecular and morphological criteria), can play an important role in bioinvasion research to identify introduced taxa, discover pathways of introduction and inform authorities to control and prevent future introductions. The present study is the first on introduced populations of Callosciurus, Asiatic tree squirrels, known as potentially invasive species in Europe (Italy, Belgium and France). We combined molecular (mitochondrial DNA markers: CoxI, D-loop) and morphometric analysis on skulls, comparing them to the widest morphological and molecular datasets ever assembled for Callosciurus. Squirrels collected in Italy and Belgium share the same haplotypes and skull characteristics, but are conspicuously different from the French population in Antibes. Genetic data revealed close similarity between French squirrels and Pallas's squirrels, Callosciurus erythraeus, from Taiwan, China. Italian and Belgian squirrels formed an independent taxonomic lineage in genetic analyses, whose taxonomic rank needs further investigation. The morphological and morphometric characteristics of these 2 populations are, however, similar to known specimens assigned to Callosciurus erythraeus. These results may indicate a common origin for the populations found in Belgium and Italy. In contrast, French specimens suggest an independent introduction event of squirrels originating from Asia.

Biodiversity threats from outside to inside: effects of alien grey squirrel (Sciurus carolinensis) on helminth community of native red squirrel (Sciurus vulgaris).

Biological invasions are among the major causes of biodiversity loss worldwide, and parasites carried or acquired by invaders may represent an added threat to native species. We compared gastrointestinal helminth communities of native Eurasian red squirrels (Sciurus vulgaris) in the presence and absence of introduced Eastern grey squirrels (Sciurus carolinensis) to detect alterations induced by the alien species. In particular, we investigated whether spillover of a North American nematode Strongyloides robustus occurs and whether prevalence of a local parasite Trypanoxyuris sciuri in red squirrels is affected by grey squirrel presence. The probability of being infected by both parasites was significantly higher in areas co-inhabited by the alien species, where 61 % of examined red squirrels (n = 49) were infected by S. robustus and 90 % by T. sciuri. Conversely, in red-only areas, the two parasites infected only 5 and 70 % of individuals (n = 60). Overall, our findings support the hypothesis that red squirrels acquire S. robustus via spillover from the alien congener and suggest that invaders' presence may also indirectly affect infection by local parasites through mechanisms diverse than spill-back and linked to the increased competitive pressure to which red squirrels are subjected. These results indicate that the impact of grey squirrel on red squirrels may have been underestimated and highlight the importance of investigating variation in macroparasite communities of native species threatened by alien competitors.

Ljungan virus and an adenovirus in Italian squirrel populations.

We report Ljungan virus infection in Eurasian red squirrels (Sciurus vulgaris) for the first time, and extend the known distribution of adenoviruses in both native red squirrels and alien gray squirrels (Sciurus carolinensis) to southern Europe.

Macroparasite fauna of alien grey squirrels (Sciurus carolinensis): composition, variability and implications for native species.

Introduced hosts populations may benefit of an "enemy release" through impoverishment of parasite communities made of both few imported species and few acquired local ones. Moreover, closely related competing native hosts can be affected by acquiring introduced taxa (spillover) and by increased transmission risk of native parasites (spillback). We determined the macroparasite fauna of invasive grey squirrels (Sciurus carolinensis) in Italy to detect any diversity loss, introduction of novel parasites or acquisition of local ones, and analysed variation in parasite burdens to identify factors that may increase transmission risk for native red squirrels (S. vulgaris). Based on 277 grey squirrels sampled from 7 populations characterised by different time scales in introduction events, we identified 7 gastro-intestinal helminths and 4 parasite arthropods. Parasite richness is lower than in grey squirrel's native range and independent from introduction time lags. The most common parasites are Nearctic nematodes Strongyloides robustus (prevalence: 56.6%) and Trichostrongylus calcaratus (6.5%), red squirrel flea Ceratophyllus sciurorum (26.0%) and Holarctic sucking louse Neohaematopinus sciuri (17.7%). All other parasites are European or cosmopolitan species with prevalence below 5%. S. robustus abundance is positively affected by host density and body mass, C. sciurorum abundance increases with host density and varies with seasons. Overall, we show that grey squirrels in Italy may benefit of an enemy release, and both spillback and spillover processes towards native red squirrels may occur.

Macroparasite community of the Eurasian red squirrel (Sciurus vulgaris): poor species richness and diversity.

The Eurasian red squirrel (Sciurus vulgaris) is the only naturally occurring tree squirrel throughout its range. We aim at improving current knowledge on its macroparasite fauna, expecting that it will have a poor parasite diversity because in species that have no sympatric congeners parasite richness should be lower than in hosts sharing their range with several closely related species, where host-switching events and lateral transmission are promoted. We examined gastro-intestinal helminth and ectoparasite communities (excluding mites) of, respectively, 147 and 311 red squirrel roadkills collected in four biogeographic regions in Italy and France. As expected, the macroparasite fauna was poor: we found five species of nematodes and some unidentified cestodes, three fleas, two sucking lice and two hard ticks. The helminth community was dominated by a single species, the oxyurid Trypanoxyuris (Rodentoxyuris) sciuri (prevalence, 87%; mean abundance, 373 ± 65 worms/host). Its abundance varied among seasons and biogeographic regions and increased with body mass in male hosts while decreased in females. The most prevalent ectoparasites were the flea Ceratophyllus (Monopsyllus) sciurorum (28%), whose presence was affected by season, and the generalist tick Ixodes (Ixodes) ricinus that was found only in France (34%). All the other helminths and arthropod species were rare, with prevalence below 10%. However, the first record of Strongyloides robustus, a common nematode of North American Eastern grey squirrels (S. carolinensis), in two red squirrels living in areas where this alien species co-inhabits, deserves further attention, since low parasite richness could result in native red squirrels being particularly vulnerable to parasite spillover.

Molecular phylogeography of European Sciurus vulgaris: refuge within refugia?

The red squirrel (Sciurus vulgaris) is a well-known forest animal distributed all over Europe. Still, we are far from having a firm knowledge of the species' phylogeography. This study investigates the genetic differentiation of S. vulgaris across the species' Eurasian range, using sequence data from the mitochondrial DNA gene (D-loop, 252 base pairs, cytochrome b, 359 base pairs), and eight variable autosomal microsatellite loci genotyped for 236 individuals. The results reveal the presence of two main mitochondrial phylogroups. The first clade comprises the individuals from the region of Calabria in southern Italy, belonging to the subspecies S. v. meridionalis, while the second clade contains the remainder of the studied individuals. Bayesian analysis of microsatellite genotypes resulted in three main clusterings corresponding to the three S. vulgaris subspecies: infuscatus, meridionalis and fuscoater. Geographical distribution of mtDNA haplotypes and mismatch analysis suggest a common refugium for the red squirrel across most of its present range from which expansion happened rather rapidly. The genotype mixing of italicus with northern populations could be a residual of postglacial expansion. The lack of mixing between the Calabrian lineage and the rest of European red squirrel haplotypes can be seen as evidence for distinct histories throughout the Pleistocene. Calabrian mtDNA probably diverged in an ice age contraction and remained isolated from the neighbouring squirrel populations until very recent times.

Anticipatory reproduction and population growth in seed predators.

Mast seeding, the intermittent, synchronous production of large seed crops by a population of plants, is a well-known example of resource pulses that create lagged responses in successive trophic levels of ecological communities. These lags arise because seed predators are thought capable of increasing reproduction and population size only after the resource pulse is available for consumption. The resulting satiation of predators is a widely cited explanation for the evolution of masting. Our study shows that both American and Eurasian tree squirrels anticipate resource pulses and increase reproductive output before a masting event, thereby increasing population size in synchrony with the resource pulse and eliminating the population lag thought to be universal in resource pulse systems.