Physiological and behavioural adjustment of a wild rodent to laboratory conditions.

Wild animals are brought to captivity for different reasons, for example to be kept in zoos and rehabilitation centres, but also for basic research. Such animals usually undergo a process of adjustment to captive conditions. While this adjustment occurs on the behavioural and the physiological level, those are usually studied separately. The aim of this study was to assess both the physiological and behavioural responses of wild wood mice, Apodemus sylvaticus, while adjusting to laboratory conditions. Over the course of four weeks, we measured in wild-caught mice brought to the laboratory faecal corticosterone metabolites and body mass as physiological parameters, stereotypic behaviour and nest-quality, as welfare-linked behavioural parameters, and four personality measures as additional behavioural parameters. The results of our study indicate that mice exhibited an adjustment in both behaviour and physiology over time in the laboratory. While the hormonal stress response decreased significantly, body mass and the proportion of stereotypic behaviours showed a tendency to increase over time. The slight increase of stereotypic behaviours, although not statistically significant, suggests the development of repetitive and non-functional behaviours as a response to laboratory conditions. However, we suggest that those behaviours might have been used by animals as a coping strategy to decrease the physiological stress response. Other behavioural parameters measured, such as boldness and nestbuilding behaviour were stable over time. The information obtained in the present study hints at a complex interplay between behavioural and physiological adjustments of wild animals to laboratory conditions, which should be considered when intending to use wild animals in experimental research.

Assessing Optimal Cell Counts in Sperm Shape Abnormality Assays in Rodents.

Rodents have been the preferred models for the evaluation of the toxicity of pollutants and drugs and their genotoxic effects, including sperm shape abnormalities. The scientific literature is dominated by studies conducted with model animals in laboratory conditions, but a generally accepted and standardized protocol addressing the optimal number of sperm cells to count is still lacking. In this study, we reviewed the literature regarding the number of counted sperm cells in such assessments, published from 1969 to 2023. To infer the number of counts providing the best cost/benefit regarding the robustness of the assay results, a new dataset involving the analysis of two populations of wild rodents was produced. We evaluated the frequency of sperm shape abnormalities in a total of 50 wild brown rats (Rattus norvegicus) captured in two port cities, aiming to detect the impact of differential sperm cell counts in the obtained results. During necropsy, the fresh epididymis tail of adult male rats was excised, and sperm cells were fixated in slides. For each animal, a total of 300, 500, 1000, and 2000 cells were sequentially counted, and head abnormalities were registered. Counting 300 sperm cells failed to detect significant differences between groups and 500 counts resulted in marginally significant differences. Only when 1000 or 2000 sperm cells were counted, significant differences emerged between groups. We propose that studies addressing sperm shape abnormalities should standardize counts to an optimal value of 1000 cells per animal, warranting robust statistical results while providing the best compromise concerning labor time.

Interaction time with conspecifics induces food preference or aversion in the wild Algerian mouse.

The social transmission of a novel food preference can avoid unnecessary costs arising from tasting nonedible foods. This type of social learning has been demonstrated in laboratory rats and mice. However, among wild animals, there may be several constraints that make it less effective. Using wild Algerian mice (Mus spretus) tested in the laboratory, we demonstrate that a preference for a novel food can be transmitted between Observer and Demonstrator individuals and that it is maintained for at least 30 days. However, only half of the Observers acquired a preference for the same food as the Demonstrators, and only when the duration of oronasal investigation was above a certain threshold (≥122 s); below this threshold (<122 s), Observers acquired a preference for the alternative food offered, which was maintained for a shorter time. Sex, size, and identity of individuals did not influence the transmission of social information. The results show that different interaction times will result in animals copying or avoiding the food choices of others. This suggests that the transmission of social information among wild animals is complex and probably influenced by many factors (e.g., dominance, familiarity, and health condition), ultimately conditioning the type of interaction between individuals and its outcome. Testing wild animals and the ecological and social constraints they face is, therefore, an important step in our understanding of how effectively social information is transmitted in nature.

Characterising Mitochondrial Capture in an Iberian Shrew.

Mitochondrial introgression raises questions of biogeography and of the extent of reproductive isolation and natural selection. Previous phylogenetic work on the Sorex araneus complex revealed apparent mitonuclear discordance in Iberian shrews, indicating past hybridisation of Sorex granarius and the Carlit chromosomal race of S. araneus, enabling introgression of the S. araneus mitochondrial genome into S. granarius. To further study this, we genetically typed 61 Sorex araneus/coronatus/granarius from localities in Portugal, Spain, France, and Andorra at mitochondrial, autosomal, and sex-linked loci and combined our data with the previously published sequences. Our data are consistent with earlier data indicating that S. coronatus and S. granarius are the most closely related of the three species, confirming that S. granarius from the Central System mountain range in Spain captured the mitochondrial genome from a population of S. araneus. This mitochondrial capture event can be explained by invoking a biogeographical scenario whereby S. araneus was in contact with S. granarius during the Younger Dryas in central Iberia, despite the two species currently having disjunct distributions. We discuss whether selection favoured S. granarius with an introgressed mitochondrial genome. Our data also suggest recent hybridisation and introgression between S. coronatus and S. granarius, as well as between S. araneus and S. coronatus.

Dissimilar use of an external heat source for thermoregulation by shrews from different geographic regions.

Ambient temperature has a substantial influence on the thermoregulation costs of small mammals due to their high surface-to-volume ratio. Shrews are among the smallest of mammals and have adopted different behavioral and physiological strategies to deal with cold temperatures. In this study, we assessed the use of an external heat source in the thermoregulatory strategy of two Crocidurinae species, Crocidura russula and C. suaveolens, and one Soricinae species, Sorex araneus. Crocidura russula inhabits western Europe and is better adapted to a Mediterranean climate; C. suaveolens inhabits central Europe; and S. araneus inhabits northern Europe and is better adapted to a Palearctic climate. We predicted that C. russula (most southern species) would spend larger amounts of time using an external heat source because it is the most cold-sensitive species, while S. araneus (most northern species) would spend less time using an external heat source or not respond to it. Shrews were experimentally tested in captivity inside a terrarium where they had access to a heat rock, which could be turned off (cold) or on (heated), depending on treatment. Our results confirmed our initial prediction: C. russula was the species that spent significantly more time on the heated rock, followed by C. suaveolens. Only a quarter of S. araneus individuals spent large amounts of time on the heat rock, which suggests this thermoregulation strategy is not generally adopted by this species, but may be rather associated with some individual personalities. We also analyzed the influence of the heat rock on rewarming from heterothermy, but heterothermy was not different between rock treatments. Overall, our results show that shrew species use external heat sources for thermoregulation according to their sensitivity to cold.

Genetic variation at the p53 locus of two ecologically divergent Microtus pine voles: identification of molecular markers for species assignment.

The Lusitanian (Microtus lusitanicus) and the Mediterranean (Microtus duodecimcostatus) pine voles are recently diverged sister species endemic of the Iberian Peninsula that can be identified with ecological and morphological characters, but in areas where the 2 species co-occur, species designation may be difficult. Genetic discrimination between M. lusitanicus and M. duodecimcostatus has not been achieved yet possibly because of their estimated recent split and an evolutionary history that includes inter-species gene flow. Following our previous observations on exons 5-7 of the p53 gene, here we analyze the potential use of the p53 genomic region as a discrimination marker of these species by extending our analyses to several kb upstream and downstream of the p53 gene and characterizing the degree of genetic differentiation in 7 markers within this region. Additionally, we fully sequenced the P53 protein of both species. We observed: (i) generally high differentiation in this region; (ii) M. duodecimcostatus showed in general higher values of nucleotide and haplotype diversities; (iii) the concatenated phylogenetic tree separates the 2 species; (iv) the 2 P53 proteins only differ in 1 amino acid; (v) 4 of the markers, 2 in p53, one in Atp1b2, and another in Wrap53, contain species-specific genetic variation thus allowing a reliable discrimination between specimens from both species, irrespective of sampling location or introgression status. We provide additional data on the putative role of p53 in the evolution of these species and present researchers with a fast and cost-effective resource for M. lusitanicus and M. duodecimcostatus identification.

Evidence of micro-evolution in Crocidura russula from two abandoned heavy metal mines: potential use of Cytb, CYP1A1, and p53 as gene biomarkers.

Heavy metals accumulated in the environment due to the mining industry may impact on the health of exposed wild animals with consequences at the population level via survival and selection of the most resistant individuals. The detection and quantification of shifts in gene frequencies or in the genetic structure in populations inhabiting polluted sites may be used as early indicators of environmental stress and reveal potential 'candidate gene biomarkers' for environmental health assessment. We had previously observed that specimens of the Greater white-toothed shrew (Crocidura russula) from two heavy metal mines in Southern Portugal (the Aljustrel and the Preguiça mines) carried physiological alterations compared to shrews from an unpolluted site. Here, we further investigated whether these populations showed genetic differences in genes relevant for physiological homeostasis and/or that are associated with pathways altered in animals living under chronic exposure to pollution, and which could be used as biomarkers. We analysed the mitochondrial cytochrome b (Cytb) gene and intronic and/or exonic regions of four nuclear genes: CYP1A1, LCAT, PRPF31, and p53. We observed (1) population differences in allele frequencies, types of variation, and diversity parameters in the Cytb, CYP1A1, and p53 genes; (2) purifying selection of Cytb in the mine populations; (3) genetic differentiation of the two mine populations from the reference by the p53 gene. Adding to our previous observations with Mus spretus, we provide unequivocal evidence of a population effect exerted by the contaminated environment of the mines on the local species of small mammals.

The Influence of the Interaction between Climate and Competition on the Distributional Limits of European Shrews.

It is known that species' distributions are influenced by several ecological factors. Nonetheless, the geographical scale upon which the influence of these factors is perceived is largely undefined. We assessed the importance of competition in regulating the distributional limits of species at large geographical scales. We focus on species with similar diets, the European Soricidae shrews, and how interspecific competition changes along climatic gradients. We used presence data for the seven most widespread terrestrial species of Soricidae in Europe, gathered from GBIF, European museums, and climate data from WorldClim. We made use of two Joint Species Distribution Models to analyse the correlations between species' presences, aiming to understand the distinct roles of climate and competition in shaping species' distributions. Our results support three key conclusions: (i) climate alone does not explain all species' distributions at large scales; (ii) negative interactions, such as competition, seem to play a strong role in defining species' range limits, even at large scales; and (iii) the impact of competition on a species' distribution varies along a climatic gradient, becoming stronger at the climatic extremes. Our conclusions support previous research, highlighting the importance of considering biotic interactions when studying species' distributions, regardless of geographical scale.

Spatial and Temporal Dynamics of Contact Zones Between Chromosomal Races of House Mice, Mus musculus domesticus, on Madeira Island.

Analysis of contact zones between parapatric chromosomal races can help our understanding of chromosomal divergence and its influence on the speciation process. Monitoring the position and any movement of contact zones can allow particular insights. This study investigates the present (2012-2014) and past (1998-2002) distribution of two parapatric house mouse chromosomal races-PEDC (Estreito da Calheta) and PADC (Achadas da Cruz)-on Madeira Island, aiming to identify changes in the location and width of their contact. We also extended the 1998-2002 sampling area into the range of another chromosomal race-PLDB (Lugar de Baixo). Clinal analysis indicates no major geographic alterations in the distribution and chromosomal characteristics of the PEDC and PADC races but exhibited a significant shift in position of the Rb (7.15) fusion, resulting in the narrowing of the contact zone over a 10+ year period. We discuss how this long-lasting contact zone highlights the role of landscape on mouse movements, in turn influencing the chromosomal characteristics of populations. The expansion of the sampling area revealed new chromosomal features in the north and a new contact zone in the southern range involving the PEDC and PLDB races. We discuss how different interacting mechanisms (landscape resistance, behaviour, chromosomal incompatibilities, meiotic drive) may help to explain the pattern of chromosomal variation at these contacts between chromosomal races.

Mapping Knowledge Gaps of Mozambique's Terrestrial Mammals.

A valuable strategy to support conservation planning is to assess knowledge gaps regarding primary species occurrence data to identify and select areas for future biodiversity surveys. Currently, increasing accessibility to these data allows a cost-effective method for boosting knowledge about a country's biodiversity. For understudied countries where the lack of resources for conservation is more pronounced to resort to primary biodiversity data can be especially beneficial. Here, using a primary species occurrence dataset, we assessed and mapped Mozambique's knowledge gaps regarding terrestrial mammal species by identifying areas that are geographically distant and environmentally different from well-known sites. By comparing gaps from old and recent primary species occurrence data, we identified: (i) gaps of knowledge over time, (ii) the lesser-known taxa, and (iii) areas with potential for spatiotemporal studies. Our results show that the inventory of Mozambique's mammal fauna is near-complete in less than 5% of the territory, with broad areas of the country poorly sampled or not sampled at all. The knowledge gap areas are mostly associated with two ecoregions. The provinces lacking documentation coincide with areas over-explored for natural resources, and many such sites may never be documented. It is our understanding that by prioritising the survey of the knowledge-gap areas will likely produce new records for the country and, continuing the study of the well-known regions will guarantee their potential use for spatiotemporal studies. The implemented approach to assess the knowledge gaps from primary species occurrence data proved to be a powerful strategy to generate information that is essential to species conservation and management plan. However, we are aware that the impact of digital and openly available data depends mostly on its completeness and accuracy, and thus we encourage action from the scientific community and government authorities to support and promote data mobilisation.

Energetics and thermal adaptation in semifossorial pine-voles Microtus lusitanicus and Microtus duodecimcostatus.

Rodents colonising subterranean environments have developed several morphological, physiological and behaviour traits that promote the success of individuals in such demanding conditions. Resting metabolic rate, thermoregulation capacity and daily energy expenditure were analysed in two semi-fossorial pine-vole species Microtus lusitanicus and Microtus duodecimcostatus inhabiting distinct areas of the Iberian Peninsula. Individuals capture location varied in habitat and soil features, allowing the comparison of energetic parameters with ecological characteristics, that can help explain the use of the subterranean environment and dependence of the burrow system. Results showed that M. duodecimcostatus has lower mass independent resting metabolic rate when compared with M. lusitanicus, which may be a response to environmental features of their habitat, such as dryer soils and lower water availability. Thermal conductance increased with body mass and was dependent on the ambient temperature. No significant differences were observed in the daily energy expenditure, but water economy data demonstrated the influence of the water available in the habitat on the energetics of voles. These species may rely on behavioural adaptations and seasonal use of burrows to cope with thermal challenges of subterranean activity and soil constraints. We found strong evidence that M. lusitanicus is able to use torpor as a response to low ambient temperatures which is a new observation among Arvicolines.

Behavioural and physiological responses of wood mice (Apodemus sylvaticus) to experimental manipulations of predation and starvation risk.

Body weight and the levels of stored body fat have fitness consequences. Greater levels of fat may provide protection against catastrophic failures in the food supply, but they may also increase the risk of predation. Animals may therefore regulate their fatness according to their perceived risks of predation and starvation: the starvation-predation trade-off model. We tested the predictions of this model in wood mice (Apodemus sylvaticus) by experimentally manipulating predation risk and starvation risk. We predicted that under increased predation risk individuals would lose weight and under increased starvation risk they would gain it. We simulated increased predation risk by playing the calls made by predatory birds (owls: Tyto alba and Bubo bubo) to the mice. Control groups included exposure to calls of a non-predatory bird (blackbird: Turdus merula) or silence. Mice exposed to owl calls at night lost weight relative to the silence group, mediated via reduced food intake, but exposure to owl calls in the day had no significant effect. Exposure to blackbird calls at night also resulted in weight loss, but blackbird calls in the day had no effect. Mice seemed to have a generalised response to bird calls at night irrespective of their actual source. This could be because in the wild any bird calling at night will be a predation risk, and any bird calling in the day would not be, because at that time the mice would normally be resting, and hence not exposed to avian predators. Consequently, mice have not evolved to distinguish different types of call but only to respond to the time of day that they occur. Mice exposed to stochastic 24h starvation events altered their behaviour (reduced activity) during the refeeding days that followed the deprivation periods to regain the lost mass. However, they only marginally elevated their food intake and consequently had reduced body weight/fat storage compared to that of the control unstarved group. This response may have been constrained by physiological factors (alimentary tract absorption capacity) or behavioural factors (perceived risk of predation). Overall the responses of the mice appeared to provide limited support for the starvation-predation trade-off model, and suggest that wood mice are much more sensitive to predation risk than they are to starvation risk.

Deep mitochondrial introgression and hybridization among ecologically divergent vole species.

The completion of speciation is typically difficult to ascertain in rapidly diverging taxa but the amount of hybridization and gene flow in sympatry or parapatry contains important information about the level of reproductive isolation achieved. Here, we examined the progress in speciation between the Mediterranean (Microtus duodecimcostatus) and the Lusitanian pine vole (M. lusitanicus), which are part of the most rapid radiation of species known in mammals. These two Iberian pine voles are classified as separate species because of differences in morphology and ecology, but relatively many ambiguous individuals can be found in sympatric conditions. Our phylogenetic analyses of rangewide data from the mitochondrial cytochrome b gene (mtDNA) demonstrated high levels of diversity and a basal separation in two parapatric lineages. However, mtDNA affiliation was at odds with morphological classification or geographical distribution of the taxa. In contrast, statistical analyses of microsatellites (nucDNA) showed two clear genetic clusters in allopatry and sympatry generally matching morphological classification. This cytonuclear discordance over a large geographic area suggests historical introgression of mtDNA from M. duodecimcostatus to M. lusitanicus. There was statistical evidence for at least two recent hybrids in the sympatry zone but gene flow is apparently low given clear-cut differences in nucDNA. Our results indicate a relatively advanced speciation process in these Iberian pine voles without fully established reproductive isolation. This situation enables use of combined population genomic and experimental approaches for the separation of patterns and mechanisms in the ongoing explosive diversification of these and other Arvicoline rodents in the future.

Of mice and 'convicts': origin of the Australian house mouse, Mus musculus.

The house mouse, Mus musculus, is one of the most ubiquitous invasive species worldwide and in Australia is particularly common and widespread, but where it originally came from is still unknown. Here we investigated this origin through a phylogeographic analysis of mitochondrial DNA sequences (D-loop) comparing mouse populations from Australia with those from the likely regional source area in Western Europe. Our results agree with human historical associations, showing a strong link between Australia and the British Isles. This outcome is of intrinsic and applied interest and helps to validate the colonization history of mice as a proxy for human settlement history.

Induction of micronuclei and sister chromatid exchange in bone-marrow cells and abnormalities in sperm of Algerian mice (Mus spretus) exposed to cadmium, lead and zinc.

As a consequence of human activities, large amounts of cadmium, lead and zinc are released in the environment, often simultaneously. The aim of this study was to investigate under experimental conditions the DNA damage induced in Algerian mice (Mus spretus) exposed to cadmium (Cd), lead (Pb) and zinc (Zn) separately, or in selected combinations. Three cytogenetic end points were considered: the frequencies of micronucleated cells (MN) and sister chromatid exchange (SCE) in the bone marrow and the frequency of sperm abnormalities. Mice were treated by intraperitoneal (i.p.) injections with 5 or 10 doses of aqueous solutions of cadmium acetate, lead acetate and zinc acetate in concentrations corresponding to 1/10 of the LD50, respectively, 21.5, 0.46 and 1.5 mg/kg bw. The control groups were injected in the same way with distilled water. With only one exception (Cd + Zn group treated with 5 doses), the results show a significant increase of MN in all groups for both treatments (5 and 10 doses). Similarly, the results concerning the SCE revealed a statistically significant increase in all treated animals, with the exception of the Zn group treated with 5 doses. The number of sperm abnormalities was significantly higher in animals treated with 5 doses, except in the group Pb + Zn. In animals treated with 10 doses the number of sperm abnormalities was always statistically higher compared with controls. This study indicates that cadmium, lead and zinc can induce MN, SCEs and sperm abnormalities in Algerian mice and that the clastogenic potential is dependent on the time of exposure and the interaction between the three elements, confirming the environmental damage that may result from the simultaneous action of several metals. Most relevant is the toxic potential for Zn, related with the dose, which may compromise its protective effect against other metal contaminations, such as cadmium.

The non-random occurrence of Robertsonian fusion in the house mouse.

Chromosomal rearrangements such as Robertsonian (Rb) fusions constitute a major phenomenon in the evolution of genome organization in a wide range of organisms. Although proximate mechanisms for the formation of Rb fusion are now well identified, the evolutionary forces that drive chromosomal evolution remain poorly understood. In the house mouse, numerous chromosomal races occur in nature, each defined by a unique combination of Rb fusions. Among the 106 different Rb fusions that were reported from natural populations, the low involvement of chromosome 19 in Rb fusions is striking, prompting the question of the randomness of chromosomal involvement in Rb fusions. We uncover a significant quadratic relationship between chromosome size and probability of fusing, which has never previously been in this species. It appears that fusions involving chromosome 19 are not particularly infrequent, given the expected low fusion probability associated with the chromosome's size. The results are discussed, assessing selective processes or constraints that may operate on chromosome size.