Widespread distribution of rodenticide resistance-conferring mutations in the Vkorc1 gene among house mouse populations in Portuguese Macaronesian islands and Iberian Atlantic areas.

Growing evidence of widespread resistance to anticoagulant rodenticides (ARs) in house mice pose significant challenges to pest control efforts. First-generation ARs were introduced in the early 1950s but resistance to these emerged later that decade. Second-generation rodenticides were then developed, with resistance being reported in the late 1970s. Research has linked resistance to ARs with mutations in the Vkorc1 gene, leading to the use of more toxic and environmentally harmful compounds. In this study, 243 tail tips of house mice from mainland Portugal and Southern Spain, the Azores and Madeira archipelagos were analysed for all 3 exons of the Vkorc1 gene. Mutations L128S, Y139C, along with the so-called spretus genotype Vkorc1spr are considered responsible for reduced susceptibility of house mice to ARs. All these sequence variants were broadly detected throughout the sampling regions. Vkorc1spr was the most often recorded among mainland populations, whereas Y139C was nearly ubiquitous among the insular populations. In contrast, L128S was only detected in mainland Portugal and four islands of the Azores archipelago. All first generation ARs such as warfarin and coumatetralyl are deemed ineffective against all Vkorc1 variants identified in this study. Second-generation bromadiolone and difenacoum should also be discarded to control populations carrying Vkorc1spr, Y139C or L128S mutations. Inadequate use of ARs in regions where resistant animals have been found in large proportions will result in the spreading of rodenticide resistance among rodent populations through the positive selection of non-susceptible individuals. Consequently, ineffectiveness of rodent control will increase and potentiate environmental contamination, hazarding non-target wildlife through secondary poisoning. We highlight the need for Vkorc1 screening as a crucial tool in rodent management, aiding in the selection of the most appropriate control/eradication method in order to prevent misuse of these toxic biocides and the spread of rodenticide resistance among house mouse populations.

Of mice and the 'Age of Discovery': the complex history of colonization of the Azorean archipelago by the house mouse (Mus musculus) as revealed by mitochondrial DNA variation.

Humans have introduced many species onto remote oceanic islands. The house mouse (Mus musculus) is a human commensal and has consequently been transported to oceanic islands around the globe as an accidental stowaway. The history of these introductions can tell us not only about the mice themselves but also about the people that transported them. Following a phylogeographic approach, we used mitochondrial D-loop sequence variation (within an 849- to 864-bp fragment) to study house mouse colonization of the Azores. A total of 239 sequences were obtained from all nine islands, and interpretation was helped by previously published Iberian sequences and 66 newly generated Spanish sequences. A Bayesian analysis revealed presence in the Azores of most of the D-loop clades previously described in the domesticus subspecies of the house mouse, suggesting a complex colonization history of the archipelago as a whole from multiple geographical origins, but much less heterogeneity (often single colonization?) within islands. The expected historical link with mainland Portugal was reflected in the pattern of D-loop variation of some of the islands but not all. A more unexpected association with a distant North European source area was also detected in three islands, possibly reflecting human contact with the Azores prior to the 15th century discovery by Portuguese mariners. Widening the scope to colonization of the Macaronesian islands as a whole, human linkages between the Azores, Madeira, the Canaries, Portugal and Spain were revealed through the sharing of mouse sequences between these areas. From these and other data, we suggest mouse studies may help resolve historical uncertainties relating to the 'Age of Discovery'.

Origin of the chromosomal radiation of Madeiran house mice: a microsatellite analysis of metacentric chromosomes.

Chromosome races of Mus musculus domesticus are characterised by particular sets of metacentric chromosomes formed by Robertsonian fusions and whole-arm reciprocal translocations. The Atlantic island of Madeira is inhabited by six chromosome races of house mice with 6-9 pairs of metacentric chromosomes. Three of these races are characterised by the metacentric 3.8 also found elsewhere in the distribution of M. m. domesticus, including Denmark and Spain. We investigated the possibility that metacentric 3.8 was introduced to Madeira during the initial colonisation, as this could have 'seeded' the cascade of chromosomal mutation that is the basis of the extraordinary chromosomal radiation observed on the island. Variation at 24 microsatellite loci mapping to three different chromosomal regions (proximal, interstitial and distal) of mouse chromosomes 3 and 8 was investigated in 179 mice from Madeira, Denmark, Portugal, Spain, Italy and Scotland. Analyses of microsatellite loci closely linked to the centromeres of these chromosomes ('proximal loci') do not support a common evolutionary origin of metacentric 3.8 among Madeiran, Danish and Spanish mouse populations. Our results suggest that Madeiran mice are genetically more similar to standard karyotype mice from Portugal than to metacentric mice from elsewhere. There is expected to be an interruption to gene flow between hybridising metacentric races on Madeira, particularly in the chromosomal regions close to the rearrangement breakpoints. Consistent with this, relating to differentiation involving chromosomes 3 and 8 on Madeira, we found greater genetic structure among races for proximal than interstitial or distal loci.

Molecular insights into the colonization and chromosomal diversification of Madeiran house mice.

The colonization history of Madeiran house mice was investigated by analysing the complete mitochondrial (mt) D-loop sequences of 156 mice from the island of Madeira and mainland Portugal, extending on previous studies. The numbers of mtDNA haplotypes from Madeira and mainland Portugal were substantially increased (17 and 14 new haplotypes respectively), and phylogenetic analysis confirmed the previously reported link between the Madeiran archipelago and northern Europe. Sequence analysis revealed the presence of four mtDNA lineages in mainland Portugal, of which one was particularly common and widespread (termed the 'Portugal Main Clade'). There was no support for population bottlenecks during the formation of the six Robertsonian chromosome races on the island of Madeira, and D-loop sequence variation was not found to be structured according to karyotype. The colonization time of the Madeiran archipelago by Mus musculus domesticus was approached using two molecular dating methods (mismatch distribution and Bayesian skyline plot). Time estimates based on D-loop sequence variation at mainland sites (including previously published data from France and Turkey) were evaluated in the context of the zooarchaeological record of M. m. domesticus. A range of values for mutation rate (mu) and number of mouse generations per year was considered in these analyses because of the uncertainty surrounding these two parameters. The colonization of Portugal and Madeira by house mice is discussed in the context of the best-supported parameter values. In keeping with recent studies, our results suggest that mutation rate estimates based on interspecific divergence lead to gross overestimates concerning the timing of recent within-species events.

Detection of antibodies against Anaplasma phagocytophilum in Algerian mice (Mus spretus), Portugal.

The recent detection of Anaplasma phagocytophilum in Portugal stimulated further research on the agent's enzootic cycle, which usually involves rodents. Thus a total 322 rodents belonging to five species, including 30 Apodemus sylvaticus (wood mouse), 65 Mus musculus (house mouse), 194 M. spretus (algerian mouse), 5 Rattus norvegicus (brown rat) and 28 R. rattus (black rat), were studied by indirect immunofluorescent assay (IFA) and/or polymerase chain reaction (PCR) for A. phagocytophilum exposure in four sampling areas of mainland and two areas of Madeira Island, Portugal. Overall, 3.6% (7/194) of M. spretus presented with IFA-positive results. Seropositive mice were detected in all three mainland sampling areas where this species was captured, with prevalence of 5.2% (5/96) and 5.0% (1/20) for the Ixodes-areas of Arrábida and Mafra, and 1.3% (1/78) for Mértola, a difference that was not statistically significant (p > 0.05). The majority of IFA-positive mice were detected in spring when considering either Arrábida alone (p = 0.026) or all M. spretus sampling areas together (p = 0.021), although the significance of this association was not evident after Bonferroni correction. Nevertheless, neither the seropositive M. spretus, nor additional samples of 10% seronegative rodents from mainland, and 16% of rodents collected in Madeira Island showed evidence of A. phagocytophilum active infections when spleen and/or lung samples were tested by PCR. Either the M. spretus results represents residual antibodies from past A. phagocytophilum infections, present infections with limited bacteremia, or cross-reactions with closely related agents deserves more investigation.

Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula.

Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals.

Patterns of genic diversity and structure in a species undergoing rapid chromosomal radiation: an allozyme analysis of house mice from the Madeira archipelago.

The chromosomal radiation of the house mouse in the island of Madeira most likely involved a human-mediated colonization event followed by within-island geographical isolation and recurrent episodes of genetic drift. The genetic signature of such processes was assessed by an allozyme analysis of the chromosomal races from Madeira. No trace of a decrease in diversity was observed suggesting the possibility of large founder or bottleneck sizes, multiple introductions and/or a high post-colonization expansion rate. The Madeira populations were more closely related to those of Portugal than to other continental regions, in agreement with the documented human colonization of the island. Such a Portuguese origin contrasts with a study indicating a north European source of the mitochondrial haplotypes present in the Madeira mice. This apparent discrepancy may be resolved if not one but two colonization events took place, an initial north European introduction followed by a later one from Portugal. Asymmetrical reproduction between these mice would have resulted in a maternal north European signature with a nuclear Portuguese genome. The extensive chromosomal divergence of the races in Madeira is expected to contribute to their genic divergence. However, there was no significant correlation between chromosomal and allozyme distances. This low apparent chromosomal impact on genic differentiation may be related to the short time since the onset of karyotypic divergence, as the strength of the chromosomal barrier will become significant only at later stages.

An assessment of time-dependent effects of lead exposure in algerian mice (Mus spretus) using different methodological approaches.

Time-dependent effects of lead (Pb) toxicity were studied in Algerian mice (Mus spretus) treated with Pb acetate via drinking water (1 g Pb acetate/L) for different periods of exposure (15, 45, and 90 d). End points included the determination of hepatic Pb concentration and the assessment of some morphophysiological, biochemical and cytogenetical parameters. A control group receiving distilled water was also monitored for comparative purposes. Hepatic Pb accumulation increased with the time of exposure and was significantly higher in treated mice when compared to controls. In association with significant body mass loss in Pb-exposed mice, for 15 and 45 d, a significant increase in the relative spleen mass was observed after 45 d of intoxication. Pb-exposed mice also showed significant decreases in red blood cells, hematocrit, and mean corpuscular hemoglobin. On the contrary, changes in plasma transferases (aspartate aminotransferase and alanine aminotransferase) and hepatic superoxide dismutase activities did not reach statistical significance. A significant increase in the frequency of micronucleated polychromatic bone marrow erythrocytes was also found in the 90-d-exposed mice, compared to nontreated mice and the other exposed groups. Exposure to Pb acetate resulted also in a slight time-dependent decrease of the polychromatic-normochromatic ratio. These results support the concept that a long-term chronic exposure to Pb induced alterations upon some morphophysiological and genetic parameters in Algerian mice.

Adaptive energetics in house mice, Mus musculus domesticus, from the island of Porto Santo (Madeira archipelago, North Atlantic).

The bioenergetic strategies of house mice (Mus musculus domesticus) from the island of Porto Santo were investigated and compared with those of mice from mainland Portugal. Energy obtained from food ingestion was 18.2% lower in Porto Santo mice than in mainland mice (1.53 vs. 1.87 kJ/g/day). The same pattern was observed for metabolisable energy intake, which was 19.2% lower in island specimens (0.87 vs. 1.08 kJ/g/day for mainland specimens). Apparent digestibility was similar in both groups of mice. Basal metabolic rate (BMR) of Porto Santo individuals was low (1.16 ml O(2)/g/h), representing only 56% of the predicted value, based on body mass, while mainland individuals exhibited a BMR closer to the expected value, corresponding to 87% of the predicted value (1.80 ml O(2)/g/h). Thermoregulatory abilities within the range of 10-28 degrees C ambient temperature did not differ between island and mainland mice. Results suggest an adaptation of Porto Santo mice to the environmental aridity of the island of Porto Santo, leading to a conservative energetic strategy.

Metabolism and thermoregulation in the Cabrera vole (Rodentia: Microtus cabrerae).

Metabolism and thermoregulation were studied for the first time in the Cabrera vole (Microtus cabrerae), an endemic and threatened rodent of the Iberian Peninsula. Low values of resting metabolic rate (RMR) were registered (1.13 mlO(2) g(-1) h(-1)) at the lower limit of the thermoneutral zone (TNZ) (around 33.5 degrees C). Body temperature increased near the TNZ up to 37.3 degrees C but remained stable, around 36 degrees C, at ambient temperatures below 25 degrees C. Values of thermal conductance remained quite stable at ambient temperatures of 10-25 degrees C (0.144-0.160 mlO(2) g(-1) h(-1) degrees C) and increased to 0.301 mlO(2) g(-1) h(-1) degrees C at 33.5 degrees C. Data revealed that M. cabrerae developed a highly adaptive ability of conserving energy and lowering the metabolic cost of thermoregulation at high ambient temperatures, allowing the body temperature to approximate that of the environment and exhibiting low resting metabolic rate and high conductance.

Hepatic elemental contents and antioxidant enzyme activities in Algerian mice (Mus spretus) inhabiting a mine area in central Portugal.

In this study the effects of heavy metals (manganese, iron, copper, zinc) and selenium exposure on the hepatic activity of antioxidant enzymes, superoxide dismutase (SOD) and glutathione S-transferases (GST), were appraised on a seasonal basis in Algerian mice (Mus spretus) inhabiting an active copper mine area. A reference population of the same species was considered for comparative purposes. Different patterns of seasonal variation were found in both populations for the manganese, iron and selenium hepatic concentrations and SOD activity. When the two populations were compared, iron and selenium concentrations were enhanced in mice from the polluted area. In addition, SOD activity was significantly decreased in summer in exposed mice, but no other significant changes in SOD and GST activities between sites throughout the year were recorded. However, when seasonal data within each group of mice were pooled, significant differences were found between sites for the average concentrations of manganese, iron and selenium, which are higher in the polluted site. In addition, significant differences were obtained for the average values of SOD and of GST activities, due to simultaneously higher GST values and slightly lower SOD values in the polluted site. The population from the reference site was more homogeneous for all parameters measured than the population from the polluted area. These results, in particular the higher variability in data collected from mice exposed to heavy metals and selenium, combined with the negative associations between biochemical markers and heavy metals, may suggest, despite the good adaptability of the mice to their habitat, biochemical stress due to diminished environmental quality.

Developmental instability in a riparian population of the Algerian mouse(Mus spretus) associated with a heavy metal-polluted area in central Portugal.

The adverse effects of heavy metals (mostly Cr, Mn, Fe, Cu, Zn, and Se) on the environment were appraised by comparing fluctuating asymmetry of dental characters (molars) in Algerian mice (Mus spretus) from polluted (Oeiras) and reference (Xévora) riparian areas, in central Portugal. The size of tooth traits was shown to be reduced in the contaminated area, and the developmental instability increased. The results suggest that Algerian mice may be used as indicators of heavy metal pollution and that fluctuating asymmetry represents a useful tool for monitoring ecological stresses, even when the contamination levels are low.

Molecular studies on the colonization of the Madeiran archipelago by house mice.

To study the colonization history of the house mouse (Mus musculus domesticus) on the Madeiran archipelago, complete mitochondrial D-loop sequences were obtained for 44 individuals from Madeira, Porto Santo and Ilhas Desertas. Altogether, 19 D-loop haplotypes were identified which formed part of a single clade in a phylogeny incorporating haplotypes from elsewhere in the range of M. m. domesticus, indicating that the Madeiras were colonized from a single source. Similarities between the sequences found in the Madeiras and those in Scandinavia and northern Germany suggest that northern Europe was the source area, and there is the intriguing possibility that the Vikings may have accidentally brought house mice to the archipelago. However, there is no record of Vikings visiting the Madeiras; on historical grounds, Portugal is the most likely source area for Madeiran mice and further molecular data from Portugal are needed to rule out that possibility.

Morphological and haematological parameters in the Algerian mouse (Mus spretus) inhabiting an area contaminated with heavy metals.

In the present study morphological (ratio of body weight to body length, weight of internal organs) and haematological parameters (cells count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration) were used for monitoring potential effects of metal contaminants on the Algerian mouse (Mus spretus). The study also provided the opportunity to evaluate the use of morphological and haematological characteristics as indicators of a metal stress. Data were taken from a riverside population inhabiting a heavy metal-contaminated site and compared with those collected from a reference population of the same species. With the exception of non-reproductive males, all mice weighed less in the metal-polluted site as compared to the reference site. Spleen and kidney masses were also less in contaminated mice but mean haemoglobin concentration was greater in the polluted group versus controls. Responses of mice from the polluted area may indicate physiological stress due to diminished environmental quality. Results also suggest that M. spretus may be used as an indicator in pollution monitoring.

Rodents and Leptospira transmission risk in Terceira island (Azores).

The role of rodents as Leptospira renal carriers in Terceira island was evaluated (1993-1995) through kidney culture and serology [microscopic aglutination test (MAT)] of 94 mice and rats. Fifty-nine animals were positive (n = 41 by serology + culturing; n = 11 serology; n = 7 culturing), presenting a wide distribution in man-made and natural areas. House mice had the highest bacteriological (82.9%) and serological (90.9%) rates, being strictly related to serovar arborea. Black rats were involved in the dispersion of all isolated L. interrogans sensu lato serovars (arborea, copenhageni and icterohaemorrhagiae). Logistic regression analysis and non-metric multi-dimensional scaling, relating Leptospira infection with biological and environmental variables, expressed that adult males Mus domesticus, sexually active and living in humid biotopes, mainly above 500 m, are the most likely reservoirs. This study emphasizes the role of house-mice in the epidemiology of leptospirosis in Terceira and the need of reducing the risk of Leptospira transmission through integrated control programmes, primarily focusing on adult house-mice in peri-domestic environments, before the breeding season.

First epidemiological data on pathogenic leptospires isolated on the Azorean islands.

Insectivores (Erinaceus europaeus) and rodents (Rattus rattus, R. norvegicus and Mus musculus) from different islands of the Azores Archipelago were found to carry three distinct Leptospira interrogans s.l. serovars (copenhageni, icterohaemorrhagiae and ballum) which have never been previously investigated there. The house mouse and the black rat were the major Leptospira reservoirs showing isolation rates ranging from 0% for both species (in Graciosa) to 88% and 33%, respectively (in Sãao Miguel). This study also showed that the majority of the animals with positive kidney cultures exhibited specific agglutinins against the isolated strains of Leptospira. The observed isolation rates in the different islands, with a very interesting island variation in prevalence, suggest that small mammals, serving as sylvatic reservoirs of pathogenic leptospires, may represent an important risk to the health of humans and livestock, particularly in the islands of Terceira and Sãao Miguel.