Francisco Petrucci-Fonseca

Mitochondrial DNA phylogeography and population history of the grey wolf Canis lupus

The grey wolf (Canis lupus) and coyote (C. latrans) are highly mobile carnivores that disperse over great distances in search of territories and mates. Previous genetic studies have shown little geographical structure in either species. However, population genetic structure is also influenced by past isolation events and population fluctuations during glacial periods. In this study, control region sequence data from a worldwide sample of grey wolves and a more limited sample of coyotes were analysed. The results suggest that fluctuating population sizes during the late Pleistocene have left a genetic signature on levels of variation in both species. Genealogical measures of nucleotide diversity suggest that historical population sizes were much larger in both species and grey wolves were more numerous than coyotes. Currently, about 300 000 wolves and 7 million coyotes exist. In grey wolves, genetic diversity is greater than that predicted from census population size, reflecting recent historical population declines. By contrast, nucleotide diversity in coyotes is smaller than that predicted by census population size, reflecting a recent population expansion following the extirpation of wolves from much of North America. Both species show little partitioning of haplotypes on continental or regional scales. However, a statistical parsimony analysis indicates local genetic structure that suggests recent restricted gene flow.

The ibex Capra pyrenaica returns to its former Portuguese range

The ibex Capra pyrenaica has recently recolonized its former Portuguese range from a contiguous Spanish protected area. The first observations of ibex in Portugal were in Peneda-Gerês National Park in 1998. In 2001 we began a survey to confirm ibex presence in Portuguese territory, and to determine the current status of the species there. There are three ibex nuclei in the general area of the international border, and they are expanding their geographical range with two of the nuclei almost restricted to Portuguese territory. In 2003 the ibex population consisted of a minimum of 75 individuals. Different founder histories have led to distinct age structures in each nucleus, but in general they exhibit the population dynamics typical of a recently reintroduced population with high reproductive potential. Priority conservation actions for this newly established population need to include increased monitoring, and reinforcement of the population with individuals from elsewhere. Effective conservation will require collaboration between staff of both Portuguese and Spanish protected areas and an integrated Spanish-Portuguese conservation plan.

Comparative proteomics of an extended spectrum β-lactamase producing Escherichia coli strain from the Iberian wolf

UNLABELLED The Iberian wolf (Canis lupus signatus) is an endangered species native to the Iberian Peninsula. Due to their predatory and wild nature, these wolves serve as important indicators of environmental contamination by antimicrobial-resistant bacteria. β-Lactam antibiotics like cefotaxime are the most commonly used antibacterial agents. Bacterial resistance to these antibiotics occurs predominantly through enzymatic inactivation by extended-spectrum beta-lactamases. Escherichia coli strain WA57, isolated from Iberian wolf feces, is a cefotaxime-resistant strain that produces extended-spectrum beta-lactamases. In this study, using 2D-GE combined with MS and bioinformatics, we report significant differences in the abundance of 40 protein spots (p<0.01) from the extracellular, periplasmic, cytoplasmic, and membrane sub-proteomes and the whole-cell proteome of WA57 exposed and non-exposed to cefotaxime. A total of 315 protein spots were collected for protein identification. The comparative proteomics presented gives an overview of the complex changes in expression and metabolism that occur when WA57 is stressed with cefotaxime. Abundance of chaperone, porin and export proteins is particularly affected showing that the stress response and transport functions might directly influence the antibiotic resistance of this strain. BIOLOGICAL SIGNIFICANCE This study highlights the importance of proteomics in detecting protein expression changes in bacterial strains exposed to stress such as that caused by cefotaxime. This approach might help us understand which pathways form barriers for antibiotics. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Iberian wolf as a reservoir of extended-spectrum β-lactamase-producing Escherichia coli of the TEM, SHV, and CTX-M groups.

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment. Moreover, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The association between antibiotic resistance and virulence determinants should be monitored, as it constitutes a serious public health problem.

Molecular structure in peripheral dog breeds: Portuguese native breeds as a case study

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for approximately 30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.

Reassortment among picobirnaviruses found in wolves

We conducted a viral metagenomics study in diarrheic free-ranging wolves in Portugal, revealing for the first time the presence of reassortant picobirnaviruses. These viruses shared identical capsid segments together with diverse RNA-dependent RNA polymerase segments. Even though causality between these picobirnaviruses and diarrhea could not be established, the study nonetheless confirms for the first time that wolves are a potential reservoir for picobirnaviruses, which might play a role as enteric pathogens.

Viral gut metagenomics of sympatric wild and domestic canids, and monitoring of viruses: Insights from an endangered wolf population

Abstract Animal host–microbe interactions are a relevant concern for wildlife conservation, particularly regarding generalist pathogens, where domestic host species can play a role in the transmission of infectious agents, such as viruses, to wild animals. Knowledge on viral circulation in wild host species is still scarce and can be improved by the recent advent of modern molecular approaches. We aimed to characterize the fecal virome and identify viruses of potential conservation relevance of diarrheic free‐ranging wolves and sympatric domestic dogs from Central Portugal, where a small and threatened wolf population persists in a highly anthropogenically modified landscape. Using viral metagenomics, we screened diarrheic stools collected from wolves (n = 8), feral dogs (n = 4), and pet dogs (n = 6), all collected within wolf range. We detected novel highly divergent viruses as well as known viral pathogens with established effects on population dynamics, including canine distemper virus, a novel bocavirus, and canine minute virus. Furthermore, we performed a 4‐year survey for the six wolf packs comprising this endangered wolf population, screening 93 fecal samples from 36 genetically identified wolves for canine distemper virus and the novel bocavirus, previously identified using our metagenomics approach. Our novel approach using metagenomics for viral screening in noninvasive samples of wolves and dogs has profound implications on the knowledge of both virology and wildlife diseases, establishing a complementary tool to traditional screening methods for the conservation of threatened species.

Hematology and serum biochemistry values of free-ranging Iberian wolves (Canis lupus) trapped by leg-hold snares

Hematology and serum biochemistry are important tools in assessing the health and physiological status of wildlife populations. Nevertheless, studies on free-ranging wolves (Canis lupus) are scarce, and no reference values are available neither for Iberian wolves nor for wolves captured with leg-hold snares. We report 37 hematology and serum biochemistry variables obtained from 26 free-ranging Iberian wolves captured with leg-hold snares between 2007 and 2014, including variables previously not reported in the literature. The values obtained are similar to the published reference intervals for Scandinavian wolves captured by darting from a helicopter, except for higher values for mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), leukocyte count, creatinine kinase (CK), γ-globulins, and total bilirubin (TBIL) and lower values for alkaline phosphatase (ALP). We propose that differences in leukocyte count, CK, and TBIL are related to the method of capture, while differences in RDW, MCHC, ALP, and γ-globulins could reflect physiological adaptations to environmental conditions, sampling, or pre-analytical artifacts. Lymphocyte count was lower and neutrophil/lymphocyte ratio was significantly higher in older, reproductive females, while ALP and phosphorus were higher in juvenile wolves. For the first time, we describe hematology and serum biochemistry values of free-ranging Iberian wolves captured with leg-hold snares. The data reported here is the first published reference for wolves captured with similar methods and for monitoring Iberian wolves populations’ physiological and health status.

Evaluating the predictive power of field variables for species and individual molecular identification on wolf noninvasive samples

Live-trapping elusive animals is often challenging, hampering the achievement of reasonable sample sizes for molecular studies. In such cases, the use of noninvasive samples (NIS) is critical in many research fields, mostly related to ecology, management and conservation of wild species. We analysed the influence of several variables potentially associated with the quality of wolf NIS—season, weather conditions, and in situ collected site and sample characteristics—on the success rates of species and individual identification performed using mtDNA and 13 microsatellites, respectively. NIS included scats, urine and saliva collected from two areas in Portugal. Scat samples exhibited the highest success rate for both species (81%) and individual identification (59%), compared with urine (63 and 30%, respectively) or saliva samples (48 and 36%, respectively). The success rate of species identification of scats was better explained by season of collection, the presence of mucous, moisture and odour. For samples with successful species identification analysis, individual identification success was best predicted by the presence of odour. Performing a preliminary selection of scat samples with the best characteristics can increase up to 13% the success rates of molecular analysis. Urine collected on snow had a higher success rate of species identification than that collected on vegetation. To our knowledge, this was the first time that wolf urine on vegetation near ground-scratching marks is used as DNA source. Saliva samples collected with different substrate types can also be used for species identification. These results contribute to optimising noninvasive sampling procedures, maximising the success of molecular ecology studies, and ultimately minimising sampling efforts and costs.

Does livestock influence the diet of Iberian ibex Capra pyrenaica in the Peneda-Gerês National Park (Portugal)?

Abstract We carried out a pilot study about the influence of the domestic goat Capra hircus on the feeding ecology of the Iberian ibex Capra pyrenaica in the Peneda-Gerês National Park (PGNP, Portugal). We mapped the grazing areas of domestic goat flocks and determined their stocking rates in two mountains of PGNP, Gerês and Amarela, and we investigated the diets of both ungulates using fecal microhistology in an area where they partially overlapped during spring and summer. Diet composition, ligneous diversity, and ligneous trophic niche were compared seasonally. Although differences in ligneous components were observed, results revealed a considerable diet overlap mainly due to graminoids consumption, especially during autumn and spring. This suggests that direct or indirect foraging competition between the goat species should not be discarded if they spatially concur. Results also emphasized distinct summer feeding strategies of C. pyrenaica in the area, and the influence of cattle (Bos taurus) and horses (Equus caballus) were hypothesized. We recommend removing feral goats from the mountain environment and monitoring grazing areas of domestic flocks. Estimates of ecological carrying capacity and future diet studies should include cattle, horses, and improvement of herb identification.