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- The potential of Aeromonas spp. from wildlife as antimicrobial resistance indicators in aquatic environmentsPublication . Grilo, Miguel; Santos, Carla Sousa; Robalo, Joana; Oliveira, ManuelaDeclines in freshwater fish populations are occurring at a fast rate, increasing the importance of ex-situ conservation programs supported by sound knowledge of population dynamics and life-history traits of the target species. We analysed the growth and age structure of wild and captive stocks of the western ruivaco Achondrostoma occidentale, a Portuguese endangered cyprinid, targeted for captive breeding and restocking since 2007. Specifically, we compared maximum size, longevity, and length-at-age among captive and wild populations, restocked and non-restocked. We found considerable variation in length-at-age and longevity between captive-bred and wild fish, with the former generally growing faster and living longer. Analysis of length-age distributions among wild populations suggested a positive effect of restocking actions in fish abundance. Results highlight the value of captive breeding for the conservation of endangered cyprinids, and reinforce the need for detailed data on life-history trait variation between captive and wild stocks to assess the efficiency of ex-situ conservation programs.
- Conservation challenges imposed by evolutionary history and habitat suitability shifts of endangered freshwater mussels under a global climate change scenarioPublication . Reis, Joaquim; Perea, Silvia; Gama, Mafalda; Mendes, Sofia L.; Sousa, Vitor C.; Lima, Cristina S.; Banha, Filipe; Gil, Maria; Alvarez, Maria Garcia; Anastácio, P.M.; Santos, Carla Sousa; Araujo, RafaelClimate change and associated shifts in temperature and precipitation patterns have become an increasing concern as drivers of ongoing biodiversity loss. The Mediterranean region is particularly vulnerable, being both a biodiversity hotspot and a region very prone to desertification. Freshwater mussels are amongst the most threatened invertebrate taxa worldwide. Unio tumidiformis is an endemic and endangered species restricted to the southern Iberian Peninsula, living in temporary Mediterranean-type streams. Freshwater mussels need a fish host for successful larval transformation, meaning U. tumidiformis must belong to the genus Squalius. The main objective of this study was to evaluate the vulnerability of U. tumidiformis to climate change, by studying its population genetics and evolutionary history, its current and future habitat suitability, and that of its hosts. Genetic population structure and diversity were assessed using Single-Nucleotide Polymorphisms through Genotyping by Sequencing and used to infer species evolutionary history. The species potential distribution was modeled using an ensemble forecasting approach, and future shifts in habitat suitability were assessed with the projected climate data layers from Worldclim. Most populations showed extreme genetic differentiation (Fst up to 0.745), even from close neighboring ones. Upper Guadiana populations were more diverse and less differentiated. We hypothesize that U. tumidiformis originated in Upper Guadiana and followed the same colonization routes as their hosts with numerous founder effects and bottlenecks. Our results also predicted a reduction of 99% of climatically suitable areas for U. tumidiformis in the Iberian Peninsula until 2040. For the fish hosts, a maximum 42% reduction in suitable areas was estimated throughout the century, with remaining adequate habitats in the north. Our results suggest that difficult conservation options are necessary, prioritizing the preservation of populations, translocations to the northern area of its historical range and stream engineering to increase resilience to droughts.
- Molecular epidemiology, virulence traits and antimicrobial resistance signatures of aeromonas spp. in the critically endangered iberochondrostoma lusitanicum follow geographical and seasonal patternsPublication . Grilo, Miguel; Isidoro, Sara; Chambel, Lélia; Marques, Carolina S.; Marques, Tiago A.; Sousa-Santos, C.; Robalo, Joana; Oliveira, ManuelaDespite the fact that freshwater fish populations are experiencing severe declines worldwide, our knowledge on the interaction between endangered populations and pathogenic agents remains scarce. In this study, we investigated the prevalence and structure of Aeromonas communities isolated from the critically endangered Iberochondrostoma lusitanicum, a model species for threatened Iberian leuciscids, as well as health parameters in this species. Additionally, we evaluated the virulence profiles, antimicrobial resistance signatures and genomic relationships of the Aeromonas isolates. Lesion prevalence, extension and body condition were deeply affected by location and seasonality, with poorer performances in the dry season. Aeromonas composition shifted among seasons and was also different across river streams. The pathogenic potential of the isolates significantly increased during the dry season. Additionally, isolates displaying clinically relevant antimicrobial resistance phenotypes (carbapenem and fluroquinolone resistance) were detected. As it inhabits intermittent rivers, often reduced to disconnected pools during the summer, the dry season is a critical period for I. lusitanicum, with lower general health status and a higher potential of infection by Aeromonas spp. Habitat quality seems a determining factor on the sustainable development of this fish species. Also, these individuals act as reservoirs of important antimicrobial resistant bacteria with potential implications for public health.
- Multilocus phylogeny and systematics of Iberian endemic Squalius (Actinopterygii, Leuciscidae)Publication . Perea, Silvia; Santos, Carla Sousa; Robalo, Joana; Doadrio, IgnacioInferring the evolutionary history of a group of species can be challenging given the many factors involved. In recent years, the increased availability of sequences of multiple genes per species has spurred the development of new methodologies to analyse multilocus data sets. Two approaches that analyse such data are concatenated supermatrix and coalescent-based species-tree analyses. In this study, we used both of these methods to infer the phylogenetic relationships of Iberian species of the genus Squalius from one mitochondrial and six nuclear genes. We found mitonuclear discordance in the phylogenetic relationships of the group. According to the mitochondrial gene analysis, all species were recovered as monophyletic except S. pyrenaicus; besides, in the concatenated supermatrix analysis of the nuclear markers, this species resolved as polyphyletic with three divergent evolutionary lineages. The coalescent-based nuclear species-tree analysis rendered a well-resolved phylogeny compared with the supermatrix analysis, which was unable to discern between S. carolitertii, S. castellanus and one of the evolutionary lineages of S. pyrenaicus. This result is likely due to the better integration of population uncertainty in the coalescent approach. Furthermore, Bayesian multilocus species delimitation analyses based on a BPP approach strongly supported the distinct nuclear lineages as different species. Nevertheless, the supermatrix analysis was able to obtain well-supported relationships in the divergent lineages with low numbers of individuals. Our study highlights the usefulness of different analytical methodologies to obtain a more complete picture of the evolutionary history of taxa, especially when discordant patterns among genes are found.
- Climatic alterations influence bacterial growth, biofilm production and antimicrobial resistance profiles in aeromonas spp.Publication . Grilo, Miguel L.; Pereira, Ana; Sousa-Santos, Carla; Robalo, Joana; Oliveira, ManuelaClimate change is expected to create environmental disruptions that will impact a wide array of biota. Projections for freshwater ecosystems include severe alterations with gradients across geographical areas. Life traits in bacteria are modulated by environmental parameters, but there is still uncertainty regarding bacterial responses to changes caused by climatic alterations. In this study, we used a river water microcosm model to evaluate how Aeromonas spp., an important pathogenic and zoonotic genus ubiquitary in aquatic ecosystems, responds to environmental variations of temperature and pH as expected by future projections. Namely, we evaluated bacterial growth, biofilm production and antimicrobial resistance profiles of Aeromonas species in pure and mixed cultures. Biofilm production was significantly influenced by temperature and culture, while temperature and pH affected bacterial growth. Reversion of antimicrobial susceptibility status occurred in the majority of strains and tested antimicrobial compounds, with several combinations of temperature and pH contributing to this effect. Current results highlight the consequences that bacterial genus such as Aeromonas will experience with climatic alterations, specifically how their proliferation and virulence and phenotypic resistance expression will be modulated. Such information is fundamental to predict and prevent future outbreaks and deleterious effects that these bacterial species might have in human and animal populations.
- Aeromonas spp. Prevalence, virulence, and antimicrobial resistance in an ex situ program for tThreatened freshwater fish—A pilot study with protective measuresPublication . Grilo, Miguel; Amaro, Guadalupe; Chambel, Lélia; Marques, Carolina S.; Marques, Tiago A.; Gil, Fátima; Sousa-Santos, Carla; Robalo, Joana; Oliveira, ManuelaEx situ breeding programs are important conservation tools for endangered freshwater fish. However, developing husbandry techniques that decrease the likelihood of disease, antimicrobial resistance, and virulence determinants acquisition during this process is challenging. In this pilot study, we conducted a captivity experiment with Portuguese nase (Iberochondrostoma lusitanicum), a critically endangered leuciscid species, to investigate the influence of simple protective measures (i.e., material disinfection protocols and animal handling with gloves) on the dynamics of a potential pathogenic genus, Aeromonas, as well as its virulence profiles and antimicrobial resistance signatures. Our findings show that antimicrobial resistance in Aeromonas spp. collected from I. lusitanicum significantly increased during the extent of the assay (5 weeks), with all isolates collected at the end of the study classified as multidrug-resistant. Additionally, humans handling fishes without protective measures were colonized by Aeromonas spp. The use of protective measures suggested a decreasing trend in Aeromonas spp. prevalence in I. lusitanicum, while bacterial isolates displayed significantly lower virulence index values when virulence phenotypical expression was tested at 22 °C. Despite this study representing an initial trial, which needs support from further research, protective measures tested are considered a simple tool to be applied in ex situ breeding programs for aquatic animals worldwide. Furthermore, current results raise concern regarding antimicrobial resistance amplification and zoonotic transmission of Aeromonas spp. in aquatic ex situ programs.