Shotgun metagenomic sequencing for conservation genomics: rock ptarmigan as a case study

The rock ptarmigan (Lagopus muta) inhabits alpine and arctic tundra throughout the northern hemisphere with its current distribution mainly determined by the last glacial maximum. In southern Europe this species is only found in fragmented populations on the main mountain ranges, where numbers are decreasing due to habitat loss, degradation and fragmentation, climate warming and human disturbance. This decline could be mitigated through conservation measures, but these require fundamental information on the evolutionary ecology, distribution and genetic diversity. Such data is difficult to collect in a species which is particularly elusive and living in extreme environments. Thanks to the technological and computational advances of recent years, understanding of species’ biology has been greatly enhanced by analyzing fecal samples, collected non-invasively in the field, using shotgun metagenomics sequencing. Although this approach is still poorly tested in wild species, ongoing studies highlight its potential for conservation and biomonitoring that should be further explored. In the present study, this approach was applied to 30 rock ptarmigan from three different areas of the Paneveggio-Pale di San Martino Natural Park, characterized by contrasting rocky substrates. The shotgun metagenomic sequencing method does not require the enrichment or targeted amplification of specific molecular markers, thus allowing both qualitative and quantitative evaluation of data on different aspects related to the species. In particular, the ongoing analyses aimed to determine: i) the genomic relationships between individuals and population, ii) the fundamental resources for the species through the study of the diet, iii) the health status of individuals through the analysis of gut microbiota and parasites. First results confirmed the utility of shotgun metagenomic sequencing for conservation purposes and provides fundamental data for improving conservation strategies for this species

Early-succession secondary forests following agropastoral abandonment are key winter habitats for the conservation of a priority bird in the European Alps

AbstractIn contrast to old-growth forests, early-successional stands remain understudied despite potentially harbouring species of conservation interest. With this work, focused on hazel grouse Tetrastes bonasia, a cryptic and indicator species known to select for close-to-natural forests, we evaluated winter densities, home range, microhabitat selection and diet, combining DNA-based mark-recapture and metabarcoding from faecal samples. In total, 216 droppings, collected over 2 years along forest transects in the Italian Alps, were successfully genotyped and 43 individuals were identified. Density estimates were similar to values reported by other studies in the Alps with an average of 4.5 and 2.4 individuals/km2 in the first and second study year, respectively, and mean home ranges estimated at 0.95 km2. According to habitat selection models and eDNA-based diet analysis, hazel grouse selected early-succession secondary-growth forests formed after the abandonment of traditional agropastoral activities. These forests, mostly composed of hazel Corylus avellana, Norway spruce Picea abies and Sorbus spp., provided winter food resources and shelter. The diet analysis also highlighted forest arthropods as a non-negligible source of food. Birds avoided areas subject to intensive browsing by ungulates; small forest roads seasonally closed to traffic had positive influence on hazel grouse (i.e. higher abundance of droppings), while roads open to traffic had no effect. Importantly, despite the high coverage of mature forest habitats of Community Interest (53% of our study area), droppings were more abundant in non-listed early-succession secondary forests with similar plant composition. Our results suggest that forest succession after agropastoral abandonment may be beneficial for some forest birds of conservation interest, while acknowledging its negative effects on the previous grassland biodiversity. Graphical abstract</jats:p

Early-succession secondary forests following agropastoral abandonment are key winter habitats for the conservation of a priority bird in the European Alps

In contrast to old-growth forests, early-successional stands remain understudied despite potentially harbouring species of conservation interest. With this work, focused on hazel grouse Tetrastes bonasia, a cryptic and indicator species known to select for close-to-natural forests, we evaluated winter densities, home range, microhabitat selection and diet, combining DNA-based mark-recapture and metabarcoding from faecal samples. In total, 216 droppings, collected over 2 years along forest transects in the Italian Alps, were successfully genotyped and 43 individuals were identified. Density estimates were similar to values reported by other studies in the Alps with an average of 4.5 and 2.4 individuals/km2 in the first and second study year, respectively, and mean home ranges estimated at 0.95 km2. According to habitat selection models and eDNA-based diet analysis, hazel grouse selected early-succession secondary-growth forests formed after the abandonment of traditional agropastoral activities. These forests, mostly composed of hazel Corylus avellana, Norway spruce Picea abies and Sorbus spp., provided winter food resources and shelter. The diet analysis also highlighted forest arthropods as a non-negligible source of food. Birds avoided areas subject to intensive browsing by ungulates; small forest roads seasonally closed to traffic had positive influence on hazel grouse (i.e. higher abundance of droppings), while roads open to traffic had no effect. Importantly, despite the high coverage of mature forest habitats of Community Interest (53% of our study area), droppings were more abundant in non-listed early-succession secondary forests with similar plant composition. Our results suggest that forest succession after agropastoral abandonment may be beneficial for some forest birds of conservation interest, while acknowledging its negative effects on the previous grassland biodiversity. Graphical abstract: [Figure not available: see fulltext.

Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection

Enzymes have been highly demanded in diverse applications such as in the food, pharmaceutical, and industrial fuel sectors. Thus, in silico bioprospecting emerges as an efficient strategy for discovering new enzyme candidates. A new program called ProspectBIO was developed for this purpose as it can find non-annotated sequences by searching for homologs of a model enzyme directly in genomes. Here we describe the ProspectBIO software methodology and the experimental validation by prospecting for novel lipases by sequence homology to Candida antarctica lipase B (CaLB) and conserved motifs. As expected, we observed that the new bioprospecting software could find more sequences (1672) than a conventional similarity-based search in a protein database (733). Additionally, the absence of patent protection was introduced as a criterion resulting in the final selection of a putative lipase-encoding gene from Ustilago hordei (UhL). Expression of UhL in Pichia pastoris resulted in the production of an enzyme with activity towards a tributyrin substrate. The recombinant enzyme activity levels were 4-fold improved when lowering the temperature and increasing methanol concentrations during the induction phase in shake-flask cultures. Protein sequence alignment and structural modeling showed that the recombinant enzyme has high similarity and capability of adjustment to the structure of CaLB. However, amino acid substitutions identified in the active pocket entrance may be responsible for the differences in the substrate specificities of the two enzymes. Thus, the ProspectBIO software allowed the finding of a new promising lipase for biotechnological application without the need for laborious and expensive conventional bioprospecting experimental steps

Condições de trabalho e transtornos mentais comuns em trabalhadores da rede básica de saúde de Botucatu (SP)

Os transtornos mentais comuns (TMC) apresentam elevada prevalência em populações gerais e de trabalhadores, com consequências individuais e sociais importantes. Este estudo, transversal e descritivo, explora a relação entre demandas psicológicas, grau de controle e presença de suporte social no trabalho e prevalência de TMC em trabalhadores da rede básica de saúde de Botucatu (SP). A coleta de dados foi feita por meio de questionário autoaplicável, não identificado, com destaque para itens relativos à demanda-controle-suporte e presença de TMC (Self Reporting Questionnaire, SRQ-20). As informações foram inseridas em banco de dados construído com Excel/Office XP 2003 e a análise estatística, efetuada com o programa SAS. Constatou-se que 42,6% dos trabalhadores apresentavam TMC. A observação de associação - alta prevalência de TMC com elevado desgaste (classificação de Karasek) e baixa prevalência de TMC com baixo desgaste - indica que, no município estudado, as condições de trabalho na atenção básica constituem fator contributivo não negligenciável ao adoecimento dos trabalhadores. Revela-se a necessidade de intervenções direcionadas ao cuidado aos trabalhadores, melhoria das condições de trabalho e aumento do suporte social no trabalho.Common mental disorders (CMD) present high prevalence among general populations and workers with important individual and social consequences. This cross-sectional and descriptive study explores the relationship between psychological job demands, job control degree and job support and prevalence of CMD among primary health care workers of Botucatu - SP. The data collection was carried out using an unidentified self-administered questionnaire, with emphasis on items relating to demand-control-support situation and occurrence of CMD (Self Reporting Questionnaire, SRQ-20). The data were stored using the software Excel / Office XP 2003, and the statistical analyses were performed in SAS system. It was evidenced that 42.6% of primary health care workers presented CMD. The observed association - high prevalence of CMD with high-strain job (Karasek model) and low prevalence of CMD with low-strain job - indicates that, in the studied city, primary health care work conditions are contributive factors to workers' illness. The survey reveals the need of interventions aiming at caring the workers and also gets better work conditions and increase social support at work