SARS-CoV-2 RNA detected in urban wastewater from Porto, Portugal: Method optimization and continuous 25-week monitoring.

Research on the emerging COVID-19 pandemic is demonstrating that wastewater infrastructures can be used as public health observatories of virus circulation in human communities. Important efforts are being organized worldwide to implement sewage-based surveillance of SARS-CoV-2 that can be used for preventive or early warning purposes, informing preparedness and response measures. However, its successful implementation requires important and iterative methodological improvements, as well as the establishment of standardized methods. The aim of this study was to develop a continuous monitoring protocol for SARS-CoV-2 in wastewater, that could be used to model virus circulation within the communities, complementing the current clinical surveillance. Specific objectives included (1) optimization and validation of a method for virus quantification; (2) monitoring the time-evolution of SARS-CoV-2 in wastewater from two wastewater treatment plants (WWTPs) in the city of Porto, Portugal. Untreated wastewater samples were collected weekly from the two WWTPs between May 2020 and March 2021, encompassing two COVID-19 incidence peaks in the region (mid-November 2020 and mid-January 2021). In the first stage of this study, we compared, optimized and selected a sampling and analysis protocol that included virus concentration through centrifugation, RNA extraction from both liquid and solid fractions and quantification by reverse transcription quantitative PCR (RT-qPCR). In the second stage, we used the selected methodology to track SARS-CoV-2 in the collected wastewater over time. SARS-CoV-2 RNA was detected in 39 and 37 out of 48 liquid and solid fraction samples of untreated wastewater, respectively. The copy numbers varied throughout the study between 0 and 0.15 copies/ng RNA and a good fit was observed between the SARS-CoV-2 RNA concentration in the untreated wastewater and the COVID-19 temporal trends in the study region. We also analyzed eight samples from the treated effluent and found no SARS-CoV-2 RNA detection after tertiary treatment and UV disinfection. In agreement with the recent literature, the results from this study support the use of wastewater-based surveillance to complement clinical testing and evaluate temporal and spatial trends of the current pandemic.

Seasonality effects on chemical composition, antibacterial activity and essential oil yield of three species of Nectandra.

In this study, we aimed to determine whether seasonality affects the content, chemical composition, and antimicrobial activity of essential oils (EOs) from the leaves of three species of Nectandra (Nectandra megapotamica, Nectandra grandiflora, and Nectandra lanceolata) native to the Atlantic rainforest, Sao Paulo state, Brazil. In addition, we identified the compounds potentially related to the antimicrobial activity. Leaves were randomly collected in the middle of winter (August), spring (November), summer (February), and autumn (May). The influence of seasonality on the content and chemical composition of EOs from the Nectandra species was evident in this study. The EOs from N. lanceolata and N. grandiflora were characterized by similarities in the chemical composition and had a higher relative proportion of oxygenated sesquiterpenes. N. megapotamica presented a different chemical profile, with plenty of monoterpenic and sesquiterpenic hydrocarbons. Changes in the EO chemical profile because of seasonality were shown by the similarities between the EOs obtained in spring and autumn and the differences between the EOs obtained in summer and winter. The EO from the leaves of N. megapotamica harvested in winter and spring showed the highest control of the growth of Escherichia coli, and this antimicrobial action can be related to the monoterpenes α-pinene and ß-pinene as well as myrcene and limonene. The minimum inhibitory concentration (MIC) of the EO from the leaves of N. lanceolata harvested in summer and autumn was lower against the gram-positive bacterium Staphylococcus aureus and can be related to the sesquiterpene hydrocarbons isobicyclogermacrenal, epi-zizanone, and germacrene B.

Microsatellite markers for Cariniana legalis (Lecythidaceae) and their transferability to C. estrellensis.

PREMISE OF THE STUDY: Microsatellite primers were developed in the neotropical tree species Cariniana legalis (Lecythidaceae) to investigate its genetic diversity, mating system, and gene flow. • METHODS AND RESULTS: We identified 96 clones containing 82 repeat motifs from a genomic library enriched for (CT)8 and (GT)8 motifs. Primer pairs were developed for 13 microsatellite loci and validated in 51 C. legalis specimens and 26 C. estrellensis specimens. Eleven loci were polymorphic, revealing a maximum of two to 15 alleles per locus in C. legalis and three to 12 in C. estrellensis. For C. legalis, the observed (H o) and expected (H e) heterozygosities ranged from 0 to 0.99 and from 0.07 to 0.90, respectively. For C. estrellensis, H o and H e ranged from 0 to 0.96 and from 0.14 to 0.91, respectively. • CONCLUSIONS: The primers identified polymorphic loci that are suitable to study genetic diversity and structure, mating system, and gene flow in C. legalis and the related species C. estrellensis.

High levels of genetic differentiation and selfing in the Brazilian cerrado fruit tree Dipteryx alata Vog. (Fabaceae).

Dipteryx alata is a native fruit tree species of the cerrado (Brazilian savanna) that has great economic potential because of its multiple uses. Knowledge of how the genetic variability of this species is organized within and among populations would be useful for genetic conservation and breeding programs. We used nine simple sequence repeat (SSR) primers developed for Dipteryx odorata to evaluate the genetic structure of three populations of D. alata located in central Brazil based on a leaf sample analysis from 101 adults. The outcrossing rate was evaluated using 300 open-pollinated offspring from 25 seed-trees. Pollen dispersal was measured by parentage analysis. We used spatial genetic structure (SGS) to test the minimal distance for harvesting seeds in conservation and breeding programs. Our data indicate that the populations studied had a high degree of genetic diversity and population structure, as suggested by the high level of divergence among populations . The estimated outcrossing rate suggested a mixed mating system, and the intrapopulation fixation index was influenced by SGS. We conclude that seed harvesting for genetic conservation and breeding programs requires a minimum distance between trees of 196 m to avoid collecting seeds from related seed-trees.

High levels of genetic differentiation and selfing in the Brazilian cerrado fruit tree Dipteryx alata Vog. (Fabaceae)

Dipteryx alata is a native fruit tree species of the cerrado (Brazilian savanna) that has great economic potential because of its multiple uses. Knowledge of how the genetic variability of this species is organized within and among populations would be useful for genetic conservation and breeding programs. We used nine simple sequence repeat (SSR) primers developed for Dipteryx odorata to evaluate the genetic structure of three populations of D. alata located in central Brazil based on a leaf sample analysis from 101 adults. The outcrossing rate was evaluated using 300 open-pollinated offspring from 25 seed-trees. Pollen dispersal was measured by parentage analysis. We used spatial genetic structure (SGS) to test the minimal distance for harvesting seeds in conservation and breeding programs. Our data indicate that the populations studied had a high degree of genetic diversity and population structure, as suggested by the high level of divergence among populations . The estimated outcrossing rate suggested a mixed mating system, and the intrapopulation fixation index was influenced by SGS. We conclude that seed harvesting for genetic conservation and breeding programs requires a minimum distance between trees of 196 m to avoid collecting seeds from related seed-trees.