A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Marine biology of the pacific lamprey Entosphenus tridentatus

Reviews in Fish Biology and Fisheries - Pacific lamprey Entosphenus tridentatus has an anadromous life cycle that begins with larvae that filter-feed in freshwater, followed by transformation into...     

Adaptive management assists reintroduction as higher tides threaten an endangered salt marsh plant

In theory, extirpated plant species can be reintroduced and managed to restore sustainable populations. However, few reintroduced plants are known to persist for more than a few years. Our adaptive‐management case study illustrates how we restored the endangered hemiparasitic annual plant, Chloropyron maritimum subsp. maritimum (salt marsh bird's beak), to Sweetwater Marsh, San Diego Bay National Wildlife Refuge, California, United States, and used monitoring and experimentation to identify the factors limiting the reintroduced population. After extirpation in 1988, reintroduction starting that year led to a resilient, genetically diverse population in 2016 (a “boom” of approximately 14,000) that rebounded from a “bust” (62 in 2014). Multiple regressions attributed 82% of the variation in population counts to tidal amplitude, rainfall, and temperature. Populations of salt marsh bird's beak crashed when the diurnal tide range peaked during the 18.6‐year lunar nodal cycle (a rarely considered factor that periodically added approximately 12 cm to tidal ranges). We explain booms as follows: During smaller tidal amplitudes, above‐average rainfall could desalinize upper intertidal soils and stimulate salt marsh bird's beak germination. Then, moderate temperature in May favors growth to reproduction in June. In addition, salt marsh bird's beak needs a short and open canopy of native perennial plants, with roots to parasitize (not non‐native annual grass pseudohosts) and nearby upland soil for a preferred pollinator, ground‐burrowing bees. Although our reintroduced salt marsh bird's beak population is an exceptional case of persistence, this rare species‐specific environmental and biological requirement makes it vulnerable to rising sea levels and global warming.     

Repeated exposure to media violence is associated with diminished response in an inhibitory frontolimbic network

Background

Media depictions of violence, although often claimed to induce viewer aggression, have not been shown to affect the cortical networks that regulate behavior.

Methodology/Principal Findings

Using functional magnetic resonance imaging (fMRI), we found that repeated exposure to violent media, but not to other equally arousing media, led to both diminished response in right lateral orbitofrontal cortex (right ltOFC) and a decrease in right ltOFC-amygdala interaction. Reduced function in this network has been previously associated with decreased control over a variety of behaviors, including reactive aggression. Indeed, we found reduced right ltOFC responses to be characteristic of those subjects that reported greater tendencies toward reactive aggression. Furthermore, the violence-induced reduction in right ltOFC response coincided with increased throughput to behavior planning regions.

Conclusions

These novel findings establish that even short-term exposure to violent media can result in diminished responsiveness of a network associated with behaviors such as reactive aggression.     

Scale-dependent effects of landscape context on urban bee diversity

Journal of Insect Conservation - As urbanization continues throughout much of the world, there is great interest in better understanding the value of urban and residential environments to...     

Response of Amazonian forests to mid-Holocene drought: A model-data comparison

There is a major concern for the fate of Amazonia over the coming century in the face of anthropogenic climate change. A key area of uncertainty is the scale of rainforest dieback to be expected under a future, drier climate. In this study, we use the middle Holocene (ca. 6000 years before present) as an approximate analogue for a drier future, given that palaeoclimate data show much of Amazonia was significantly drier than present at this time. Here, we use an ensemble of climate and vegetation models to explore the sensitivity of Amazonian biomes to mid-Holocene climate change. For this, we employ three dynamic vegetation models (JULES, IBIS, and SDGVM) forced by the bias-corrected mid-Holocene climate simulations from seven models that participated in the Palaeoclimate Modelling Intercomparison Project 3 (PMIP3). These model outputs are compared with a multi-proxy palaeoecological dataset to gain a better understanding of where in Amazonia we have most confidence in the mid-Holocene vegetation simulations. A robust feature of all simulations and palaeodata is that the central Amazonian rainforest biome is unaffected by mid-Holocene drought. Greater divergence in mid-Holocene simulations exists in ecotonal eastern and southern Amazonia. Vegetation models driven with climate models that simulate a drier mid-Holocene (100–150 mm per year decrease) better capture the observed (palaeodata) tropical forest dieback in these areas. Based on the relationship between simulated rainfall decrease and vegetation change, we find indications that in southern Amazonia the rate of tropical forest dieback was ~125,000 km² per 100 mm rainfall decrease in the mid-Holocene. This provides a baseline sensitivity of tropical forests to drought for this region (without human-driven changes to greenhouse gases, fire, and deforestation). We highlight the need for more palaeoecological and palaeoclimate data across lowland Amazonia to constrain model responses.     

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1′-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1′-modification.     

Type I interferons and MAVS signaling are necessary for tissue resident memory CD8+ T cell responses to RSV infection

Respiratory syncytial virus (RSV) can cause bronchiolitis and viral pneumonia in young children and the elderly. Lack of vaccines and recurrence of RSV infection indicate the difficulty in eliciting protective memory immune responses. Tissue resident memory T cells (T_RM) can confer protection from pathogen re-infection and, in human experimental RSV infection, the presence of lung CD8⁺ T_RM cells correlates with a better outcome. However, the requirements for generating and maintaining lung T_RM cells during RSV infection are not fully understood. Here, we use mouse models to assess the impact of innate immune response determinants in the generation and subsequent expansion of the T_RM cell pool during RSV infection. We show that CD8⁺ T_RM cells expand independently from systemic CD8⁺ T cells after RSV re-infection. Re-infected MAVS and MyD88/TRIF deficient mice, lacking key components involved in innate immune recognition of RSV and induction of type I interferons (IFN-α/β), display impaired expansion of CD8⁺ T_RM cells and reduction in antigen specific production of granzyme B and IFN-γ. IFN-α treatment of MAVS deficient mice during primary RSV infection restored T_RM cell expansion upon re-challenge but failed to recover T_RM cell functionality. Our data reveal how innate immunity, including the axis controlling type I IFN induction, instructs and regulates CD8⁺ T_RM cell responses to RSV infection, suggesting possible mechanisms for therapeutic intervention.