Although the presence/absence of aquatic invertebrates using environmental DNA (eDNA) has been established for several species, inferring population densities has remained problematic. The invasive American signal crayfish, Pacifastacus leniusculus (Dana), is the leading cause of decline in the UK's only native crayfish species, Austropotamobius pallipes (Lereboullet). Methods to detect species at low abundances offer the opportunity for the early detection, and potential eradication, of P. leniusculus before population densities reach threatening levels in areas occupied by A. pallipes. Using a factorial experimental design with aquaria, we investigated the impacts of biomass, sex ratio, and fighting behavior on the amount of eDNA released by P. leniusculus, with the aim to infer density per aquarium depending on treatments. The amount of target eDNA in water samples from each aquarium was measured using the quantitative Polymerase Chain Reaction. We show that the presence of eggs significantly increases the concentration of crayfish eDNA per unit of mass, and that there is a significant relationship between eDNA concentration and biomass when females are egg‐bearing. However, the relationship between crayfish biomass and eDNA concentration is lost in aquaria without ovigerous females. Female‐specific tanks had significantly higher eDNA concentrations than male‐specific tanks, and the prevention of fighting did not impact the amount of eDNA in the water. These results indicate that detection and estimate of crayfish abundance using eDNA may be more effective while females are ovigerous. This information should guide further research for an accurate estimation of crayfish biomass in the field depending on the season. Our results indicate that detection and quantification of egg‐laying aquatic invertebrate species using eDNA could be most successful during periods when eggs are developing in the water. We recommend that practitioners consider the reproductive cycle of target species when attempting to study or detect aquatic species using eDNA in the field.

1. Reliable and accurate biodiversity census methods are essential for monitoring ecosystem health and assessing potential ecological impacts of future development projects. Although metabarcoding is increasingly used to study biodiversity across ecological research, morphology-based identification remains the preferred approach for marine ecological impact assessments. Comparing metabarcoding to morphology-based protocols currently used by ecological surveyors is essential to determine whether this DNA-based approach is suitable for long-term monitoring of the marine ecosystems. 2. We compared metabarcoding and morphology-based approaches for the analysis of invertebrates in low diversity intertidal marine sediment samples. We used a recently developed bioinformatics pipeline and two taxonomic assignment methods to resolve and assign amplicon sequence variants (ASVs) from Illumina amplicon data. We analysed the community composition recovered by both methods and tested the effects, on the levels of diversity detected by the metabarcoding method, of sieving samples prior to DNA extraction. 3. Metabarcoding of the mitochondrial marker cytochrome c oxidase I (COI) gene recovers the presence of more taxonomic groups than the morphological approach. We found that sieving samples results in lower alpha diversity detected and suggests a community composition that differs significantly from that suggested by un-sieved samples in our metabarcoding analysis. We found that while metabarcoding and morphological approaches detected similar numbers of species, they are unable to identify the same set of species across samples. 4. Synthesis and applications. We show that metabarcoding using the cytochrome c oxidase I (COI) marker provides a more holistic, community-based, analysis of benthic invertebrate diversity than a traditional morphological approach. We also highlight current gaps in reference databases and bioinformatic pipelines for the identification of intertidal benthic invertebrates that need to be addressed before metabarcoding can replace traditional methods. Ultimately, with these limitations taken into consideration, resolving community-wide diversity patterns with metabarcoding could improve the management of non-protected marine habitats in the United Kingdom.

SUMMARYAquaporins (AQPs) are membrane proteins that form water channels, allowing rapid movement of water across cell membranes. AQPs have been reported in species of all life kingdoms and in almost all tissues, but little is known about them in insects. Our purpose was to explore the occurrence of AQPs in the ovary of the phylogenetically basal insect Blattella germanica (L.) and to study their possible role in fluid homeostasis during oogenesis. We isolated an ovarian AQP from B. germanica (BgAQP) that has a deduced amino acid sequence showing six potential transmembrane domains, two NPA motifs and an ar/R constriction region, which are typical features of the AQP family. Phylogenetic analyses indicated that BgAQP belongs to the PRIP group of insect AQPs, previously suggested to be water specific. However, ectopic expression of BgAQP in Xenopus laevis oocytes demonstrated that this AQP transports water and modest amounts of urea, but not glycerol, which suggests that the PRIP group of insect AQPs may have heterogeneous solute preferences. BgAQP was shown to be highly expressed in the ovary, followed by the fat body and muscle tissues, but water stress did not significantly modify the ovarian expression levels. RNA interference (RNAi) reduced BgAQP mRNA levels in the ovary but the oocytes developed normally. The absence of an apparent ovarian phenotype after BgAQP RNAi suggests that other functionally redundant AQPs that were not silenced in our experiments might exist in the ovary of B. germanica. Supplementary material available online at

Background Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0–171.2] to 180.0 [135.4–227.9] mmHg and the ventilatory ratio from 1.73 [1.33–2.25] to 1.96 [1.61–2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01–1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01–1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93–1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation.