Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (C t ) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.

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A putative driver of global amphibian decline is the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd). While Bd has been documented across continental Africa, its distribution in West Africa remains ambiguous. We tested 793 West African amphibians (one caecilian and 61 anuran species) for the presence of Bd. The samples originated from seven West African countries - Bénin, Burkina Faso, Côte d'Ivoire, Ghana, Guinea, Liberia, Sierra Leone - and were collected from a variety of habitats, ranging from lowland rainforests to montane forests, montane grasslands to humid and dry lowland savannahs. The species investigated comprised various life-history strategies, but we focused particularly on aquatic and riparian species. We used diagnostic PCR to screen 656 specimen swabs and histology to analyse 137 specimen toe tips. All samples tested negative for Bd, including a widespread habitat generalist Hoplobatrachus occipitalis which is intensively traded on the West African food market and thus could be a potential dispersal agent for Bd. Continental fine-grained (30 arc seconds) environmental niche models suggest that Bd should have a broad distribution across West Africa that includes most of the regions and habitats that we surveyed. The surprising apparent absence of Bd in West Africa indicates that the Dahomey Gap may have acted as a natural barrier. Herein we highlight the importance of this Bd-free region of the African continent - especially for the long-term conservation of several threatened species depending on fast flowing forest streams (Conraua alleni (“Vulnerable”) and Petropedetes natator (“Near Threatened”)) as well as the “Critically Endangered” viviparous toad endemic to the montane grasslands of Mount Nimba (Nimbaphrynoides occidentalis).

Determining the boundaries between species and deciding when to describe new species are challenging practices that are particularly difficult in groups with high levels of geographic variation. The coast horned lizards (Phrynosoma blainvillii, Phrynosoma cerroense and P. coronatum) have an extensive geographic distribution spanning many distinctive ecological regions ranging from northern California to the Cape Region of Baja California, Mexico, and populations differ substantially with respect to external morphology across much of this range. The number of taxa recognized in the group has been reevaluated by herpetologists over 20 times during the last 180 years, and typically without the aid of explicit species delimitation methods, resulting in a turbulent taxonomy containing anywhere from one to seven taxa. In this study, we evaluate taxonomic trends through time by ranking 15 of these species delimitation models (SDMs) using coalescent analyses of nuclear loci and SNPs in a Bayesian model comparison framework. Species delimitation models containing more species were generally favoured by Bayesian model selection; however, several three-species models outperformed some four- and five-species SDMs, and the top-ranked model, which contained five species, outperformed all SDMs containing six species. Model performance peaked in the 1950s based on marginal likelihoods estimated from nuclear loci and SNPs. Not surprisingly, SDMs based on genetic data outperformed morphological taxonomies when using genetic data alone to evaluate models. The de novo estimation of population structure favours a three-population model that matches the currently recognized integrative taxonomy containing three species. We discuss why Bayesian model selection might favour models containing more species, and why recognizing more than three species might be warranted.

Both cryptic and aposematic colour patterns can reduce predation risk to prey. These distinct strategies may not be mutually exclusive, because the impact of prey coloration depends on a predator's sensory system and cognition and on the environmental background. Determining whether prey signals are cryptic or aposematic is a prerequisite for understanding the ecological and evolutionary implications of predator-prey interactions. This study investigates whether coloration and pattern in an exceptionally polymorphic toad, Rhinella alata, from Barro Colorado Island, Panama reduces predation via background matching, disruptive coloration, and/or aposematic signaling. When clay model replicas of R. alata were placed on leaf litter, the model's dorsal patternbut not its colour -affected attack rates by birds. When models were placed on white paper, patterned and un-patterned replicas had similar attack rates by birds. These results indicate that dorsal patterns in R. alata are functionally cryptic and emphasize the potential effectiveness of disruptive coloration in a vertebrate taxon.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Physiological function in ectotherms is tightly linked to body temperature. As a result, the thermal sensitivity of physiological function may evolve to optimize fitness across different thermal environments. One hypothesis for the evolution of thermal sensitivity, coadaptation, predicts that optimal temperatures for performance should evolve to match the temperatures that an organism experiences in nature. Another hypothesis, countergradient variation, posits that genetic variation can compensate for decreased performance in cool environments, leading to physiological phenotypes that do not track environmental temperatures. On Mo'orea, French Polynesia, thermal ecology and physiology were studied in two morphologically similar skinks that differ in habitat use. Previous studies show that Emoia impar tends to inhabit closed-canopy and interior habitats that are cooler compared to those inhabited by Emoia cyanura, but these differences had not been quantified on Mo'orea. The goal of this study was to determine whether this pattern of habitat partitioning exists on Mo'orea and relates to interspecific differences in thermal physiology and to evaluate whether the evolution of thermal sensitivity supports coadaptation or countergradient variation. I found that E. impar inhabits closed-canopy habitats with cooler substrates and with higher altitudes compared to habitats of E. cyanura. Although the two species do not differ significantly in critical thermal minimum, E. impar has a significantly lower preferred body temperature and critical thermal maximum than does E. cyanura. Despite a preference for cooler habitats and temperatures, E. impar has a warmer optimal temperature for sprint speed and sprints faster than E. cyanura at all temperatures, which supports the countergradient model of thermal adaptation. These results are robust to three different curve-fitting functions and support the view that generalist/specialist trade-offs do not universally constrain the evolution of performance curves.