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Modern metagenomic environmental DNA studies are almost completely reliant on next-generation sequencing, making evaluations of these methods critical. We compare two next-generation sequencing techniques – amplicon and shotgun – on water samples across four of Brazil’s major river floodplain systems (Amazon, Araguaia, Paraná, and Pantanal). Less than 50% of phyla identified via amplicon sequencing were recovered from shotgun sequencing, clearly challenging the dogma that mid-depth shotgun recovers more diversity than amplicon-based approaches. Amplicon sequencing also revealed ~27% more families. Overall the amplicon data were more robust across both biodiversity and community ecology analyses at different taxonomic scales. Our work doubles the sampling size in similar environmental studies, and novelly integrates environmental data (e.g., pH, temperature, nutrients) from each site, revealing divergent correlations depending on which data are used. While myriad variants on NGS techniques and bioinformatic pipelines are available, our results point to core differences that have not been highlighted in any studies to date. Given the low number of taxa identified when coupling shotgun data with clade-based taxonomic algorithms, previous studies that quantified biodiversity using such bioinformatic tools should be viewed cautiously or re-analyzed. Nonetheless, shotgun has complementary advantages that should be weighed when designing projects.

Indirect methods for conducting faunal inventories present great promise, and genomic inventories derived from environmental sources (eDNA) are improving. Invertebrate ingested DNA (iDNA) from terrestrial leeches in the family Haemadipsidae has shown potential for surveying vertebrates and biodiversity monitoring in protected areas. Here we present an initial, and critical, evaluation of the limitations and biases of current iDNA protocols for biodiversity monitoring using both standard and NGS barcoding approaches. Key findings include the need for taxon relevant multi-locus markers and reference databases. In particular, the limitations of available reference databases have profound potential to mislead and bias eDNA and iDNA results if not critically interpreted. Nevertheless, there is great potential for recovery of amplifiable DNA from gut contents of invertebrate museum specimens which may reveal both temporal patterns and cryptic diversity in protected areas with increased efficiency. Our analyses of ingested DNA (iDNA) from both freshly stored and previously collected (legacy) samples of terrestrial leeches successfully identified vertebrates from Myanmar, Australia and Madagascar and indicate the potential to characterize microbial communities, pathogen diversity and interactions at low cost.

The common bed bug ( Cimex lectularius ) has been a persistent pest of humans for thousands of years, yet the genetic basis of the bed bug’s basic biology and adaptation to dense human environments is largely unknown. Here we report the assembly, annotation and phylogenetic mapping of the 697.9-Mb Cimex lectularius genome, with an N50 of 971 kb, using both long and short read technologies. A RNA-seq time course across all five developmental stages and male and female adults generated 36,985 coding and noncoding gene models. The most pronounced change in gene expression during the life cycle occurs after feeding on human blood and included genes from the Wolbachia endosymbiont, which shows a simultaneous and coordinated host/commensal response to haematophagous activity. These data provide a rich genetic resource for mapping activity and density of C. lectularius across human hosts and cities, which can help track, manage and control bed bug infestations. The common bedbug is a pest for humans, yet its molecular biology is poorly understood. Here, the authors sequence the common bedbug genome and profile gene expression across all life stages to show major changes in gene expression after feeding on human blood.

Fossil rodent middens are powerful tools in paleoecology. In arid parts of western North America, packrat (Neotoma spp.) middens preserve plant and animal remains for tens of thousands of years. Midden contents are so well preserved that fragments of endogenous ancient DNA (aDNA) can be extracted and analyzed across millennia. Here, we explore the use of shotgun metagenomics to study the aDNA obtained from packrat middens up to 32,000 C14 years old. Eleven Illumina HiSeq 2500 libraries were successfully sequenced, and between 0.11% and 6.7% of reads were classified using Centrifuge against the NCBI “nt” database. Eukaryotic taxa identified belonged primarily to vascular plants with smaller proportions mapping to ascomycete fungi, arthropods, chordates, and nematodes. Plant taxonomic diversity in the middens is shown to change through time and tracks changes in assemblages determined by morphological examination of the plant remains. Amplicon sequencing of ITS2 and rbcL provided minimal data for some middens, but failed at amplifying the highly fragmented DNA present in others. With repeated sampling and deep sequencing, analysis of packrat midden aDNA from well‐preserved midden material can provide highly detailed characterizations of past communities of plants, animals, bacteria, and fungi present as trace DNA fossils. The prospects for gaining more paleoecological insights from aDNA for rodent middens will continue to improve with optimization of laboratory methods, decreasing sequencing costs, and increasing computational power. Past biodiversity revealed by ancient DNA metagenomics of North American fossil packrat middens. Here, we test shotgun sequencing of ancient DNA to identify the biological components of Late Pleistocene packrat middens. These methods are the first step in establishing robust paleogenomic methods to the analysis of well‐preserved midden materials.

The Bermuda fireworm Odontosyllis enopla exhibits an extremely tight circalunar circadian behavior that results in an impressive bioluminescent mating swarm, thought to be due to a conventional luciferase-mediated oxidation of a light-emitting luciferin. In addition, the four eyes become hypertrophied and heavily pigmented, and the nephridial system is modified to store and release gametes and associated secretions. In an effort to elucidate transcripts related to bioluminescence, circadian or circalunar periodicity, as well as epitoky-related changes of the eyes and nephridial system, we examined the transcriptomic profile of three female O. enopla during a bioluminescent swarm in Ferry Reach, Bermuda. Using the well-characterized luciferase gene of the Japanese syllid Odontosyllis undecimdonta as a reference, a complete best-matching luciferase open reading frame (329 amino acids in length) was found in all three individuals analyzed in addition to numerous other paralogous sequences in this new gene family. No photoproteins were detected. We also recovered a predicted homolog of 4-coumarate-CoA ligase (268 amino acids in length) that best matched luciferase of the firefly Luciola with the best predicted template being the crystal structure of luciferase for Photinus pyralis, the common eastern firefly. A wide variety of genes associated with periodicity were recovered including predicted homologs of clock, bmal1, period, and timeless. Several genes corresponding to putative epitoky-related changes of the eyes were recovered including predicted homologs of a phototransduction gene, a retinol dehydrogenase and carotenoid isomerooxygenase as well as a visual perception related retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase. Genes correlating to putative epitoky-related changes of the nephridia included predicted homologs of nephrocystin-3 and an egg-release sex peptide receptor.

Pyrosomes are tunicates in the phylum Chordata, which also contains vertebrates. Their gigantic blooms play important ecological and biogeochemical roles in oceans. Pyrosoma , meaning “fire-body”, derives from their brilliant bioluminescence. The biochemistry of this light production is unknown, but has been hypothesized to be bacterial in origin. We found that mixing coelenterazine—a eukaryote-specific luciferin—with Pyrosoma atlanticum homogenate produced light. To identify the bioluminescent machinery, we sequenced P. atlanticum transcriptomes and found a sequence match to a cnidarian luciferase (RLuc). We expressed this novel luciferase (PyroLuc) and, combined with coelenterazine, it produced light. A similar gene was recently predicted from a bioluminescent brittle star, indicating that RLuc-like luciferases may have evolved convergently from homologous dehalogenases across phyla (Cnidaria, Echinodermata, and Chordata). This report indicates that a widespread gene may be able to functionally converge, resulting in bioluminescence across animal phyla, and describes and characterizes the first putative chordate luciferase.

Language is a remarkably efficient tool for transmitting information. Yet human speakers make statements that are inefficient, imprecise, or even contrary to their own beliefs, all in the service of being polite. What rational machinery underlies polite language use? Here, we show that polite speech emerges from the competition of three communicative goals: to convey information, to be kind, and to present oneself in a good light. We formalize this goal tradeoff using a probabilistic model of utterance production, which predicts human utterance choices in socially sensitive situations with high quantitative accuracy, and we show that our full model is superior to its variants with subsets of the three goals. This utility-theoretic approach to speech acts takes a step toward explaining the richness and subtlety of social language use.

Premise Common steps in phylogenomic matrix production include biological sequence concatenation, morphological data concatenation, insertion/deletion (indel) coding, gene content (presence/absence) coding, removing uninformative characters for parsimony analysis, recording with reduced amino acid alphabets, and occupancy filtering. Existing software does not accomplish these tasks on a phylogenomic scale using a single program. Methods and Results BAD2matrix is a Python script that performs the above‐mentioned steps in phylogenomic matrix construction for DNA or amino acid sequences as well as morphological data. The script works in UNIX‐like environments (e.g., LINUX, MacOS, Windows Subsystem for LINUX). Conclusions BAD2matrix helps simplify phylogenomic pipelines and can be downloaded from https://github.com/dpl10/BAD2matrix/tree/master under a GNU General Public License v2.

The SARS-CoV-2 coronavirus is wreaking havoc globally, yet, as a novel pathogen, knowledge of its biology is still emerging. Climate and seasonality influence the distributions of many diseases, and studies suggest at least some link between SARS-CoV-2 and weather. One such study, building species distribution models (SDMs), predicted SARS-CoV-2 risk may remain concentrated in the Northern Hemisphere, shifting northward in summer months. Others have highlighted issues with SARS-CoV-2 SDMs, notably: the primary niche of the virus is the host it infects, climate may be a weak distributional predictor, global prevalence data have issues, and the virus is not in population equilibrium. While these issues should be considered, we believe climate’s relationship with SARS-CoV-2 is still worth exploring, as it may have some impact on the distribution of cases. To further examine if there is a link to climate, we build model projections with raw SARS-CoV-2 case data and population-scaled case data in the USA. The case data were from across March 2020, before large travel restrictions and public health policies were impacting cases across the country. We show that SDMs built from population-scaled case data cannot be distinguished from control models (built from raw human population data), while SDMs built on raw case data fail to predict the known distribution of cases in the U.S. from March. The population-scaled analyses indicate that climate did not play a central role in early U.S. viral distribution and that human population density was likely the primary driver. We do find slightly more population-scaled viral cases in cooler areas. Ultimately, the temporal and geographic constraints on this study mean that we cannot rule out climate as a partial driver of the SARS-CoV-2 distribution. Climate’s role on SARS-CoV-2 should continue to be cautiously examined, but at this time we should assume that SARS-CoV-2 will continue to spread anywhere in the U.S. where governmental policy does not prevent spread.

Replication is the cornerstone of science – but when and why? Not all studies need replication, especially when resources are limited. We propose that a decision-making framework based on Bayesian philosophy of science provides a basis for choosing which studies to replicate.