gauseR: Simple methods for fitting Lotka‐Volterra models describing Gause’s “Struggle for Existence”

Point 1: The ecological models of Alfred J. Lotka and Vito Volterra have had an enormous impact on ecology over the past century. Some of the earliest—and clearest—experimental tests of these models were famously conducted by Georgy Gause in the 1930s. Although well known, the data from these experiments are not widely available and are often difficult to analyze using standard statistical and computational tools.Point 2: Here, we introduce the gauseR package, a collection of tools for fitting Lotka‐Volterra models to time series data of one or more species. The package includes several methods for parameter estimation and optimization, and includes 42 datasets from Gause''s species interaction experiments and related work. Additionally, we include with this paper a short blog post discussing the historical importance of these data and models, and an R vignette with a walk‐through introducing the package methods. The package is available for download at github.com/adamtclark/gauseR.Point 3: To demonstrate the package, we apply it to several classic experimental studies from Gause, as well as two other well‐known datasets on multi‐trophic dynamics on Isle Royale, and in spatially structured mite populations. In almost all cases, models fit observations closely and fitted parameter values make ecological sense.Point 4: Taken together, we hope that the methods, data, and analyses that we present here provide a simple and user‐friendly way to interact with complex ecological data. We are optimistic that these methods will be especially useful to students and educators who are studying ecological dynamics, as well as researchers who would like a fast tool for basic analyses.     

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.     

Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques

Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g. changes in redox states and bond lengths). In situ experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ experiments (e.g. on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ and in situ samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.     

Gruffi: an algorithm for computational removal of stressed cells from brain organoid transcriptomic datasets

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single‐cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.     

Taking practical learning in STEM education home: Examples from do‐it‐yourself experiments in plant biology

Practical teaching can give authentic learning experiences and teach valuable skills for undergraduate students in the STEM disciplines. One of the main ways of giving students such experiences, laboratory teaching, is met with many challenges such as budget cuts, increased use of virtual learning, and currently the university lockdowns due to the COVID‐19 pandemic. We highlight how at‐home do‐it‐yourself (DIY) experiments can be a good way to include physical interaction with your study organism, system, or technique to give the students a practical, authentic learning experience. We hope that by outlining the benefits of a practical, at‐home, DIY experiment we can inspire more people to design these teaching activities in the current remote teaching situation and beyond. By contributing two examples in the field of plant biology we enrich the database on experiments to draw inspiration from for these teaching methods.     

An open spatial capture–recapture model for estimating density,movement, and population dynamics from line‐transect surveys

The purpose of many wildlife population studies is to estimate density, movement, or demographic parameters. Linking these parameters to covariates, such as habitat features, provides additional ecological insight and can be used to make predictions for management purposes. Line‐transect surveys, combined with distance sampling methods, are often used to estimate density at discrete points in time, whereas capture–recapture methods are used to estimate movement and other demographic parameters. Recently, open population spatial capture–recapture models have been developed, which simultaneously estimate density and demographic parameters, but have been made available only for data collected from a fixed array of detectors and have not incorporated the effects of habitat covariates. We developed a spatial capture–recapture model that can be applied to line‐transect survey data by modeling detection probability in a manner analogous to distance sampling. We extend this model to a) estimate demographic parameters using an open population framework and b) model variation in density and space use as a function of habitat covariates. The model is illustrated using simulated data and aerial line‐transect survey data for North Atlantic right whales in the southeastern United States, which also demonstrates the ability to integrate data from multiple survey platforms and accommodate differences between strata or demographic groups. When individuals detected from line‐transect surveys can be uniquely identified, our model can be used to simultaneously make inference on factors that influence spatial and temporal variation in density, movement, and population dynamics.     

Benefits and limitations of a new genome‐based PCR‐RFLP genotyping assay (GB‐RFLP): A SNP‐based detection method for identification of species in extremely young adaptive radiations

High‐throughput DNA sequencing technologies make it possible now to sequence entire genomes relatively easily. Complete genomic information obtained by whole‐genome resequencing (WGS) can aid in identifying and delineating species even if they are extremely young, cryptic, or morphologically difficult to discern and closely related. Yet, for taxonomic or conservation biology purposes, WGS can remain cost‐prohibitive, too time‐consuming, and often constitute a “data overkill.” Rapid and reliable identification of species (and populations) that is also cost‐effective is made possible by species‐specific markers that can be discovered by WGS. Based on WGS data, we designed a PCR restriction fragment length polymorphism (PCR‐RFLP) assay for 19 Neotropical Midas cichlid populations (Amphilophus cf. citrinellus), that includes all 13 described species of this species complex. Our work illustrates that identification of species and populations (i.e., fish from different lakes) can be greatly improved by designing genetic markers using available “high resolution” genomic information. Yet, our work also shows that even in the best‐case scenario, when whole‐genome resequencing information is available, unequivocal assignments remain challenging when species or populations diverged very recently, or gene flow persists. In summary, we provide a comprehensive workflow on how to design RFPL markers based on genome resequencing data, how to test and evaluate their reliability, and discuss the benefits and pitfalls of our approach.     

Sea lice and salmon population dynamics: effects of exposure time for migratory fish

The ecological impact of parasite transmission from fish farms is probably mediated by the migration of wild fishes, which determines the period of exposure to parasites. For Pacific salmon and the parasitic sea louse, Lepeophtheirus salmonis, analysis of the exposure period may resolve conflicting observations of epizootic mortality in field studies and parasite rejection in experiments. This is because exposure periods can differ by 2–3 orders of magnitude, ranging from months in the field to hours in experiments. We developed a mathematical model of salmon–louse population dynamics, parametrized by a study that monitored naturally infected juvenile salmon held in ocean enclosures. Analysis of replicated trials indicates that lice suffer high mortality, particularly during pre-adult stages. The model suggests louse populations rapidly decline following brief exposure of juvenile salmon, similar to laboratory study designs and data. However, when the exposure period lasts for several weeks, as occurs when juvenile salmon migrate past salmon farms, the model predicts that lice accumulate to abundances that can elevate salmon mortality and depress salmon populations. The duration of parasite exposure is probably critical to salmon–louse population dynamics, and should therefore be accommodated in coastal planning and management where fish farms are situated on wild fish migration routes.     

Enabling high‐throughput biology with flexible open‐source automation

Our understanding of complex living systems is limited by our capacity to perform experiments in high throughput. While robotic systems have automated many traditional hand‐pipetting protocols, software limitations have precluded more advanced maneuvers required to manipulate, maintain, and monitor hundreds of experiments in parallel. Here, we present Pyhamilton, an open‐source Python platform that can execute complex pipetting patterns required for custom high‐throughput experiments such as the simulation of metapopulation dynamics. With an integrated plate reader, we maintain nearly 500 remotely monitored bacterial cultures in log‐phase growth for days without user intervention by taking regular density measurements to adjust the robotic method in real‐time. Using these capabilities, we systematically optimize bioreactor protein production by monitoring the fluorescent protein expression and growth rates of a hundred different continuous culture conditions in triplicate to comprehensively sample the carbon, nitrogen, and phosphorus fitness landscape. Our results demonstrate that flexible software can empower existing hardware to enable new types and scales of experiments, empowering areas from biomanufacturing to fundamental biology.     

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.     

A pipeline for effectively developing highly polymorphic simple sequence repeats markers based on multi‐sample genomic data

Simple sequence repeats (SSRs) are widely used genetic markers in ecology, evolution, and conservation even in the genomics era, while a general limitation to their application is the difficulty of developing polymorphic SSR markers. Next‐generation sequencing (NGS) offers the opportunity for the rapid development of SSRs; however, previous studies developing SSRs using genomic data from only one individual need redundant experiments to test the polymorphisms of SSRs. In this study, we designed a pipeline for the rapid development of polymorphic SSR markers from multi‐sample genomic data. We used bioinformatic software to genotype multiple individuals using resequencing data, detected highly polymorphic SSRs prior to experimental validation, significantly improved the efficiency and reduced the experimental effort. The pipeline was successfully applied to a globally threatened species, the brown eared‐pheasant (Crossoptilon mantchuricum), which showed very low genomic diversity. The 20 newly developed SSR markers were highly polymorphic, the average number of alleles was much higher than the genomic average. We also evaluated the effect of the number of individuals and sequencing depth on the SSR mining results, and we found that 10 individuals and ~10X sequencing data were enough to obtain a sufficient number of polymorphic SSRs, even for species with low genetic diversity. Furthermore, the genome assembly of NGS data from the optimal number of individuals and sequencing depth can be used as an alternative reference genome if a high‐quality genome is not available. Our pipeline provided a paradigm for the application of NGS technology to mining and developing molecular markers for ecological and evolutionary studies.     

Camera traps and guard observations as an alternative to researcher observation for studying anthropogenic foraging

Foraging by wildlife on anthropogenic foods can have negative impacts on both humans and wildlife. Addressing this issue requires reliable data on the patterns of anthropogenic foraging by wild animals, but while direct observation by researchers can be highly accurate, this method is also costly and labor‐intensive, making it impractical in the long‐term or over large spatial areas. Camera traps and observations by guards employed to deter animals from fields could be efficient alternative methods of data collection for understanding patterns of foraging by wildlife in crop fields. Here, we investigated how data on crop‐foraging by chacma baboons and vervet monkeys collected by camera traps and crop guards predicted data collected by researchers, on a commercial farm in South Africa. We found that data from camera traps and field guard observations predicted crop loss and the frequency of crop‐foraging events from researcher observations for crop‐foraging by baboons and to a lesser extent for vervets. The effectiveness of cameras at capturing crop‐foraging events was dependent on their position on the field edge. We believe that these alternatives to direct observation by researchers represent an efficient and low‐cost method for long‐term and large‐scale monitoring of foraging by wildlife on crops.     

How low can you go? Introducing SeXY: sex identification from low‐quantity sequencing data despite lacking assembled sex chromosomes

Accurate sex identification is crucial for elucidating the biology of a species. In the absence of directly observable sexual characteristics, sex identification of wild fauna can be challenging, if not impossible. Molecular sexing offers a powerful alternative to morphological sexing approaches. Here, we present SeXY, a novel sex‐identification pipeline, for very low‐coverage shotgun sequencing data from a single individual. SeXY was designed to utilize low‐effort screening data for sex identification and does not require a conspecific sex‐chromosome assembly as reference. We assess the accuracy of our pipeline to data quantity by downsampling sequencing data from 100,000 to 1000 mapped reads and to reference genome selection by mapping to a variety of reference genomes of various qualities and phylogenetic distance. We show that our method is 100% accurate when mapping to a high‐quality (highly contiguous N50 > 30 Mb) conspecific genome, even down to 1000 mapped reads. For lower‐quality reference assemblies (N50 < 30 Mb), our method is 100% accurate with 50,000 mapped reads, regardless of reference assembly quality or phylogenetic distance. The SeXY pipeline provides several advantages over previously implemented methods; SeXY (i) requires sequencing data from only a single individual, (ii) does not require assembled conspecific sex chromosomes, or even a conspecific reference assembly, (iii) takes into account variation in coverage across the genome, and (iv) is accurate with only 1000 mapped reads in many cases.     

Preparation,Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.     

The Human Lagging Strand DNA Polymerase δ Holoenzyme Is Distributive

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5′-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ^AA. The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.     

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.^1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression^3-6 and other characteristics of cultures at steady-state equilibrium.⁷ Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.⁸ A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.^9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. We present here the design and operation of a relatively simple, low cost array of miniature chemostats—or ministats—and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.     

A human liver microphysiology platform for investigating physiology,drug safety,and disease models

This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 μM troglitazone or 210 μM nimesulide produced acute toxicity within 2–4 days, whereas 28 μM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.