An efficient single-cell transcriptomics workflow for microbial eukaryotes benchmarked on Giardia intestinalis cells.

BACKGROUND: Most diversity in the eukaryotic tree of life is represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, some protists remain difficult to maintain in cultures, which further influences what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists is often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed. RESULTS: In this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol originally developed for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinalis was used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we reduced the reaction volume and purified the amplified cDNA with alternative beads to test different cost-reducing changes of Smart-seq2. Neither improved the procedure, and reducing the volumes by half led to significantly fewer genes detected. We also added a 5' biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency. CONCLUSIONS: The presented single-cell RNA sequencing workflow represents an efficient method to explore the diversity and cell biology of individual protist cells.

Warmer nights offer no respite for a defensive mutualism.

Ecologically relevant symbioses are widespread in terrestrial arthropods but based on recent findings these specialized interactions are likely to be especially vulnerable to climate warming. Importantly, empirical data and climate models indicate that warming is occurring asynchronously, with night-time temperatures increasing faster than daytime temperatures. Daytime (DTW) and night-time warming (NTW) may impact ectothermic animals and their interactions differently as DTW results in greater daily temperature variation and moves organisms nearer to their thermal limits, while NTW avoids thermal limits and may relieve constraints of cooler night-time temperatures; a nuance that has largely been ignored in the literature. In laboratory experiments, we investigated how the timing of warming influences a widespread defensive mutualism involving the pea aphid Acyrthosiphon pisum, and its heritable symbiont, Hamiltonella defensa, which protects against an important natural enemy, the parasitic wasp Aphidius ervi. Three aphid sublines were experimentally created from single aphid genotype susceptible to A. ervi: one line infected with a highly protective H. defensa strain, one infected with a moderately protective strain and one without any facultative symbiont. We examined aphid fitness in the presence and absence of parasitoids and when exposed to an average 2.5°C increase occurring across three warming scenarios (night-time vs. daytime vs. uniform) relative to no-warming controls. An increase of 2.5°C, as predicted to occur by the IPCC before 2100, was sufficient to disable the aphid defensive mutualism regardless of the timing of warming; a surprising result given that the daily maxima for control and NTW scenarios were identical. We also found that warming negatively impacted (a) symbiont-mediated interactions between host and parasitoid more than symbiont-free ones; (b) species interactions (host-parasitoid) more than each participant independently and (c) aphids more than parasitoids even though higher trophic levels are generally predicted to be more affected by warming. Here we show that 2.5°C warming, regardless of timing, negatively impacted a common microbe-mediated defensive mutualism. While this was a laboratory-based study, results suggest that temperature increases predicted in the near-term may disrupt the many ecological symbioses present in terrestrial ecosystems.

Opposite effects of daytime and nighttime warming on top-down control of plant diversity.

Ecological analyses of climate warming explore how rising mean temperature will affect the species composition of communities and their associated functioning. Experimentation usually presumes that warming arises from simultaneous increase in daily maximum (daytime) and minimum (nighttime) temperatures. Yet evidence shows that mean warming arises largely from increasing nighttime temperatures. We report on a 3-yr experiment that compared the effects of daytime and nighttime warming on a community comprising herbaceous plants, grasshopper herbivores and predatory spiders. We warmed experimental mesocosms 3-4°C above ambient control treatments during the daytime (06:00-18:00 h) or nighttime (18:00-06:00 h). Daytime warming caused spiders to seek a thermal refuge low in the plant canopy and away from grasshopper prey, which allowed grasshoppers to spend more time feeding on a competitively dominant plant species. Nighttime had the opposite effect, where spider activity increased causing grasshoppers to reduce feeding. Two consecutive years of daytime warming resulted in a suppression of the competitive dominant plant and increased the diversity and evenness of the plant community, whereas nighttime warming had opposite effects. These results show that ignoring the nuanced effects of asymmetrical warming may lead to inaccurate conclusions about the net effects of climate change on ecosystems.

Covariance between predation risk and nutritional preferences confounds interpretations of giving-up density experiments.

Giving-up density (GUD) experiments have been a foundational method to evaluate perceived predation risk, but rely on the assumption that food preferences are absolute, so that areas with higher GUDs can be interpreted as having higher risk. However, nutritional preferences are context dependent and can change with risk. We used spiders and grasshoppers to test the hypothesis that covariance in nutritional preferences and risk may confound the interpretation of GUD experiments. We presented grasshoppers with carbohydrate-rich and protein-rich diets, in the presence and absence of spider predators. Predators reduced grasshopper preference for the protein-rich food, but increased their preference for the carbohydrate-rich food. We then measured GUDs with both food types under different levels of risk (spider density, 0-5). As expected, GUDs increased with spider density indicating increasing risk, but only when using protein-rich food. With carbohydrate-rich food, GUD was independent of predation risk. Our results demonstrate that predation risk and nutritional preferences covary and can confound interpretation of GUD experiments.

Why and How to Create Nighttime Warming Treatments for Ecological Field Experiments.

While average global temperatures are increasing, a disproportionate amount of warming can be attributed to increasing nighttime temperatures rather than increasing daytime temperatures. Theory predicts that the timing of warming can generate different effects on organisms and their interactions within ecosystems. This occurs because an organism's response to warming depends on the current temperature. For example, warming when temperatures are low may have positive effects on an organism, while warming when temperatures are already high may have negative effects on an organism. Most field experiments that examine the ecological effects of climate warming employ warming methodologies that disproportionately elevate daytime warming treatments. The bias towards daytime warming treatments may arise because daytime temperatures can be manipulated with relatively simple and inexpensive technology that capitalizes on solar energy, such as open-top chambers that create a "greenhouse effect" or shade structures that reduce temperatures. However, these popular methods are ineffective when solar radiation is absent, and thus do not create warming treatments that accurately mimic the temporal patterns of climate warming. To encourage the investigation of nighttime warming's effect on ecosystems, we discuss why daytime and nighttime warming may have different effects on organisms, then present a review of methods that can be employed to elevate nighttime temperature in terrestrial field experiments. For each method, we offer a brief explanation, an evaluation of its pros and cons, and citations for further reference, as well as empirical data when possible. While some are impractical, we attempt to provide a comprehensive list of potential nighttime warming methods in hopes of stimulating ideas and discussions.

Combined effects of night warming and light pollution on predator-prey interactions.

Interactions between multiple anthropogenic environmental changes can drive non-additive effects in ecological systems, and the non-additive effects can in turn be amplified or dampened by spatial covariation among environmental changes. We investigated the combined effects of night-time warming and light pollution on pea aphids and two predatory ladybeetle species. As expected, neither night-time warming nor light pollution changed the suppression of aphids by the ladybeetle species that forages effectively in darkness. However, for the more-visual predator, warming and light had non-additive effects in which together they caused much lower aphid abundances. These results are particularly relevant for agriculture near urban areas that experience both light pollution and warming from urban heat islands. Because warming and light pollution can have non-additive effects, predicting their possible combined consequences over broad spatial scales requires knowing how they co-occur. We found that night-time temperature change since 1949 covaried positively with light pollution, which has the potential to increase their non-additive effects on pea aphid control by 70% in US alfalfa. Our results highlight the importance of non-additive effects of multiple environmental factors on species and food webs, especially when these factors co-occur.

The body size dependence of trophic cascades.

Trophic cascades are indirect positive effects of predators on resources via control of intermediate consumers. Larger-bodied predators appear to induce stronger trophic cascades (a greater rebound of resource density toward carrying capacity), but how this happens is unknown because we lack a clear depiction of how the strength of trophic cascades is determined. Using consumer resource models, we first show that the strength of a trophic cascade has an upper limit set by the interaction strength between the basal trophic group and its consumer and that this limit is approached as the interaction strength between the consumer and its predator increases. We then express the strength of a trophic cascade explicitly in terms of predator body size and use two independent parameter sets to calculate how the strength of a trophic cascade depends on predator size. Both parameter sets predict a positive effect of predator size on the strength of a trophic cascade, driven mostly by the body size dependence of the interaction strength between the first two trophic levels. Our results support previous empirical findings and suggest that the loss of larger predators will have greater consequences on trophic control and biomass structure in food webs than the loss of smaller predators.

A bioenergetic framework for the temperature dependence of trophic interactions.

Changing temperature can substantially shift ecological communities by altering the strength and stability of trophic interactions. Because many ecological rates are constrained by temperature, new approaches are required to understand how simultaneous changes in multiple rates alter the relative performance of species and their trophic interactions. We develop an energetic approach to identify the relationship between biomass fluxes and standing biomass across trophic levels. Our approach links ecological rates and trophic dynamics to measure temperature-dependent changes to the strength of trophic interactions and determine how these changes alter food web stability. It accomplishes this by using biomass as a common energetic currency and isolating three temperature-dependent processes that are common to all consumer-resource interactions: biomass accumulation of the resource, resource consumption and consumer mortality. Using this framework, we clarify when and how temperature alters consumer to resource biomass ratios, equilibrium resilience, consumer variability, extinction risk and transient vs. equilibrium dynamics. Finally, we characterise key asymmetries in species responses to temperature that produce these distinct dynamic behaviours and identify when they are likely to emerge. Overall, our framework provides a mechanistic and more unified understanding of the temperature dependence of trophic dynamics in terms of ecological rates, biomass ratios and stability.

Species interactions and a chain of indirect effects driven by reduced precipitation.

Climate change can affect species directly and indirectly by altering interactions between species within communities. These indirect effects can ramify through a community and affect many species, including some that may not have been directly affected by the perturbation. Identifying these chains of indirect effects is difficult, and most studies only follow indirect effects across two or three species. Here, we use a factorial field experiment to demonstrate that precipitation affects spotted aphids through a complex chain of indirect interactions that are mediated by other herbivores and a generalist predator. We experimentally simulated drought, which reduced water content in alfalfa plants. While water stress in alfalfa had no direct effect on spotted aphids, it lowered the population growth rate of pea aphids, another common alfalfa pest. Because ladybeetle predators were attracted to high pea aphid densities, predator densities were lower in drought treatments. Consequently, spotted aphid densities were released from top-down control (apparent competition) in drought treatments and reached densities three times higher than spotted aphids in ambient treatments with high pea aphid densities. Thus, drought affected spotted aphids in the interaction chain: drought --> alfalfa --> pea aphids --> predators --> spotted aphids. This result illustrates the lengthy path that indirect effects of climate change may take through a community, as well as the importance of community-level experiments in determining the net effect of climate change.

Direct and indirect effects of warming on aphids, their predators, and ant mutualists.

Species exist within communities of other interacting species, so an exogenous force that directly affects one species can indirectly affect all other members of the community. In the case of climate change, many species may be affected directly and subsequently initiate numerous indirect effects that propagate throughout the community. Therefore, the net effect of climate change on any one species is a function of the direct and indirect effects. We investigated the direct and indirect effects of climate warming on corn leaf aphids, a pest of corn and other grasses, by performing an experimental manipulation of temperature, predators, and two common aphid-tending ants. Although warming had a positive direct effect on aphid population growth rate, warming reduced aphid abundance when ants and predators were present. This occurred because winter ants, which aggressively defend aphids from predators under control temperatures, were less aggressive toward predators and less abundant when temperatures were increased. In contrast, warming increased the abundance of cornfield ants, but they did not protect aphids from predators with the same vigor as winter ants. Thus, warming broke down the ant-aphid mutualism and counterintuitively reduced the abundance of this agricultural pest.

Local adaptation to temperature conserves top-down control in a grassland food web.

A fundamental limitation in many climate change experiments is that tests represent relatively short-term 'shock' experiments and so do not incorporate the phenotypic plasticity or evolutionary change that may occur during the gradual process of climate change. However, capturing this aspect of climate change effects in an experimental design is a difficult challenge that few studies have accomplished. I examined the effect of temperature and predator climate history in food webs composed of herbaceous plants, generalist grasshopper herbivores and spider predators across a natural 4.8°C temperature gradient spanning 500 km in northeastern USA. In these grasslands, the effects of rising temperatures on the plant community are indirect and arise via altered predator-herbivore interactions. Experimental warming had no direct effect on grasshoppers, but reduced predation risk effects by causing spiders from all study sites to seek thermal refuge lower in the plant canopy. However, spider thermal tolerance corresponded to spider origin such that spiders from warmer study sites tolerated higher temperatures than spiders from cooler study sites. As a consequence, the magnitude of the indirect effect of spiders on plants did not differ along the temperature gradient, although a reciprocal transplant experiment revealed significantly different effects of spider origin on the magnitude of top-down control. These results suggest that variation in predator response to warming may maintain species interactions and associated food web processes when faced with long term, chronic climate warming.

SNAPSHOT USA 2019: a coordinated national camera trap survey of the United States.

With the accelerating pace of global change, it is imperative that we obtain rapid inventories of the status and distribution of wildlife for ecological inferences and conservation planning. To address this challenge, we launched the SNAPSHOT USA project, a collaborative survey of terrestrial wildlife populations using camera traps across the United States. For our first annual survey, we compiled data across all 50 states during a 14-week period (17 August-24 November of 2019). We sampled wildlife at 1,509 camera trap sites from 110 camera trap arrays covering 12 different ecoregions across four development zones. This effort resulted in 166,036 unique detections of 83 species of mammals and 17 species of birds. All images were processed through the Smithsonian's eMammal camera trap data repository and included an expert review phase to ensure taxonomic accuracy of data, resulting in each picture being reviewed at least twice. The results represent a timely and standardized camera trap survey of the United States. All of the 2019 survey data are made available herein. We are currently repeating surveys in fall 2020, opening up the opportunity to other institutions and cooperators to expand coverage of all the urban-wild gradients and ecophysiographic regions of the country. Future data will be available as the database is updated at eMammal.si.edu/snapshot-usa, as will future data paper submissions. These data will be useful for local and macroecological research including the examination of community assembly, effects of environmental and anthropogenic landscape variables, effects of fragmentation and extinction debt dynamics, as well as species-specific population dynamics and conservation action plans. There are no copyright restrictions; please cite this paper when using the data for publication.

Climate warming and predation risk during herbivore ontogeny.

Phenological effects of climate change are expected to differ among species, altering interactions within ecological communities. However, the nature and strength of these effects can vary during ontogeny, so the net community-level effects will be the result of integration over an individual's lifetime. I resolved the mechanism driving the effects of warming and spider predation risk on a generalist grasshopper herbivore at each ontogenetic stage and quantified the treatment effects on a measure of reproductive fitness. Spiders caused nymphal grasshoppers to increase the proportion of herbs in their diet, thus having a positive indirect effect on grasses and a negative indirect effect on herbs. Warming strengthened the top-down effect by affecting spiders and grasshoppers differently. In cooler, ambient conditions, grasshoppers and spiders had a high degree of spatial overlap within the plant canopy. Grasshopper position was unaffected by temperature, but spiders moved lower in the canopy in response to warming. This decreased the spatial overlap between predator and prey, allowing nymphal grasshoppers to increase daily feeding time. While spiders decreased grasshopper growth and reproductive fitness in ambient conditions, spiders had no effect on grasshopper fitness in warmed treatments. The study demonstrates the importance of considering the ontogeny of behavior when examining the effects of climate change on trophic interactions.

Behavioral plasticity mitigates the effect of warming on white-tailed deer.

Climate change is expected to create novel environments in which extant species cannot persist, therefore leading to the loss of them and their associated ecological functions within the ecosystem. However, animals may employ behavioral mechanisms in response to warming that could allow them to maintain their functional roles in an ecosystem despite changed temperatures. Specifically, animals may shift their activity in space or time to make use of thermal heterogeneity on the landscape. However, few studies consider the role of behavioral plasticity and spatial or temporal heterogeneity in mitigating the effects of climate change. We conducted experiments to evaluate the potential importance of behavior in mediating the net effects of warming on white-tailed deer (Odocoileus virginianus). We used shade structures to manipulate the thermal environment around feeding stations to monitor deer feeding activity and measure total consumption. In individual experiments where deer only had access to unshaded feeders, deer fed less during the day but compensated by increasing feeding during times when temperature was lower. In group experiments where deer had access to both shaded and unshaded feeders, deer often fed during the day but disproportionally preferred the cooler, shaded feeders. Our results suggest that deer can capitalize on temporal and spatial heterogeneity in the thermal environment to meet nutritional and thermal requirements, demonstrating the importance of behavioral plasticity when predicting the net effects of climate change.

A framework and standardized terminology to facilitate the study of predation-risk effects.

The very presence of predators can strongly influence flexible prey traits such as behavior, morphology, life history, and physiology. In a rapidly growing body of literature representing diverse ecological systems, these trait (or "fear") responses have been shown to influence prey fitness components and density, and to have indirect effects on other species. However, this broad and exciting literature is burdened with inconsistent terminology that is likely hindering the development of inclusive frameworks and general advances in ecology. We examine the diverse terminology used in the literature, and discuss pros and cons of the many terms used. Common problems include the same term being used for different processes, and many different terms being used for the same process. To mitigate terminological barriers, we developed a conceptual framework that explicitly distinguishes the multiple predation-risk effects studied. These multiple effects, along with suggested standardized terminology, are risk-induced trait responses (i.e., effects on prey traits), interaction modifications (i.e., effects on prey-other-species interactions), nonconsumptive effects (i.e., effects on the fitness and density of the prey), and trait-mediated indirect effects (i.e., the effects on the fitness and density of other species). We apply the framework to three well studied systems to highlight how it can illuminate commonalities and differences among study systems. By clarifying and elucidating conceptually similar processes, the framework and standardized terminology can facilitate communication of insights and methodologies across systems and foster cross-disciplinary perspectives.

Self-perpetuating ecological-evolutionary dynamics in an agricultural host-parasite system.

Ecological and evolutionary processes may become intertwined when they operate on similar time scales. Here we show ecological-evolutionary dynamics between parasitoids and aphids containing heritable symbionts that confer resistance against parasitism. In a large-scale field experiment, we manipulated the aphid's host plant to create ecological conditions that either favoured or disfavoured the parasitoid. The result was rapid evolutionary divergence of aphid resistance between treatment populations. Consistent with ecological-evolutionary dynamics, the resistant aphid populations then had reduced parasitism and increased population growth rates. We fit a model to quantify costs (reduced intrinsic rates of increase) and benefits of resistance. We also performed genetic assays on 5 years of field samples that showed persistent but highly variable frequencies of aphid clones containing protective symbionts; these patterns were consistent with simulations from the model. Our results show (1) rapid evolution that is intertwined with ecological dynamics and (2) variation in selection that prevents traits from becoming fixed, which together generate self-perpetuating ecological-evolutionary dynamics.

Infectious Diseases, Livestock, and Climate: A Vicious Cycle?

Ruminant livestock are a significant contributor to global methane emissions. Infectious diseases have the potential to exacerbate these contributions by elevating methane outputs associated with animal production. With the increasing spread of many infectious diseases, the emergence of a vicious climate-livestock-disease cycle is a looming threat.

Experimental warming transforms multiple predator effects in a grassland food web.

This experimental study tests new theory for multiple predator effects on communities by using warming to alter predator habitat use and hence direct and indirect interactions in a grassland food web containing two dominant spider predator species, a dominant grasshopper herbivore and grass and herb plants. Experimental warming further offers insight into how climate change might alter direct and indirect effects. Under ambient environmental conditions, spiders used habitat in spatially complementary locations. Consistent with predictions, the multiple predator effect on grasshoppers and on plants was the average of the individual predator effects. Warming strengthened the single predator effects. It also caused the spider species to overlap lower in the vegetation canopy. Consistent with predictions, the system was transformed into an intraguild predation system with the consequent extinction of one spider species. The results portend climate caused loss of predator diversity with important consequences for food web structure and function.

Climate warming strengthens indirect interactions in an old-field food web.

Climate change is expected to alter trophic interactions within food chains, but predicting the fate of particular species is difficult because the predictions hinge on knowing exactly how climate influences direct and indirect interactions. We used two complementary approaches to examine how climate change may alter trophic interactions within an old-field food web composed of herbaceous plants, grasshopper herbivores, and spider predators. We synthesized data spanning 15 years of experimentation during which interannual mean growing season temperature varied by 2 degrees C and precipitation by 2.5 cm. We also manipulated temperature within mesocosms to test the affect of temperature on primary production and strength of direct and indirect trophic interactions. Both approaches produced similar results: plant production was not directly affected by temperature or precipitation, but the strength of top-down indirect effects on grasses and forbs increased by 30-40% per 1 degrees C. Hence, the net effect of climate change was to strengthen top-down control of this terrestrial system.