Fantastic beasts and how to study them: rethinking experimental animal behavior.

Humans have been trying to understand animal behavior at least since recorded history. Recent rapid development of new technologies has allowed us to make significant progress in understanding the physiological and molecular mechanisms underlying behavior, a key goal of neuroethology. However, there is a tradeoff when studying animal behavior and its underlying biological mechanisms: common behavior protocols in the laboratory are designed to be replicable and controlled, but they often fail to encompass the variability and breadth of natural behavior. This Commentary proposes a framework of 10 key questions that aim to guide researchers in incorporating a rich natural context into their experimental design or in choosing a new animal study system. The 10 questions cover overarching experimental considerations that can provide a template for interspecies comparisons, enable us to develop studies in new model organisms and unlock new experiments in our quest to understand behavior.

The Bacterial Gq Signal Transduction Inhibitor FR900359 Impairs Soil-Associated Nematodes.

The cyclic depsipeptide FR900359 (FR) is derived from the soil bacterium Chromobacterium vaccinii and known to bind Gq proteins of mammals and insects, thereby abolishing the signal transduction of their Gq protein-coupled receptors, a process that leads to severe physiological consequences. Due to their highly conserved structure, Gq family of proteins are a superior ecological target for FR producing organisms, resulting in a defense towards a broad range of harmful organisms. Here, we focus on the question whether bacteria like C. vaccinii are important factors in soil in that their secondary metabolites impair, e.g., plant harming organisms like nematodes. We prove that the Gq inhibitor FR is produced under soil-like conditions. Furthermore, FR inhibits heterologously expressed Gαq proteins of the nematodes Caenorhabditis elegans and Heterodera schachtii in the micromolar range. Additionally, in vivo experiments with C. elegans and the plant parasitic cyst nematode H. schachtii demonstrated that FR reduces locomotion of C. elegans and H. schachtii. Finally, egg-laying of C. elegans and hatching of juvenile stage 2 of H. schachtii from its cysts is inhibited by FR, suggesting that FR might reduce nematode dispersion and proliferation. This study supports the idea that C. vaccinii and its excreted metabolome in the soil might contribute to an ecological equilibrium, maintaining and establishing the successful growth of plants.

Tuning molecular motor transport through cytoskeletal filament network organization.

Within cells, crosslinking proteins organize cytoskeletal filaments both temporally and spatially to create dynamic and structurally diverse networks. Molecular motors move on these networks for both force generation and transport processes. How the transport statistics depend on the network architecture remains poorly characterized. Using cross-linking proteins (α-actinin, fimbrin, fascin, or filamin) and purified actin, we create cytoskeletal networks with diverse microscopic architectures. We track the motion of myosin II motor proteins moving on these networks and calculate transport statistics. We observe that motor dynamics change predictably based on the bundling of filaments within the underlying networks and discuss implications for network function.

Stochastic feeding dynamics arise from the need for information and energy.

Animals regulate their food intake in response to the available level of food. Recent observations of feeding dynamics in small animals showed feeding patterns of bursts and pauses, but their function is unknown. Here, we present a data-driven decision-theoretical model of feeding in Caenorhabditis elegans Our central assumption is that food intake serves a dual purpose: to gather information about the external food level and to ingest food when the conditions are good. The model recapitulates experimentally observed feeding patterns. It naturally implements trade-offs between speed versus accuracy and exploration versus exploitation in responding to a dynamic environment. We find that the model predicts three distinct regimes in responding to a dynamical environment, with a transition region where animals respond stochastically to periodic signals. This stochastic response accounts for previously unexplained experimental data.

Distinct unfolded protein responses mitigate or mediate effects of nonlethal deprivation of C. elegans sleep in different tissues.

BACKGROUND: Disrupting sleep during development leads to lasting deficits in chordates and arthropods. To address lasting impacts of sleep deprivation in Caenorhabditis elegans, we established a nonlethal deprivation protocol. RESULTS: Deprivation triggered protective insulin-like signaling and two unfolded protein responses (UPRs): the mitochondrial (UPRmt) and the endoplasmic reticulum (UPRER) responses. While the latter is known to be triggered by sleep deprivation in rodent and insect brains, the former was not strongly associated with sleep deprivation previously. We show that deprivation results in a feeding defect when the UPRmt is deficient and in UPRER-dependent germ cell apoptosis. In addition, when the UPRER is deficient, deprivation causes excess twitching in vulval muscles, mirroring a trend caused by loss of egg-laying command neurons. CONCLUSIONS: These data show that nonlethal deprivation of C. elegans sleep causes proteotoxic stress. Unless mitigated, distinct types of deprivation-induced proteotoxicity can lead to anatomically and genetically separable lasting defects. The relative importance of different UPRs post-deprivation likely reflects functional, developmental, and genetic differences between the respective tissues and circuits.

C. elegans is not a robust model organism for the magnetic sense.

Magnetoreception is defined as the ability to sense and use the Earth's magnetic field, for example to orient and direct movements. The receptors and sensory mechanisms underlying behavioral responses to magnetic fields remain unclear. A previous study described magnetoreception in the nematode Caenorhabditis elegans, which requires the activity of a single pair of sensory neurons. These results suggest C. elegans as a tractable model organism for facilitating the search for magnetoreceptors and signaling pathways. The finding is controversial, however, as an attempt to replicate the experiment in a different laboratory was unsuccessful. We here independently test the magnetic sense of C. elegans, closely replicating the assays developed in the original publication. We find that C. elegans show no directional preference in magnetic fields of both natural and higher intensity, suggesting that magnetotactic behavior in the worm is not robustly evoked in a laboratory setting. Given the lack of a robust magnetic response under controlled conditions, we conclude that C. elegans is not a suitable model organism to study the mechanism of the magnetic sense.

The role of food odor in invertebrate foraging.

Foraging for food is an integral part of animal survival. In small insects and invertebrates, multisensory information and optimized locomotion strategies are used to effectively forage in patchy and complex environments. Here, the importance of olfactory cues for effective invertebrate foraging is discussed in detail. We review how odors are used by foragers to move toward a likely food source and the recent models that describe this sensory-driven behavior. We argue that smell serves a second function by priming an organism for the efficient exploitation of food. By appraising food odors, invertebrates can establish preferences and better adapt to their ecological niches, thereby promoting survival. The smell of food pre-prepares the gastrointestinal system and primes feeding motor programs for more effective ingestion as well. Optimizing resource utilization affects longevity and reproduction as a result, leading to drastic changes in survival. We propose that models of foraging behavior should include odor priming, and illustrate this with a simple toy model based on the marginal value theorem. Lastly, we discuss the novel techniques and assays in invertebrate research that could investigate the interactions between odor sensing and food intake. Overall, the sense of smell is indispensable for efficient foraging and influences not only locomotion, but also organismal physiology, which should be reflected in behavioral modeling.

Automatically tracking feeding behavior in populations of foraging C. elegans.

Caenorhabditis elegans feeds on bacteria and other small microorganisms which it ingests using its pharynx, a neuromuscular pump. Currently, measuring feeding behavior requires tracking a single animal, indirectly estimating food intake from population-level metrics, or using restrained animals. To enable large throughput feeding measurements of unrestrained, crawling worms on agarose plates at a single worm resolution, we developed an imaging protocol and a complementary image analysis tool called PharaGlow. We image up to 50 unrestrained crawling worms simultaneously and extract locomotion and feeding behaviors. We demonstrate the tool's robustness and high-throughput capabilities by measuring feeding in different use-case scenarios, such as through development, with genetic and chemical perturbations that result in faster and slower pumping, and in the presence or absence of food. Finally, we demonstrate that our tool is capable of long-term imaging by showing behavioral dynamics of mating animals and worms with different genetic backgrounds. The low-resolution fluorescence microscopes required are readily available in C. elegans laboratories, and in combination with our python-based analysis workflow makes this methodology easily accessible. PharaGlow therefore enables the observation and analysis of the temporal dynamics of feeding and locomotory behaviors with high-throughput and precision in a user-friendly system.

A small worm called C. elegans is constantly hungry. It spends all its time looking for food or eating. Hunger and environmental factors, like light, influence its feeding behavior. Studying these worms has helped scientists learn how feeding affects health, longevity, and aging. Feeding studies might also help scientists learn how the nervous system works and how it controls feeding. Most studies have used one of two approaches. Scientists may measure how much food a group of C. elegans eat by measuring food before and after it is offered to the worms. Or they restrain individual worms and measure the movement of a tube-like muscle, called the pharynx, which the animals use to vacuum up food. Restraining the worms can alter their behavior or brain activity, and studying group feeding habits may miss individual differences, so neither is optimal. Ideally, scientists could measure the feeding activity of many free-ranging worms, but because the movements of the pharynx are small, that too can be a challenge. Bonnard, Liu et al. developed a software tool that automatically detects and measures feeding behavior in a group of about 30 free-ranging C. elegans simultaneously. In the experiments, Bonnard, Liu et al. genetically engineered worms expressing a fluorescent protein in their pharynx, making it possible to measure its movements with a microscope. They used the microscope to capture images of 30-50 animals at a time as they foraged for food in a dish. Then, they used the software to analyze the data they collected. Over three days and five imaging sessions, Bonnard and Liu et al. tracked the feeding behavior of about 1,000 animals under different conditions. The experiments show that the pharynx grows rapidly during early worm development when the worms quadruple their length, but the rate of pharynx muscle contractions stays the same. They also showed the technique could measure feeding behaviors in animals with different genetic backgrounds, ages, or those engaged in behaviors like mating. The tool allows for larger and longer-term studies of worm feeding behaviors than previous approaches. Bonnard, Liu et al. made their software, called PharaGlow, available for use by other researchers. The tool may make feeding measurements a routine part of C. elegans studies. It will allow scientists to gain new insights into the role of feeding in a range of processes, including aging, fitness, mating, and overall health. Follow-up studies could determine if these findings are general strategies that also apply to other animals.

Decoding locomotion from population neural activity in moving C. elegans.

We investigated the neural representation of locomotion in the nematode C. elegans by recording population calcium activity during movement. We report that population activity more accurately decodes locomotion than any single neuron. Relevant signals are distributed across neurons with diverse tunings to locomotion. Two largely distinct subpopulations are informative for decoding velocity and curvature, and different neurons' activities contribute features relevant for different aspects of a behavior or different instances of a behavioral motif. To validate our measurements, we labeled neurons AVAL and AVAR and found that their activity exhibited expected transients during backward locomotion. Finally, we compared population activity during movement and immobilization. Immobilization alters the correlation structure of neural activity and its dynamics. Some neurons positively correlated with AVA during movement become negatively correlated during immobilization and vice versa. This work provides needed experimental measurements that inform and constrain ongoing efforts to understand population dynamics underlying locomotion in C. elegans.

Turning away from danger.

The flexible escape behavior exhibited by C. elegans in response to threats relies on a combination of feedback and feedforward circuits.

Serotonin-dependent kinetics of feeding bursts underlie a graded response to food availability in C. elegans.

Animals integrate physiological and environmental signals to modulate their food uptake. The nematode C. elegans, whose food uptake consists of pumping bacteria from the environment into the gut, provides excellent opportunities for discovering principles of conserved regulatory mechanisms. Here we show that worms implement a graded feeding response to the concentration of environmental bacteria by modulating a commitment to bursts of fast pumping. Using long-term, high-resolution, longitudinal recordings of feeding dynamics under defined conditions, we find that the frequency and duration of pumping bursts increase and the duration of long pauses diminishes in environments richer in bacteria. The bioamine serotonin is required for food-dependent induction of bursts as well as for maintaining their high rate of pumping through two distinct mechanisms. We identify the differential roles of distinct families of serotonin receptors in this process and propose that regulation of bursts is a conserved mechanism of behaviour and motor control.

A scalable method for automatically measuring pharyngeal pumping in C. elegans.

BACKGROUND: The nematode Caenorhabditis elegans is widely used for studying small neural circuits underlying behavior. In particular, the rhythmic feeding motions collectively termed pharyngeal pumping are regulated by a nearly autonomous network of 20 neurons of 14 types. Despite much progress achieved through laser ablation, genetics, electrophysiology, and optogenetics, key questions regarding the regulation of pumping remain open. NEW METHOD: We describe the implementation and application of a scalable automated method for measuring pumping in controlled environments. Our implementation is affordable and flexible: key hardware and software elements can be modified to accommodate different requirements. RESULTS: We demonstrate prolonged measurements under controlled conditions and the resulting high quality data. We show the scalability of our method, enabling high throughput, and its suitability for maintaining static and dynamic conditions. When food availability was oscillated, pumping rates were low as compared to steady conditions and pumping activity was not reliably modulated in response to changes in food concentration. COMPARISON WITH EXISTING METHOD: The prevailing method for measuring rates of pumping relies on scoring by visual inspection of short recordings. Our automated method compares well with manual scoring. It enables detailed statistical characterization under experimental conditions not previously accessible and minimizes unintentional bias. CONCLUSIONS: Our approach adds a powerful tool for studying pharyngeal pumping. It enhances the experimental versatility of assaying genetic and pharmacological manipulations and the ability to characterize the resulting behavior. Both the experimental setup and the analysis can be readily adapted to additional challenging motion detection problems.

Disturbance Regimes Predictably Alter Diversity in an Ecologically Complex Bacterial System.

Diversity is often associated with the functional stability of ecological communities from microbes to macroorganisms. Understanding how diversity responds to environmental perturbations and the consequences of this relationship for ecosystem function are thus central challenges in microbial ecology. Unimodal diversity-disturbance relationships, in which maximum diversity occurs at intermediate levels of disturbance, have been predicted for ecosystems where life history tradeoffs separate organisms along a disturbance gradient. However, empirical support for such peaked relationships in macrosystems is mixed, and few studies have explored these relationships in microbial systems. Here we use complex microbial microcosm communities to systematically determine diversity-disturbance relationships over a range of disturbance regimes. We observed a reproducible switch between community states, which gave rise to transient diversity maxima when community states were forced to mix. Communities showed reduced compositional stability when diversity was highest. To further explore these dynamics, we formulated a simple model that reveals specific regimes under which diversity maxima are stable. Together, our results show how both unimodal and non-unimodal diversity-disturbance relationships can be observed as a system switches between two distinct microbial community states; this process likely occurs across a wide range of spatially and temporally heterogeneous microbial ecosystems. IMPORTANCE: The diversity of microbial communities is linked to the functioning and stability of ecosystems. As humanity continues to impact ecosystems worldwide, and as diet and disease perturb our own commensal microbial communities, the ability to predict how microbial diversity will respond to disturbance is of critical importance. Using microbial microcosm experiments, we find that community diversity responds to different disturbance regimes in a reproducible and predictable way. Maximum diversity occurs when two communities, each suited to different environmental conditions, are mixed due to disturbance. This maximum diversity is transient except under specific regimes. Using a simple mathematical model, we show that transient unimodality is likely a common feature of microbial diversity-disturbance relationships in fluctuating environments.