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.

Megapixel camera arrays enable high-resolution animal tracking in multiwell plates.

Tracking small laboratory animals such as flies, fish, and worms is used for phenotyping in neuroscience, genetics, disease modelling, and drug discovery. An imaging system with sufficient throughput and spatiotemporal resolution would be capable of imaging a large number of animals, estimating their pose, and quantifying detailed behavioural differences at a scale where hundreds of treatments could be tested simultaneously. Here we report an array of six 12-megapixel cameras that record all the wells of a 96-well plate with sufficient resolution to estimate the pose of C. elegans worms and to extract high-dimensional phenotypic fingerprints. We use the system to study behavioural variability across wild isolates, the sensitisation of worms to repeated blue light stimulation, the phenotypes of worm disease models, and worms' behavioural responses to drug treatment. Because the system is compatible with standard multiwell plates, it makes computational ethological approaches accessible in existing high-throughput pipelines.

Measuring Caenorhabditis elegans Spatial Foraging and Food Intake Using Bioluminescent Bacteria.

For most animals, feeding includes two behaviors: foraging to find a food patch and food intake once a patch is found. The nematode Caenorhabditis elegans is a useful model for studying the genetics of both behaviors. However, most methods of measuring feeding in worms quantify either foraging behavior or food intake, but not both. Imaging the depletion of fluorescently labeled bacteria provides information on both the distribution and amount of consumption, but even after patch exhaustion a prominent background signal remains, which complicates quantification. Here, we used a bioluminescent Escherichia coli strain to quantify C. elegans feeding. With light emission tightly coupled to active metabolism, only living bacteria are capable of bioluminescence, so the signal is lost upon ingestion. We quantified the loss of bioluminescence using N2 reference worms and eat-2 mutants, and found a nearly 100-fold increase in signal-to-background ratio and lower background compared to loss of fluorescence. We also quantified feeding using aggregating npr-1 mutant worms. We found that groups of npr-1 mutants first clear bacteria from within the cluster before foraging collectively for more food; similarly, during large population swarming, only worms at the migrating front are in contact with bacteria. These results demonstrate the usefulness of bioluminescent bacteria for quantifying feeding and generating insights into the spatial pattern of food consumption.

Comparison of solitary and collective foraging strategies of Caenorhabditis elegans in patchy food distributions.

Collective foraging has been shown to benefit organisms in environments where food is patchily distributed, but whether this is true in the case where organisms do not rely on long-range communications to coordinate their collective behaviour has been understudied. To address this question, we use the tractable laboratory model organism Caenorhabditis elegans, where a social strain (npr-1 mutant) and a solitary strain (N2) are available for direct comparison of foraging strategies. We first developed an on-lattice minimal model for comparing collective and solitary foraging strategies, finding that social agents benefit from feeding faster and more efficiently simply owing to group formation. Our laboratory foraging experiments with npr-1 and N2 worm populations, however, show an advantage for solitary N2 in all food distribution environments that we tested. We incorporated additional strain-specific behavioural parameters of npr-1 and N2 worms into our model and computationally identified N2's higher feeding rate to be the key factor underlying its advantage, without which it is possible to recapitulate the advantage of collective foraging in patchy environments. Our work highlights the theoretical advantage of collective foraging owing to group formation alone without long-range interactions and the valuable role of modelling to guide experiments. This article is part of the theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'.

Shared behavioral mechanisms underlie C. elegans aggregation and swarming.

In complex biological systems, simple individual-level behavioral rules can give rise to emergent group-level behavior. While collective behavior has been well studied in cells and larger organisms, the mesoscopic scale is less understood, as it is unclear which sensory inputs and physical processes matter a priori. Here, we investigate collective feeding in the roundworm C. elegans at this intermediate scale, using quantitative phenotyping and agent-based modeling to identify behavioral rules underlying both aggregation and swarming-a dynamic phenotype only observed at longer timescales. Using fluorescence multi-worm tracking, we quantify aggregation in terms of individual dynamics and population-level statistics. Then we use agent-based simulations and approximate Bayesian inference to identify three key behavioral rules for aggregation: cluster-edge reversals, a density-dependent switch between crawling speeds, and taxis towards neighboring worms. Our simulations suggest that swarming is simply driven by local food depletion but otherwise employs the same behavioral mechanisms as the initial aggregation.

Non-muscle myosin II is required for correct fate specification in the Caenorhabditis elegans seam cell divisions.

During development, cell division often generates two daughters with different developmental fates. Distinct daughter identities can result from the physical polarity and size asymmetry itself, as well as the subsequent activation of distinct fate programmes in each daughter. Asymmetric divisions are a feature of the C. elegans seam lineage, in which a series of post-embryonic, stem-like asymmetric divisions give rise to an anterior daughter that differentiates and a posterior daughter that continues to divide. Here we have investigated the role of non-muscle myosin II (nmy-2) in these asymmetric divisions. We show that nmy-2 does not appear to be involved in generating physical division asymmetry, but nonetheless is important for specifying differential cell fate. While cell polarity appears normal, and chromosome and furrow positioning remains unchanged when nmy-2 is inactivated, seam cell loss occurs through inappropriate terminal differentiation of posterior daughters. This reveals a role for nmy-2 in cell fate determination not obviously linked to the primary polarity determination mechanisms it has been previously associated with.

The C. elegans TPR Containing Protein, TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis.

Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.

Goose RIG-I functions in innate immunity against Newcastle disease virus infections.

Mammalian retinoic acid-inducible gene I (RIG-I) is a chief antiviral gene sensing viral RNA molecules including Newcastle disease virus (NDV). In this study, goose RIG-I gene (gRIG-I) was identified. The 2805 bp-long gene encodes a gRIG-I protein that exhibits 93.8% amino acid identity to duck RIG-I. DF-1 chicken fibroblast cells transfected with full-length of gRIG-I or CARD domain of gRIG-I plasmids respond significantly to the agonist of 21-mer 5'ppp RNA, evident through enhancement of IFN-ß promoter activity. Goose RIG-I transfected 293T/17 cells were then tested for the response to NDV infection, resulting in up-regulated activity of IFN-ß promoter, and mRNA levels of IRF-3 and IFIT1, but decreased virus titer. Similar results were obtained in transfected DF-1 chicken fibroblast cells and goose embryo fibroblast cells in response to NDV infections Animal experiments further support a role of gRIG-I in goose innate immunity against NDV infections by showing increased gRIG-I mRNA levels and decreased virus titer in geese lung and air sac post-infection.