Variation in social feeding behavior and interactions among Caenorhabditis nematodes

Abstract

The ability to respond to complex stimuli and environmental cues is essential for organisms to survive and reproduce. Responding to a wide range of stimuli requires a neuronal network that can integrate cues and execute behavioral responses. Evolution of behaviors occurs ubiquitously in most established ecological niches, even among closely related species. To uncover the genetic and neuronal drivers of evolving behaviors, we have taken advantage of the large and relatively ancient divergence in the Caenorhabditis genus to ask how different Caenorhabditis nematodes respond to environmental stimuli and whether behavioral traits are shared or distinct. Here, we assayed foraging behaviors of twelve members of the Caenorhabditis clade, including members of both the elegans and japonica supergroup, and the basal taxon C. monodelphis. For each species, we analyzed social feeding and bordering behaviors, which are well characterized in C. elegans. These behaviors are the functional readout of complex sensory integration of multiple sensory cues including pheromones, touch, O₂/CO₂ concentration, and attractive and noxious stimuli. We hypothesized that the evolutionary divergence between species would correlate to divergence in these behaviors. We observed a wide variation in social aggregate feeding and bordering behaviors of hermaphrodite and female animals, but the variation did not correlate with evolutionary relatedness of the species. Addition of male animals with female or hermaphrodite animals of the same species increased aggregation behavior of subset of species, but not others. Combination of a second species with C. elegans significantly reduced aggregate feeding behavior of C. elegans, but not the other species. Intraspecies and interspecies interactions therefore modifies behavioral paradigms. Overall, we find that foraging and social feeding behaviors vary widely across Caenorhabditis species, likely due to species-specific responses and integration of environmental and context sensory cues. In general, the clade represents a compelling model to dissect evolution of behavior across diverse environments and a large timescale.

Figure 1.. The Caenorhabditis clade of nematodes is an evolutionary and diverse lineage.

(A) Phylogenetic tree of Caenorhabditis clade showing the two large subgroups (elegans and japonica) and the basal taxon monodelphis. Species included in behavioral assays are highlighted and color-coded based on reproductive strategy (red = hermaphroditic or orange = gonochoristic). Figure was adapted from Stevens et al., 2020.

Figure 2.. Aggregation behavior is diverse across Caenorhabditis clade.

(A) Representative images of select Caenorhabditis species aggregation feeding and bordering behaviors. (B) Quantification of number of aggregating animals in 13 members of the Caenorhabditis clade. Species are ordered based on the provided phylogeny (and for subsequent figures). Significant variation is present among members of the clade, and also within individual species. The 4 elegans groups assayed represent the negative control (N2 ‘Bristol’), positive control npr-1(ad609), modulated positive control npr-1(ad609);nrx-1(wy778), and wild isolate CB4856 ‘Hawaiian’, respectively. Bar graphs represent the mean values across biological replicates (each replicate indicated by a dot). Error bars show stand error of mean. One-way ANOVA with Tukey’s post-hoc test was used for statistical comparisons, with p-values indicated in standard format ( p=0.05(*), p=0.01(**), p=0.001(***), p= <0.001 (****)). Hermaphroditic species are labeled in red, while gonochoristic species are labeled in black. (C) Quantification of percent solitary animals on food border in 13 members of Caenorhabditis clade. Significant variation is present among members of the clade, and also within individual species. Graphical representations and statistical analysis are consistent with previous panel.

Figure 3.. Statistical analysis of phenotypes.

(A) Heat-map of ANOVA-generated p-values of all pairwise comparisons in aggregation assay. (B) Heat-map of ANOVA-generated p-values of all pairwise comparisons in bordering assay. (C) XY data plot showing clustering off species when comparing aggregation and bordering phenotypes. As anticipated, elegans N2 and monodelphis, and elegans npr-1 represent the low and high extremes of the cluster, respectively. Consistent with the figures above, hermaphroditic species are labeled in red.

Figure 4.. Ecological drivers modulate social feeding behaviors in a species-specific manner.

(A) Representative images and (B) quantification of number of aggregating animals of Caenorhabditis species in mixed sex populations. tropicalis and monodelphis both showed significant differences in aggregation when males were added to the population. Bar graphs represent the mean values across biological replicates (each replicate indicated by a dot). Error bars show stand error of mean. Standard T-test was used for individual comparisons; p-values are indicated in standard format ( p=0.05(*), p=0.01(**), p=0.001(***), p= <0.001 (****)). Hermaphroditic species are labeled in red, while gonochoristic species are labeled in black. (C) Quantification of 30 animal single-species controls. D) Quantification of mixed species assays, where 15 elegans myo-2p+ animals were mixed with 15 elegans myo-2p-, monodelphis, or nigoni. Combining elegans and nigoni significantly lowers aggregation of myo-2 expressing elegans compared to their elegans alone control (left), while addition of monodelphis caused an insignificant but trending reduction (left). monodelphis and nigoni have significantly lower aggregation than their elegans myo-2p− control (right). (E) Representative images of mixed species assays. myo-2p expressing elegans are indicated by arrows (top left window).

Figure 5.. Protein alignment of NPR-1 orthologues in amongst Caenorhabditis nematode species.

(A) NPR-1 orthologues were queried from existing Caenorhabditis genomes or assemblies based on sequence similarity. A single orthologue was identified in each species with the exception of nigoni which was excluded due to the lack of a confident orthologue. Overall, NPR-1 is relatively well conserved across the clade.