Nematodes respond to a multitude of environmental cues. For example, the social behaviours clumping and bordering were described as a mechanism of hyperoxia avoidance in Caenorhabditis elegans and Pristionchus pacificus. A recent study in P. pacificus revealed a novel regulatory pathway that inhibits social behaviour in a response to an as yet unknown environmental cue. This environmental signal is recognised by ciliated neurons, as mutants defective in intraflagellar transport (IFT) proteins display social behaviours. The IFT machinery represents a large protein complex and many mutants in genes encoding IFT proteins are available in C. elegans. However, social phenotypes in C. elegans IFT mutants have never been reported. Here, we examined 15 previously isolated C. elegans IFT mutants and found that most of them showed strong social behaviour. These findings indicate conservation in the inhibitory mechanism of social behaviour between P. pacificus and C. elegans.
Nematodes respond to a multitude of environmental cues, in part to avoid or minimise environmental stresses (Bargmann, 2006). In Pristionchus pacificus, social behaviour, namely clumping and bordering at the edge of the bacterial food lawn, has been described as a mechanism to avoid hyperoxic conditions in the laboratory (Moreno et al., 2016). Interestingly, there is substantial natural variation in this behaviour, indicating the adaptation of different strains to different environments. Most of the P. pacificus strains isolated from the wild are solitary and distribute equally in the lawn of food, whereas the strains of one phylogenetic clade that is endemic to high-altitude locations on La Réunion Island display social behaviour under laboratory conditions (Moreno et al., 2016).
In the model nematode Caenorhabditis elegans similar traits have evolved independently. All C. elegans natural isolates analysed so far display social behaviour in the laboratory (Macosko et al., 2009; Frézal & Félix, 2015). However, the wild type lab strain N2 became solitary during an early domestication event that was associated with a gain-of-function mutation in the neuropeptide Y-like receptor encoded by the npr-1 gene (de Bono & Bargmann, 1998; Rogers et al., 2003; Gray et al., 2004; Weber et al., 2010). Interestingly, the polymorphism in Cel-npr-1 is associated with several traits (Andersen et al., 2014; Sterken et al., 2015), whereas Ppa-npr-1 is not responsible for the natural variation in social behaviour among P. pacificus strains (Moreno et al., 2016, 2017). These findings indicated that social behaviour in P. pacificus and C. elegans represents a phenotypic convergence with different evolutionary histories and distinct genetic control (Moreno et al., 2016, 2017).
A recent study in P. pacificus revealed the existence of an additional regulatory pathway that inhibits social behaviour in response to a yet unknown environmental signal. Interestingly, this environmental signal is recognised by ciliated neurons, and mutations in genes encoding intraflagellar transport (IFT) proteins promote social behaviour (Moreno et al., 2017). The IFT machinery represents a protein complex of six sub-complexes that has been intensively studied in C. elegans and consists of the IFT-sub-complex A and B, the BBSome, the homodimeric and heterodimeric kinesin motors and the dynein motor (Inglis et al., 2006; Taschner & Lorentzen, 2016). Whilst many C. elegans mutants for IFT-encoding proteins are available, a social phenotype as seen for IFT mutants in P. pacificus has never been described in C. elegans IFT mutants. Here we examined 15 previously isolated C. elegans IFT mutants (Inglis et al., 2006; Taschner & Lorentzen, 2016). Surprisingly, most of these mutants showed social behaviour, which is regulated by [O2] levels similar to P. pacificus IFT mutants. These findings indicate conservation in the inhibitory mechanism of social behaviour between P. pacificus and C. elegans.
IFT mutants cause social behaviours in C. elegans
As P. pacificus IFT mutants display social behaviour (Moreno et al., 2017), we tested whether a similar mechanism is present in C. elegans. We screened the social behaviour of 15 IFT C. elegans mutants, including representatives of all six IFT sub-complexes (see Table S1 in the Supplementary Material). These mutants were produced in previous studies and obtained from the Caenorhabditis Genetics Center (see Table S1). Surprisingly, 12 out of the 15 mutants showed strong social behaviours (Fig. 1A; Table S2 in the Supplementary Material), including osm-3, which was previously reported to be solitary (de Bono et al., 2002). By contrast, dyf-1 and osm-1 mutants remained solitary, which indicates a certain degree of specificity in the function of these genes within the IFT complex or neural network. The different phenotype of dyf-1 and osm-3 mutants is unexpected since DYF-1 has been shown to be specifically required for the OSM-3 kinesin to dock onto and move IFT particles (Ou et al., 2005). In addition, the mutant of the kinesin motor component klp-20 also showed solitary behaviour, suggesting that in C. elegans only the OSM-3-kinesin motor is required for the transport of the receptor involved in the inhibition of the solitary behaviour. Taken together, these results suggest that an IFT-mediated inhibitory mechanism of social behaviour is conserved between P. pacificus and C. elegans. However, the designation of specific sensory neurons as candidates for executing this inhibition is difficult since most of the IFT genes are generally expressed in amphid and phasmid neurons (Tabish et al., 1995; Collet et al., 1998; Wicks et al., 2000; Efimenko et al., 2006; Kunitomo & Lino, 2008; Burghoorn et al., 2012).
C. elegans IFT mutants modulate social behaviour in response to oxygen
To test whether social behaviour of IFT mutants is regulated by [O2], we selected the three most outcrossed IFT mutants (xbx-1, ifta-1 and che-11) and performed bordering assays using an aerotaxis chamber. In addition, we tested the social strain CB4856 and the npr-1(null) ad609 allele. In all lines tested, the social behaviour showed the following dynamics: 10% [O2] induced a strong inhibition of bordering from 90-100% to 20-40%, while the increase to 21% [O2] induced bordering between 90-100% again (Fig. 1B; Table S3 in the Supplementary Material). Thus, C. elegans IFT mutants avoid hyperoxic conditions, similarly to P. pacificus IFT mutants and social strains.
daf-22 mutants are solitary in both C. elegans and P. pacificus nematodes
Finally, we tested if ascarosides trigger the inhibition of social behaviour, given the known role of some ascarosides (ascr#2, ascr#3 and ascr#5) in inducing repulsion in N2 hermaphrodites (Macosko et al., 2009; Ludewig & Schroeder, 2013). We tested the social behaviour of the ascaroside-defective mutant daf-22 produced in solitary strains of C. elegans and P. pacificus (Golden & Riddle, 1985; Srinivasan et al., 2008; Markov et al., 2016). Both the Cel-daf-22 mutant and the Ppa-daf-22.1;Ppa-daf-22.2 double mutant remained solitary (Fig. 1D; Table S4 in the Supplementary Material), further supporting our hypothesis that ascarosides are not the trigger for solitary behaviour in C. elegans N2 and P. pacificus RS2333.
Taken together, our findings show that, similar to P. pacificus, a cilia-mediated inhibitory mechanism of social behaviour is present in C. elegans. Mutants defective in IFT proteins show social behaviour, a phenotype that was previously unnoticed. We propose that opposite inputs regulate social behaviour in nematodes: hyperoxic environmental conditions induce social behaviour, while an unknown input (X) integrated through sensory-cilia induces solitary behaviour (Fig. 1E). However, as long as the environmental input remains unknown, a full understanding of the response to hyperoxic conditions and the ecological relevance of solitary foraging remain unclear.
Materials and methods
All strains were maintained under standard conditions at 20°C on NGM-agar plates seeded with Escherichia coli OP50 (Sulston & Hodgkin, 1988). We used the following strains: P. pacificus daf-22.1(tu489);daf-22.2(tu504) double mutant, C. elegans N2 (Bristol) and CB4856 (Hawaii), npr-1(ad609) and daf-22(m130) C. elegans mutants and 15 IFT C. elegans mutants (see Table S1).
The assay for quantification of bordering and clumping behaviours was performed as previously indicated (Moreno et al., 2016). Bordering was measured as the percentage of animals located within 2 mm of the edge of the bacterial lawn, while clumping was measured as the percentage of animals in contact with two or more other animals along at least 50% of their body length. The regulation of bordering behaviour by [O2] was analysed by performing the assay in a custom-fabricated Plexiglas chamber as previously indicated (Zimmer et al., 2009; Moreno et al., 2016), with three replicates completed per strain/mutant. Nematodes were exposed to shifting [O2] levels from 21% to 10% and back to 21% after 15-min intervals.
Statistical analyses were performed in the computing environment R ver. 3.1.3 (R Core Team, 2015). Replicates of bordering and clumping assays and aerotaxis assays were used to calculate means and standard errors (SEM). Two-sample equal variance Student’s t-test, with Bonferroni corrections for multiple hypothesis testing, was used to confirm significant differences in bordering and clumping averages between wild type and mutant strains, and before and after [O2] shifts.
We thank Drs A. Streit and J. Lightfoot for reading the manuscript and for discussions. All strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). This work was funded by the Max Planck Society.
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Caenorhabditis elegans IFT mutant alleles employed in this study.
Statistical values from pairwise comparisons for mean bordering and clumping between Caenorhabditis elegans N2, CB5846 and 15 IFT mutants.
Statistical values from pairwise comparisons for mean bordering and clumping at different oxygen concentrations ([O2]) for CB4856, one npr-1(null) allele and three IFT mutant alleles.
Statistical values from pairwise comparisons for mean bordering and clumping between the reference strains of Caenorhabditis elegans and Pristionchus pacificus and the corresponding daf-22 mutants.