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Sleep is a reversible state, characterized by the inhibition of periodic behaviors that occur during waking hours. Caenorhabditis elegans demonstrates stress-induced sleep following exposure to environmental stressors, like noxious heat or ultraviolet irradiation. During this time, animals inhibit movement, feeding, and defecation, behavioral quiescence largely controlled by neuropeptide signaling from the ALA and RIS sleep interneurons. Here, we tested whether egg retention and/or production which occurs during suboptimal environmental conditions, is regulated by the ALA and/or RIS, or other neuropeptides. We find that during stress-induced sleep, worms reduce egg-laying behavior and egg production (i.e., fertility). While the behavior is modestly modified in the absence of the ALA and RIS, as well as some neuropeptides, fertility is regulated by other mechanisms. 

Sleep is ancient and genetically conserved across phylogeny. Neuropeptide signaling plays a fundamental role in the regulation of sleep for mammals, fish, and invertebrates like Caenorhabditis elegans. Developmentally timed-sleep and stress-induced sleep of C. elegans are controlled by distinct and overlapping neuropeptide pathways. The RPamide neuropeptides nlp-2, nlp-22, and nlp-23, play antagonistic roles during the regulation of developmentally-timed sleep, however, their role in stress-induced sleep has not been explored. These genes are linked on the X chromosome, which has made genetic analyses challenging. Here we used CRISPR to generate new alleles of nlp-22 and nlp-23, nlp-22;nlp-23 double mutants, and nlp-2;nlp-22;nlp-23 triple mutants. Confirming previous studies, we find that nlp-22 is required for developmentally-timed sleep, and show that nlp-23 is also required. However, all three genes are dispensable for stress-induced sleep. 

Orcokinin neuropeptides are conserved among ecdysozoans, but their functions are incompletely understood. Here, we report a role for orcokinin neuropeptides in the regulation of sleep in the nematode Caenorhabditis elegans. The C. elegans orcokinin peptides, which are encoded by the nlp-14 and nlp-15 genes, are necessary and sufficient for quiescent behaviors during developmentally timed sleep (DTS) as well as during stress-induced sleep (SIS). The five orcokinin neuropeptides encoded by nlp-14 have distinct but overlapping functions in the regulation of movement and defecation quiescence during SIS. We suggest that orcokinins may regulate behavioral components of sleep-like states in nematodes and other ecdysozoans. 

ABSTRACT Genome-wide association studies (GWAS) can identify genetic variants responsible for naturally occurring and quantitative phenotypic variation. Association studies therefore provide a powerful complement to approaches that rely on de novo mutations for characterizing gene function. Although bacteria should be amenable to GWAS, few GWAS have been conducted on bacteria, and the extent to which nonindependence among genomic variants (e.g., linkage disequilibrium [LD]) and the genetic architecture of phenotypic traits will affect GWAS performance is unclear. We apply association analyses to identify candidate genes underlying variation in 20 biochemical, growth, and symbiotic phenotypes among 153 strains of Ensifer meliloti . For 11 traits, we find genotype-phenotype associations that are stronger than expected by chance, with the candidates in relatively small linkage groups, indicating that LD does not preclude resolving association candidates to relatively small genomic regions. The significant candidates show an enrichment for nucleotide polymorphisms (SNPs) over gene presence-absence variation (PAV), and for five traits, candidates are enriched in large linkage groups, a possible signature of epistasis. Many of the variants most strongly associated with symbiosis phenotypes were in genes previously identified as being involved in nitrogen fixation or nodulation. For other traits, apparently strong associations were not stronger than the range of associations detected in permuted data. In sum, our data show that GWAS in bacteria may be a powerful tool for characterizing genetic architecture and identifying genes responsible for phenotypic variation. However, careful evaluation of candidates is necessary to avoid false signals of association. IMPORTANCE Genome-wide association analyses are a powerful approach for identifying gene function. These analyses are becoming commonplace in studies of humans, domesticated animals, and crop plants but have rarely been conducted in bacteria. We applied association analyses to 20 traits measured in Ensifer meliloti , an agriculturally and ecologically important bacterium because it fixes nitrogen when in symbiosis with leguminous plants. We identified candidate alleles and gene presence-absence variants underlying variation in symbiosis traits, antibiotic resistance, and use of various carbon sources; some of these candidates are in genes previously known to affect these traits whereas others were in genes that have not been well characterized. Our results point to the potential power of association analyses in bacteria, but also to the need to carefully evaluate the potential for false associations.