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.

Download Full-text

Measuring C. elegans spatial foraging and food intake using bioluminescent bacteria

10.1101/759928 ◽

2019 ◽

Cited By ~ 1

Author(s):

Siyu Serena Ding ◽

Karen S. Sarkisyan ◽

Andre E. X. Brown

Keyword(s):

Food Intake ◽

Foraging Behaviour ◽

Light Emission ◽

Fold Increase ◽

Food Patch ◽

Background Signal ◽

Bioluminescent Bacteria ◽

C Elegans ◽

Tightly Coupled ◽

Lower Background

ABSTRACTFor most animals, feeding includes two behaviours: 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 behaviours. However, most methods of measuring feeding in worms quantify either foraging behaviour or food intake but not both. Imaging the depletion of fluorescently labelled 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 each other before foraging collectively for more food; similarly, during high density swarming, only worms at the migrating front are in contact with bacteria. These results demonstrate the usefulness of bioluminescent bacteria for quantifying feeding and suggest a hygiene hypothesis for the function of C. elegans aggregation and swarming.

Download Full-text

Insights into the Involvement of Spliceosomal Mutations in Myelodysplastic Disorders from Analysis of SACY-1/DDX41 in Caenorhabditis elegans

Genetics ◽

10.1534/genetics.119.302973 ◽

2020 ◽

Vol 214 (4) ◽

pp. 869-893 ◽

Cited By ~ 1

Author(s):

Tatsuya Tsukamoto ◽

Micah D. Gearhart ◽

Seongseop Kim ◽

Gemechu Mekonnen ◽

Caroline A. Spike ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Myelodysplastic Syndromes ◽

Missense Mutations ◽

Synthetic Lethal ◽

C Elegans ◽

Link Type ◽

Dead Box ◽

Synthetic Lethal Interactions ◽

Biochemical Analyses ◽

Cellular Phenotypes

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1. Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3′ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.

Download Full-text

Dehydrated Caenorhabditis elegans Stocks Are Resistant to Multiple Freeze-Thaw Cycles

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401825 ◽

2020 ◽

Vol 10 (12) ◽

pp. 4505-4512

Author(s):

Patrick D. McClanahan ◽

Richard J. McCloskey ◽

Melanie Ng Tung Hing ◽

David M. Raizen ◽

Christopher Fang-Yen

Keyword(s):

Caenorhabditis Elegans ◽

Freeze Thaw ◽

C Elegans ◽

Link Type ◽

Defective Mutant ◽

Genetic Stocks ◽

Dauer Larvae

Ultracold preservation is widely used for storage of genetic stocks of Caenorhabditis elegans. Current cryopreservation protocols are vulnerable to refrigeration failures, which can result in the loss of stock viability due to damage during re-freezing. Here we present a method for preserving worms in a dehydrated and frozen form that retains viability after multiple freeze-thaw cycles. After dehydration in the presence of trehalose or glycerol, C. elegans stocks can be frozen and thawed multiple times while maintaining viability. While both dauer and non-dauer larvae survive desiccation and freezing, the dauer defective mutant daf-16 does not survive desiccation. Our technique is useful for storing stocks in a manner robust to freezer failures, and potentially for shipping strains between laboratories.

Download Full-text

Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400781 ◽

2019 ◽

Vol 10 (1) ◽

pp. 267-280 ◽

Cited By ~ 7

Author(s):

Michael A. Q. Martinez ◽

Brian A. Kinney ◽

Taylor N. Medwig-Kinney ◽

Guinevere Ashley ◽

James M. Ragle ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Protein Degradation ◽

Single Cell ◽

Water Soluble ◽

Growth Media ◽

Synthetic Analog ◽

Rapid Degradation ◽

Target Proteins ◽

C Elegans ◽

Link Type

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegansSKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.

Download Full-text

Sex and Mitonuclear Adaptation in Experimental Caenorhabditis elegans Populations

Genetics ◽

10.1534/genetics.119.301935 ◽

2019 ◽

Vol 211 (3) ◽

pp. 1045-1058 ◽

Cited By ~ 8

Author(s):

Riana I. Wernick ◽

Stephen F. Christy ◽

Dana K. Howe ◽

Jennifer A. Sullins ◽

Joseph F. Ramirez ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Mitochondrial Gene ◽

Large Population ◽

Functional Genomic ◽

Partial Recovery ◽

Mitochondrial Dna Mutation ◽

Link Protein ◽

Dna Mutation ◽

Link Type ◽

High Fitness

To reveal phenotypic and functional genomic patterns of mitonuclear adaptation, a laboratory adaptation study with Caenorhabditis elegans nematodes was conducted in which independently evolving lines were initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1. Following 60 generations of evolution in large population sizes with competition for food resources, two distinct classes of lines representing different degrees of adaptive response emerged: a low-fitness class that exhibited minimal or no improvement compared to the gas-1 mutant ancestor, and a high-fitness class containing lines that exhibited partial recovery of wild-type fitness. Many lines that achieved higher reproductive and competitive fitness levels were also noted to evolve high frequencies of males during the experiment, consistent with adaptation in these lines having been facilitated by outcrossing. Whole-genome sequencing and analysis revealed an enrichment of mutations in loci that occur in a gas-1-centric region of the C. elegans interactome and could be classified into a small number of functional genomic categories. A highly nonrandom pattern of mitochondrial DNA mutation was observed within high-fitness gas-1 lines, with parallel fixations of nonsynonymous base substitutions within genes encoding NADH dehydrogenase subunits I and VI. These mitochondrial gene products reside within ETC complex I alongside the nuclear-encoded GAS-1 protein, suggesting that rapid adaptation of select gas-1 recovery lines was driven by fixation of compensatory mitochondrial mutations.

Download Full-text

DAF-16 and SMK-1 Contribute to Innate Immunity During Adulthood in Caenorhabditis elegans

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401166 ◽

2020 ◽

Vol 10 (5) ◽

pp. 1521-1539 ◽

Cited By ~ 1

Author(s):

Daniel R. McHugh ◽

Elena Koumis ◽

Paul Jacob ◽

Jennifer Goldfarb ◽

Michelle Schlaubitz-Garcia ◽

...

Keyword(s):

Innate Immunity ◽

Immune System ◽

Caenorhabditis Elegans ◽

Host Defense ◽

Insulin Signaling ◽

Bacterial Pathogens ◽

C Elegans ◽

Link Type ◽

Age Dependent ◽

Over Time

Aging is accompanied by a progressive decline in immune function termed “immunosenescence”. Deficient surveillance coupled with the impaired function of immune cells compromises host defense in older animals. The dynamic activity of regulatory modules that control immunity appears to underlie age-dependent modifications to the immune system. In the roundworm Caenorhabditis elegans levels of PMK-1 p38 MAP kinase diminish over time, reducing the expression of immune effectors that clear bacterial pathogens. Along with the PMK-1 pathway, innate immunity in C. elegans is regulated by the insulin signaling pathway. Here we asked whether DAF-16, a Forkhead box (FOXO) transcription factor whose activity is inhibited by insulin signaling, plays a role in host defense later in life. While in younger C. elegansDAF-16 is inactive unless stimulated by environmental insults, we found that even in the absence of acute stress the transcriptional activity of DAF-16 increases in an age-dependent manner. Beginning in the reproductive phase of adulthood, DAF-16 upregulates a subset of its transcriptional targets, including genes required to kill ingested microbes. Accordingly, DAF-16 has little to no role in larval immunity, but functions specifically during adulthood to confer resistance to bacterial pathogens. We found that DAF-16-mediated immunity in adults requires SMK-1, a regulatory subunit of the PP4 protein phosphatase complex. Our data suggest that as the function of one branch of the innate immune system of C. elegans (PMK-1) declines over time, DAF-16-mediated immunity ramps up to become the predominant means of protecting adults from infection, thus reconfiguring immunity later in life.

Download Full-text

The Mitochondrial Na+/Ca2+ Exchanger Inhibitor CGP37157 Preserves Muscle Structure and Function to Increase Lifespan and Healthspan in Caenorhabditis elegans

Frontiers in Pharmacology ◽

10.3389/fphar.2021.695687 ◽

2021 ◽

Vol 12 ◽

Author(s):

Paloma García-Casas ◽

Pilar Alvarez-Illera ◽

Eva Gómez-Orte ◽

Juan Cabello ◽

Rosalba I. Fonteriz ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Regular Structure ◽

Fold Increase ◽

Maximum Speed ◽

Tor Pathway ◽

C Elegans ◽

Promising Target ◽

Metabolism Enzymes ◽

And Function ◽

Induced Changes

We have reported recently that the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 extends lifespan in Caenorhabditis elegans by a mechanism involving mitochondria, the TOR pathway and the insulin/IGF1 pathway. Here we show that CGP37157 significantly improved the evolution with age of the sarcomeric regular structure, delaying development of sarcopenia in C. elegans body wall muscle and increasing the average and maximum speed of the worms. Similarly, CGP37157 favored the maintenance of a regular mitochondrial structure during aging. We have also investigated further the mechanism of the effect of CGP37157 by studying its effect in mutants of aak-1;aak-2/AMP-activated kinase, sir-2.1/sirtuin, rsks-1/S6 kinase and daf-16/FOXO. We found that this compound was still effective increasing lifespan in all these mutants, indicating that these pathways are not involved in the effect. We have then monitored pharynx cytosolic and mitochondrial Ca2+ signalling and our results suggest that CGP37157 is probably inhibiting not only the mitochondrial Na+/Ca2+ exchanger, but also Ca2+ entry through the plasma membrane. Finally, a transcriptomic study detected that CGP37157 induced changes in lipid metabolism enzymes and a four-fold increase in the expression of ncx-6, one of the C. elegans mitochondrial Na+/Ca2+ exchangers. In summary, CGP37157 increases both lifespan and healthspan by a mechanism involving changes in cytosolic and mitochondrial Ca2+ homeostasis. Thus, Ca2+ signalling could be a promising target to act on aging.

Download Full-text

Signaling by AWC Olfactory Neurons Is Necessary for Caenorhabditis elegans’ Response to Prenol, an Odor Associated with Nematode-Infected Insects

Genetics ◽

10.1534/genetics.120.303280 ◽

2020 ◽

Vol 216 (1) ◽

pp. 145-157

Author(s):

Tiffany Baiocchi ◽

Kyle Anesko ◽

Nathan Mercado ◽

Heenam Park ◽

Kassandra Kin ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Behavioral Ecology ◽

Natural Variation ◽

Isoamyl Alcohol ◽

Life Cycles ◽

C Elegans ◽

Link Type ◽

Genome Wide ◽

Ecological Differences

Chemosensation plays a role in the behaviors and life cycles of numerous organisms, including nematodes. Many guilds of nematodes exist, ranging from the free-living Caenorhabditis elegans to various parasitic species such as entomopathogenic nematodes (EPNs), which are parasites of insects. Despite ecological differences, previous research has shown that both EPNs and C. elegans respond to prenol (3-methyl-2-buten-1-ol), an odor associated with EPN infections. However, it is unclear how C. elegans responds to prenol. By utilizing natural variation and genetic neuron ablation to investigate the response of C. elegans to prenol, we found that the AWC neurons are involved in the detection of prenol and that several genes (including dcap-1, dcap-2, and clec-39) influence response to this odorant. Furthermore, we identified that the response to prenol is mediated by the canonically proposed pathway required for other AWC-sensed attractants. However, upon testing genetically diverse isolates, we found that the response of some strains to prenol differed from their response to isoamyl alcohol, suggesting that the pathways mediating response to these two odorants may be genetically distinct. Further, evaluations leveraging natural variation and genome wide association revealed specific genes that influence nematode behavior and provide a foundation for future studies to better understand the role of prenol in nematode behavioral ecology.

Download Full-text

Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

Genetics ◽

10.1534/genetics.119.302655 ◽

2019 ◽

Vol 213 (4) ◽

pp. 1197-1207 ◽

Cited By ~ 1

Author(s):

Ivana Sfarcic ◽

Theresa Bui ◽

Erin C. Daniels ◽

Emily R. Troemel

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Promoter Activity ◽

Fluorescent Protein ◽

Developmental Stages ◽

Fluorescent Reporters ◽

C Elegans ◽

Link Type ◽

Highly Sensitive ◽

Green Fluorescent

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

Download Full-text

MAP Kinase Signaling Antagonizes PAR-1 Function During Polarization of the Early Caenorhabditis elegans Embryo

Genetics ◽

10.1534/genetics.109.106716 ◽

2009 ◽

Vol 183 (3) ◽

pp. 965-977 ◽

Cited By ~ 16

Author(s):

Annina C. Spilker ◽

Alexia Rabilotta ◽

Caroline Zbinden ◽

Jean-Claude Labbé ◽

Monica Gotta

Keyword(s):

Cell Division ◽

Caenorhabditis Elegans ◽

Map Kinase ◽

Cell Fate ◽

Asymmetric Cell Division ◽

Asymmetric Distribution ◽

Kinase Signaling ◽

C Elegans ◽

Link Type ◽

Map Kinase Signaling

PAR proteins (partitioning defective) are major regulators of cell polarity and asymmetric cell division. One of the par genes, par-1, encodes a Ser/Thr kinase that is conserved from yeast to mammals. In Caenorhabditis elegans, par-1 governs asymmetric cell division by ensuring the polar distribution of cell fate determinants. However the precise mechanisms by which PAR-1 regulates asymmetric cell division in C. elegans remain to be elucidated. We performed a genomewide RNAi screen and identified six genes that specifically suppress the embryonic lethal phenotype associated with mutations in par-1. One of these suppressors is mpk-1, the C. elegans homolog of the conserved mitogen activated protein (MAP) kinase ERK. Loss of function of mpk-1 restored embryonic viability, asynchronous cell divisions, the asymmetric distribution of cell fate specification markers, and the distribution of PAR-1 protein in par-1 mutant embryos, indicating that this genetic interaction is functionally relevant for embryonic development. Furthermore, disrupting the function of other components of the MAPK signaling pathway resulted in suppression of par-1 embryonic lethality. Our data therefore indicates that MAP kinase signaling antagonizes PAR-1 signaling during early C. elegans embryonic polarization.

Download Full-text