A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Summary

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

Abstract

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.

We have developed an assay that measures a nematode's ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.

Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

Introduction

Caenorhabditis elegans has been used as a model for the study of neurobiology for decades due to the relative ease in analyzing the circuitry of a nervous system composed of only 302 neurons¹. Provided that the organism is reliant on responding to environmental cues, much of the nervous system is dedicated to regulating the integration of environmental signals². Despite the simplicity of its nervous system, C. elegans can detect and respond to diverse environmental signals including repellents³, attractants⁴, temperature⁵, and even ....

Protocol

1. Preparation of Experimental Organisms

1. Pick 10 L4 staged nematodes per strain 24 h prior to commencing the assay to ensure that organisms are young adults when tested. For each mutant or control nematode tested, pick 10 L4s (10 for the control and 10 for the assay).
   1. Maintain L4 organisms using standard methods^26,27 for 24 h on standard agar plates seeded with OP50 Escherichia coli. If organisms are lost during the subsequent wash steps, compensate by increasing the starting sample size (i.e. pick 20 worms rather than 10).
      Note: Behavior is an....

Results

We utilized wild type (N2), npr-9(tm1652), and an npr-9 overexpression strain, i.e. npr-9(GF) (IC836 -npr-9::npr-9;sur-5::gfp;odr-1::rfp), to evaluate responses to starvation and copper aversion. Wild type organisms are capable of detecting and responding to the aversive copper barrier, while npr-9(GF) mutants do not initiate an aversive response to the copper over the 4 h assay²¹. After 30 min of starvation, ro.......

Discussion

This assay design modifies the food race assay²⁴ to include a copper solution to create an aversive midline barrier and around the edge of the plate to prevent a loss of nematodes. Organisms are tested for their ability to cross the aversive barrier and reach a food patch over a 4 h period. In the context of npr-9(GF), we have utilized this assay to evaluate how starvation conditions could affect aversive responses and the detection of food. Provided that we had previously characterized <.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
M9 Solution [3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Autoclave to sterilize before use.]			Produced in lab
Cupric Sulfate	Sigma	C-1297	Use water to appropriately suspend to a concentration of 0.5M