Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Summary

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Abstract

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity¹ and vibration². In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus³. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.

EIW assays involve counting the number of eggs retained in the uterus of C. elegans⁴. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine^5-9.

Introduction

Caenorhabditis elegans, a microscopic, free-living roundworm, is a model organism traditionally used to study developmental and cell signaling processes because of its transparent anatomy, well-characterized development, fully-sequenced genome, short generation-time, and genetic homology to humans. More recently, C. elegans has become a model organism in the field of environmental toxicology and innate immunity^{10, 11}.

These self-fertilizing hermaphroditic worms become sexually mature within two to three days of hatching from the egg. During its life cycle, C. elegans passes through four larval stages (L1-L4), before reaching adulthood. One isolated hermaphrodite can produce, on average, 300 offspring within three days of peak fecundity. In reproductively mature C. elegans hermaphrodites, fertilized eggs are retained within the uterus for several hours before being laid. The normal number of eggs stored in the uterus at any one time (during peak fecundity) is between ten and fifteen¹². The number of eggs in the uterus is a function of both the rate of egg production and the rate of egg laying. Fertilized eggs are expelled from the uterus by the contraction of sixteen vulval muscles arranged around the opening of the vulva¹³. Hermaphrodite-specific motorneurons (HSN's) and VC motorneurons synapse onto vulval muscles affecting muscle contraction and thus egg-laying behavior^5,7,13,14. Expulsion of eggs from the uterus occurs due to coordinated activity of neurons and muscles.

Lab cultures of C. elegans are typically raised on a diet of nonpathogenic Escherichia coli OP50. In the natural environment, C. elegans come into contact with a variety of food sources, such as pathogenic bacteria, that can be potentially harmful. When exposed to harmful substances in the environment, C. elegans retain eggs until the environment becomes more favorable. Presumably this egg retention is an effort to protect their progeny.

In this egg in worm (EIW) assay, C. elegans are exposed to the potentially pathogenic bacteria, Enterococcus faecalis, which is found in the environment. Exposure to pathogenic forms of E. faecalis can cause persistent intestinal infection and even death in C. elegans¹⁵. Exposure to other forms of pathogenic bacteria have been shown to affect egg retention^16,17, however the effect was not quantified. In addition, the effect of mildly pathogenic strains of E. faecalis, strains that are not immediately lethal, on egg-laying behavior has not been studied.

EIW assays involve counting the number of eggs retained in the uterus of C. elegans⁴. Even though C. elegans are transparent, eggs accumulating in the uterus can be difficult to quantify in an intact animal. The EIW assay involves bleaching gravid adult C. elegans that were exposed to bacteria for a fixed period of time. The bleach solution dissolves the outer cuticle leaving the eggs behind. The eggs are refractive to the effects of bleach due to the presence of a protective eggshell. After bleaching, one is very easily able to count the number of eggs released from the uterus of the worms upon bleaching.

The assay described is a simple, inexpensive, and quick method to quantify the number of eggs in the uterus at one time, and thus quantify the effects of E. faecalis on egg retention. This assay may be used to quantify the effect of other types of bacteria, environmental toxins or drugs on egg retention. This assay also has the potential to be used as a screen for bacterial pathogenicity.

Protocol

1) Preparation of Nematode Growth Media (NGM)

1. To make 50 plates, add 1.5 g NaCl, 8.5 g Ultrapure Agar, and 1.25 g peptone to a 1 L flask.
2. Add 487.5 ml of dH₂O to flask. Swirl gently to mix and cover opening of flask with a piece of aluminum foil.
3. Sterilize the solution by autoclaving at standard conditions (121 psi, 120 °C, 20 min).
4. Allow the solution to cool to 45 °C in a water bath.
5. To the solution, add the following in order: 500 µl cholesterol from a 5 mg/ml stock solution (dissolved in ethanol), 500 µl 1 M CaCl₂, 500 µl 1 M MgSO₄, and 12.5 ml KPO₄ buffer (54.15 g KH₂PO₄ and 17.8 g K₂HPO₄ in 500 ml of dH₂O) as described in¹⁸.
6. Using sterile pipettes, add 10 ml of agar solution to each 60 mm polystyrene Petri dish. Allow the media to solidify at room temperature overnight.

2) Preparation of B Broth E. coli Media

1. To make five 100 ml cultures, add 5.0 g of Bacto Tryptone, 2.5 g yeast extract, 5.0 g NaCl to a beaker.
2. Add sterile, distilled water to a final volume of 500 ml. Heat the solution on a hot plate, while stirring, until the solutes have dissolved.
3. Pour 100 ml of B broth into five glass bottles (at least 200 ml volume).
4. Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 °C, 20 min).
5. Allow B broth to cool to room temperature before use.

3) Seeding C. elegans Maintenance Plates

1. After allowing NGM plates to dry at room temperature for at least one week, inoculate one 100 ml B broth culture with a few colonies of E. coli (OP50).
2. Grow E. coli culture at 37 °C overnight.
3. Pipette one-two drops (approximately 100 µl) of OP50 culture onto the center of each NGM plate. Plates are typically stacked lid-side up. When pipetting (seeding), begin by pipetting the culture onto the plate at the bottom of the stack. Work your way up each stack. Try not to move the plates after seeding, because it is important that the culture not get too close to the edges of the plate.
4. Allow the seeded plates to dry at room temperature for at least one week before using.
5. Seeded plates are stored (inverted) in sealed plastic containers at room temperature.

4) Maintaining C. elegans Strains

1. To maintain well-fed C. elegans stocks, use a worm pick to move three L4s or gravid adults to a new, seeded NGM plate every 3-4 days.
2. Store plates (inverted) preferably inside an incubator at 15- 22 °C. Cultures in the assays described were stored at 20 °C. C. elegans development is temperature-dependent. As maintenance temperature increases, time between developmental stages decreases.

5) Preparation of Tryptic Soy Agar (TSA) E. faecalis Culture Media

1. To make 50 plates, measure 500 ml of sterile, distilled water into a flask and bring to a boil, while stirring, on a hotplate.
2. Add 20 g of powdered TSA agar to flask and allow solution to dissolve.
3. Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 °C, 20 min).
4. Allow the solution to cool to 45 °C in a water bath.
5. Using sterile pipettes, add 10 ml of agar solution to each 60 mm polystyrene Petri dish. Allow the media to solidify at room temperature overnight.

6) Preparation of Tryptic Soy Broth (TSB) E. faecalis Culture Media

1. To make 250 culture tubes, measure 500 ml of sterile, distilled water into a flask and warm on a hotplate while stirring.
2. Add 15 g of TSB agar powder to the flask and allow the powder to dissolve.
3. Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 °C, 20 min).
4. Allow solution to cool to room temperature, then pipette 2 ml into sterile, 5 ml test tubes.

7) Preparation of Brain Heart Infusion (BHI) Media, with Streptomycin, for E. faecalis Culture

1. Measure 500 ml of sterile, distilled water into a flask and bring to a boil, while stirring, on a hotplate.
2. Add 26 g of BHI medium to the flask and allow the solution to dissolve.
3. Sterilize the solutions by autoclaving at standard conditions (121 psi, 120 °C, 20 min).
4. Allow solution to cool to 45 °C in a water bath.
5. Using sterile technique, add 0.5 ml of a 10 mg/ml streptomycin stock solution and swirl to mix.
6. Add 10 ml of the solution to each 60 mm polystyrene Petri dish.
7. Allow media to solidify at room temperature overnight. Store plates (inverted) at 4 °C until a few hours before use.

8) Maintaining E. faecalis Strains

1. To maintain E. faecalis stocks, use a sterile loop to streak individual colonies onto separate tryptic soy agar (TSA) plates.
2. Grow cultures at 37 °C overnight. Store plates (inverted) in a sealed bag or box at room temperature for several weeks.

9) Preparing E. faecalis Plates for EIW Assay

1. Inoculate a 2 ml TSB culture with a colony of E. faecalis and incubate overnight at 37 °C.
2. Pipette 20 µl of E. faecalis culture onto the center of a prepared BHI-streptomycin plate, and incubate overnight at 37 °C.

10) Preparing E. coli Plates for EIW Assay (Control Plates)

1. Inoculate a 100 ml B-broth culture with a colony of E. coli OP50 and incubate overnight at 37 °C
2. Pipette 20 µl of the OP50 culture onto the center of an unseeded NGM plate and allow the seeded plates to dry at room temperature overnight.

11) Egg in Worm Assay

1. Pick 15- 20 staged-L4 C. elegans (Figure 1) onto the center of a lawn of bacteria on a prepared BHI-Strep-E. faecalis plate. Be careful not to transfer a lot of OP50 to the BHI plate. Pick the same number of L4s to a control NGM-OP50 plate.
2. Incubate plates for 40 hr at 20 °C.
3. Prepare 20% bleach solution. Mix a commercial bleach (6.0% sodium hypochlorite) with the appropriate volume of distilled water.
4. Add 10 µl drops of bleach solution to ten distinct locations on a plastic lid (of a worm or bacterial plate).
5. Using a worm pick, transfer one worm into each bleach drop. Gently swirl the tip of the pick in the bleach droplet to make sure the worm has washed off the end of the pick (confirm by viewing the droplet under a dissecting microscope).
6. Allow the cuticle to dissolve for approximately 10 min or until the worms burst open, expelling the eggs (Figure 2). Be careful not to transfer eggs from the plate.
7. Using a dissecting microscope, count the eggs in each drop of bleach.

Results

This assay allows one to quantify the number of retained eggs within C. elegans after exposure to E. faecalis. L4 staged worms (characterized by transparent open space above their vulva, Figure 1) were exposed to E. faecalis through adulthood. After forty hours of exposure to E. faecalis, bleaching was performed. As the cuticle disintegrated in the bleach droplet, the eggs became more apparent (Figure 2). The number of retained eggs was easily quantifi...

Discussion

The most critical steps in successfully performing this assay are: 1) using well-fed stocks of C. elegans, 2) culturing single types of bacteria on the assay plate, 3) accurately identifying staged L4 worms for exposure to E. faecalis, 4) keeping exposure time to E. faecalis consistent across all trials and 5) bleaching time should not exceed ten minutes to prevent egg disintegration.

For this assay it is important to pick healthy, well-fed worms. Maternal starvation...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar, ultrapure	Affymetrix	10906
Bacto Peptone	Becton Dickinson	211677
Bacto Tryptone	Becton Dickinson	211705
Brain Heart Infusion dehydrated medium	Carolina Biological Supply	781781
C. elegans, N2 strain	Caenorhabditis Genetics Center		http://www.cbs.umn.edu/cgc
Cholesterol	Alfa Aesar	A11470
Culture plates for C. elegans	Tritech Research Inc.	T3308
Culture plates for E. faecalis	Fisher Scientific-Fisherbrand	875713
E. coli (OP50)	Caenorhabditis Genetics Center		http://www.cbs.umn.edu/cgc
E. faecalis strains			provided by J. Middleton. All isolates were confirmed as enterococci
by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;
all strains grew at 44.5 ºC and were catalase negative and hydrolyzed esculin. A simplified dichotomous key based on pigmentation and fermentation reactions for six sugars (arabinose, mannitol, methyl-α-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed presumptive identification of all E. faecalis strains (Efs lacks pigmentation and is arabinose, MGP and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains were confirmed using the API 20 STREP system (Biomerieux).
Microscope	Motic	SMZ 168B	any microscope with transmitted illumination and 50X magnification should be sufficient
Streptomycin sulfate	Fisher BioReagents	BP910-50
Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco	Becton Dickinson	236950
Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL	Becton Dickinson	211768
Yeast extract	Acros	61180-1000