The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors

Summary

Here, a system is reported for studying the collective behaviors of nematodes by culturing them in bulk using dog food agar medium. This system allows researchers to propagate large numbers of dauer worms and can be applied to Caenorhabditis elegans and other related species.

Abstract

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals have been investigated in the fields of both biology and physics. In the laboratory, researchers have used various model animals such as the fruit fly and zebrafish for approximately a century, but it has remained a major challenge to study large-scale complex collective behavior orchestrated by these genetically tractable model animals. This paper presents a protocol to create an experimental system of collective behaviors in Caenorhabditis elegans. The propagated worms climb on the lid of the Petri plate and show collective swarming behavior. The system also controls worm interactions and behaviors by changing the humidity and light stimulation. This system allows us to examine the mechanisms underlying collective behaviors by changing environmental conditions and examining the effects of individual-level locomotion on collective behaviors using mutants. Thus, the system is useful for future research in both physics and biology.

Introduction

Both nonscientists and scientists are fascinated with animals' collective behaviors, as in flocks of birds and schools of fish. Collective behaviors have been analyzed in a broad range of fields, including physics, biology, mathematics, and robotics. In particular, active matter physics is a growing research field that focuses on systems composed of self-propelled elements, that is, dissipative systems, such as flocks of birds, schools of fish, biofilms of motile bacteria, cytoskeletons composed of active molecules, and groups of self-propelled colloids. The theory of active matter physics maintains that however complex the behaviors of individuals are, the collective motions of enormous numbers of living things are governed by a small number of simple rules. For example, the Vicsek model, a candidate for a unified description of the collective motion of self-propelled particles, predicts that short-range alignment interaction of moving objects is required to form a long-range ordered phase with eccentric fluctuation in 2D, as in herds of animals¹. Top-down experimental approaches pertaining to the physics of active matter are developing rapidly. Previous experiments confirmed the formation of a long-range ordered phase in Escherichia coli². Other recent works employed cells^3,4, bacteria⁵, motile colloids⁶, or moving proteins^7,8. Simple minimal models such as the Vicsek model successfully described these real phenomena. In contrast with these unicellular experimental systems, collective behaviors by animals are usually observed in the wild, as no one could hope to perform controlled experiments with 10,000 real birds or fish.

Biologists share the same interest as physicists: how individuals interact with each other and functionally behave as a group. One of the traditional research fields for analyzing individual behavior is neuroscience, in which the mechanisms underlying behavior have been examined at the neuronal and molecular levels. Many neuroscientific bottom-up approaches have been developed thus far. Top-down approaches in physics and bottom-up approaches in biology can be facilitated using model animals such as the fruit fly, the worm Caenorhabditis elegans, and the mouse⁹. However, there have been few findings on the large-scale collective behavior of these model animals in the laboratory¹⁰; it is still difficult to prepare genetically tractable model animals on a large scale in the laboratory. Therefore, in current research on collective behaviors in biology and physics, it has been difficult for scientists who usually do research in the laboratory to study animals' collective behaviors.

In this study, we established a method for the large-scale cultivation of nematodes to study their collective behaviors. This system allows us to change environmental conditions and examine the effect of individual-level locomotion on collective behaviors using mutants¹⁰. In active matter physics, the parameters of the mathematical model can be controlled in both experiments and simulations, which enables verification of that model for identifying unified descriptions. Genetics is used to understand the neural circuit mechanism underlying collective behavior¹¹.

Protocol

1. Preparation of worms

NOTE: Prepare the wild-type N2 Bristol strain¹² and ZX899 strain (lite-1(ce314); ljIs123[mec-4p::ChR2, unc-122p::RFP])¹³ for the observation of collective behaviors and optogenetic experiments, respectively. Maintain the ZX899 strain under dark conditions.

1. Deposit four well-fed adult worms on a 60 mm plate containing 14 mL of nematode growth medium (NGM) with agar and seeded with E. coli OP50¹².
2. Grow F1 worms to starvation in the NGM plate at 23 °C for 7 days. The yield of F1 worms is approximately 100 worms/plate at this point. The stages of worms comprise a mixed population of dauer and starved L1 larvae.

2. Preparation of dog food agar (DFA) medium plates

1. Autoclave a glass bottle containing 2 g of powdered dog food and 5 mL of 1% agar medium and cool it to room temperature (Figure 1A).
   NOTE: Other dog foods from different manufacturers can be used in this experiment.

3. Inoculation of worms to DFA medium plates

1. Transfer small amounts (approximately 0.5 g) of DFA medium onto the center of an NGM plate seeded with E. coli OP50 (Figure 1B). For optogenetic experiments, pour 40 µL of 50 µM all-trans-retinal, the cofactor of channel rhodopsin 2, onto the DFA before inoculation of worms.
2. Collect the starved worms from four NGM plates using autoclaved water.
3. Place a small fragment of dog food (approximately 0.5 g) on the DFA medium, approximately 2 mm away from the lid of the plate.
4. Illuminate the NGM plate with ultraviolet light for 15 min inside a clean bench to prevent contamination.
5. Inoculate the collected worms (approximately 400 worms) onto the DFA medium on NGM plates. Do not seal the plate with parafilm to avoid increasing the humidity inside the Petri plate and generating water droplets that trap worms on a lid.
6. Propagate the worms at 23 °C and allow them to climb up to the lid of the plate for approximately 10-14 days.
   ​NOTE: Because the number of worms on the lids hardly increased after 10-14 days, it was presumed that the worms had likely run out of food.

4. Observation of collective behavior

1. On the day of the experiment, place a new NGM plate that did not include E. coli and dog food agar medium onto an aluminum plate on the stage of a macro zoom microscope (Figure 2A). Keep the bottom of this new NGM plate at 23 °C using a Peltier temperature controller unit for at least 5 min (Figure 2B). Then, replace the lid of this new NGM plate with the lid of the plate that the worms climbed up onto. Use the objective lens (x2, NA = 0.5) as a low-magnification objective (Figure 2A).
2. Increase the temperature of the bottom of the Petri plate from 23 °C to 26 °C to alter the humidity inside that plate (Figure 2).
3. Acquire images of the inner surface of the plate lid with the camera at 20 frame s⁻¹ (Supplementary Video S1).
4. Save the acquired images in the Tagged Image File format.

5. Optogenetic experiment

1. Use a 100 W mercury lamp to deliver blue light, filtered with a filter set. Control the illumination time precisely using an electromagnetic shutter system (Figure 2B).
2. Maintain ZX899 on DFA under these conditions for 5 min before blue light illumination.
3. Illuminate ZX899 worms attached to the lid of a Petri plate on the microscope stage maintained at 23 °C.
4. Acquire images of the inner surface of the plate lid with a camera at 20 frame s⁻¹ (Supplementary Video S2).

Results

Here, wild-type dauer worms were used for collective behavior observations. Worms were cultivated at 23 °C for approximately 10-14 days and climbed up to the inner surface of the lid of a DFA medium plate. On the experimental day, only the lid was transferred to a new NGM plate without E. coli and DFA medium. The bottom of this Petri plate was initially kept at 23 °C using the Peltier system, and then its temperature was increased to 26 °C. A movie was taken under the microscope.

Discussion

In this study, we show a protocol for preparing a system for the large-scale collective behavior of C. elegans in the laboratory. The DFA-based method was originally established with Caenorhabditis japonica¹⁴ and Neoaplectana carpocapsae Weiser¹⁵, both of which are non-model animals. However, this method was not applied to investigate collective behaviors. The C. elegans is a genetically tractable model animal¹¹

Materials

Name	Company	Catalog Number	Comments
Escherichia coli and C. elegans strains
E. coli OP50	Caenorhabditis Genetics Center	OP50	Food for C. elegans. Uracil auxotroph. E. coli B.
lite-1(ce314); ljIs123[mec-4p::ChR2, unc-122p::RFP]	author	ZX899	lite-1(ce314) mutant carrying the genes expressing ChR2 and RFP under the control of the mec-4 and unc-122 promoter, respectively
N2 Bristrol	Caenorhabditis Genetics Center		Wild-type C. elegans strain
For worm cultivation
Agar purified, powder	Nakarai tesque	01162-15	For preparation of NGM plates
All-trans retinal	Sigma-Aldrich	R2500	For optogenetics
Bacto pepton	Becton Dickinson	211677	For preparation of NGM plates
Calcium chloride	Wako	036-00485	For preparation of NGM plates
Cholesterol	Wako	034-03002	For preparation of NGM plates
di-Photassium hydrogenphosphate	Nakarai tesque	28727-95	For preparation of NGM plates
Dog food	Nihon Pet Food	VITA-ONE	For preparation of dog food agar medium
LB broth, Lennox	Nakarai tesque	20066-95	For culture of E. coli OP50
Magnesium sulfate anhydrous	TGI	M1890	For preparation of NGM plates
Petri dishes (60 mm)	Nunc	150270	For preparation of NGM plates
Potassium Dihydrogenphosphate	Nakarai tesque	28720-65	For preparation of NGM plates
Sodium Chloride	Nakarai tesque	31320-05	For preparation of NGM plates
Observation
Computer	CT solution	CS6229	Windows10 Pro with Intel Xeon Gold 6238R CPU and 768 GB of RAM
CMOS Camera	Hamamatsu photonics	ORCA-Lightning C14120-20P	For data acquisition
CMOS Camera	Olympus	DP74	For data acquisition
Microscope with SZX-MGFP set	Olympus	MVX10	For data acquisition
x2 Objective lens	Olympus	MV PLAPO 2XC	Working distance 20 mm and numerical aperture 0.5
Shutter control
Shutter	OptoSigma	BSH2-RIX	For controlling temporal pattern of  light illumination
Shutter controller	OptoSigma	SSH-C2B-A	For controlling temporal pattern of  light illumination
Temperature control
Peltier temperature controller unit	VICS	WLVPU-30	For controlling humidity inside a Petri plate
UNI-THEMO CONTROLLER	Ampere	UTC-100	For controlling humidity inside a Petri plate
Data acquisition software
HCImage	Hamamatsu photonics		For data acquisition