A simple protocol for cultivating the bacterivorous soil nematode Caenorhabditis elegans in its natural ecology in the laboratory

Abstract

The complex interplay between an animal and its surrounding environment requires constant attentive observation in natural settings. Moreover, how ecological interactions are affected by an animal's genes is difficult to ascertain outside the laboratory. Genetic studies with the bacterivorous nematode Caenorhabditis elegans have elucidated numerous relationships between genes and functions, such as physiology, behaviors, and lifespan. However, these studies use standard laboratory culture that does not reflect C. elegans true ecology. C. elegans is found growing in nature and reproduced in large numbers in soils enriched with rotting fruit or vegetation, a source of abundant and diverse microbes that nourish the thriving populations of nematodes. We developed a simple mesocosm we call soil-fruit-natural-habitat that simulates the natural ecology of C. elegans in the laboratory. Apples were placed on autoclaved potted soils, and after a soil microbial solution was added, the mesocosm was subjected to day-night, temperature, and humidity cycling inside a growth chamber. After a period of apple-rotting, C elegans were added, and the growing worm population was observed. We determined optimal conditions for the growth of C. elegans and then performed an ecological succession experiment observing worm populations every few days. Our data showed that the mesocosm allows abundant growth and reproduction of C. elegans that resembles populations of the nematode found in rotting fruit in nature. Overall, our study presents a simple protocol that allows the cultivation of C. elegans in a natural habitat in the laboratory for a broad group of scientists to study various aspects of animal and microbial ecology.

Keywords: C. elegans; bacterial predator; ecological succession; nematode cultivation; nematode ecology; soil ecology; soil microbe.

Figure 1

Setting up the soil-fruit-natural-habitat mesocosm. An SFNH mesocosm was set-up by adding autoclaved peat and loam soil-mix in a plastic pot. In total, 5 mL of microbial solution (MS) was added on top of the soil before the halved apple was put in place. The set-up is then placed inside the growth chamber where SFNH with C. elegans was incubated; shown are the temperature (red) and humidity (black) cycling parameters. After 6 days of the apple-rotting period, 30 L1-stage C. elegans larvae were added by lifting the rotten apple (some soils are attached) and pipetting M9 buffer containing the larvae on the surface of the soil. (A) 15 days experiment from setting-up SFNH (day 0) to harvest day (day 15) where the 9 days C. elegans population was harvested is conducted. (B) Sterile synchronization process of L1-stage C. elegans larvae. Created with BioRender.com.

Figure 2

Microbial solution (MS) preparation. To replace the microbiome that was stripped off the soil due to autoclaving, we isolated the microbiome from a previous SFNH where C. elegans and other nematodes grew abundantly. (Top) The soil and apple samples from this mesocosm were placed into conical tubes with 0.85% NaCl separately. (Middle) The mixture was centrifuged, and the supernatant was used as the microbial solution (MS). To make sure that all nematodes, insects, and eggs from the apple and soil samples were killed, the MS was incubated at 37°C for 14 h, frozen overnight at −70°C, and then thawed the next day. (Bottom) Afterward, an aliquot of the MS was plated in NGM agar plates to check for any nematode growth. Upon clearance, the solution was also plated in nutrient broth, R2A, and PDA agar plates to confirm the presence of microorganisms. Created with BioRender.com.

Figure 3

Harvesting of C. elegans from the SFNH mesocosm. (A) Baermann funnel technique was used to isolate the worms from both the soil and the apple. Muslin cloth was used to line the inner surface of the funnel, and then, the apple and/or soil was placed on it. M9 buffer was poured afterward, and the set-up was let to stand for 4 h to give time for the worms to swim to the clamped silicon tubes. The buffer was then collected into 50 mL conical tubes and used for worm population analysis. (B) The funnels are placed on a funnel-holder rack while letting it stand for 4 h. Created with BioRender.com.

Figure 4

C. elegans population growth is affected by soil composition. The results of SFNH mesocosm experiment where worms were incubated for 9 days. Reproductive fitness of the worm was observed highest when the soil is a mixture of 50% highly organic peat and 50% loam soil. Different letters denote differences in the mean using One-way ANOVA (p-value < 0.5) and Duncan’s Multiple Range Test (DMRT; alpha value = 0.5). Error bars are standard deviations (n = 3).

Figure 5

Dilution of microbial solution affects C. elegans population growth in SFNH mesocosm. In total, 5 mL dilutions of the original MS were added at starting of each SFNH experiment, and C. elegans populations were counted 9 days after adding the worms. Different letters denote differences in the mean using one-way ANOVA (p-value < 0.05) and DMRT (alpha value = 0.05). Error bars are standard deviations (n = 3).

Figure 6

A combination of rotting apple and MS in the SFNH mesocosm provides greater C. elegans population growth. (A) For the experiment to analyze the significance of the apple in the SFNH mesocosm, four treatments were set up where either the apple, MS, or both were removed to observe the role of the apple in the mesocosm. (B) Data shows that the combination of rotting apple and MS allows for higher worm growth. C. elegans populations were counted 9 days after adding the worms. This figure also shows that worm growth, albeit much lower, was observed even when apple was not present in the mesocosm. Different letters denote differences of the mean using one-way ANOVA (p-value > 0.5) and DMRT (alpha value = 0.5). Error bars are standard deviations (n = 9). (C) Based on the results, 6 days apple rotting is enough to support C. elegans growth and reproduction in the SFNH mesocosm. C. elegans populations were counted 9 days after adding the worms. Different letters denote differences of the mean using one-way ANOVA (p-value < 0.5) and DMRT (alpha value = 0.5). Error bars are standard deviations (n = 3).

Figure 7

Ecological succession assay in the SFNH mesocosm. (A) Timeline for ecological succession (ES) assay where worm population was harvested at an increment of 3 days of growth. (B) Apple rotting in SFNH from the day of set up (Day 0) to harvest day (Day 21). (C) An increase in the C. elegans population was observed on day 15 of the ES assay, and a subsequent decline in the average population was observed from day 18 onwards. It was also important to note that some pots were observed (21D) to have more worms than the rest of the replicates for the same day treatment. Each pot is an individual ecological environment on its own and microenvironmental factors surrounding each pot might have caused this high number of worm population. Solid sphere = 1 pot, n = 18 pots for each day. Red lines = upper standard deviation; blue horizontal line = mean.

Figure 8

Comparison of developmental stages in plate and SFNH grown C. elegans. (A) Illustration of the protocol performed to compare the growth and development of C. elegans in SFNH versus the traditional OP50 plate. (B) Comparison of the average number of worm per pot among plate-grown and SFNH-grown C. elegans. (C) Percentages of different worm stages across the experimental set-ups show that similar to plate cultivation, SFNH cultivation also results in a higher L1 larvae percentage compared with L3–L4 and adult stages. YA, young adult; L1, L1 larvae; the number before letters indicates the number of worm(s) initially added, and the number of days indicates the cultivation period. Different letters denote differences in the mean using one-way ANOVA (p-value < 0.01) and Tukey’s HSD (alpha value = 0.05). Error bars are standard deviations (n = 3). Created with BioRender.com.

Figure 9

Possible applications of the SFNH protocol. The SFNH protocol application can further the understanding of C. elegans biology. This protocol can be used to understand how the nematode behaves and reacts to the factors present in its natural habitat. This protocol can also be used in climatic change effects on soil ecology and the changes in soil microbial ecology. Finally, the agricultural application of this protocol can also help understand the effects of pesticides, microplastic, and other exogenous factors on the natural fauna of the agricultural ecosystem without spreading harm to the environment. Created with BioRender.com.