Deformities in C. elegans Intestine Due to Infection

Introduction

The aim of this experiment was to study the effect of bacterial infection on the morphology of Caenorhabditis elegans intestine. We used two different species of bacteria, Escherichia coli (strain OP50) and Pseudomonas aeruginosa (strain PA14). OP50 was the control, since it is the standard laboratory food for C. elegans. PA14 is known to be pathogenic to C. elegans. We infected L4 worms orally and observed the changes in their intestine after imaging with an epifluorescence microscope. We also performed a quantitative analysis of the intestinal morphology by calculating a deformation index for the intestine.

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Materials and Methods

Materials

1. C. elegans master plate
2. 2 transfer plates with only agar
3. 3 E. coli plates (GFP tagged)
4. 3 P. aeruginosa plates (GFP tagged)
5. Parafilm
6. Two eyelash picks — made with eyelashes, toothpicks, 200 µL pipette tips, parafilm, and fevicol
7. A dissecting microscope
8. 3% agar
9. Levamisole
10. Glass slides and coverslips
11. Epifluorescence microscope
12. Incubator

Methods

Worm Picking

Using a master plate, we gently picked around 80 L4 worms using an eyelash pick and transferred them to a plain agar plate; 40 worms per plate. We left the worms on the plate for 20–30 minutes, so that the bacteria coating their body would be removed when they moved in the agar. The L4 worms were identifiable by a vulval opening. A dissection microscope was used to pick the worms.

Infection

We then transferred the worms to the experimental plates. For the control group, we transferred worms to three E. coli plates, 10 worms each. For the experimental group, we transferred worms to three P. aeruginosa plates, 10 worms each. We left the worms on the plates for 48 hours at 25°C.

Imaging

The worm intestines had been tagged with mCherry, and both bacteria were tagged with GFP. On a glass slide, we dropped some 3% agar to create a gel pad for the worms. The worms were anesthetized using levamisole, and a coverslip was placed on top. We imaged the worms using an epifluorescence microscope and captured the images at 40X magnification.

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Results

Figure 1: Section of a C. elegans intestine fed E. coli OP50 (standard food). The intestine is mostly smooth, without kinks or inflammation.	Figure 2: Section of a C. elegans intestine fed pathogenic P. aeruginosa PA14. The intestine appears noticeably more swollen and deformed.

Deformation Index

To quantify the degree to which the worm had been affected, we defined a "deformation index" (DI). This was calculated by taking the ratio of the length of the intestine to the length of the worm:

$$\text{DI}=\frac{\text{Length of Intestine}}{\text{Length of Worm}}$$

In case of no or minimal deformation, the worm length should be approximately equal to the length of the intestine, giving a ratio of around 1. In case of high deformation, the length of the intestine will be much longer than the length of the worm, resulting in a ratio significantly greater than 1.

Analysis

The images were analyzed using Fiji. We used the segmented line tool to trace the length of the intestine and the worm, and then obtained the length in pixels using the measure command. Three sections of each worm had been imaged. The DI for each section was calculated for the top and bottom respectively, yielding 6 DI values per worm. The average DI for each worm was then calculated ($n=3$ worms per bacterial exposure):

Bacteria	Average DI
OP50	1.0979
OP50	1.1424
OP50	1.1507
PA14	1.0865
PA14	1.5180
PA14	1.5740

Graphical Distribution

Figure 3: Average Deformation Index per worm. Individual replicates in blue; green coordinate indicates group mean $\pm$ standard deviation.

Statistical Significance

A one-tailed Welch's $t$-test was performed to determine whether there was a significant difference between the PA14 and OP50 infections. This test was chosen because the sample standard deviations of the two groups were unequal:

• $H_0$: There is no significant difference between the intestinal DIs of worms fed the different bacteria.
• $H_1$: ${\text{DI}}_{\text{PA14}}>{\text{DI}}_{\text{OP50}}$
• Significance Level: $\alpha =0.05$

$$t=\frac{{\overline{X}}_1-{\overline{X}}_2}{\sqrt{\frac{s_1^2}{n_1}+\frac{s_2^2}{n_2}}}$$

Where ${\overline{X}}_1$ and ${\overline{X}}_2$ are the sample means, $s_1$ and $s_2$ are the sample standard deviations, and $n_1$ and $n_2$ are the sample sizes.

$$t=\frac{1.392838693-1.13033354}{\sqrt{\frac{{0.2667850071}^2}{3}+\frac{{0.02840530039}^2}{3}}}\approx 1.695$$

The degrees of freedom $\nu$ for Welch's $t$-test are calculated using the Welch–Satterthwaite equation:

$$\nu =\frac{{(\frac{s_1^2}{n_1}+\frac{s_2^2}{n_2})}^2}{\frac{{\left(\frac{s_1^2}{n_1}\right)}^2}{n_1-1}+\frac{{\left(\frac{s_2^2}{n_2}\right)}^2}{n_2-1}}\approx 2.045\approx 2$$

From the $t$-distribution table, the critical value for a one-tailed test with 2 degrees of freedom at a 0.05 significance level is 2.920:

$$P(T>2.920)=0.05⟹P(T>1.695)>0.05$$

Figure 4: $t$-distribution with 2 degrees of freedom, plotted in Desmos.

Therefore, at $\alpha =0.05$, we do not have enough statistical evidence to reject the null hypothesis.

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Discussion

Visibly, there appears to be a clear morphological difference between the intestines of the two groups of worms. However, the statistical analysis does not reject the null hypothesis, primarily attributable to the small sample size ($n=3$ per group) and substantial within-group variance in the PA14 cohort (standard deviation $\approx 10\times$ higher than OP50 due to an apparent outlier).

Additionally, worm stage staging errors during picking (e.g. mix of L3 and L4 worms maturing together over 48 hours) could introduce morphological variability.

NOTE

Biological Observations on Barrier Dysfunction:
In several images, ingested bacteria were observed outside the intestinal boundary in both OP50 and PA14 worms. Intestinal barrier dysfunction is an evolutionarily conserved hallmark of aging across organisms (Salazar et al., 2023), so bacteria outside the lumen in OP50 controls likely reflects senescence, whereas in PA14 worms it is directly attributable to pathogen-mediated epithelial damage (Tan et al., 1999).

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References

1. Cambridge International. List of Formulae and Statistical Tables. PDF link
2. Desmos. t-distribution Calculator. Desmos
3. Salazar, A.M., Aparicio, R., Clark, R.I., Rera, M. and Walker, D.W. (2023). Intestinal barrier dysfunction: an evolutionarily conserved hallmark of aging. Disease Models & Mechanisms, 16(4). DOI
4. Tan, M.W., Rahme, L.G., Sternberg, P.W. and Ausubel, F.M. (1999). Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. PNAS, 96(5), pp. 2408–2413. DOI