Caenorhabditis elegans, Dougherty, 1953, Dougherty, 1953

Le, Tho Son, Nguyen, Thi Hong Gam, Ha, Bich Hong, Huong, Bui Thi Mai, Nguyen, Thi Thu Hang, Vu, Kim Dung, Ho, Tu Cuong & Wang, John, 2022, Reproductive Span of Caenorhabditis elegans Is Extended by Microbacterium sp., Journal of Nematology 54 (1), pp. 1-21 : 4-10

publication ID	https://doi.org/ 10.2478/jofnem-2022-0010
DOI	https://doi.org/10.5281/zenodo.12639537
persistent identifier	https://treatment.plazi.org/id/2D2AB30D-FFD0-FF93-A93A-FCE86DE9F90C
treatment provided by	Felipe
scientific name	Caenorhabditis elegans
status

Treatment

Isolation and screening of bacteria affecting RS of C. elegans View in CoL

We hypothesized that the different bacteria encountered and eaten by C. elegans in the wild could affect its physiology, and in particular RS. To this end, we first isolated bacteria from rotting vegetation, substrates from which nematodes would often be found, from two national parks in Vietnam. We isolated 64 bacterial strains from 50 sampling sites in Cat Tien National Park and 45 strains from 44 sites in Cuc Phuong National Park ( Tables S2 View Table 2 and S 3 View Table S ). These 109 strains were qualitatively characterized based on colony size, color, thickness, and prevalence on the original screening plate ( Table S3 View Table S ).

Next, we used a simple 1-plate assay (see “Methods”) to screen all 109 isolates for an effect on RS of C. elegans in comparison to the E. coli OP 50 control. The RS of C. elegans on OP50 was about 3–4 days. Qualitatively, 108 strains also had similar RS in the 3- to 4-day range. One strain, CFBb37, seemed to extend RS to approximately 7 days. We note that we found a nematode, Caenorhabditis briggsae , co-occurring in the field sample containing CFBb37 ( Table S2 View Table 2 ). We also noticed that this and six other bacterial strains reduced the body size and another delayed the onset of first brood production in C. elegans ( Fig. S1 View Figure 1 and Table S2 View Table 2 ). To determine their genus or species identity we sequenced the 16S rDNA barcode for these eight bacterial strains as well as four others without obvious effects on C. elegans ( Table S2 View Table 2 ). This revealed that CFBb37 is a Microbacterium sp. , which is in the Actinobacteria phylum. Most of the other strains belonged to the phylum Proteobacteria (n = 8) while the rest were in the phyla Bacteroidetes (n = 1) or Firmicutes (n = 2). We focused on Microbacterium sp. CFBb37 for the rest of this study.

Confirmation of RS extension by Microbacterium sp. CFBb37

To confirm the screening results, we determined more precisely the RS of C. elegans when fed with Microbacterium sp. CFBb37, the control OP50, and two additional identified strains ( Acinetobacter sp. CFBb9 and Serratia sp. CFBb17). In this assay we moved pools of adults (n = 6–15) to fresh plates daily, which allowed determining both the presence of brood and counting the number of brood per day. The worms growing on OP50 as well as Acinetobacter sp. CFBb9 and Serratia sp. CFBb17 had similar RS durations, which was 4 days (P <0.001; Table 1 View Table 1 ). In contrast, worms growing on Microbacterium sp. CFBb37 had an RS of 8 days (P <0.001), confirming the RS extension from the screen. Qualitatively, average daily brood counts appeared to decline exponentially from day 2 onward (day 2, n = 47; day 7 and later, n <4) ( Table 1 View Table 1 ).

In the above assay, the number of parents per plate varied both within and among bacterial strains. While unlikely to be of major effect, we decided to eliminate the potential confounding effects of parental interactions and differing parental density by re-examining brood presence (and brood sizes, see below) using one parental worm per plate. We found that the average RS for worms growing on Microbacterium sp. CFBb37 was 79% greater than on OP50 (7.29 ± 0.25 days; mean ± 1 standard error (SE) versus 4.08 ± 0.16; P <0.001; Fig. 1A and S1 View Figure 1 ; Table 2 View Table 2 ). The combined data clearly indicate that CFBb37 induces RS extension in C. elegans .

Daily count of eggs in the uterus of C. elegans

Our assays for RS above were sometimes difficult (e.g., looking for rare laid eggs near the end of the RS period) or required moving the worms to new plates everyday as well as waiting for the progeny to hatch. A simplification of the RS assay would be to directly look for eggs in the uteruses (“uterus egg assay”) of the focal adult worm at 400× magnification under a DIC compound microscope each day. The presence of at least one egg would indicate fertility. The lack of eggs could indicate reproductive senescence or that any eggs were recently laid. Examining many adult worms each day should help distinguish between the two possibilities, although there would still be a bias for incorrectly scoring a batch of worms (i.e., day) as reproductively senescent.

To test whether the uterus egg assay gives comparable results to our original RS assays, we placed C. elegans L1s on either CFBb37 or OP50 and raised them to adulthood. Then, each day we counted the number of eggs in the uterus of 29–37 worms. For each day, if this number of eggs was greater than zero (using a one-sample t -test), then we considered that day to be a fertile day (i.e., not reproductively senescent).

Using this assay, the worms on CFBb37 had an RS of 8 days while those on OP50 had an RS of 4 days ( Table S5 View Table S ). The results from counting eggs in the uteruses were qualitatively concordant with the earlier brood-based assays (7.29 and 4.08 days, respectively; Table 2 View Table 2 ). Thus, although this approach has an inherent bias for reproductive senescence (i.e., scoring shorter RS), we suggest that the simpler uterus egg assay provides a reasonably good estimate for the RS, and can distinguish differences in RS, at least for those separated by several days.

Reduction of brood size by

Microbacterium sp. CFBb37

While confirming the RS extension by Microbacterium sp. CFBb37 on pools of C. elegans , we noticed that this bacterial strain reduced the average total brood size compared with OP50 (141.6 vs 251.2; P <0.001; Table 1 View Table 1 ). As above, to remove the potential confounding effects of pooled worms, we recounted the brood sizes using one parental worm per plate. We found that the average total brood size on Microbacterium sp. CFB 37 was less than on OP50 (137.63 mean ± 5.74 SE versus 266.28 ± 6.42; P <0.001, Table 2 View Table 2 ). Partitioning the data into daily brood sizes over the first 8 days (i.e., the number of days with data for both bacterial strains, Table 2 View Table 2 ) revealed that worms fed with OP50 had greater brood sizes than those fed with Microbacterium sp. CFB 37 during the first 3 days (29–125 more eggs/day) while the opposite (2–19 fewer eggs/day) occurred during the last 5 days (all P <0.001, Table 2 View Table 2 ). Although there were more days where worms growing on CBFb37 had greater brood sizes than OP50, the magnitude of the differences was smaller during these 5 days, explaining the lower average total brood sizes for Microbacterium sp. CFB 37.

Lifespan extension by Microbacterium sp. CFBb37

Bacterial diet has been shown to affect the (somatic) lifespan of C. elegans in many cases (Zhang et al., 2017; Kumar et al., 2020). Thus, we tested whether Microbacterium sp. CFBb37 could also alter the C. elegans lifespan. We found that worms fed with Microbacterium sp. CFBb37 lived longer than those fed with OP50 (26.77 ± 0.86 days, mean ± 1 SE, versus 17.26 ± 0.57; P <0.001; Fig. 2 View Figure 2 and Table S4 View Table S ).

aFirst, an average brood number per individual was calculated per pool, then the average of the pools was reported.

bN, number of pools tested; n, total numbers of individuals tested.

cAverage brood size per worm in pools is the sum of all the daily brood sizes.

dRS is the number of days with average daily individual brood counts>0 based on one-sample t -test.

RS, reproductive span.

To determine at what general “time period” lifespan extension was occurring, we partitioned the total lifetime into the time spent in the larval, reproductive, and post-reproductive periods. For Microbacterium sp. CFBb37, C. elegans spent an average of 2.89 days as larvae, 7.29 days as reproductive adults, and 16.59 days as post-reproductive adults. On OP50, these durations were 2.05, 4.08, and 11.13 days, respectively. Thus, worms fed with CFBb37 spent longer periods of time in all three stages. This suggests that the total lifespan of the worms on CFBb37 was likely a combination of slower growth as larvae plus lifespan extension during reproductive and post-reproductive stages.

Extension of RS in closely related species

The extension of RS by Microbacterium sp. CBFb37 (compared with E. coli OP 50) in C. elegans could be a species-specific effect. Alternatively, Microbacterium sp. CBFb37 may also extend RS in other worm species. We tested this by examining RS using both the uterus egg assay and the presence of brood assay for two additional Caenorhabditis species ( C. briggsae CFB 233 and C. tropicalis BRC 20400) and another Rhabditidae family species, Protorhabditis sp. CFB 231. C. briggsae CFB 233 and Protorhabditis sp. CFB 231 were isolated from two different field samples that were each also the source of two bacterial strains ( Tables S2 View Table 2 and S 3 View Table S ). The sexual system of Protorhabditis sp. CFB 231 could be self-fertile hermaphrodites, because their spermathecae contain sperm ( Fig. S2 View Figure 2 ). However, other parthenogenetic Protorhabditis species also produce sperm ( Fradin et al., 2017; Grosmaire et al., 2019), so additional genetic analysis will be needed to determine the sexual system of this species.

For all three species and for both assays, we found that feeding on Microbacterium sp. BCFb37 extended the RS compared with OP50 (all P <0.001, log-rank test; Figures 1B–D and S1 View Figure 1 ; Tables 2 View Table 2 and S 5 View Table S ). For C. briggsae the RS extension was 1.64 additional days (+41%), for C. tropicalis 2.59 days (+109%), and for Protorhabditis sp. 3.09 days (+86%). Together, these results suggest that Microbacterium sp. CBFb37 can likely extend RS broadly across the Caenorhabditis genus and perhaps, at least, across the Rhabditidae family.

(Continued)

an, the total number of tested individuals in each test.

bAverage brood size per worm was the total brood of all hermaphrodites (n) divided by n.

cAverage RS. N.A., not applicable.

RS, reproductive span; SE, standard error.

Brood sizes of closely related species on Microbacterium sp. CFBb37

The extension of RS in C. elegans by Microbacterium sp. CBFb37 was associated with a decrease in brood size. We next examined whether the extension of RS in the three other species was also associated with a consistent decrease in brood size. We found that the brood sizes of both C. briggsae (16.00 ± 1.88 Microbacterium sp. CBFb37 versus 65.40 ± 7.09 E. coli OP 50; P <0.001; Table 2 View Table 2 ) and Protorhabditis sp. (22.08 ± 2.26 versus 161.38 ± 10.05; P <0.01; Table 2 View Table 2 ) on Microbacterium sp. CFBb37 was less than on E. coli OP 50. However, C. tropicalis brood size on Microbacterium sp. CFBb37 was more than on E. coli OP 50 (25.25 ± 3.59 versus 10.78 ± 1.90; P <0.01; Table 2 View Table 2 ). Together, these results suggest that the interaction between Microbacterium sp. CFBb37 and the nematodes is not simple.

C. elegans
	OP50		CFBb37		Different
Reproductive day number	Eggs laid per day	Greater than zero P	Eggs laid per day	Greater than zero P	between the strains P (paired
	(mean ± SE)	(one-sample t- test)	(mean ± SE)	(one-sample t- test)	t- test)
Day 1	32.68 ± 5.04	<0.001	3.76 ± 1.74	<0.001	<0.01
Day 2	149.75 ± 4.71	<0.001	24.64 ± 2.53	<0.001	<0.001
Day 3	78.55 ± 6.46	<0.001	23.49 ± 2.12	<0.001	<0.001
Day 4	3.25 ± 0.81	<0.001	22.05 ± 2.31	<0.001	<0.001
Day 5	1.40 ± 0.77	>0.05	15.10 ± 1.75	<0.001	<0.001
Day 6	0.58 ± 0.55	>0.05	7.93 ± 1.14	<0.001	<0.001
Day 7	0.05 ± 0.03	>0.05	4.39 ± 0.79	<0.001	<0.001
Day 8	0.03 ± 0.03	>0.05	1.88 ± 0.51	<0.001	<0.01
Day 9	0		0.56 ± 0.23	<0.05	N.A.
Day 10			0.07 ± 0.04	>0.05	N.A.
Day 11			0.03 ± 0.03	>0.05	N.A.
Day 12			0
n a	40		41
Average brood size per wormb	266.28 ± 6.42		137.63 ± 5.74		<0.001
RS (day) c	4.08 ± 0.16		7.29 ± 0.25		<0.001 (log-rank test)
C. briggsae
Day 1	11.59 ± 1.33	<0.001	2.11 ± 0.34	<0.001	<0.001
Day 2	18.21 ± 1.89	<0.001	3.80 ± 0.57	<0.001	<0.001
Day 3	22.56 ± 3.46	<0.001	3.17 ± 0.56	<0.001	<0.001
Day 4	12.00 ± 2.52	<0.001	2.03 ± 0.50	<0.001	<0.001
Day 5	0.88 ± 0.28	<0.01	2.30 ± 0.50	<0.001	0.0109
Day 6	0.16 ± 0.10	>0.05	1.69 ± 0.44	<0.001	<0.01
Day 7	0		0.50 ± 0.17	<0.001	N.A.
Day 8			0.22 ± 0.07	<0.01	N.A.
Day 9			0.08 ± 0.08	>0.05	N.A.
Day 10			0.08 ± 0.08	>0.05	N.A.
Day 11			0
na	32		36
Average brood size per wormb	65.40 ± 7.09		16.00 ± 1.88		<0.001
RS (day) c	3.97 ± 0.25		5.61 ± 0.39		<0.001 (log-rank test)
C. tropicalis
Day 1	4.37 ± 0.78	<0.001	4.75 ± 0.65	<0.001	1.0

Table 1. Average daily individual brood countsa for C. elegaNS on three environmental bacterial species and the OP50 control.

	Acinetobacter sp. CFBb9 (N = 4, n = 29) b		Serratia sp. CFBb17 (N = 4, n = 39) b		Microbacterium sp. CFBb37 (N = 3, n = 37) b		Escherichia coli OP 50 (N = 5, n = 33) b
Reproductive day number	Eggs laid per individual	Greater than zero P (one-sample t- test)	Eggs laid per individual	Greater than zero P (one-sample t- test)	Eggs laid per individual	Greater than zero P (one-sample t- test)	Eggs laid per individual	Greater than zero P (one-sample t- test)
Day 1	41.24	<0.001	69.30	<0.001	35.16	<0.001	151.69	<0.001
Day 2	162.34	<0.001	165.74	<0.001	47.00	<0.001	95.39	<0.001
Day 3	44.51	<0.001	39.23	<0.001	24.08	<0.001	2.96	<0.001
Day 4	2.07	<0.001	0.95	<0.001	13.59	<0.001	1.12	<0.001
Day 5	0		0		8.86	<0.001	0
Day 6	0		0		6.16	<0.001	0
Day 7					3.51	<0.001
Day 8					1.49	<0.001
Day 9					1.02	<0.001
Day 10					0.24	<0.001
Day 11					0.51	<0.001
Day 12					0
Day 13					0
Average brood size per wormc	250.16		275.22		141.62		251.16
RS (days) d	4		4		11		3.8