Reproductive Span of Caenorhabditis elegans Is Extended by Microbacterium sp.

Tho Son Le; Thi Hong Gam Nguyen; Bich Hong Ha; Bui Thi Mai Huong; Thi Thu Hang Nguyen; Kim Dung Vu; Tu Cuong Ho; John Wang

doi:10.2478/jofnem-2022-0010

Figures & Tables

RS of individual nematodes on Microbacterium sp. CFBb37 and the E. coli OP50 control. (A) C. elegans, (B) C. briggsae, (C) C. tropicalis, and (D) Protorhabditis sp. on CFBb37 had longer RS than on OP50 (P < 0.001, log-rank test). Full RS data in Table 2. RS, reproductive span.

Lifespan assays of C. elegans on Microbacterium sp. CFBb37 and the OP50 control. C. elegans grown on CFBb37 survived longer than OP50 (P < 0.001, log-rank test). Detailed lifespan data are in Tables S4 and S5.

Summary of RS extension in the Caenorhabditis species (orange) and the outgroup Protorhabditis sp. (light blue). Red lines indicate inferred ancestral evolutionary lineages with extended RS. We suggest that other species within the genus Caenorhabditis are likely to have extended RS but this is untested. For simplification, some species are not listed and are grouped together in triangles, with the numbers of known species indicated. Phylogeny is adapted from Kiontke and Fitch (2005) and Felix et al. (2014) with the position of Protorhabditis sp. CFB231 placed next to P. sp. 1 species based on the 18S ribosomal RNA gene sequence (Zhang et al., 2000; Morgulis et al., 2008). Protorhabditis sp. CFB231 is self-fertile but the sexual system, hermaphroditic or parthenogenetic, is unknown (indicated by a question mark). RS, reproductive span.

Size comparison of the four tested nematode species grown on Microbacterium sp. CFBb37 and Escherichia coli OP50. Images are representative samples.

Protorhabditis sp. CFB231 make sperm. (A) DIC image of spermathecal region (outlined). One oocyte is apparently transiting the spermatheca. (B) DAPI staining for nuclei. (C) Merged images of DIC and DAPI. DIC, differential interference contrast.

Total average brood and RS of tested nematode species_

Reproductive day number	OP50		CFBb37		Different between the strains P (paired t-test)
Reproductive day number	Eggs laid per day (mean ± SE)	Greater than zero P (one-sample t-test)	Eggs laid per day (mean ± SE)	Greater than zero P (one-sample t-test)	Different between the strains P (paired t-test)
C. elegans
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 worm^b	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
n^a	32		36
Average brood size per worm^b	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
Day 2	4.81 ± 1.37	<0.001	4.79 ± 0.92	<0.001	0.8652
Day 3	1.22 ± 0.39	<0.01	7.20 ± 1.71	<0.001	<0.01
Day 4	0.37 ± 0.24	>0.05	3.88 ± 1.05	<0.01	<0.01
Day 5	0		3.29 ± 0.92	<0.01	N.A.
Day 6			0.95 ± 0.29	<0.01	N.A.
Day 7			0.33 ± 0.14	<0.05	N.A.
Day 8			0.04 ± 0.04	>0.05	N.A.
Day 9			0		N.A.
n^a	27		24
Average brood size per worm^b	10.78 ± 1.90		25.25 ± 3.59		<0.01
RS (day)^c	2.37 ± 0.20		4.96 ± 0.36		<0.001 (log-rank test)
Protorhabditis sp.
Day 1	45.00 ± 5.02	<0.001	2.70 ± 0.30	<0.001	<0.001
Day 2	95.15 ± 7.18	<0.001	4.21 ± 0.49	<0.001	<0.001
Day 3	18.06 ± 4.95	<0.001	3.23 ± 0.52	<0.001	<0.01
Day 4	2.00 ± 0.69	<0.01	3.12 ± 0.56	<0.001	>0.05
Day 5	0.88 ± 0.70	>0.05	2.38 ± 0.43	<0.001	N.A.
Day 6	0.24 ± 0.18	>0.05	1.36 ± 0.28	<0.001	N.A.
Day 7	0.06 ± 0.06	>0.05	1.09 ± 0.24	<0.001	N.A.
Day 8	0		1.00 ± 0.24	<0.001	N.A.
Day 9			1.17 ± 0.33	<0.01	N.A.
Day 10			0.53 ± 0.18	<0.01	N.A.
Day 11			0.53 ± 0.19	<0.01	N.A.
Day 12			0.42 ± 0.14	<0.01	N.A.
Day 13			0.14 ± 0.06	<0.05	N.A.
Day 14			0.10 ± 0.07	>0.05	N.A.
Day 15			0.06 ± 0.04	>0.05	N.A.
			0
n^a	34		47
Average brood size per worm^b	161.38 ± 10.05			22.08 ± 2.26	<0.01
RS (day)^c	3.59 ± 0.20			6.68 ± 0.61	<0.001 (log-rank test)

Bacterial isolates with description of colonial morphology, and geography of sample sites_

Cat Tien National Park				Cuc Phuong National Park
Strain	Morphology of colony^a	North	East	Strain	Morphology of colony^a	North	East
CFBb1	Small	11°23′57.5″	107°20′21.4″	CFBb65	Big	20°17′40.4″	105°39′46.6″
CFBb2	Big	11°23′54.4″	107°20′21.4″	CFBb66	Big	–	–
CFBb3	Small	–	–	CFBb67	Small	–	–
CFBb4	Irregular	11°24′22.8″	107°24′22″	CFBb68	Small	–	–
CFBb5	Irregular	–	–	CFBb69	Big	20°17′40″	105°39′57.5″
CFBb6	Big	–	–	CFBb70	Small	–	–
CFBb7	Big	–	–	CFBb71	Small	20°17′41.3″	105°39′59.6″
CFBb8	Small	–	–	CFBb72	Small	–	–
CFBb9	Big	11°24′20.9″	107°24′22.3″	CFBb73	Big	–	–
CFBb10	Irregular	11°24′21.4″	107°24′25.6″	CFBb74	Big	–	–
CFBb11	Big	11°24′20.9″	107°24′22.3″	CFBb75	Small	–	–
CFBb12	Small	11°24′20.9″	107°24′22.1″	CFBb76	Irregular	20°20′56.7″	105°35′48.1″
CFBb13	Big	11°24′19.6″	107°24′22.1″	CFBb77	Big	–	–
CFBb14	Small	–	–	CFBb78	Small	20°15′51.2″	105°41′57.6″
CFBb15	Big	11°24′19″	107°24′22.2″	CFBb79	Big	20°20′56.6″	105°35′47.4″
CFBb16	Big	11°24′18.6″	107°24′22.3″	CFBb80	Small	–	–
CFBb17	Irregular	11°24′21.4″	107°24′21.4″	CFBb81	Irregular	–	–
CFBb18	Big	–	–	CFBb82	Big	20°15′7.6″	105°42′45″
CFBb19	Big	11°28′46.4″	107°22′52.9″	CFBb83	Small	–	–
CFBb20	Small	–	–	CFBb84	Big	20°14′53.5″	105°42′34.3″
CFBb21	Medium	11°23′18.7″	107°21′3.4″	CFBb85	Small	–	–
CFBb22	Medium	–	–	CFBb86	Small	–	–
CFBb23	Big	11°29′7.2″	107°22′58″	CFBb87	Medium	20°14′59.8″	105°42′28.1″
CFBb24	Small	–	–	CFBb88	Medium	–	–
CFBb25	Medium	11°27′56.1″	107°22′43.5″	CFBb89	Big	–	–
CFBb26	Big, yellow	11°27′0.9″	107°21′27.1″	CFBb90	Small	–	–
CFBb27	Small	–	–	CFBb91	Big	20°14′56.7″	105°42′21.1″
CFBb28	Big	11°27′1.5″	107°21′27.5″	CFBb92	Big	–	–
CFBb29	Small	–	–	CFBb93	Big	20°14′39.1″	105°42′25.3″
CFBb30	Big, red	11°26′56.6″	107°21′35.9″	CFBb94	Small	–	–
CFBb31	Big	–	–	CFBb95	Big	20°20′56.7″	105°35′46.5″
CFBb32	Big	11°23′18.8″	107°21′3.6″	CFBb96	Small	–	–
CFBb33	Small	–	–	CFBb97	Medium	20°20′58.4″	105°35′36.5″
CFBb34	Big	11°27′31.9″	107°20′43.1″	CFBb98	Medium	–	–
CFBb35	Big	11°27′5″	107°27′25.6″	CFBb99	Big	–	–
CFBb36	Irregular	11°27′3″	107°21′51.7″	CFBb100	Big	–	–
CFBb37	Small	11°27′5″	107°27′25.6″	CFBb101	Small	–	–
CFBb38	Small	–	–	CFBb102	Big	20°21′0.5″	105°35′36.3″
CFBb39	Big	11°27′17.9″	107°22′5.6″	CFBb103	Big	–	–
CFBb40	Big	11°27′17.9″	107°22′5.6″	CFBb104	Small	–	–
CFBb41	Small	11°27′17.9″	107°22′5.6″	CFBb105	Small	–	–
CFBb42	Big	11°26′47.8″	107°26′19.8″	CFBb106	Big	–	–
CFBb43	Small	11°26′47.8″	107°26″19.8″	CFBb107	Small	–	–
CFBb44	Big	11°27′50.4″	107°27′43.5″	CFBb108	Big	20°20′54.1″	105°35′48.5″
CFBb45	Irregular	11°27′51.8″	107°27′42.1″	CFBb109	Small	–	–
CFBb46	Big	–	–
CFBb47	Small	–	–
CFBb48	Big	11°23′19.3″	107°21′3.7″
CFBb49	Small	–	–
CFBb50	Big, pink	11°26′14.5″	107°25′24.2″
CFBb51	Small	–	–
CFBb52	Big	11°26′12.1″	107°25′25.7″
CFBb53	Small	11°26′12.1″	107°25′25.7″
CFBb54	Small	11°26′12.1″	107°25′25.7″
CFBb55	Big	11°25′23″	107°25′41.4″
CFBb56	Small	–	–
CFBb57	Medium	11°23′26″	107°21′1.8″
CFBb58	Big	11°24′49″	107°25′27.7″
CFBb59	Big	–	–
CFBb60	Small	–	–
CFBb61	Small	–	–
CFBb62	Big	–	–
CFBb63	Medium	11°24′48.2″	107°25′25.9″
CFBb64	Small	11°23′39.7″	107°21′47.9″

Counts of surviving and lifespan of C_ elegans on Microbacterium sp_ CFBb37 and the OP50 control_

Day of lifespan	Number of surviving worms
Day of lifespan	OP50	CFBb37
1	73	60
2	73	60
3	73	60
4	73	60
5	73	60
6	73	60
7	73	60
8	73	60
9	72	59
10	72	59
11	69	59
12	66	59
13	63	58
14	46	54
15	39	53
16	36	52
17	25	52
18	18	49
19	16	48
20	14	48
21	13	47
22	12	47
23	11	45
24	7	43
25	6	43
26	6	42
27	4	36
28	3	30
29	3	25
30	1	24
31	1	16
32	0	11
33		6
34		1
35		0
Censored	7	0
Lifespan (days) (mean ± 1SE)	17.26 ± 0.57	26.77 ± 0.86
P (log-rank test)		<0.001

Average daily individual brood countsa for C_ eleganson three environmental bacterial species and the OP50 control_

Reproductive day number	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 OP50 (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	250.16		275.22		141.62		251.16
size per worm^c
RS (days)^d	4		4		11		3.8

Wild-type isolates of nematodes in Cat Tien National Park_

Species name	Strain number	GenBank accession No.	Best BLAST hit^a	Query coverage	Identity (%)	Bit score (max/total)	E value
Caenorhabditis briggsae	CFB233	MZ457559	Caenorhabditis briggsae AF16	98%	100%	1,544/1,544	0.0
Protorhabditis sp.	CFB231	MZ474671	Protorhabditis sp. 1 GVDU-2019	90%	95.35	1,229/1,229	0.0

Bacterial strains isolated from rotting vegetation samples collected in Cat Tien National Park and their effects on nematodes_

Strain^a	Species	Isolation site	GenBank accession No.	Phenotype
CFBb3	Pseudomonas sp.	11°23′54.4″N107°20′21.4″E	OM456346	Late laying egg
CFBb9^b	Acinetobacter sp.	11°24′20.9″N107°24′22.3″E	MZ467312	Normal
CFBb11	Acinetobacter sp.	11°24′20.9″N107°24′22.3″E	OM456348	Small body
CFBb17	Serratia sp.	11°24′21.4″N107°24′21.4″E	MZ457333	Small body
CFBb35	Serratia sp.	11°27′5″N107°27′25.6″E	OM629173	Normal
CFBb37	Microbacterium sp	11°27′5″N107°27′25.6″E	MZ453404	RS; small body
CFBb39	Sphingobacterium sp.	11°27′17.9″N107°22′5.6″E	OM892000	Normal
CFBb40	Bacillus sp.	11°27′17.9″N107°22′5.6″E	OM456347	Normal
CFBb42	Serratia sp.	11°26′47.8″N107°26′19.8″E	OM456350	Small body
CFBb53	Bacillus cereus	11°26′12.1″N107°25′25.7″E	OM883917	Small body
CFBb54	Enterobacter sp.	11°26′12.1″N107°25′25.7″E	OM456349	Small body
CFBb57	Serratia sp.	11°23′26″N107°21′1.8″E	OM892003	Small body

Counts of daily uterus eggs of close nematode species_

Nematode	Reproductive day number	OP50			CFBb37			Different between the strains P (paired t-test)
Nematode	Reproductive day number	Number of tested hermaphrodites	Number of eggs in uterus/individual (mean ± SE)	Greater than zero P (one-sample t-test)	Number of tested hermaphrodites	Number of eggs in uterus/individual (mean ± SE)	Greater than zero P (one-sample t-test)	Different between the strains P (paired t-test)
C. elegans	Day 1	32	3.94 ± 0.68	<0.001	35	1.57 ± 0.34	<0.001	0.0051
	Day 2	32	25.25 ± 1.12	<0.001	31	3.13 ± 0.32	<0.001	<0.001
	Day 3	29	5.38 ± 0.98	<0.001	42	4.02 ± 0.33	<0.001	0.06615
	Day 4	30	1.13 ± 0.34	<0.01	32	3.69 ± 0.33	<0.001	<0.001
	Day 5	37	0.03 ± 0.03	>0.05	34	3.5 ± 0.46	<0.001	<0.001
	Day 6				37	3.22 ± 0.51	<0.001
	Day 7				34	2.29 ± 0.36	<0.001
	Day 8				35	0.43 ± 0.16	<0.01
C. briggsae	Day 1	39	3.30 ± 0.73	<0.001	39	0.51 ± 0.09	<0.001	<0.001
	Day 2
	Day 3	34	4.09 ± 0.39	<0.001	35	0.6 ± 0.15	<0.001	<0.001
	Day 4	39	2.38 ± 0.24	<0.001	32	0.81 ± 0.14	<0.001	<0.001
	Day 5	35	0.2 ± 0.07	<0.01	35	1.46 ± 0.15	<0.001	<0.001
	Day 6	34	0.09 ± 0.05	>0.05	31	1.45 ± 0.19	<0.001	<0.001
	Day 7				32	0.63 ± 0.15	<0.001
C. tropicalis	Day 1	33	2.09 ± 0.22	<0.001	32	0.28 ± 0.09	<0.001	<0.001
	Day 2	32	3.47 ± 0.23	<0.001	37	2.76 ± 0.32	<0.001	0.2149
	Day 3	38	1.87 ± 0.23	<0.001	36	3.19 ± 0.4	<0.001	0.0044
	Day 4	28	0.64 ± 0.23	<0.05	38	2.6 ± 0.24	<0.001	<0.001
	Day 5	36	0.19 ± 0.13	>0.05	34	2.94 ± 0.36	<0.001	<0.001
	Day 6				36	1.53 ± 0.2	<0.001
	Day 7				27	1.63 ± 0.22	<0.001
	Day 8				17	0.82 ± 0.16	<0.01
P. sp.	Day 1	34	3.23 ± 0.55	<0.001	32	3.28 ± 0.26	<0.001	0.595
	Day 2	36	3.67 ± 0.63	<0.001	38	1.61 ± 0.18	<0.001	<0.001
	Day 3	39	1.38 ± 0.24	<0.001	36	1.58 ± 0.12	<0.001	0.629
	Day 4	36	0.19 ± 0.03	<0.05	36	1.56 ± 0.12	<0.001	<0.001
	Day 5	34	0.15 ± 0.03	>0.05	40	1.80 ± 0.16	<0.001	<0.001
	Day 6				35	1.06 ± 0.13	<0.001	<0.001
	Day 7				53	0.81 ± 0.10	<0.001	<0.001

Reproductive Span of Caenorhabditis elegans Is Extended by Microbacterium sp.

Figures & Tables

Figure 1

Figure 2

Figure 3

Figure S1

Figure S2

Total average brood and RS of tested nematode species_

Bacterial isolates with description of colonial morphology, and geography of sample sites_

Counts of surviving and lifespan of C_ elegans on Microbacterium sp_ CFBb37 and the OP50 control_

Average daily individual brood countsa for C_ eleganson three environmental bacterial species and the OP50 control_

Wild-type isolates of nematodes in Cat Tien National Park_

Bacterial strains isolated from rotting vegetation samples collected in Cat Tien National Park and their effects on nematodes_

Counts of daily uterus eggs of close nematode species_

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