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Mosser et al. page - 1 -

BACTERIA-INDUCED INTERNAL EGG HATCHING FREQUENCY IS PREDICTIVE OF LIFESPAN IN 1 CAENORHABDITIS ELEGANS POPULATIONS 2 3 Thomas MOSSER, Ivan MATIC, Magali LEROY* 4 Laboratory of Evolutive, Medical and Molecular Genetics, Inserm, U1001, Université Paris 5 Descartes, Sorbonne Paris Cité, Faculté De Médecine Paris Descartes, Paris, France. 6 7 * To whom correspondence should be addressed: Inserm U1001, Université Paris Descartes, 8 Sorbonne Paris Cité, Faculté De Médecine Paris Descartes, 156 rue de Vaugirard, F-75730 Paris 9 cedex 15, France. Tel. +33 140 615 321, Fax. +33 140 615 322, Email: [email protected] 10 11 RUNNING TITLE: 12 BAGGING IS PREDICITIVE OF LIFESPAN IN C. ELEGANS 13 14 ABSTRACT 15 Internal egg hatching in Caenorhabditis elegans is induced by exposure to bacteria. This study 16 demonstrates that wormbags frequency determination allows for an advanced insight into the 17 degree of bacterial pathogenicity, and is highly predictive of worm populations survival. 18 Therefore, wormbagging frequency can be regarded as a reliable population-wide stress reporter. 19 20 KEYS WORDS: C. elegans, Host-bacteria interaction, Internal hatching, Wormbag, Lifespan 21 22

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.06357-11 AEM Accepts, published online ahead of print on 16 September 2011

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Caenorhabditis elegans worms normally lay eggs that hatch outside of the parental body. 23 However, eggs can also be retained and hatch inside the parental body called a “bag of worms” or 24 wormbag, the process being called wormbagging (23, 27). Internal hatching is modulated by 25 genes (29), and is not restricted to the widely used laboratory strain N2 (20). Internal hatching is 26 rare when worms are maintained under standard laboratory conditions. However, transfer from 27 solid to liquid medium (24), axenic (25) and adverse environmental conditions such as starvation 28 (9), exposure to toxic compounds (8, 28), and bacteria (1, 21) can increase the frequency of 29 wormbags. It was proposed that wormbagging could be an adaptive response as, under starvation 30 conditions, the parental body can provide enough food for larvae to reach the resistant dauer 31 stage (10, 16). Retention of progeny could also provide physical protection and transport for 32 small larvae, which can be released in more favorable environments. 33

C. elegans is a widely used model organism for host-pathogen interaction studies (2, 6, 34 13, 17, 26). The impact of the bacteria on worms is evaluated through analysis of life history 35 traits such as survival. In survival assays, wormbags are generally disregarded or internal 36 hatching prevented by use of genetically sterile mutants or ablation of germline cells, despite the 37 established role of the germline in both lifespan and immune response (3), or by chemical 38 sterilization, e.g. 5-fluorodeoxyuridine treatment (11), that can also affects bacteria and 39 significantly modify host-pathogen interactions. 40

This study investigates: (i) how bacteria modulate wormbags frequency, (ii) the 41 correlation of wormbags frequency with lifespan at the population level, and (iii) the impact of 42 internal hatching on the nematode reproductive fitness. C. elegans N2 (ancestral) strain was 43 exposed to gram-negative Escherichia coli commensal and pathogenic strains: control strain 44 OP50 (7, 19), ED1A (18), IAI13 (22), MG1655 (5), IAI1(22), F11 (18), and 536 (18). The 45 virulence of these strains was previously established using C. elegans sterile fer-15 strain (14). 46


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The gram-positive Enterococcus faecalis strain OG1RF was also used (15). All assays were 47 performed at 25°C on monoxenic-seeded nematode growth medium agar plates with age-48 synchronized L4 worms developed on OP50 (4). The proportion of wormbags was determined 49 over the 4-day reproductive period (Fig 1A). Wormbags frequency varied from 8% on OP50 to 50 59% on the pathogenic 536 strain (Fig. 1B). 51

Wormbags frequency can be modulated by food availability (9). However, this does not 52 seem to be the case in this study. The number of live cells in bacterial lawns is significantly 53 different (unpaired t-test, p<0.0001) between OP50 (mean ± SD, 3.83x109 ± 8.46x108 CFU/lawn) 54 and ED1A (1.15x1010 ± 9.56x108 CFU/lawn) strains, while they induce similar bagging 55 frequencies (Fig. 1B). On the contrary, ED1A and 536 (1.45x1010 ± 5.12x108 CFU/lawn) strains 56 generate similar amount of live cells but induce different bagging frequencies (Fig. 1B). Thus, the 57 nature of the bacterial strains, not the quantity of cells, modulates wormbags frequency. 58

Next, the correlation between bagging frequency and lifespan was established. Lifespan 59 was defined as the time from the end of L4 stage until death of the adult worm (4). When 60 wormbags were included in the analysis the bagging frequency correlates significantly with the 61 mean lifespan (Fig. 2A), as well as median and 25% survival (Fig. S1A and B). This could be 62 expected because wormbags have a reduced average survival and their proportion in the 63 population can be very high and increases with bacterial pathogenicity (Fig. 1B). However, the 64 correlations remain significant between wormbags frequency and survival even when wormbags 65 were censured from the survival analyses (Fig. 2B, S1C and D). Attenuated lipopolysaccharide-66 truncated E. coli mutants were constructed by gene replacement through homologous 67 recombination using pKD3 template plasmid (12), and 68 gTgAATTTACTgACAgTgAgTACTgATCTCATCAgTATTTTgTgTAggCTggAgCTgCTTC 69 and 70


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CATAgAggAAgAATgCTAgCAAAAAgAgCACCAgCATgACCATATgAATATCCTCCTTAg 71 primers for the rfe gene. The correlation between wormbags frequency and lifespan is also 72 significant in worm populations exposed to attenuated strains independently of whether 73 wormbags are included or censured (Fig. 2C and D). Therefore, wormbags frequency is highly 74 predictive of the survival of worm populations exposed to different bacterial strains. In addition, 75 wormbags frequency provides an advanced insight into the population lifespan because 90% of 76 wormbags appear by the third day (Fig 1A), while complete survival studies can last over two 77 weeks. 78

Internal hatching can result in early death of individuals, reducing population survival 79 significantly when wormbags frequency reaches 20% (Fig. 2A-D, S1A-D). However, it is not 80 necessarily lethal to worms. Some wormbags expulse internally hatched larvae and live as long as 81 some non-wormbags. Because pathogenic bacteria induce high frequency of wormbags, to 82 disregard or prevent wormbagging when investigating host-pathogen interaction can lead to 83 important underestimations of the bacterial pathogenicity. Exclusion of wormbags from survival 84 assays resulted in the overestimations of worm populations survival by up to 27%. 85

Internal hatching decreases survival, but it also has a potential to reduce reproductive 86 output, i.e., fitness, because retained larvae could damage gonads. Offspring production and 87 survival were quantified on E. coli 536 and E. faecalis OG1RF, the two strains inducing 88 wormbags frequencies greater than 50%. Individual nematodes were maintained in 12-well plates 89 and transferred every 24h into new plate wells. Plates were further incubated 24h to allow for 90 eggs hatching. All internally hatched larvae were released or freed themselves by the end of the 91 reproductive period. Reproductive output was measured as the total number of larvae produced 92 per individual worm (4). Wormbags had as much or more progeny than non-wormbags (Fig. 3A). 93 The impact of internal hatching vs. external hatching on the offsprings survival was also 94


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evaluated. Internally hatched worms survive as well or better than externally hatched worms (Fig. 95 3B). Thus, internal hatching does not negatively impact brood size nor offsprings survival. 96

Because wormbagging is induced by exposure to pathogens and because its frequency 97 correlates with the survival of non-bagging worms, it cannot be neglected as an irrelevant by-98 product and can be regarded as a population-wide stress reporter. This study shows that 99 wormbags frequency can be used as proxy for reliable survival determination and allows for an 100 advanced insight in the degree of bacterial pathogenicity. 101 102 103


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We acknowledge Dr. J.J. Ewbank (Centre d'Immunologie de Marseille-Luminy, 104 INSERM/CNRS/Université de la Méditerranée, Marseille, France) for providing C. elegans 105 strain N2, Dr. U. Dobrindt (Institut für Molekulare Infektionsbiologie, Bayerische Julius-106 Maximilians-Universität Würzburg, Würzburg, Germany) for providing E. coli bacterial mutant 107 strain 536∆waaG, and Dr. P. Courvalin (Unité des Agents Antibactériens, Institut Pasteur, Paris, 108 France) for providing bacterial strain E. faecalis OG1RF. 109 This work was supported in part by a fellowship from the Nestlé Fondation to M.L. and an ANR 110 grant ANR-06-BLAN-0406/AGING to I.M. 111 112 LITERATURE CITED 113 114 1. Aballay, A., P. Yorgey, and F. M. Ausubel. 2000. Salmonella typhimurium proliferates 115

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FIGURE LEGENDS 194 195 FIG. 1. Timeline and frequency of bacteria-induced internal egg hatching. 196 (A) Cumulative percentage of wormbags appearance over the course of C. elegans reproductive 197 period. Data are compiled, all bacterial strains included, from all experiments of wormbags 198 frequency determination (N=55). Graphed: mean ± SD. (B) Frequency of wormbags in C. 199 elegans populations exposed to E. coli OP50 (N=381), ED1A (N=291), IAI13 (N=245), MG1655 200 (N=582), IAI1 (N=378), F11 (N=235), 536 (N=905), and E. faecalis OG1RF (N=313) strains. 201 Data are compiled from at least three independent experiments. Bagging frequencies were 202 analyzed using one-way ANOVA followed by Dunnett’s Multiple Comparison Test to OP50. 203 Graphed: mean ± SEM, ns: not significant, *: p<0.05, ***: p<0.0001. 204 205 FIG. 2. Correlation of the wormbags frequency with the mean lifespan. 206 (A-D) Correlation between bagging frequency and lifespan in C. elegans. Worm populations 207 (N=+WB/-WB) were exposed to E. coli OP50 (N=381/ 344), ED1A (N=291/ 261), IAI13 208 (N=245/193), MG1655 (N=582/402), IAI1 (N=378/211), F11 (N=235/120), 536 (N=905/368), 209 and E. faecalis OG1RF (N=313/144) strains (A and B), and E. coli attenuated 210 lipopolysaccharides-truncated 536∆rfe (N=210/167), 536∆waaG (N=214/198), and IAI1∆rfe 211 (N=153/133) mutants (C and D). Correlations between the wormbags frequency and the mean 212 lifespan were calculated using linear regression to mean survival when wormbags are included (A 213 and C) and when wormbags are censured (B and D) from the survival assays. Data are compiled 214 from at least three independent experiments. Graphed: mean ± SEM. 215 216 FIG. 3. Impact of internal hatching on C. elegans fitness. 217


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(A) Average brood size per individual non-wormbags (NWB, open symbols) and wormbags 218 (WB, full symbols) exposed to E. coli 536 (circle symbols, NWB N=127 and WB N=171) and E. 219 faecalis OG1RF (square symbols, NWB N=61 and WB N=54) strains. Average brood size are as 220 expected at 25°C. Data are compiled from at least two independent experiments. Unpaired t-test, 221 ns: not significant, *:p<0.05. Graphed: mean ± SD. (B) Survival of the internally hatched 222 (Larvae, full symbols) and externally hatched (Eggs, open symbols) offsprings on E. coli 536 223 (dashed lines, Eggs N=328 and Larvae N=189) and E. faecalis OG1RF (plain lines, Eggs N=127 224 and Larvae N=122) strains. Data were compiled from at least two independent experiments. 225 Survival assays were analyzed using the Gehan-Breslow-Wilcoxon test comparing conditions by 226 pairs and allowing for unequal hazard ratios. Graphed: mean ± SEM, ns: not significant, 227 *:p<0.05. 228 229


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