Description
Caenorhabditis elegans
naturally thrives in a soil environment where they feed on bacteria and are in constant association with a diverse range of microbes (Barker
et al.
1994).
C. elegans
egg laying is delayed or reduced when animals are infected with
Burkholderia pseudomallei
,
Burkholderia thailandensis
,
Staphylococcus aureus
, and
Serratia marcescens
(Mallo
et al.
2002; O’Quinn
et al.
2002; Irazoqui
et al.
2010). These changes in egg laying may be a protective response to pathogenic bacteria. Mutants of TGF-β-like DBL-1 signaling pathway also display reduced brood size (Luo
et al.
2009; Roberts
et al.
2010).  While the peak of egg-laying activity seen in normal animals between days two and four is depressed in
dbl-1
pathway mutants, the reproductive span of these
dbl-1
pathway mutants is increased to up to 13 days (Luo
et al.
2009). To determine if the egg-laying observed during infection is DBL-1 pathway-dependent, we tested the effect of the DBL-1 signaling pathway on egg laying when
C. elegans
were fed on representative Gram-negative (
S. marcescens
) and Gram-positive bacteria (
Staphylococcus epidermidis
).
Similar to previously published reports, we found that loss of DBL-1 pathway signaling decreases brood size and increases reproductive span in normal laboratory conditions (
E. coli
strain OP50 and 20°C incubation) (Figure 1A and B) (Luo
et al.
2009; Roberts
et al.
2010).
Here, we report three new results. First, brood size reductions caused by infection and by loss of DBL-1 signaling are independent (Figure 1A). Wild-type and
dbl-1(nk3)
animals both significantly decrease their brood size when grown on
S. marcescens
(
p
= 0.005 and
p
< 0.001, respectively).
dbl-1
mutant animals laid even fewer eggs than the wild-type animals on
S. marcescens
, suggesting that the reduced brood size phenotype is independently affected by both
S. marcescens
exposure and by loss of DBL-1 (
p
< 0.001).  While the decrease in brood size of wild-type animals on
S. epidermidis
was not significant (
p
= 0.115), the decreased brood size of
dbl-1(nk3)
animals was significant on this pathogenic bacterial strain (
p
= 0.045). Indeed, the decreases in brood size upon infection with either
S. marcescens
or
S. epidermidis
in both wild-type and
dbl-1(nk3)
populations are similar (
p
= 0.57), suggesting the pathogenic bacteria affect brood size independent of DBL-1. Because
dbl-1(nk3)
populations display a further reduced brood size upon infection by either pathogen compared to the wild type, the negative effects of pathogen exposure and loss of DBL-1 signaling on brood size appear to be additive (
p
< 0.05).
Second, while the wild-type population on
S. marcescens
survived until all animals ceased laying eggs, all
dbl-1(nk3)
animals died on
S. marcescens
by Day 5. These results explain why the extended reproductive span normally seen in
dbl-1(nk3)
populations was not observed on
S. marcescens
. These results also support previous reports of decreased viability of
dbl-1
mutant animals on another variety of
S. marcescens
, Db11 (Mallo
et al.
2002).
Third,
S. epidermidis
affects egg-laying patterns similar to loss of
dbl-1
function. Initially, both wild-type and
dbl-1(nk3)
strains on
S. epidermidis
have reduced eggs laid in the first four days compared to strains grown on the
E. coli
control. Loss of DBL-1 further reduced the number of eggs laid during each of these days, suggesting that this phenotype is independently affected by both
S. epidermidis
exposure and by loss of DBL-1 (
p
= 0.004).  Then, both wild-type and
dbl-1(nk3)
strains on
S. epidermidis
have similar extended reproductive spans, extending to at least day 13 (one tenacious wild-type hermaphrodite laid embryos until day 15). This
S. epidermidis
-induced reproductive span extension appears to be independent of DBL-1 signaling, because the numbers of eggs laid by both wild-type and
dbl-1(nk3)
populations between days 5 and 16 were similar at these time points (
p
= 0.509).
Reagents
Strains were maintained on EZ media plates at 20°C (0.55 g Tris-Cl, 0.24 g Tris base, 3.1 g BD Bacto
TM
Peptone, 8 mg cholesterol, 2 g sodium chloride, 20 g agar, in water to 1 L (E. Lambie, personal communication). The
C. elegans
strains used were N2 and NU3
dbl-1(nk3).
The Gram-negative bacterial strains used were
Escherichia coli
OP50 (CGC) and
Serratia marcescens
(Carolina Biological Supply Company). The Gram-positive bacterial strain used was
Staphylococcus epidermidis
(ATCC 49134).
S. marcescens
and
S. epidermidis
were provided by A. J. Hammett, TWU. All bacterial strains were grown for 9 hours in tryptic soy broth at 37°C before plating on EZ media plates.