Materials and Methods
(a) Nematode cultivation and phenotypes
Stock cultures of all nematode strains were kept on 6-cm plates with 1.7% nematode
growth medium (NGM) agar and fed with a lawn of Escherichia coli OP50 grown in
400 µl L-Broth. Because the mouth dimorphism is a polyphenism in P. pacificus, both
the Eu and St forms were available from a single isogenic culture, albeit with different
frequencies as described below. Forms were selected for experiments by scoring their
phenotypes as previously described [24]. Characters distinguishing the Eu form from
the St form were the presence vs. absence of a subventral tooth, a claw-like vs.
triangular dorsal tooth, and a wide vs. narrow mouth (Fig. 1). Phenotypes were
determined using a Zeiss Discovery V.12 stereomicroscope, supplemented where
necessary with differential interference contrast (DIC) microscopy on a Zeiss
Axioskop.
All P. pacificus individuals subjected to functional and fitness assays were
hermaphrodites. In experiments that introduced males as a source of sperm for
assayed individuals, males were always well-fed young adults from bacterial culture.
Assayed hermaphrodites were derived from multiple sibships, although inbred
cultures of P. pacificus, which were perpetuated every generation from small numbers
of hermaphrodites, ensured high homozygosity for each strain. Furthermore, the strain
was maintained as a well-fed culture on bacteria for at least five generations prior to
the experiment to control for any possible epigenetic effects. Therefore, assayed
individuals were isolated from identical environmental and highly homozygous
genetic backgrounds.
(b) Fecundity on a heterogeneous adult diet
As an alternative test for fecundity differences of the two forms on a prey diet, we
measured the fecundity of mothers when offered bacteria following such a diet.
Additionally, to test whether fecundity differences could be due to the availability of
self-sperm, we offered a subset of prey-fed hermaphrodites both bacteria and mates.
In the experiment, J4 hermaphrodites of P. pacificus RS5205 were fed for two days
on prey as described above. After this feeding regimen, mothers of both mouth-forms
were placed on individual NGM plates with (i) an excess (400 µl) of OP50, (ii) an
excess of OP50 and one adult male of the same strain, or (iii) neither OP50 nor males.
Nematodes were allowed to feed and, where possible, mate for three days, after which
all living offspring were counted.
(c) Differential fecundity on a bacterial diet
Finally, to test whether differences in adult feeding-structures could impact lifetime
fecundity on a strictly bacterial diet, we transferred pre-adult stage (J4)
hermaphrodites of P. pacificus strain RS5205 individually to NGM plates seeded with
an excess (400 µl) of OP50. The mouth phenotype of hermaphrodites was determined
during the assay, upon nematodes reaching maturity. Hermaphrodites were allowed to
lay eggs for two days and were transferred to new plates every two days until egglaying ceased. Hatched larvae across all plates were counted. Additionally, we tested
for differences of fecundity on a bacterium associated with P. pacificus in the wild,
Pseudomonas fluorescens strain “Pento3” [22]. This assay was performed as on
OP50, except prior to the assay nematode cultures were treated by bleaching to
remove OP50 from stock culture plates and reared from eggs for one generation on P.
fluorescens. The entire experiment was performed twice, each with sample sizes of n
= 14-15 for each form on each food source. After confirming no significant
differences between trials, results were pooled for a total sample size of n = 29-30 for
each category.
(d) Statistical analyses
For the predation assay, proportions of successful Eu and St worms within each strain
were compared using Pearson’s χ2 test as implemented in R. Differences in predation
measures (encounters per minute, attacks per encounter, and time required to kill)
between Eu and St worms were carried out via Kruskal-Wallis ANOVAs, as
Kolmogornov-Smirnov tests revealed departures from normality for most variables.
To test for differences between the two forms in brood size on bacterial diets we
performed a parametric test (Student’s t-test for independent samples), as distributions
did not deviate from normality. Kolmogornov-Smirnov, Kruskal-Wallis, and t-tests
were conducted using Statistica v.9 (Statsoft). Kaplan-Maier survival curves were
calculated and compared via log-rank tests, as implemented in Minitab 14.
Differences in brood size (over specific days, and in total) between Eu and St worms,
and between starved and prey-fed worms, were examined using Kruskal-Wallis
ANOVAs as described above.
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Figure S1. Predatory behaviour of Pristionchus pacificus JU482, as measured by
encounters of prey per minute and attacks per prey individual encountered. Box plots
show the median (centre line), the lower and upper quartiles (box bounds), and the range
limits (whiskers). (a) Encounters per minute. For both successful and non-successful acts
of predation, Eu individuals did not encounter prey more often than St individuals. (b)
Attacks per encounter. Neither mouth-form showed more attacks for a given number of
opportunities to make them.
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Figure S2. Fecundity (brood size) when fed ad libitum on bacteria. Food sources were
Escherichia coli OP50 and the naturally associated strain Pseudomonas fluorescens
“Pento3.” No differences were detected between eurystomatous (Eu) and stenostomatous
(St) individuals, indicating no fecundity advantage for either form in the presence of
abundant bacterial food.
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