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NATURE|Vol 442|24 August 2006
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Kin preference in a social microbe
Given the right circumstances, even an amoeba chooses to be altruistic towards its relatives.
a
1
Proportion of fluorescent spores
Kin recognition helps cooperation to evolve
in many animals1, but it is uncertain whether
microorganisms can also use it to focus altruistic behaviour on relatives. Here we show that
the social amoeba Dictyostelium purpureum
prefers to form groups with its own kin in
situations where some individuals die to
assist others. By directing altruism towards
kin, D. purpureum should generally avoid
the costs of chimaerism2,3 experienced by the
related D. discoideum.
Dictyostelium normally live as asexually
reproducing, unicellular amoebae in forest
soils. But when starved of their bacterial food
source, they aggregate in thousands to form
a multicellular, motile ‘slug’. This eventually
becomes a fruiting body4,5, in which some amoebae in the group differentiate to form spores and
other amoebae die to form a stalk structure that
assists the dispersal of these spores. Stalk cells
are therefore sacrificed to aid the others.
Because multicellularity in social amoebae is
accomplished by aggregation of cells, fruiting
bodies could consist of one or more clones. In
the model organism Dictyostelium discoideum,
genetically distinct clones can mix to form chimaeras, and one clone may sometimes exploit
another by contributing less than its proportional share to the sterile stalk2. However, tests
with several clones of other species suggest that
mixing may not be the rule6–8.
We therefore tested for kin discrimination in
D. purpureum. We performed 14 pairwise mixing experiments between isolates collected at
different locations in the Houston Arboretum,
Texas. One isolate of each pair was labelled
with a fluorescent dye. Cells of the two isolates
were plated in equal proportions at a density
of about 2ǂ106 cells per cm2 and allowed to
complete social development in the absence of
bacteria. We then assessed the proportions of
the two isolates in some 500 spores for each of
10 fruiting bodies.
Randomization tests revealed that 12 of
14 pairwise experiments showed evidence of
strong kin discrimination: although different
isolates aggregated together, individual fruiting
bodies consisted of predominantly one isolate
or the other, with significantly higher variances
in the proportion of fluorescent spores than
were found in randomly mixed clonal controls
(Fig. 1a, and see supplementary information).
Although two pairs of isolates did not discriminate in favour of kin, perhaps because of shared
identity at one or more recognition alleles, kin
b 13, 18
d 18, 18
8 hrs
8 hrs
c 13, 18
10 hrs
e 18, 18
10 hrs
0.8
0.6
0.4
0.2
0
13, 18 18, 13
13, 13 18, 18
Figure 1 | Kin discrimination during social development in the amoeba Dictyostelium purpureum.
a, Scatter plot showing the proportion of fluorescently labelled spores in individual fruiting bodies
formed at high amoeba density for isolates 13 and 18 (see text; bold, fluorescently labelled isolate).
A greater variance in experimental (red circles) than in control treatments (blue circles) indicates
clonal sorting (P<0.0002). b–e, Fluorescent micrographs taken with the same field of view at different
times of development at high density. b, Initial aggregates contain two isolates (13, labelled green; 18,
unlabelled); c, these isolates then separate individually as ‘slugs’ that emerge from the aggregates.
d,e, In controls of pure isolate 18, where half of the cells are fluorescently labelled, labelled and
unlabelled cells mix equally at aggregation (d), and at the slug stage (e). Arrows designate individual
aggregates (b, d) and emerging slugs (c, e). Scale bar, 1 mm.
discrimination was significant overall (Wilcoxon signed-rank test (WSRT), Zǃǁ3.111,
nǃ14, Pǃ0.0019). The calculated relatedness
in fruiting bodies from experimental mixes
rose to 0.81, compared with the value of 0.50
expected for complete mixing.
We monitored the timing of kin recognition
by using fluorescence microscopy (Fig. 1b–e).
Different clones mixed during early aggregation (Fig. 1b) but separated when they emerged
as slugs (Fig. 1c); however, single-clone controls remained mixed under the same conditions (Fig. 1d, e). These results indicate that
kin discrimination is not due to differences in
developmental timing between clones.
Strict exclusion of non-kin carries the risk of
suboptimal group sizes when kin are rare9. To
determine whether kin-discriminating clones
of D. purpureum would mix with non-kin if kin
were less abundant, we did 11 experiments at
an amoeba density low enough to make fruiting bodies scarce (about 2ǂ105 cells per cm2).
There was less sorting in these low-density
experiments than in the high-density ones
(WSRT, Zǃǁ2.667, nǃ11, Pǃ0.0076; see
supplementary information). This effect was
lost when high- and low-density experiments
were done simultaneously in six pairwise mixtures (WSRT, Zǃǁ1.572, nǃ6, Pǃ0.1159);
©2006 Nature Publishing Group
however, the more important result is unambiguous. Dictyostelium purpureum preferentially
associates with kin, and this remains true even
at low density when partners are hard to find.
This kin discrimination means D. purpureum
should avoid the disadvantages of forming
chimaeras, and indeed only one clone was
consistently cheated (see supplementary information). Our findings support the application
of kin-selection theory to microorganisms and
provide further evidence that social microbes
can show sophisticated behaviour10 previously
thought to occur only in higher organisms.
Natasha J. Mehdiabadi*, Chandra N. Jack*,
Tiffany Talley Farnham*, Thomas G. Platt*†,
Sara E. Kalla*, Gad Shaulsky‡,
David C. Queller*, Joan E. Strassmann*
*Department of Ecology and Evolutionary Biology,
Rice University, Houston, Texas 77005, USA
e-mail: [email protected]
‡Department of Molecular and Human
Genetics, Baylor College of Medicine, Houston,
Texas 77030, USA
†Present address: Department of Biology, Indiana
University, Bloomington, Indiana 47405, USA
1. Fletcher, D. J. C. & Michener, C. D. Kin Recognition in
Animals (Wiley, Chichester, 1987).
2. Strassmann, J. E., Zhu, Y. & Queller, D. C. Nature 408,
965–967 (2000).
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3. Foster, K. R., Fortunato, A., Strassmann, J. E. & Queller, D. C.
Proc. R. Soc. Lond. B 269, 2357–2362 (2004).
4. Kessin, R. H. Dictyostelium — Evolution, Cell Biology, and the
Development of Multicellularity (Cambridge Univ. Press,
Cambridge, 2001).
5. Bonner, J. T. The Cellular Slime Molds 3rd edn (Princeton
Univ. Press, Princeton, 1967).
6. Buss, L. W. Proc. Natl Acad. Sci. USA 79, 5337–5341 (1982).
7. Bonner, J. T. & Adams, M. S. J. Embryol. Exp. Morphol. 6,
346–356 (1958).
NATURE|Vol 442|24 August 2006
8. Kaushik, S., Katoch, B. & Nanjundiah, V. Behav. Ecol.
Sociobiol. 59, 521–530 (2006).
9. Reeve, H. K. Am. Nat. 133, 407–435 (1989).
10. Crespi, B. J. Trends Ecol. Evol. 16, 178–183 (2001).
Supplementary information accompanies this
communication on Nature’s website.
Received 16 January; accepted 29 June 2006.
Competing financial interests: declared none.
doi:10.1038/442881a
GENE EXPRESSION
Long-term gene silencing by RNAi
We have therefore investigated the heritability
of gene silencing by RNAi over many generations in C. elegans and used an RNAi screen to
identify genes that may influence this inheritance. (For details of methods, see supplementary information.)
We injected wild-type Bristol N2 worms
with a double-stranded RNA that targets the
C. elegans gene ceh-13 for one generation.
The Ceh-13 phenotype, in which the worm
is small and dumpy, persisted in some animals indefinitely. Inheritance was not fully
penetrant: only about 30% of the progeny of
Ceh-13 worms inherited the phenotype. Wildtype siblings never had progeny with the Ceh13 phenotype, and crossing worms that had
a Ceh-13 phenotype with unaffected males
showed that the trait is dominant. A single episode of RNAi can therefore induce heritable
silencing that is not fully
a
penetrant and behaves in
a dominant fashion.
To show that this is a
general phenomenon, we
targeted 171 other genes
by using a single treatment of RNAi and found
13 that could be inheritably silenced (among them
were dpy-6, dpy-28 and
++
–
+/–
+
unc-73; data not shown).
+
++
+/–
–
100
b
We also showed that
a single transgenic copy
80
of a gene (gfp) express60
ing green fluorescent
protein (GFP) could be
40
silenced, and the silenc20
ing inherited. We used
animals expressing GFP
0
F1 F2 F5 F4 F5 F10 F12 F15 F20
under the control of a
Generation after feeding
germline-specific proFigure 1 | RNA interference triggers inheritable silencing of a transgene
moter and created interencoding green fluorescent protein (GFP). a, Four degrees of GFP
ference by feeding them
expression in Caenorhabditis elegans are revealed using Nomarski optics bacteria that express
(top panels) and ultraviolet illumination (bottom panels): ++, very
double-stranded RNA
bright, only observed in untreated transgenic NL3630 worms; ǁ, +/ǁ
homologous to gfp; progand +, weaker GFP signals from progeny of an RNAi-treated worm that
eny that did not express
did not express GFP. b, Worms were fed on bacteria expressing doubleGFP were then transstranded RNA targeting the transgene gfp. Ten independent lines were
ferred to new plates. In
quantified for 20 generations. Shown is the mean percentage of worms
all siblings, GFP expreswith the indicated amount of GFP expression in the progeny of a worm
that did not express GFP.
sion was reduced relative
Unfed
strain
Worms with indicated level
of GFP expression (%)
Small RNA molecules participate in a variety of
activities in the cell: in a process known as RNA
interference (RNAi), double-stranded RNA
triggers the degradation of messenger RNA that
has a matching sequence; the small RNA intermediates of this process can also modify gene
expression in the nucleus1. Here we show that a
single episode of RNAi in the nematode
Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger. Our
findings may prove useful in the ongoing development of RNAi to treat disease.
It has been shown that phenotypes induced
in C. elegans by RNAi can last for two or three
generations2. Because the generation time of
a worm is only three days, however, it is not
clear whether this effect can be explained simply by a slow dilution of the silencing factors.
882
©2006 Nature Publishing Group
to wild-type expression (Fig. 1). We detected
animals that had reduced GFP expression over
80 generations.
Is RNAi the mechanism behind the initial
silencing? There are two features of effective
interference in C. elegans: it targets exons, not
introns, and it depends on the canonical RNAi
genes rde-1 and rde-4 (ref. 3) (see supplementary
information). Tests for both show that RNAi is
responsible for the effect, and this is further supported by our observation that genes are more
likely to be indefinitely silenced in worms with
a mutation in eri-1 (results not shown), which
are hypersensitive to RNAi (ref. 4).
But RNAi probably does not underlie the
inheritance mechanism — rde-1 and rde-4 are
dispensable. To investigate further, we used a
candidate RNAi screen to identify genes that
affect the maintenance of silencing and found
four that abolish inheritance when knocked
out: hda-4 (a class II histone deacetylase),
K03D10.3 (a histone acetyltransferase of the
MYST family), isw-1 (a homologue of the yeast
chromatin-remodelling ATPase ISW1) and
mrg-1 (a chromo-domain protein) (see supplementary information).
The fact that these genes are all involved in
chromatin remodelling suggests that the inheritance of RNAi-induced phenotypes is due to
silencing at the transcriptional level. It may be
that this is achieved by modification of specific
residues in histone tails, because culturing
worms in the presence of the histone deacetylase inhibitor trichostatin A relieves silencing.
Earlier work has revealed a link between
RNAi and transcriptional silencing5 and inheritance of silencing for one generation in mice6.
We have shown that RNAi can induce silencing effects that, once established, are inherited
indefinitely over generations of sexual reproduction, in the absence of the trigger and of
RNAi machinery.
Nadine L. Vastenhouw*, Karin Brunschwig†,
Kristy L. Okihara*, Fritz Müller†,
Marcel Tijsterman*, Ronald H. A. Plasterk*‡
*Hubrecht Laboratory–KNAW, ‡University of
Utrecht, Uppsalalaan 8, 3584 CT Utrecht,
the Netherlands
e-mail: [email protected]
†Institute of Zoology, University of Fribourg,
Pérolles, 1700 Fribourg, Switzerland
1. Matzke, M. A. & Birchler, J. A. Nature Rev. Genet. 6, 24–35
(2005).
2. Grishok, A., Tabara, H. & Mello, C. C. Science 287, 2494–
2497 (2000).
3. Tabara, H. et al. Cell 99, 123–132 (1999).
4. Kennedy, S., Wang, D. & Ruvkun, G. Nature 427, 645–649
(2004).
5. Lippman, Z. & Martienssen, R. Nature 431, 364–370 (2004).
6. Rassoulzadegan, M. et al. Nature 441, 469–474 (2006).
Supplementary information accompanies this
communication on Nature’s website.
Received 7 February; accepted 15 June 2006.
Competing financial interests: declared none.
doi:10.1038/442882a
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