Symbiotic Bacterium Modifies Aphid Body Color
Tsutomu Tsuchida, et al.
Science 330, 1102 (2010);
DOI: 10.1126/science.1195463

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REPORTS

The value of the maximal growth rate lc(kt) =kt ⋅ (rmax – r0) ≈ 2.85 hour–1 (based on the average rmax) corresponds well to the doublingtime of ~20 min for typical E. coli strains in richmedia. Moreover, Eq. 5 quantitatively accounts
for the correlation observed between growth ratel and nutritional capacity kn (fig. S3A).
This theory can be inverted to predict the effect of protein expression on cell growth. Unnecessary protein expression leads to diminishedgrowth (19). Understanding the origin of thisgrowth inhibition is of value in efforts to increase
the yield of heterologous protein in bacteria (20)and to understand the fitness benefit of gene
regulation (21, 22). Aside from protein-specifictoxicity, several general causes of growth inhibition have been suggested, including diversion of
metabolites (23), competition among sigma
factors for RNA polymerases (24), and competition among mRNA for ribosomes (19, 25).
We modeled the expression of unnecessary
protein (of mass fraction fU) as an additional(neutral) component of the proteome that effectively causes a reduction of rmax to rmax – fU/r
(Fig. 4A). Equation 5 then predicts a linear
reduction of the growth rate,lðfU Þ ¼ lðfU ¼ 0Þ ⋅ ½1 − ðfU =fc Þð6Þextrapolating toward zero growth at fc = r ·(rmax – r0) ≈ 0.48. The prediction quantitatively
described the observed growth defect caused byinducible expression of b-galactosidase (Fig. 4B),
as well as previous results obtained for various
proteins and expression vectors (Fig. 4C) (19, 26),
without any adjustable parameters. These results
suggest that growth reduction is a simple consequence of ribosome allocation subject to the constraints of Eqs. 1, 3, and 4.
Robust empirical correlations of the RNA/
protein ratio with the growth rate (Figs. 1A and2A and figs. S1 and S2) revealed underlying
constraints of cellular resource allocation and led
to the formulation of a simple growth theory thatprovided quantitative predictions and unifying
descriptions of many important but seemingly
unrelated aspects of bacterial physiology. Like
Ohm’s law, which greatly expedited the design ofelectrical circuits well before electricity was
understood microscopically, the empirical correlations described here may be viewed as microbial “growth laws,” the use of which may
facilitate our understanding of the operation anddesign of complex biological systems well before
all the underlying regulatory circuits are elucidated at the molecular level.

1102

References and Notes
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2. H. Youk, A. van Oudenaarden, Nature 462, 875 (2009).
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6. A. P. Arkin, D. A. Fletcher, Genome Biol. 7, 114 (2006).
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8. R. Kwok, Nature 463, 288 (2010).
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J. Gen. Microbiol. 19, 592 (1958).
10. H. Bremer, P. P. Dennis, in Escherichia coli and Salmonella,
F. C. Neidhardt, Ed. (ASM Press, Washington, DC, 1996),
pp. 1553–1569.
11. O. Maaløe, in Biological Regulation and Development,
R. F. Goldberger, Ed. (Plenum, New York, 1979),
pp. 487–542.
12. J. Forchhammer, L. Lindahl, J. Mol. Biol. 55, 563 (1971).
13. See supporting material on Science Online.
14. T. Ruusala, D. Andersson, M. Ehrenberg, C. G. Kurland,
EMBO J. 3, 2575 (1984).
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Genet. 38, 749 (2004).
16. A. L. Koch, Can. J. Microbiol. 34, 421 (1988).
17. A particular protein species may belong to multiple
classes; sometimes this is a result of expression frommultiple promoters that are differently regulated.
18. J. Monod, Annu. Rev. Microbiol. 3, 371 (1949).
19. H. Dong, L. Nilsson, C. G. Kurland, J. Bacteriol. 177,
1497 (1995).
20. C. P. Chou, Appl. Microbiol. Biotechnol. 76, 521 (2007).

21. E. Dekel, U. Alon, Nature 436, 588 (2005).
22. D. M. Stoebel, A. M. Dean, D. E. Dykhuizen, Genetics
178, 1653 (2008).
23. B. R. Glick, Biotechnol. Adv. 13, 247 (1995).
24. A. Farewell, K. Kvint, T. Nyström, Mol. Microbiol. 29,
1039 (1998).
25. J. Vind, M. A. Sørensen, M. D. Rasmussen, S. Pedersen,
J. Mol. Biol. 231, 678 (1993).
26. W. E. Bentley, N. Mirjalili, D. C. Andersen, R. H. Davis,
D. S. Kompala, Biotechnol. Bioeng. 35, 668 (1990).
27. R. Mikkola, C. G. Kurland, Biochimie 73, 1551 (1991).
.28. We thank H. Bremer, L. Csonka, P. Dennis, M. Ehrenberg,
P. Geiduschek, M. Schaechter, A. Tadmor, andmembers of the Hwa lab for suggestions and discussions;D. Hughes for the Sm mutant strains; and P.-h. Lee
and B. Willumsen for the use of unpublished data.
Supported by NIH grant RO1GM77298, NSF grant
MCB0746581, and the NSF-supported Center for
Theoretical Biological Physics (grant PHY0822283).
M.S. was supported by a Natural Sciences andEngineering Research Council of Canada fellowship.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/330/6007/1099/DC1
Materials and Methods
SOM Text
Figs. S1 to S6Tables S1 to S7
References
20 May 2010; accepted 6 October 201010.1126/science.1192588

Symbiotic Bacterium Modifies
Aphid Body Color
Tsutomu Tsuchida,1*† Ryuichi Koga,2† Mitsuyo Horikawa,3 Tetsuto Tsunoda,3 Takashi Maoka,4Shogo Matsumoto,1 Jean-Christophe Simon,5 Takema Fukatsu2*
Color variation within populations of the pea aphid influences relative susceptibility to predators and parasites.
We have discovered that infection with a facultative endosymbiont of the genus Rickettsiella changes the insects’body color from red to green in natural populations. Approximately 8% of pea aphids collected in Western
Europe carried the Rickettsiella infection. The infection increased amounts of blue-green polycyclic quinones,
whereas it had less of an effect on yellow-red carotenoid pigments. The effect of the endosymbiont on body color
is expected to influence prey-predator interactions, as well as interactions with other endosymbionts.
he world is full of colors, and many animals
have color vision, recognizing their environment, habitat, food, enemies, rivals,
and mates by visual cues. Body color is thus an
ecologically important trait, often involved in species recognition, sexual selection, mimicry, aposematism, and crypsis (1, 2). In the pea aphid
Acyrthosiphon pisum, red and green color morphs
are found in the same populations. Early work
has shown that the aphid body color is genetically determined, with red being dominant overT

1
Molecular Entomology Laboratory, RIKEN Advanced Science
Institute, Wako 351-0198, Japan. 2National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 3058566, Japan. 3Faculty of Pharmaceutical Sciences, Tokushima
Bunri University, Tokushima 770-8514, Japan. 4Research Institute for Production Development, Kyoto 606-0805, Japan.
5Institut National de la Recherche Agronomique, UMR 1099
BiO3P, Institut National de la Recherche Agronomique (INRA)/
Agrocampus Ouest/Université Rennes 1, BP 35327, 35653 Le
Rheu Cedex, France.

*To whom correspondence should be addressed. E-mail:t-tsuchida@riken.jp (T.T.); t-fukatsu@aist.go.jp (T.F.)†These authors contributed equally to this work.

19 NOVEMBER 2010

VOL 330SCIENCEgreen (3). Ecological studies show that ladybird
beetles tend to consume red aphids on green plants(4), and parasitoid wasps preferentially attack green
aphids (5). The predation and parasitism pressuresappear to maintain the color variation in natural
aphid populations (1, 4). An unexpected recent
discovery showed that the aphid genome contains
several genes for carotenoid synthesis not found inanimal genomes. The genes are of fungal origin
and appear to have been acquired in the evolutionary history of aphids via ancient lateraltransfer. One of the genes is involved in synthesisof red color pigments, and the presence or absenceof the gene is responsible for the red or green
coloration of the aphids (6). Here, we report another factor affecting aphid color polymorphism:a previously unrecognized endosymbiont thatmodifies insect body color in natural populations.
While screening pea aphid strains from natural populations collected in France, we foundseveral strains of green aphids producing red nymphs.
As the nymphs grew, their body color changed
from reddish to greenish, and the adults becamewww.sciencemag.org

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is identical to the description of an electric circuitwith two resistors (fig. S6), with Eqs. 1 and 4
being analogous to Ohm’s law. Solving these equations simultaneously leads to the Michaelis-Menten
relation known empirically for the dependenceof cell growth on nutrient level (18)knlðkt , kn Þ ¼ lc ðkt Þ ⋅ð5Þ
kt þ kn

REPORTS

0 day old

6 day old

11 day old

E

A Rickettsiella- &Hamiltonellainfected strain(RA04)

B

0 day old

6 day old

11 day old

0 day old

7 day old

12 day old

Rickettsiella-freestrain(10TVamp)

F

Rickettsiellatransfected strain(10TVamp/RA04acg)

Rickettsiella-infected& Hamiltonellaeliminated strain(RA04acg)
0 day oldC

red aphids nor originally green aphids were affected (Fig. 1, fig. S1, and table S1). The bodycoloring patterns in the experimentally infectedaphid strains were similar to those in the naturally
Rickettsiella-infected strains (Fig. 1). Quantitative
polymerase chain reaction (PCR) analyses revealed
that the intensity of green color was positively
correlated with the infection density of Rickettsiellafor different host and endosymbiont genotypes(fig. S1). These results indicate that the Rickettsiellainfection is responsible for green body color in atleast some green pea aphids in natural populations.

By antibiotic treatments (7), we successfully
eliminated the Hamiltonella/Serratia infection fromthe aphids without affecting Rickettsiella andBuchnera infections. Their body-coloring patterns
did not change after the treatments (Fig. 1B). When
we injected diverse aphid strains, which harbored
only Buchnera, with hemolymph from the Rickettsiellainfected strains (7), the aphids produced both
Rickettsiella-infected and -uninfected offspring. Notably, Rickettsiella-infected red aphids of distinct
genotypes consistently changed body color to green
as they developed, whereas neither uninfected

6 day old

11 day old

G
Rickettsiella-freestrain(4TVamp)D

Rickettsiella-freestrain(YR2amp)H

Rickettsiellatransfected strain(4TVamp/RA04acg)

Rickettsiellatransfected strain(YR2amp/RA04acg)

Fig. 1. (A to H) Rickettsiella-induced body-color change in pea aphids of different genetic backgrounds. Scale bars, 1 mm. For details of the aphid
strains, see table S1.
A

90

VF02F (Le Rheu, France) [AB522697]
TP05e (Le Rheu, France) [AB522699]
RA04 (Le Rheu, France) [AB522705]
From
Rp74 (Ranspach, France) [AB522701]
100
pea
P136 (Mauzé Le Mignon, France) [AB522702]
aphid Rickettsiella
MIL08 (Mirecourt, France) [AB522700]
JOV01H (Jena, Germany) [AB522703]
100
GCt10 (Caumont, France) [AB522704]
VS01 (Le Rheu, France) [AB522698]
Rickettsiella popilliae [EU180598]
100
Rickettsiella melolonthae [EF408231]
68
Rickettsiella grylli [U97547]
Symbiont of Amblyomma americanum (tick) [AY939824]
76
CoxiellaSymbiont of Haemaphysalis longicornis (tick) [AY342035]
100
Coxiella burnetii [AY342037]
Legionella feeleii [X73406]
Legionella
Legionella pneumophila [NC 009494]
100
57
Legionella lytica [NR 026334]
Escherichia coli [NC 000913]
0.05

B

C

D

20 µm

200 µm 50 µm
oe

E

F

G

em
em

oe
oe

em
50 µm

em em

400 µm 50 µm

em

I

H

Downloaded from www.sciencemag.org on January 10, 2011

green (Fig. 1A and table S1). A survey of endosymbiotic microbiota in these aphid strains fromdistinct geographic origins and bearing different
genotypes (7) identified g-proteobacterial facultativeendosymbionts (one of which is a Hamiltonellaor Serratia-like organism) that confer protection
to their host aphids against parasitoid wasps (8).
In addition, we found a previously unrecognizedaphid endosymbiont of the genus Rickettsiella,
whose members are insect pathogens that are
phylogenetically related to the human pathogens
Coxiella and Legionella (Fig. 2A) (9).

m

*

*

*
Fig. 2. (A) Phylogenetic analysis of Rickettsiella endosymbionts from European*
pea aphids on the basis of 16S ribosomal RNA gene sequences. A maximum
vac
1 µm
likelihood phylogeny inferred from 1384 aligned nucleotide sites is shown with*bootstrap values. (B to G) In situ hybridization of Rickettsiella. (B) A matureJ*
embryo (blue) containing many primary bacteriocytes harboring Buchnera (green)
*
lu
*
vac
*
and a secondary bacteriocyte harboring Rickettsiella (red) that together constitute
*
*
*
*
*
*
*
*
a huge bacteriome. (C) Enlarged image of the secondary bacteriocyte. (D) Sheath
*
*
*
*
*
*
cells harboring Rickettsiella adhering to the periphery of primary bacteriocytes
* * *
*
*
*
* *
* *
(white arrowheads). (E) Oenocytes (oe) infected with Rickettsiella. (F) Posterior part
* *
*
*
* * *
of an ovary, where ovariole pedicels are heavily infected with Rickettsiella (yellow** *arrowheads). em, embryo. (G) Enlarged image of an ovariole pedicel. (H to J)bbb*
Electron microscopy images of Rickettsiella. (H) Image of a secondary bacteriocytehem
1 µm
harboring Rickettsiella and a primary bacteriocyte harboring Buchnera. vac,*vacuole; b, Buchnera; asterisks, Rickettsiella. (I) Enlarged image of a Rickettsiellacell. m, mitochondrion. (J) Image of the wall of an ovariole pedicel infected with Rickettsiella. hem, hemocoel; lu, lumen of ovariole pedicel; nu, nucleus.

www.sciencemag.org

SCIENCEVOL 330

19 NOVEMBER 2010

m

nu

5 µm

1103

Diagnostic PCR surveys detected 7.9% [28 of
353 insects (28/353)] Rickettsiella infection inWestern European populations of A. pisum (fig.
S2A). Fitness measurements revealed that infection
status did not affect growth rate and body size for
two aphid genotypes, although we observed significantly larger body size and faster growth with
Rickettsiella infection for one aphid genotype(fig. S3). Similarly to Hamiltonella and Serratia(10, 11), Rickettsiella resided in secondary bacteriocytes and sheath cells in vivo and was also
found intracellularly and extracellularly in various tissues and the hemolymph (Fig. 2, B to J). In
natural populations, not all green aphids were
infected with Rickettsiella, and some strains of
red aphids were found with Rickettsiella infection(fig. S2, B and C). It appears that the combinationof aphid genotype and the endosymbiont contribute to body color (3, 6): Some endosymbiont
genotypes may fail to induce green coloration,
whereas some host genotypes may attenuate or
inhibit the activity of Rickettsiella. Similar interactions have been documented for other facultative endosymbionts in the pea aphid (12, 13).
Aphid body color mainly consists of two majorgroups of pigment molecules: (i) yellow-red colors
from carotenoid pigments such as b-carotene,lycopene, and torulene (6, 14) and (ii) blue-green
and other pigments from structurally complex
polycyclic quinones and their glycosides, calledaphins or aphinins (14, 15). When the naturally
red aphid strain was infected with Rickettsiellaand became green, we observed some changes incarotenoid compositions, but the differences were
not sufficient to account for the degree of green
pigmentation (fig. S4). Moran and Jarvik (6) havefound that a fungus-derived carotenoid desaturase
gene, tor, is present in red aphid clones but isabsent in green aphid clones and is responsible for
production of the red carotenoids. Our quantitative reverse transcription–PCR assay showed that
expression levels of the tor gene were not significantly affected by the Rickettsiella infection (fig. S5).
We recovered almost all of the green pigments
into the butanol fraction by water-butanol extraction of the aphids (7). Thin-layer chromatography ofthe extracts separated one major and several minor green bands, which presumably represented
polycyclic quinone glycosides like aphinins (14),although their exact structures were not determined(7). The intensity of the green bands was greater inthe Rickettsiella-infected green-aphid strains thanin the original red-aphid strains (fig. S6A). Densitometric quantification of the major band revealed
a 2.4- to 4.6-fold increase of the green pigment in
the Rickettsiella-infected aphid strains comparedwith the uninfected strains (fig. S6B). We presume
that Rickettsiella does not synthesize new green
pigments for itself but stimulates aphid metabolism to increase green-pigment production (fig. S7).
The murky hue of the Rickettsiella-induced green
aphids (Fig. 1 and fig. S1) is probably a result of the
combination of the green pigments and the reddish
carotenoid pigments. Genome sequencing of the
Rickettsiella endosymbiont and transcriptomic

1104

analysis of the infected and uninfected aphid
hosts should provide insights into the molecularand metabolic interactions between host andendosymbiont that lead to the body-color change.
Previous studies have identified a variety of
biological roles for facultative endosymbionts inthe pea aphid, including tolerance to high temperature, resistance against natural enemies, andplant adaptation (8, 16–19). We have added another endosymbiont relationship that potentiallyaffects a host trait of ecological importance inways that have yet to be verified. For example,
the induced green color may reduce the predation
risk by ladybird beetles. Notably, Rickettsiella isfrequently found in co-infections with either Hamiltonella [55.6% (35/63)] or Serratia [20.6% (13/63)]
endosymbionts (fig. S2D), both of which areprotective against parasitoid wasps (8) and may
act to offset the risk of green aphids attracting
parasitoids.
References and Notes
1. G. D. Ruxton, T. N. Sherratt, M. P. Speed, Avoiding
Attack: The Evolutionary Ecology of Crypsis, Warning
Signals and Mimicry (Oxford Univ. Press, Oxford, 2005).
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material on Science Online.
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Proc. Natl. Acad. Sci. U.S.A. 100, 1803 (2003).

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.20. We thank J. Peccoud, Y. Outreman, S. Stoeckel, L. Mieuzet,
J. Jaquiéry, and W. Weisser for aphid sampling; J. Bonhomme,
L. Mieuzet, and J. Makino for aphid rearing and genotyping;and X.-Y. Meng and S. Hanada for help with electron
microscopy. This work was supported by the Japan-France
Integrated Action Program SAKURA of the Japan Society
for the Promotion of Science (to T.F. and J.-C.S.); the Grantin-Aid for Scientific Research on Innovative Areas(22128007) of the Ministry of Education, Culture, Sports,
Science and Technology, Japan (to T.F.); and funds from
INRA AIP Bioresources (to J.-C.S.). T.T. was supported by a
RIKEN special postdoctoral research fellowship.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/330/6007/1102/DC1
Materials and Methods
Figs. S1 to S7Table S1
References
22 July 2010; accepted 8 October 201010.1126/science.1195463

PiggyBac Transposon Mutagenesis:
A Tool for Cancer Gene
Discovery in Mice
Roland Rad,1 Lena Rad,1 Wei Wang,1 Juan Cadinanos,1,2 George Vassiliou,1 Stephen Rice,1
Lia S. Campos,1 Kosuke Yusa,1 Ruby Banerjee,1 Meng Amy Li,1 Jorge de la Rosa,2 Alexander Strong,1
Dong Lu,1 Peter Ellis,1 Nathalie Conte,1 Fang Tang Yang,1 Pentao Liu,1 Allan Bradley1*
Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here,
we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery
in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers
whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements
introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable ofgenome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous
retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-relatedtranscription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty havedifferent integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to becompatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Micewith different transposon types, copy numbers, and chromosomal locations support wide applicability.
enetic screening in higher organisms has
been hampered for decades by the lackof efficient insertional mutagenesis tools.
Retroviruses have been used for cancer gene discovery in mice, but their application has beenG

19 NOVEMBER 2010

VOL 330SCIENCElimited to the study of hematopoietic and mammary tumors because of viral tropism for thesetissues (1). DNA transposons, which are the key
insertional mutagens in lower organisms, were inactivated in vertebrate genomes millions of years

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REPORTS