Ryuichi Koga (Senior Researcher), Takema Fukatsu (Leader) and others of the Symbiotic Evolution and Biological Functions Research Group, the Bioproduction Research Institute (Director: Yoichi Kamagata) of the National Institute of Advanced Industrial Science and Technology (AIST; President: Tamotsu Nomakuchi), in collaboration with Tsutomu Tsuchida (RIKEN Special Postdoctoral Researcher) of the Advanced Science Institute (Director: Kohei Tamao), RIKEN (President: Ryoji Noyori) and other groups, discovered a novel symbiotic bacterium of the genus Rickettsiella in European natural populations of the pea aphid Acyrthosiphon pisum, and demonstrated that the symbiont induces a drastic color change of the host aphids: originally red insects turned into green when get infected.

Body color is an ecologically important trait, often involved in species recognition, sexual selection, mimicry, aposematism, and crypsis. However, there has been no report on such a phenomenon that the important biological trait, body color, is drastically changed by a symbiotic microorganism. This finding provides a new viewpoint to the ecology and adaptation of insects and other organisms in general.

This study was published online in Science, an American scientific journal, on November 19, 2010 (JST).

Figure 1 : The green pea aphid (left) is, although genetically identical to the red aphid (right), infected with a novel symbiont of the genus Rickettsiella, which modifies the aphid body color from red to green.

Microorganisms are much more diverse than animals and plants. The biological functions of microorganisms have been utilized in various fields, such as pharmaceuticals, agriculture, chemicals, foods, fuels, and detergents, to support our daily lives, and they are therefore of great economic value. Most of those bioactive substances have been discovered and produced by using microbiological techniques that are based on isolation and cultivation. On the other hand, "unculturable microorganisms" are commonly present in nature and hard to handle with conventional techniques. Recent studies have shown that unculturable microorganisms live symbiotically in the majority of diverse insect species, which are attracting attention as untapped genetic resources.

Furthermore, the question as to how living organisms have acquired diverse biological functions during the process of evolution is of scientific interest and curiosity. Understanding of the mechanisms whereby insects have acquired novel biological functions through symbiotic bacteria will answer this question and take us a step closer to understanding the origin of evolutionary novelties in diverse organisms.

Thus far, we have studied a variety of unculturable symbiotic bacteria at AIST, and unveiled novel biological functions of symbiotic bacteria in the pea aphid (AIST press release on March 25, 2004; AIST press release on September 28, 2010). In the past several years, in collaboration with Dr. Jean-Christophe Simon of the French National Institute for Agricultural Research, we have generated a number of European pea aphid strains of different genetic backgrounds that were artificially infected with different symbiotic bacteria. Using these aphid strains, we have promoted a joint project to comprehensively explore the biological effects of symbiotic bacteria on the host aphid under different combinations of host and symbiont genotypes. The findings reported here were unexpectedly obtained in the course of this project.

This study was supported by a grant from the Japanese-French Scientific Collaboration Project (SAKURA Program) of the Japan Society for the Promotion of Science, and by Grants-in-Aid for Scientific Research on Innovative Areas ("Genetic Bases for the Evolution of Complex Adaptive Traits") from the Ministry of Education, Culture, Sports, Science, and Technology.

Many animals have color vision, thereby recognizing the environment, habitat, food, enemies, rivals, and mates. The body color of animals is thus an ecologically important trait, often involved in species recognition, sexual selection, mimicry, aposematism, and crypsis.

More than 4000 species of aphids are known, including many important agricultural pests, some of which exhibit body color polymorphism within the same species. For example, red and green aphids coexist in European and American populations of the pea aphid (Fig. 1). Ecological studies have suggested that different color preferences of different predators are involved in the coexistence of individuals with different colors in the same species. Major predators of aphids are ladybird beetles and parasitoid wasps. Ladybird beetles tend to prey on red aphids on green plants, while parasitoid wasps attack green aphids more frequently.

Previous studies have shown that the red body color of the pea aphid is genetically dominant over green. Furthermore, a recent study based on the draft genome sequence of the pea aphid has shown that a carotenoid desaturase obtained by lateral gene transfer from a fungus determines the body color. Aphids with this gene can synthesize red carotenoid pigments and become red, whereas aphids become green if this gene is absent.

In collaboration with the French National Institute for Agricultural Research, we collected pea aphid strains originating from different field populations in Europe, and obtained several green aphid strains producing red nymphs. Nymphs of these strains changed the body color from reddish to greenish as they grew, and fourth instar nymphs and adult aphids became completely green (Fig. 2).

Figure 2 : From left to right: aphids of the CGt10 strain 0, 4, 11, and 15 days after birth: The body color changes from red to green during the developmental process.

Analyses of symbiotic microbiota in these aphid strains identified two facultative symbiotic bacteria in addition to the essential symbiotic bacterium Buchnera. One of the facultative symbionts was either Hamiltonella or Serratia. The other was a facultative symbiotic bacterium of the genus Rickettsiella, which had previously been unknown from aphids (Fig. 3). We investigated 353 individuals originating from European pea aphid populations, 28 (7.9%) of which were found to be infected with Rickettsiella, indicating its wide distribution in natural aphid populations (Fig. 4).

Figure 3 : In the aphid body, Rickettsiella cells (red) are localized in secondary bacteriocytes (A, arrow) and small flat sheath cells (B, arrowhead), showing a different distribution from that of Buchnera cells (green) localized in primary bacteriocytes. Blue indicates aphid cell nuclei. (C) A transmission electron microscopic image of a Rickettsiella cell. m indicates mitochondrion.

Figure 4 : Rickettsiella infection rates in French pea aphid populations

Figure 5 : Procedures for establishment of Rickettsiella–infected and uninfected aphid strains under the same genetic background

We micro-injected body fluid from Rickettsiella–infected aphids into aphids of an uninfected strain. The offspring of the injected aphids were individually maintained, whereby a number of aphid strains with the same genetic background (the only difference being whether or not they were infected with Rickettsiella) were created (Fig. 5). Strikingly, the body color of aphids in the Rickettsiella–infected red strains consistently changed to green (Fig. 6).

Figure 6 : Aphid body color change from red to green by Rickettsiella infection. Top, appearance of the aphids; bottom, quantified values reflecting body color (hue angle). Small hue angle values represent red whereas large hue angle values show green. Approximate body colors are shown on the right side of each graph.

On the other hand, the body color of aphids in originally green strains was not affected when infected with Rickettsiella. We created Rickettsiella–infected strains and uninfected strains under three aphid genotypes, and compared their body weight, growth rate, number of progeny, and lifespan. The comparison showed no significant differences, indicating that Rickettsiella infection changes the aphid body color from red to green without apparent adverse effects.

The body color of aphids mainly consists of yellow-to-red carotenoid pigments, and polycyclic quinone pigments of various colors, from green to blue. We performed pigment analyses of Rickettsiella–induced green aphids and uninfected red aphids, and found no significant differences in composition and quantity of the carotenoid pigments. Meanwhile, although Rickettsiella infection caused no change in the green pigment composition, the pigment quantity exhibited over 3-fold increase in the infected aphids than in uninfected aphids. This suggests that Rickettsiella infection somehow activates the production of green pigments in the host aphid, which causes the body color change.

The change in body color from red to green due to Rickettsiella infection probably makes the aphids less vulnerable to feeding by ladybird beetles but more vulnerable to attack by parasitoid wasps. Interestingly, most (about 80%) of the Rickettsiella–infected aphids were also infected with the symbiotic bacterium Hamiltonella or Serratia. It was reported that Hamiltonella and Serratia give resistance to parasitoid wasps by killing the eggs and larvae laid in the aphid body. This suggests the possibility that Rickettsiella changes the body color of the aphids, making them less vulnerable to feeding by ladybird beetles, and co-infects with a symbiotic bacterium protective against parasitoid wasps that prefer green aphids, thereby increasing the aphids' survival rate and, as a result, its own survival rate.

No previous studies have reported symbiotic bacteria that change the body color of normal individuals. This is the world's first report to show the involvement of symbiotic bacteria in the change in animal body color.

We will sequence the genome of Rickettsiella, and will analyze the gene expression changes in the host aphid after Rickettsiella infection by using a next-generation DNA sequencer. From these studies, we hope to understand the molecular mechanism by which infection with the symbiotic bacterium Rickettsiella affects gene expression and metabolism in the host aphid and causes the drastic phenotypic change in the host insect.

We would also like to verify the hypothesis that Rickettsiella makes aphids less vulnerable to feeding by ladybird beetles by changing their body color. It should also be tested that Rickettsiella infection increases the survival rate of the host aphid as well as its own survival rate by co-infecting with Hamiltonella or Serratia, which gives resistance to parasitoid wasps.

Understanding the mechanism by which symbiotic microorganisms change the body color of the host organism may potentially lead to the development of efficient methods for production of dyes and pigments, and new techniques to control the color and appearance of organisms.

Tsutomu Tsuchida, Ryuichi Koga, Mitsuyo Horikawa, Tetsuto Tsunoda, Takashi Maoka, Shogo Matsumoto, Jean-Christophe Simon and Takema Fukatsu (2010) Symbiotic bacterium modifies aphid body color. Science 330: 1102-1104.