Ryo Futahashi (Senior Researcher) and Takema Fukatsu (Prime Senior Researcher of the Bioproduction Research Institute and Leader of the group) of the Symbiotic Evolution and Biological Functions Research Group, the Bioproduction Research Institute (Director: Tomohiro Tamura) of the National Institute of Advanced Industrial Science and Technology (AIST; President: Ryoji Chubachi), have discovered an extraordinary diversity of opsin genes that encode light sensors involved in color vision in dragonflies, in collaboration with Shunsuke Yajima (Professor) of the Faculty of Applied Bio-Science, Tokyo University of Agriculture (NODAI; President: Katsumi Takano), Ryouka Kawahara-Miki (Postdoctoral Fellow) of the NODAI Genome Research Center, Kentaro Arikawa (Professor) and Michiyo Kinoshita (Lecturer) of the School of Advanced Sciences, The Graduate University for Advanced Studies (SOKENDAI; President: Yasunobu Okada) and others.

We humans have a trichromatic color vision based on three opsin genes sensitive to blue, green, or red light. Opsin genes play key roles in color vision; for example, insects possess an opsin gene for ultraviolet (UV) light, which allows the insects to recognize UV light that is invisible to human beings. It is known that 3-5 opsin genes are involved in color vision in many animals. In this research, the researchers discovered that dragonflies possess surprisingly many (15-33) opsin genes, and that most of them are differentially expressed between adults and larvae, as well as between the dorsal and ventral regions of adult compound eyes. This is new knowledge about the diversity and evolution of color vision in animals.

This study was published online in an American scientific journal, Proceedings of the National Academy of Science USA, on February 24, 2015 (Japan Time).

A flying Anax parthenope, whose compound eyes are highly developed

The visual sense is crucial for many animals including human beings, and numerous studies have been conducted in both basic and applied research fields. Light is converted to electric signals in photoreceptor cells of the eye, and these signals are processed in the brain. Opsin proteins, which function as “light sensors” in the photoreceptor cells, are encoded by opsin genes. Specific types of opsin gene produce “light sensors” that are sensitive to specific wavelength light. For example, human beings possess three opsin genes for light sensors sensitive to blue, green, or red light, and can see light from purple to red, but not UV light. Honey bees and fruit flies, on the other hand, possess an opsin gene for UV light, but not for red light, enabling them to see UV light, but not red light. Thus, opsin genes are related closely to color vision. It has been thought that 3-5 opsin proteins are involved in color vision in most animals.

Dragonflies are diurnal insects with compound eyes that consist of thousands of small eyes (ommatidia). Many dragonflies, such as red dragonflies, have bright body color. Because their auditory and olfactory senses are degenerated, they are more strongly dependent on visual sense compared with other insects. Molecular bases underlying color vision in dragonflies, however, have not been extensively studied.

AIST has been making efforts to elucidate sophisticated biological functions in various insects. On biological function and ecological importance of insect body color, there are notable achievements such as “Symbiotic bacterium modifies insect body color” (AIST press release on November 19, 2010) and “Molecular basis of body color change in red dragonflies” (AIST press release on July 10, 2012).

NODAI is well experienced in gene analysis of various organisms using next-generation sequencers as a “hub for genome analysis of biological resources”. SOKENDAI is distinguished in the analysis of the molecular basis and physiological phenomena related to color vision in animals. This collaborative study to elucidate the molecular basis of the diversity of color vision in dragonflies has been achieved by teamwork of the institutions taking advantage of each field of expertise.

This study was supported by Grants-in-Aid for Scientific Research by the Japan Society for the Promotion of Sciences and Ministry of Education, Culture, Sports, Science and Technology.

Using the red dragonfly Sympetrum frequens, the researchers investigated the spectral sensitivity of compound eyes (Fig. 1). Dragonflies possess a pair of large compound eyes and three ocelli on their head. The compound eyes are mainly responsible for vision, whereas the ocelli are involved in maintaining horizontal balance (Fig. 1a). In S. frequens, the dorsal region of the compound eyes is structurally different from the ventral region: the dorsal ommatidia have larger facets and orange screening pigment, whereas the ventral ommatidia have smaller facets and dark purple screening pigment (Figs. 1a and b). Electrophysiological analysis revealed that the dorsal eye region was sensitive mainly to a short-wavelength range between UV (300 nm) and blue-green light (500 nm), whereas the ventral eye region was sensitive to a broader wavelength range from UV to red light (620 nm) (Fig. 1c). This result suggests that the color vision of S. frequens differs between the dorsal and ventral regions of their compound eyes.

Figure 1: Morphology, anatomy and spectral sensitivity of adult compound eyes of Sympetrum frequens (a) Frontal view of adult head; (b) Section of a compound eye; (c) Spectral sensitivity of dorsal and ventral regions of adult compound eyes

Because the number of opsin genes in dragonflies was unknown, the researchers performed comprehensive transcriptomic analysis of adult and larval heads of S. frequens by using a next-generation sequencer. As a result, as many as 20 opsin genes were identified (Fig. 2a). Insect opsin proteins can be classified into two types, visual and nonvisual opsins, based on their amino acid sequences. Visual opsins are subdivided into UV type, short-wavelength (SW: blue light) type, and long-wavelength (LW: green to red light) type. Opsin genes of S. frequens were categorized into 1 UV type, 5 SW types, 10 LW types, and 4 nonvisual types, and the number of visual opsin genes was extraordinarily large compared with other insects (Fig. 2a). Furthermore, a strikingly large number (15-33) of opsin genes were identified from various dragonfly species.

Figure 2: Evolutionary change in the number of opsin genes among insects and a summary of opsin expression pattern of S. frequens (a) The number of opsin genes of S. frequens in comparison with those in the genomes of diverse insects (b) The number of opsin genes that are expressed in the dorsal or ventral regions of the adult compound eye, adult head region containing ocelli, or in the larval head of S. frequens. Each gene was expressed at a specific life stage and in a specific region.

The researchers next analyzed the expression pattern of opsin genes in the dorsal or ventral regions of the adult compound eye, adult head containing ocelli, or in the larval head. Most of the opsin genes were expressed only at a specific life stage and in a specific region. For example, each opsin gene of S. frequens was expressed in either larvae or adults as shown in Fig. 2b. Furthermore, most of the opsin genes in adults were expressed only in one of the following three regions: the dorsal region of the compound eye, the ventral region of the compound eye, or the region around the ocelli (Fig. 2b). Thus, dragonflies expressed completely different types of opsin genes between adults and larvae as well as between dorsal and ventral regions of adult compound eyes. These differences possibly produce a difference in the light sensitivity. Four nonvisual opsin genes were scarcely expressed in the larval and adult visual organs.

Larval dragonflies are sedentary under the water, whereas adults actively fly in the air; therefore, it is anticipated that larvae are less dependent on their visual sense or color vision than adults are. In addition, the dorsal region of the adult compound eye is mainly used to recognize objects against the sky, whereas the ventral region is mainly used to recognize the environment on the ground, mates, and food.

In order to adapt to various types of light environment, dragonflies may have diversified their opsin genes in a stage and region specific manner (Fig. 3). Larvae express a relatively small number of opsin genes reflecting less visual dependence, whereas adults express many opsin genes in their compound eyes. In addition, even in the same adult compound eye, the dorsal region, which perceives the SW-skewed light directly from the sky, expresses more SW opsin genes, whereas the ventral region, which perceives reflected light from objects on the ground, expresses more LW opsin genes (Figs. 2b and 3).

The repertoire of opsin genes differed among dragonfly species. Therefore, the opsin genes may have evolved according to the habitat or behavior of each species; for example, the larvae of sand-burrowing species lack SW opsin gene expression in larval eyes.

Figure 3: Differential expression of opsin genes in S. frequens

The researchers demonstrated that dragonflies utilize different sets of opsin genes depending on types of light environment, which can be achieved by an extraordinary increase in the number of opsin genes. Further investigation may lead to understanding the benefits of using different gene sets for each environment. By further characterization of each gene, the adaptation mechanisms to various types of light environment would be more thoroughly understood.

The researchers plan to analyze opsin gene expression in each photoreceptor cell to elucidate the detailed characteristics of the gene and to investigate the molecular evolution of color vision and its adaptation to various types of light environment.