Extraordinary diversity of visual opsin genes in dragonflies

Abstract

Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15-33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage- and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies.

Keywords: color vision; dragonfly; molecular evolution; opsin.

Fig. 1.

Adult and larval eyes of zygopteran, anisozygopteran, and anisopteran dragonflies. Images courtesy of Akira Ozono.

Fig. 2.

Morphology, anatomy and spectral sensitivity of adult compound eyes of Sympetrum frequens. (A) Frontal view of adult head. An, antenna; DCE, dorsal region of compound eye; LO, lateral ocellus; MO, median ocellus; VCE, ventral region of compound eye. (B) Unstained dorso-ventral section of a compound eye. (C) Unstained superficial section of the dorsal region of a compound eye, wherein photoreceptor cells and/or screening pigment cells accumulate orange pigment. (D) Unstained superficial section of the ventral region of a compound eye, wherein photoreceptor cells and/or screening pigment cells accumulate dark purple pigment. (E) Spectral sensitivity of the dorsal and ventral regions of adult eyes measured by electroretinography. Each symbol indicates the mean value of four dark-adapted individuals with SE (n = 8).

Fig. 3.

Identification of 20 opsin genes in three dragonflies of the family Libellulidae. (A) A neighbor-joining phylogeny of 20 opsin genes each from S. frequens (red shade), O. albistylum (blue shade), and L. fulva (gray shade) inferred from 869 aligned amino acid sites. On each node, bootstrap values are indicated in the order of neighbor-joining method/maximum-likelihood method. Accession numbers or annotation IDs are shown in brackets. Classification of the opsin genes is indicated on the right side. Insect visual opsin genes are highlighted in purple for UV type, in blue for SW type, and in green for LW type, respectively. (B) Gene clusters of SW and LW opsin genes on the genome of L. fulva.

Fig. 4.

Opsin genes of the red dragonfly S. frequens in comparison with those encoded in the genomes of diverse insects. Numbers of opsin genes of pteropsin type (ptero), RGR-like (RGR), arthropsin type (arth), Rh7-like (Rh7), UV type, SW type, and LW type are mapped on the insect phylogeny (75). Numbers of insect visual opsins (visual) are highlighted by purple for UV type, blue for SW type, and green for LW type.

Fig. 5.

Expression levels of 20 opsin genes in adult and larval visual organs of S. frequens and O. albistylum. (A–D) Nonvisual opsin genes. (E) Visual opsin gene of UV type. (F–J) Visual opsin genes of SW type. (K–T) Visual opsin genes of LW type. D, dorsal region of adult eyes; L, larval whole head; O, adult head region containing ocelli; O.a., O. albistylum; S.f., S. frequens; V, ventral region of adult eyes. The numbers indicate FPKM values.

Fig. 6.

Opsin genes in diverse dragonflies. (A) Adult dragonflies representing 12 species and 11 families examined in this study. (B) Numbers of opsin genes of pteropsin type, RGR-like, arthropsin type, Rh7-like, UV type, SW type, and LW type mapped on the dragonfly phylogeny (76). Numbers of insect visual opsins (visual) are highlighted by purple for UV type, blue for SW type, and green for LW type. SW genes are further categorized into groups a, b, and c; so are LW genes into groups A, B, C, D, E, and F (also see Fig. S2). Estimated gains and losses of the opsin genes in the evolutionary course of the dragonflies are indicated on the branches.

Fig. 7.

Expression profiles of opsin genes in diverse dragonflies. (A) Expression levels of visual opsin genes in adult and larval visual organs of 12 dragonfly species. Phylogenetic relationship of the dragonflies is shown on the top: A.mel., Asiagomphus melaenops; A.par., Anax parthenope; A.sie., Anotogaster sieboldii; E.sup., Epiophlebia superstes; I.asi., Ischnura asiatica; I.per., Indolestes peregrinus; M.amp., Macromia amphigena; M.cos., Mnais costalis; O.alb., Orthetrum albistylum; S.fre., Sympetrum frequens; S.uchi., Somatochlora uchidai; T.pry., Tanypteryx pryeri. Molecular phylogeny of SW-type opsin genes is shown on the upper left, where a, b, and c indicate the SW gene groups a, b, and c (see also Fig. S2A). Molecular phylogeny of LW-type opsin genes is shown on the lower left, where A, B, C, D, E, and F indicate the LW gene groups A, B, C, D, E and F (see also Fig. S2B). Gene expression levels are displayed by heat map presentation in connection to the dragonfly phylogeny and the opsin gene phylogenies, in which D, V, O, and L indicate expression levels in dorsal region of adult eyes, ventral region of adult eyes, adult head region containing ocelli, and larval whole head, respectively. In the color spectrum bar (Upper Left), magenta and green indicate positive and negative values relative to the average FPKM per gene per dragonfly species, whereas gray indicates no detectable expression with FPKM < 1. On the bottom, ecological traits for each dragonfly species are indicated according to refs. and . For larval microhabitat: mud is crawling, or burrowing in muddy water bottom of open lowland ponds, marsh, rice paddies, or rivers; sand is burrowing in sandy water bottom of mountain streams or small rivers under dense tree cover; pit is digging pits on wet mountain slopes under forest cover; stone is hiding under stones in rocky water bottom of mountain streams under forest cover; and weed is hanging on water plants in open ponds or rivers. For adult twilight flying activity: +++, very active; ++, active; +, moderate; –, not observed. (B) A pit-dwelling larva of T. pryeri (arrow). (C) A bottom-burrowing larva of A. sieboldii (arrow); Inset shows an exposed larva. (D) Adults of A. parthenope in twilight flight.

Fig. 8.

Phylogenetic relationship of opsin genes of insects (red shade), mantis shrimps (yellow shade), water flea (blue shade), and human. A neighbor-joining phylogeny inferred from 979 aligned amino acid sites is shown. On each node, bootstrap values are indicated in the order of neighbor-joining method/maximum-likelihood method. Accession numbers or annotation IDs are in brackets. Classification of the opsin genes is indicated on the right side.