Evolution and Expression Plasticity of Opsin Genes in a Fig Pollinator, Ceratosolen solmsi

Ceratosolen solmsi. PLoS ONE 8(1): e53907. doi:10.1371/journal.pone.0053907 Evolution and Expression Plasticity of Opsin Genes in a Fig Pollinator, Ceratosolen solmsi Bo Wang 0 Jin-Hua Xiao 0 Sheng-Nan Bian 0 Li-Ming Niu 0 Robert W. Murphy 0 Da-Wei Huang 0 Keith A. Crandall, George Washington University, United States of America 0 1 Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences , Beijing , China , 2 Graduate School of the Chinese Academy of Sciences , Beijing , China , 3 Plant Protection College, Shandong Agricultural University , Tai'an, China , 4 Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences , Danzhou, Hainan , China , 5 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences , Kunming, China, 6 Department of Natural History , Royal Ontario Museum , Toronto, Ontario , Canada Figs and fig pollinators have co-evolved species-specific systems of mutualism. So far, it was unknown how visual opsin genes of pollinators have evolved in the light conditions inside their host figs. We cloned intact full-length mRNA sequences of four opsin genes from a species of fig pollinator, Ceratosolen solmsi, and tested for selective pressure and expressional plasticity of these genes. Molecular evolutionary analysis indicated that the four opsin genes evolved under different selective constraints. Subsets of codons in the two long wavelength sensitive opsin (LW1, LW2) genes were positively selected in ancestral fig pollinators. The ultraviolet sensitive opsin (UV) gene was under strong purifying selection, whereas a relaxation of selective constrains occurred on several amino acids in the blue opsin. RT-qPCR analysis suggested that female and male fig pollinators had different expression patterns possibly due to their distinct lifestyles and different responses to light within the syconia. Co-evolutionary history with figs might have influenced the evolution and expression plasticity of opsin genes in fig pollinators. - Funding: This project was supported by the National Natural Science Foundation of China (NSFC grant no. 31090253, 31172072, 31210103912), partially by Major Innovation Program of Chinese Academy of Sciences (KSCX2-EW-Z-2), a grant (No. O529YX5105) from the Key Laboratory of the Zoological Systematics and Evolution of the Chinese Academy of Sciences and National Science Fund for Fostering Talents in Basic Research (Special subjects in animal taxonomy, NSFCJ0930004), and by a Natural Sciences and Engineering Research Council (Canada) Discovery Grant (3148). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Opsin genes encode proteins that are members of the G protein coupled receptors (GPCR). These proteins, which have molecular masses of 3050 kDa, can form visual pigments with a covalently bonded light-absorbing chromophore, typically the 11-cis-retinal chromophore [18]. In animals, photosensitivity, which serves many purposes including vision, is mainly conferred by visual pigments [9]. Insects occur in various habitats and experience a great diversity of visual conditions. Not surprising, insects have a concomitantly diverse array of visual receptors [2]. Many studies reveal adaptations of insects to their specific light environment or behavior by analyzing the opsin genes [1020]. Figs (family Moraceae) and their insect pollinators (family Agaonidae) form one of the best known examples of obligate mutualism [21]. Female wasps enter receptive fruits (syconium), the unique, closed inflorescence of figs, to lay eggs and the process results in the pollination of flowers [22]. As the larvae develop, they induce galls. Male fig wasps cannot leave the cavity of fig fruits, whereas the new generation of female pollinators is responsible for colonizing new hosts [22,23]. This fine-tuned mutualistic system is at least tens of millions of years old [2426]. Sex-specific morphological and behavioral specializations in the fig wasps are associated with life cycles of their hosts [22,23]. The most remarkable specialization is the extreme extent of sexual dimorphism. Female pollinators possess functional wings and compound eyes, while males are apterous and have vestigial compound eyes and antennae. Additionally, ocelli are absent in male pollinators [23]. Phenotypic adaptation has a genetic basis and this should be reflected in gene sequences and/or patterns of gene expression. Herein, we explore how opsin genes of fig pollinators have evolved and are expressed. Molecular evolutionary analyses and reverse transcription quantitative PCR (RT-qPCR) experiments on Ceratosolen solmsi are employed to answer a suite of questions in fig pollinators. (1) What opsin genes do fig pollinators express? (2) How did opsin gene sequences evolve in fig pollinators? (3) What are the circadian rhythms of opsin gene expression in fig pollinators? (4) Does the expression of opsin genes differ between females and males and if so how? Materials and Methods The sampling of living material involved in our experiments includes figs (Ficus hispida) and fig pollinators (C. solmsi). All necessary permits were obtained for the field sampling. Collection permits were provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. Sample Collection and Experimental Design Fig fruits of F. hispida were collected from Danzhou (N.19u3092999, E.109u299699), Hainan province, China in October 2010 and October 2011. All fig fruits were collected at the same developmental stage several days before becoming ripe. Identity of the fig pollinator C. solmsi was confirmed using morphological traits ascertained with a Nikon SMZ80 microscope. We subjected the fig fruits to different treatments of light and then collected adult fig pollinators. Half of the fig fruits were kept in a darkroom (0:24 L:D), and the others were kept in an environmental chamber for light treatment with a daily light cycle (,15:9 L:D). After two days of treatments, every 3 hours (3:00, 6:00, 9:00, 12:00, 15:00, 18:00, 21:00, 24:00) figs were flash-frozen in liquid nitrogen. Subsequently, female and male pollinators were removed from the inside of the syconia and immersed into Sample Protector (TaKaRa, China). In addition, to evaluate how opsin gene expression changes outside the fig fruits, we also collected females that had emerged from the syconia under light treatment at each time point and exposed them to light for 3 additional hours before flash-freezing. Males were not submitted to this treatment because they seldom emerge from the syconia. Individuals for all insect samples were at the same developmental stage. In tot (...truncated)