Colour vision of Ischnura heterosticta (Insecta: Odonata): Role in sexual selection, communication and visual plasticity Shao-chang Huang BSc, MSc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Queensland Brain Institute Abstract Sensory systems are important for any life task of an animal. Vision, and colour vision in particular, is essential for visual–based insects, such as Odonata (damselflies and dragonflies), many of which display colour patterns on their bodies. Numerous behavioural studies suggest that the diverse colour patterns function as a means for intersexual, intrasexual, interspecific, or intraspecific recognition and play a role in sexual selection, particularly in ischnuran damselflies that have sex- limited polymorphism. However, to date there are no comprehensive studies linking behavioural to electrophysiological evidence to support the role of colour patterns and colour vision in mate choice in this group of insects. In my Ph.D. thesis, I investigated the function of body colouration and colour vision in sexual selection and communication of the Australian polymorphic damselfly, Ischnura heterosticta, and examined the mechanisms underlying its colour vision and colour discrimination ability. In an observational study (Chapter 2), I surveyed I. heterosticta reproductive behaviours and daily activity patterns in the field, providing the first detailed account of their colour morphs and behavioural biology. Andromorph females are blue like males, and gynomorph females have colour morphs in green, intermediate, and grey. Mating pairs, usually with gynomorphs, are formed after dawn and mating can last up to 3-4 hours. Oviposition occurs in the days after mating, and ovipositing females are subjected to aggressive male harassment, which varies with female colouration. These data provided the biological foundation regarding colour-based sexual selection in I. heterosticta investigated in the following chapters. In the next set of experiments (Chapter 3), I discovered a unique irreversible ontogenetic colour change in females of I. heterosticta: blue andromorphs are sexually immature individuals that emerge from nymphs. After 4 to 7 days, they turn into green-grey gynomorphs to signal their sexual maturity and advertise readiness to mate. Gynomorphs are the preferred mating partners, which suggests that blue andromorphs avoid unnecessary long mating during sexual immaturity through their male-mimicking colouration. This discovery provided the first indication that colour plays a key role in mate choice and that female polymorphism in I. heterosticta may be maintained via colour signals. In the next study (Chapter 4), I tested whether and how male mating preference was associated with colour cues by manipulating female body colours artificially. The outcomes strongly suggest that female body colouration is the key visual signal for mate choice in I. heterosticta. Males always preferred gynomorph females irrespective of female ratio or prior mating experience. Only under low ambient light males of I. heterosticta occasionally misidentified blue andromorphs as mating ii partners, likely as consequence of the insufficient light. This finding provided the behavioural indication that ischnuran damselflies rely on colour vision for mate recognition. The next study (Chapter 5) examined the morphological structure of the compound eyes of I. heterosticta using histological approaches. The diameter of an ommatidium is about 10 μm, and its length can be up to 400 μm depending on the area of the retina. Several retinula cells were identified distributed along the ommatidium. This provided essential background information on the visual anatomy in this species, and the anatomical indication that I. heterosticta may have colour vision, which was investigated in the next chapter. I used electrophysiological approaches (Chapter 6) to investigate the spectral sensitivities of the visual system of I. heterosticta. The results showed that they have trichromatic vision, being able to detect UV, blue and green light. Contrast calculations confirmed that their colour discrimination ability enables them to distinguish the spectral differences of individual female morphs. These results were congruent with the behavioural findings from previous chapters, showing that I. heterosticta use colour signals for mate choice in context of sexual selection. The final study (Chapter 7) examined the molecular basis of colour vision in I. heterosticta, and investigated whether it is plastic and dependent on light experience. I identified three types of opsin genes in I. heterosticta, corresponding to three wavelengths of light (UV, blue and green), confirming that this species is trichromatic. I found that during development opsins are differentially expressed in nymphs (aquatic) and adults (terrestrial/aerial), and opsin expression also changes when individuals are reared or kept under different ambient light conditions. These findings were further supported by electrophysiological experiments, showing an equivalent change in response. This demonstrates that colour vision in I. heterosticta is plastic and that ambient light induces visual plasticity. This plasticity facilitates adaptation to the changing visual environments during development, and ensures that adults can detect the crucial colour cues that are key for mate choice and reproduction. This thesis is the first comprehensive study examining the functional role of colour vision and body colouration in a polymorphic ischnuran damselfly. This body of work also contributes important new insights into the mechanisms underlying sexual selection, evolution and maintenance of female-limited polymorphism in I. heterosticta. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. Shao-chang Huang iv Publications during candidature Conference Abstracts Huang, S.-c., Reinhard, J., Chiou, T.-h. and Marshall, J. Colour vision influences mate choice in the polymorphic damselfly Ischnura heterosticta. 10th International Congress of Neuroethology, College Park, USA, 2012. Huang, S.-c. and Reinhard, J. Female body colour determines mating preference in the polymorphic damselfly Ischnura heterosticta. Conference of the Australasian Society for the Study of Animal Behaviour (ASSAB), Geelong, Australia, 2012. Huang, S.-c., Marshall, J. and Reinhard, J. Prey catching ability in damselflies: function of ommatidia structure, viewing angle, and visual resolution. 2nd QBI-MCN Symposium on Systems Neuroscience, Munich, Germany, 2012. Huang, S.-c., Reinhard, J., Chiou, T.-h. and Marshall, J. Colour vision influences mate choice in the polymorphic damselfly Ischnura heterosticta. Applied Vision Association (AVA) Animal Vision Meeting, Brighton, UK, 2012. Huang, S.-c., Reinhard, J. and Chiou, T.-h. Colour vision in the damselfly Ischnura heterosticta: a function in sexual selection. 32nd Annual Meeting of the Australian Neuroscience Society (ANS), Gold Coast, Australia, 2012. Huang, S.-c., Reinhard, J., Chiou, T.-h. and Marshall, J. Colour vision in the damselfly Ischnura heterosticta: a function in sexual selection. Colour and Colour Vision Workshop, Sydney, Australia, 2011. Huang, S.-c. and Reinhard, J. Love enters through the eyes: Sexual selection via female colour polymorphism in the damselfly Ischnura heterosticta (Insecta: Odonata). Conference of the Australasian Society for the Study of Animal Behaviour (ASSAB), Adelaide, Australia, 2011. v Peer-reviewed papers Huang, S.-c., Chiou, T.-h, Marshall, J. and Reinhard, J. (2014) Spectral sensitivities and color signals in a polymorphic damselfly. PLoS One 9: e87972. Huang, S.-c. and Reinhard, J. (2012) Color change from male-mimic to gynomorphic: a new aspect of signaling sexual status in damselflies (Odonata, Zygoptera). Behavioral Ecology 23: 1269-1275. Huang, S.-c., Reinhard, J. and Norval, G. (2012) The reproductive biology and daily activity patterns of Ischnura heterosticta (Burmeister) in eastern Australia (Zygoptera: Coenagrionidae). Odonatologica 41: 99-107. Manuscripts under review Huang,