Molecular Tuning of Rhodopsins for High Visual Sensitivity in Different Light Environments : Variation in Absorbance Spectrum and Opsin Sequence within and between Species

Visual pigments of different animal species must have evolved at some stage to match the prevailing light environments, since all visual functions depend on their ability to absorb available photons and transduce the event into a reliable neural signal. There is a large literature on correlation between the light environment and spectral sensitivity between different fish species. However, little work has been done on evolutionary adaptation between separated populations within species. More generally, little is known about the rate of evolutionary adaptation to changing spectral environments. The objective of this thesis is to illuminate the constraints under which the evolutionary tuning of visual pigments works as evident in: scope, tempo, available molecular routes, and signal/noise trade-offs. Aquatic environments offer Nature s own laboratories for research on visual pigment properties, as naturally occurring light environments offer an enormous range of variation in both spectral composition and intensity. The present thesis focuses on the visual pigments that serve dim-light vision in two groups of model species, teleost fishes and mysid crustaceans. The geographical emphasis is in the brackish Baltic Sea area with its well-known postglacial isolation history and its aquatic fauna of both marine and fresh-water origin. The absorbance spectrum of the (single) dim-light visual pigment were recorded by microspectrophotometry (MSP) in single rods of 26 fish species and single rhabdoms of 8 opossum shrimp populations of the genus Mysis inhabiting marine, brackish or freshwater environments. Additionally, spectral sensitivity was determined from six Mysis populations by electroretinogram (ERG) recording. The rod opsin gene was sequenced in individuals of four allopatric populations of the sand goby (Pomatoschistus minutus). Rod opsins of two other goby species were investigated as outgroups for comparison. Rod absorbance spectra of the Baltic subspecies or populations of the primarily marine species herring (Clupea harengus membras), sand goby (P. minutus), and flounder (Platichthys flesus) were long-wavelength-shifted compared to their marine populations. The spectral shifts are consistent with adaptation for improved quantum catch (QC) as well as improved signal-to-noise ratio (SNR) of vision in the Baltic light environment. Since the chromophore of the pigment was pure A1 in all cases, this has apparently been achieved by evolutionary tuning of the opsin visual pigment. By contrast, no opsin-based differences were evident between lake and sea populations of species of fresh-water origin, which can tune their pigment by varying chromophore ratios. A more detailed analysis of differences in absorbance spectra and opsin sequence between and within populations was conducted using the sand goby as model species. Four allopatric populations from the Baltic Sea (B), Swedish west coast (S), English Channel (E), and Adriatic Sea (A) were examined. Rod absorbance spectra, characterized by the wavelength of maximum absorbance (λmax), differed between populations and correlated with differences in the spectral light transmission of the respective water bodies. The greatest λmax shift as well as the greatest opsin sequence difference was between the Baltic and the Adriatic populations. The significant within-population variation of the Baltic λmax values (506-511 nm) was analyzed on the level of individuals and was shown to correlate well with opsin sequence substitutions. The sequences of individuals with λmax at shorter wavelengths were identical to that of the Swedish population, whereas those with λmax at longer wavelengths additionally had substitution F261F/Y in the sixth transmembrane helix of the protein. This substitution (Y261) was also present in the Baltic common gobies and is known to redshift spectra. The tuning mechanism of the long-wavelength type Baltic sand gobies is assumed to be the co-expression of F261 and Y261 in all rods to produce ≈ 5 nm redshift. The polymorphism of the Baltic sand goby population possibly indicates ambiguous selection pressures in the Baltic Sea. The visual pigments of all lake populations of the opossum shrimp (Mysis relicta) were red-shifted by 25 nm compared with all Baltic Sea populations. This is calculated to confer a significant advantage in both QC and SNR in many humus-rich lakes with reddish water. Since only A2 chromophore was present, the differences obviously reflect evolutionary tuning of the visual protein, the opsin. The changes have occurred within the ca. 9000 years that the lakes have been isolated from the Sea after the most recent glaciation. At present, it seems that the mechanism explaining the spectral differences between lake and sea populations is not an amino acid substitution at any other conventional tuning site, but the mechanism is yet to be found.

Viimeisten vuosikymmenten aikana näön ekologista tutkimusta on tehty vertailemalla eri lajeja ja niiden elinympäristöjä. Nyt valmistuneessa tutkimuksessa valoympäristön vaikutusta näköpigmentin evolutiiviseen säätymiseen tarkasteltiin uudella tavalla. Tutkimukseen valittiin saman kala- ja katkalajin erilaisissa valoympäristöissä eläviä populaatioita. Työssä keskityttiin valoympäristön vaikutuksen arviointiin pimeänäön pigmenttimolekyyli, rodopsiinin osalta: sen aminohapporakenteeseen ja spektraaliherkkyyteen eli siihen mitä aallonpituuksia pigmenttimolekyyli vastaanottaa. Samalla arvioitiin evolutiivisten muutosten nopeutta, jos eläimen valoympäristö muuttuu. Tutkimusympäristöksi valittiin Itämeri, jonka jääkausiin liittyvät kehitysvaiheet ja eläinlajiston muuttohistoria tunnetaan tarkasti. Menetelminä käytettiin 1) mikrospektrofotometriaa näköpigmenttien spektraaliherkkyyden mittaamiseksi ja 2) sekvensointia näköpigmentin aminohapporakenteen selvittämiseksi. Tutkimuksen kalamallilajina käytettiin hietatokkoa ja sen neljää Euroopan rannikolla esiintyvää populaatiota. Populaatioiden välillä havaittiin pieniä, mutta merkityksellisiä eroja spektraaliherkkyysarvossa ja rodopsiiniproteiinin aminohapporakenteessa. Tulokset voidaan selittää hyvin valoympäristön erojen perusteella. Lisäksi havaittiin, että Itämeren hietatokkopopulaatio koostui kahdesta pienemmästä alapopulaatiosta, joilla näön spektraaliherkkyys ja proteiinin aminohapporakenne erosivat toisistaan. Ilmeisesti Itämeren vaihtelevat elinolosuhteet ylläpitävät Itämeren hietatokkopopulaation kahtiajakautumista. Toisella mallilajilla, Mysis jäännehalkoisjalkaisella, havaittiin myös valoympäristöön sopeutumista. Mysiksen järvipopulaation spektraaliherkkyys oli selvästi erilaiselle aallonpituusalueelle kuin Itämeressä elävällä populaatiolla vastaten siten paremmin järven valoympäristöä. Tutkimukseen liittyi myös 22 Itämeressä esiintyvän kalalajin spektraaliherkkyysmittaukset. Näiltä lajeilta mitattiin lisäksi niiden järvessä tai meressä elävän populaation spektraaliherkkyysarvo, jotta valoympäristöön sopeutumista voitiin arvioida. Tutkituista lajeista hietatokon, silakan ja kampelan näköpigmentit ovat sopeutuneet Itämeren valaistusolosuhteisiin. Sen sijaan järvien erilaisin valoympäristöihin sopeutumista ei havaittu. Meriympäristöstä kotoisin olevilla lajeilla muutos on todennäköisesti tapahtunut rodopsiiniproteiinin aminohappojärjestyksessä. Muutokset näköpigmenttimolekyylin rakenteessa ovat olleet nopeita. Ne ovat todennäköisesti tapahtuneet viimeisten 9000 vuoden kuluessa, edellisen jääkauden jälkeen.