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Cave-adapted animals are an excellent model for studying the molecular mechanisms underlying the convergent evolution of various traits such as pigment loss or albinism. Characterizing the nature of biological pigments in phylogenetically closest surface relatives is the first step that must be taken to understand this phenomenon. To date, the only information on the mechanisms of pigment regression in troglobites has been published for melanin pigment, while the mechanisms of ommochrome loss remain largely unknown. Ommochromes are tryptophan derivatives and generally a less studied class of biological pigments found exclusively in protostomes including crustaceans. Our study was focused on surface and cave crustaceans of the order Isopoda. Using a biochemical approach to extract and detect ommochromes in conjunction with liquid chromatography coupled to mass spectrometry (UPLC- MS), we identified for the first time and to the best of our knowledge ommochromes in three groups of isopods: Sphaeromatidae (Flabellifera), family Trichoniscidae (Oniscidea), and genus Proasellus (Asellota). Using the same method, we quantified the precursors of ommochrome synthesis in both surface and cave representatives to determine which step of the biosynthetic pathway may be defective in albino cave dwellers. In general, our results show an accumulation of tryptophan in cave dwellers, suggesting that the disruption of ommochrome synthesis is likely at the very beginning of this anabolic cascade. Our data suggest that isopods are a good group to study the precise molecular mechanisms underlying albinism as a consequence of ommochrome deficiency. To address this issue, we will apply RNA-Seq and assemble de novo transcriptomes for several pairs of surface-cave relatives. We plan to compare transcriptomic data for candidate genes to understand expression changes and/or possible inactivating mutations that may be responsible for albinism in different isopod lineages.

Albinism or pigment loss is an adaptation to a subterranean lifestyle and one of the prime examples of convergent evolution. It occurs in diverse cave-adapted animal groups regardless of the type of pigment they contain. The causes of melanin loss have been described in only a couple of cave-dwellers, while the mechanisms of albinism as a consequence of any other pigment deficiency are completely unexplored. Ommochromes are tryptophan derivates and a less studied class of pigments found in a wide range of invertebrates. Our research was focused on related crustaceans of the order Isopoda. Using a biochemical approach to extract and detect ommochromes in conjunction with liquid chromatography coupled to mass spectrometry (UPLC-MS) the type of biological pigments in urface isopods was determined. We identified for the first time and to the best of our knowledge ommochromes in three groups of surface isopods: families Sphaeromatidae (Flabellifera) and Trichoniscidae (Oniscidea) and the genus Proasellus (Asellota). Additionally, we quantified the precursors of ommochrome synthesis in closely related surface and cave species. Our results suggest that the disruption of ommochrome synthesis is preferentially at the beginning of this anabolic cascade since we observed the accumulation of tryptophan in cave dwellers. These results suggest that isopods are a good model to study the molecular mechanisms underlying albinism as a consequence of ommochrome deficiency. To resolve that issue, we will look for the expression changes and possible inactivating mutations in the candidate genes using a comparative transcriptomic approach on several pairs of surface-cave relatives.

Špiljske životinje pokazuju mnoštvo konvergentnih prilagodbi, a jedna od najpoznatijih je albinizam ili gubitak pigmentacije. Do sada je proučavan uglavnom s aspekta gubitka melanina, dok je evolucija albinizma kao posljedica nestanka bilo kojeg drugog pigmenta, uključujući i omokrome, neistražena. Omokromisu derivati triptofana i česti pigmenti kod beskralježnjaka, uključujući i jednakonožne rakove. Da bise razumjela evolucija albinizma kod špiljskih jednakonožnih rakova, prvo je potrebno potvrditi prisustvo omokroma kod najbližih nadzemnih srodnika. Koristeći tekućinsku kromatografiju u kombinacijis masenom spektrometrijom, prvi put smo, prema našim saznanjima, identificirali omokrome kod triskupine nadzemnih rakova: obitelji Sphaeromatidae (Flabellifera) i Trichoniscidae (Oniscidea) i rodu Proasellus (Asellota). Nadalje, kvantificiralismo prekursore sinteze omokroma u nadzemnim išpiljskim vrstama kako bismo odredili koji je korak biosinteze blokiran kod albino vrsta. Naširezultati ukazuju da je blokada sinteze omokroma dominantno na početku ove anaboličke kaskade budući da smo primijetili nakupljanje triptofana kod špiljskih vrsta. Kako bismo otkrili gene i mutacije koje bi mogle biti odgovorne za gubitak omokroma, sastavilismo de novo transkriptome za nekoliko parova nadzemnih išpiljskih srodnika. Naši preliminarnirezultati pokazuju smanjenu ekspresiju glavnih gena uključenih u sintezu omokroma kod albino špiljskih vrsta iz roda Proasellus.

Gubitak pigmentacije ili albinizam regresivna je osobina životinja prilagođenih životu u špiljama te dobar primjer konvergentne evolucije. Da bi se objasnio ovaj fenomen u troglobiontima, prvo je potrebno okarakterizirati tip bioloških pigmenata u filogenetski najbližim vanjskim srodnicima. Albinizam je do sada proučavan uglavnom s aspekta gubitka melanina, dok točan mehanizam gubitka omokroma ostaje uglavnom nepoznat. Omokromi su derivati triptofana i općenito su slabije proučavana klasa bioloških pigmenata, koja je do sada opisana u protostomičnim organizmima kao što su glavonošci, plošnjaci te člankonošci - kukci i pauci. Prisutnost ovih pigmenata kod rakova, ranije je bila predložena u podredovima jednakonožnih (izopodnih) rakova: Asellota i Oniscidea. Korištenjem biokemijskog pristupa ekstrakcije i detekcije omokroma zajedno sa HPLC- MS analizom, po prvi smo put identificirali omokrome i njihove prekursore u još jednom podredu izopodnih rakova - Flabellifera (porodica Sphaeromatidae). Uz to, prisutnost omokroma potvrđena je kod porodice Trichoniscidae (Oniscidea), kao i rodu Proasellus (Asellota). Štoviše, spomenute pigmentirane skupine izopodnih rakova imaju podzemne albino srodnike na području dinarskog krša, što čini ove parove vrsta dobrim modelom za proučavanje molekularnih mehanizama albinizma kao posljedice nedostatka pigmenta omokroma.

The evolutionary reason why many animals display color changes remains a key question. Solving it requires an integrative understanding of the biological significance of colors. In this work, I investigate the structure–function relationship of color-changing ommochrome pigments and their organelles using analytical chemistry, quantum modelling and subcellular multimodal imaging. This interdisciplinary approach reveals that the photochemical and intracellular bases of ommochromes determine both their color-changing capacities and their chemical reactivity, among other pleiotropic functions. My results support the idea that color changes of ommochromes are a multiscale property that affects biologically relevant functions besides coloration, such as redox and metal homeostasis.; L’évolution de la capacité à changer de couleur chez les animaux est un sujet majeur. Son étude requiert une compréhension intégrée de l’importance biologique des couleurs. Dans ces travaux, j’explore la relation structure–fonction, en termes de changement de couleur, des pigments appelés ommochromes et de leurs organites. J’utilise pour cela la chimie analytique, la modélisation quantique et l’imagerie multimodale à l’échelle subcellulaire. Cette approche interdisciplinaire révèle que les bases photochimiques et intracellulaires des ommochromes déterminent, à la fois, leurs capacités à changer de couleur et leur réactivité chimique. Mes résultats renforcent l’idée que les changements de couleur des ommochromes sont une propriété multi-échelle qui a des effets sur des fonctions biologiques au-delà de la coloration, comme l’homéostasie redox et métallique.