京都大学 / 新制・課程博士 / 博士(理学) / 甲第23042号 / 理博第4719号 / 新制||理||1676(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 沼田 英治, 教授 曽田 貞滋, 准教授 森 哲 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The intertidal zone is a dynamic environment that fluctuates with the 12.4-h tidal and 24-h light/dark cycle to predictably alter food availability, temperature, air exposure, wave action, oxygen partial pressure, and osmotic conditions. Intertidal sessile bivalves exhibit behavioral or physiological changes to minimize the persistent challenges of fluctuating environmental conditions, such as adjusting gaping behavior and heart rate. At the cellular level, transcriptomic studies on mussels’ baseline circadian and circatidal rhythms have determined that the circadian rhythm is the dominant transcriptional rhythm. However, as proteins reflect the basic molecular phenotype of an organism and their abundance may differ greatly from that of mRNA, these methods could fail to detect important cyclical changes in the proteome that cope with the regular stress of tidal rhythms. For this study, we acclimated intertidal Mytilus californianus to circadian (12:12 h light/dark cycle) and circatidal (6:6 h tidal cycle) conditions in a tidal simulator and sampled gill tissue from mussels every 2 h for 48 h for proteomic analysis. Approximately 86% of the proteins that were detected exhibited rhythmicity over the time course. The circadian cycle primarily determined the cyclic abundance of energy metabolism proteins, pivoting around the transition to the nighttime high tide. The tidal cycle contributed to alterations in cytoskeletal components, ER protein processing and vesicular trafficking, extracellular matrix and immune proteins, and oxidative stress and chaperoning proteins. We also found evidence that post-translational modifications may be important for driving these rhythms, as acetylation and phosphorylation motifs were enriched in the rhythmic proteins and we identified rhythms in elements of methylation, mitochondrial peptide processing, and acylation. These dynamic changes in proteins across numerous functional categories indicate that the combination of circadian and tidal cycles drive complex cellular changes to coordinate processes in a changing environment. This variation clearly shows that differential expression studies and biomonitoring efforts cannot assume a static baseline of cellular conditions in intertidal mussels.

Muitos trabalhos sobre comportamento consideram que os ritmos observados são simplesmente uma resposta aos estímulos ambientais. No entanto, desde o século XVIII, sabe-se que a temporização é real e muitos organismos exibem ritmos endógenos que são arrastados por zeitgebers ou \"doadores de tempo\". Para a grande maioria dos seres vivos, o principal zeitgeber é o ciclo claro/escuro (CE). No entanto, para os organismos das zonas entremarés, o ciclo das marés é tão importante quanto o ciclo de luz. A maré e seus componentes agindo como zeitgeber têm sido estudados com invertebrados e pouca atenção tem sido dada aos peixes que vivem nas zonas entremarés. Uma das espécies mais comuns destes ambientes é o Bathygobius soporator, conhecido como amborê, e o presente trabalho teve como objetivo verificar a presença de ritmicidade em sua atividade locomotora, avaliando as contribuições endógenas e exógenas dos ciclos de luminosidade, da variação da coluna d\'água e da turbulência da água sobre este ritmo. Para isso, os amborês foram mantidos em aquários individuais onde as condições abióticas puderam ser controladas de acordo com os experimentos em andamento. A atividade locomotora foi registrada com câmeras de segurança e fontes de luz infravermelha acopladas a um vídeo \"time-lapse\" que fez o registro de um quadro a cada trinta segundos. As distâncias percorridas a cada trinta segundos foram categorizadas em quatro classes discretas, e esses valores foram agrupados a cada trinta minutos, compondo 48 pontos a cada 24 horas. A análise das séries temporais foi feita através do programa \"El Temps\" que gerou actogramas e periodogramas de Lomb-Scargle, que permitem identificar ritmos com períodos significativos. Diante dos resultados obtidos, pôde-se concluir que o ciclo CE e de variação na coluna d\'água são zeitgebers, enquanto a turbulência é um agente mascarador para a ritmicidade locomotora dos amborês. Concluiu-se, também, que os relógios biológicos nos amborês não são rígidos e possuem acoplamento extremamente frágil, gerando uma alta plasticidade na expressão dos ritmos endógenos e exógenos, o que está de acordo com o modo de vida dos amborês observados no ambiente natural. / Many organisms\' rhythms are considered to be simple reactions to the cyclical changes in the environment. However, the endogenous rhythms entrained by zeitgebers are well known and have been described for many organisms since the 18th century. Although the light/dark cycle (LD) is the main zeitgeber for most living beings, the tidal cycles are as important for the intertidal organisms. It has been studied as a zeitgeber mainly for invertebrates, and little attention has been given to the intertidal fish. Bathygobius soporator, also known as frillfin goby, is one of the most abundant species in the tropical intertidal zone, and this project was designed to verify the rhythmicity in its locomotor activity, and measure how the LD cycle, the cyclical change in water level, and the turbulence cycle affect the endogenous and/or exogenous aspects of this rhythm. The gobies were kept in individual aquariums where all abiotic conditions were controlled according to the requirements of each experiment. The locomotor activity was recorded with security cameras and infrared light bulbs connected to a time-lapse video recorder that taped one frame every thirty seconds. The distance moved by each fish every thirty seconds was categorized into four discrete classes. The values assigned to these classes of movements were added into thirty minutes blocks, thus giving 48 numbers for every 24 hours. The collected data was analyzed by the \"El Temps\" software that provided actograms and Lomb-Scargle periodograms which allowed the detection of rhythms and significant periods. The results led us to conclude that LD cycle and the cyclical change in water level are zeitgebers to locomotor activity of B. soporator, and that the turbulence is a masking agent. We could also affirm that the frillfin gobies\' biological clocks are weak and have a very fragile coupling, giving the fish a malleable expression of their endogenous and exogenous rhythms. This concurs with the natural history of B. soporator.

This dissertation investigated larval release and larval behavior of the Caribbean spiny lobster Panulirus argus and the spotted spiny lobster P. guttatus. These species were examined under laboratory conditions to determine the phase relationship between larval release and natural environmental cycles. P. argus displayed a nocturnal tidal rhythm, while P. guttatus displayed a circadian rhythm in larval release. P. argus releases larvae near the time of nocturnal high slack water, while P. guttatus released larvae near the time of sunrise. The role of 'pumping pheromones' in controlling larval release behaviors was tested by measuring the pumping response of ovigerous P. argus to (1) hatch water, (2) homogenized-embryo water, (3) embryo-conditioned water, and (4) water containing homogenized-egg cases. Lobsters with late-stage embryos displayed increased pleopod pumping with increased concentration of hatch water. Water individually conditioned with homogenized late-stage embryos, intact late-stage embryos, and homogenized egg-cases induced pumping activity in females with late-stage embryos, indicating the presence of a chemical cue. I quantified pumping responses upon exposure to synthetic peptides to determine if they mimicked pheromones that induce larval release behaviors. Pumping behavior was evoked by oligopeptides with a basic amino acid at the carboxy-terminus, preceded by several neutral amino acids. Carboxyl-terminal arginine peptides serve as pheromone mimics. I investigated whether these peptides originate from the action of trypsin-like enzymes by conducting a bioassay measuring pumping activity of ovigerous P. argus subjected to increasing concentrations of trypsin, trypsin inhibitor, and a combination of the two. Pumping activity increased with increasing concentrations of trypsin and trypsin inhibitor, while behaviors ceased when ovigerous females were subjected to a complex of the two. Pheromones are generated by trypsin-like enzymes assisting in the degradation of the egg membranes at the time of hatching. Vertical swimming behaviors of stage-I phyllosoma larvae of P. argus and P. guttatus were observed under laboratory conditions. P. argus larvae displayed a pattern of twilight vertical migration, while P. guttatus larvae displayed nocturnal diel vertical migration (DVM). Rhythms persisted for 5-6 cycles under constant conditions, indicating that an endogenous rhythm in activity plays a proximate role in DVM for both species. / Dissertation

Muitos trabalhos sobre comportamento consideram que os ritmos observados são simplesmente uma resposta aos estímulos ambientais. No entanto, desde o século XVIII, sabe-se que a temporização é real e muitos organismos exibem ritmos endógenos que são arrastados por zeitgebers ou \"doadores de tempo\". Para a grande maioria dos seres vivos, o principal zeitgeber é o ciclo claro/escuro (CE). No entanto, para os organismos das zonas entremarés, o ciclo das marés é tão importante quanto o ciclo de luz. A maré e seus componentes agindo como zeitgeber têm sido estudados com invertebrados e pouca atenção tem sido dada aos peixes que vivem nas zonas entremarés. Uma das espécies mais comuns destes ambientes é o Bathygobius soporator, conhecido como amborê, e o presente trabalho teve como objetivo verificar a presença de ritmicidade em sua atividade locomotora, avaliando as contribuições endógenas e exógenas dos ciclos de luminosidade, da variação da coluna d\'água e da turbulência da água sobre este ritmo. Para isso, os amborês foram mantidos em aquários individuais onde as condições abióticas puderam ser controladas de acordo com os experimentos em andamento. A atividade locomotora foi registrada com câmeras de segurança e fontes de luz infravermelha acopladas a um vídeo \"time-lapse\" que fez o registro de um quadro a cada trinta segundos. As distâncias percorridas a cada trinta segundos foram categorizadas em quatro classes discretas, e esses valores foram agrupados a cada trinta minutos, compondo 48 pontos a cada 24 horas. A análise das séries temporais foi feita através do programa \"El Temps\" que gerou actogramas e periodogramas de Lomb-Scargle, que permitem identificar ritmos com períodos significativos. Diante dos resultados obtidos, pôde-se concluir que o ciclo CE e de variação na coluna d\'água são zeitgebers, enquanto a turbulência é um agente mascarador para a ritmicidade locomotora dos amborês. Concluiu-se, também, que os relógios biológicos nos amborês não são rígidos e possuem acoplamento extremamente frágil, gerando uma alta plasticidade na expressão dos ritmos endógenos e exógenos, o que está de acordo com o modo de vida dos amborês observados no ambiente natural. / Many organisms\' rhythms are considered to be simple reactions to the cyclical changes in the environment. However, the endogenous rhythms entrained by zeitgebers are well known and have been described for many organisms since the 18th century. Although the light/dark cycle (LD) is the main zeitgeber for most living beings, the tidal cycles are as important for the intertidal organisms. It has been studied as a zeitgeber mainly for invertebrates, and little attention has been given to the intertidal fish. Bathygobius soporator, also known as frillfin goby, is one of the most abundant species in the tropical intertidal zone, and this project was designed to verify the rhythmicity in its locomotor activity, and measure how the LD cycle, the cyclical change in water level, and the turbulence cycle affect the endogenous and/or exogenous aspects of this rhythm. The gobies were kept in individual aquariums where all abiotic conditions were controlled according to the requirements of each experiment. The locomotor activity was recorded with security cameras and infrared light bulbs connected to a time-lapse video recorder that taped one frame every thirty seconds. The distance moved by each fish every thirty seconds was categorized into four discrete classes. The values assigned to these classes of movements were added into thirty minutes blocks, thus giving 48 numbers for every 24 hours. The collected data was analyzed by the \"El Temps\" software that provided actograms and Lomb-Scargle periodograms which allowed the detection of rhythms and significant periods. The results led us to conclude that LD cycle and the cyclical change in water level are zeitgebers to locomotor activity of B. soporator, and that the turbulence is a masking agent. We could also affirm that the frillfin gobies\' biological clocks are weak and have a very fragile coupling, giving the fish a malleable expression of their endogenous and exogenous rhythms. This concurs with the natural history of B. soporator.

Les rythmes biologiques constituent une propriété fondamentale de la vie, permettant aux organismes d’appréhender leur environnement et d’en anticiper les changements. Ces rythmes possèdent une double origine : une horloge moléculaire génère ces rythmes, qui sont ensuite synchronisés par des facteurs environnementaux. Si les organismes terrestres sont essentiellement soumis au rythme d’alternance jour/nuit, les espèces marines côtières et estuariennes occupent un biotope plus changeant encore : ils sont en effet également confrontés au rythme des marées. Pourtant, leurs rythmes biologiques sont à ce jour encore mal connus et les mécanismes moléculaires de(s) (l’) horloge(s) sous-jacente(s) ne sont pas caractérisés. Parallèlement, les efflorescences d’algues toxiques, en constante augmentation depuis 1970, constituent un problème écologique majeur, tant local qu’international. Les objectifs du présent travail consistaient à caractériser les rythmes de référence de l’huître Crassostrea gigas et d’en déterminer l’origine (moléculaire, zeitgebers). Il s’agissait ensuite d’étudier les perturbations potentielles de ces rythmes par l’algue toxique Alexandrium minutum, qui produit des toxines paralytiques et est régulièrement présente dans de nombreuses mers du globe. Les travaux réalisés ont permis de mettre en évidence l’existence d’un rythme d’activité valvaire circadien, faible et dual, et n’a pas permis de supporter l’hypothèse de l’existence d’une horloge circatidale. Nous avons formulé l’hypothèse que, chez C. gigas, le rythme tidal est soit d’origine exogène, soit produit par l’horloge circadienne synchronisée par les marées. Les analyses moléculaires réalisées sur le gène circadien cryptochrome dans le muscle adducteur - effecteur du mouvement des valves - ont montré que ce gène oscille à une fréquence tidale dans le muscle strié, favorisant notre seconde hypothèse. Par ailleurs, au-delà des gènes de l’horloge, l’algue A. minutum réprime l’expression de gènes impliqués dans différentes voies métaboliques importantes : la lutte contre le stress oxydant (cat, gpx), la respiration mitochondriale (cox1), l’immunité (ilk), la détoxification (mdr). Finalement nos analyses ont permis de mettre en évidence un impact génotoxique d’A. minutum chez C. gigas. / Biological rhythms constitute a fundamental property of life, allowing organisms to anticipate and adapt to their changing environment. These rhythms present a double origin: they are generated by a molecular clock and synchronized by environmental cues. Whereas terrestrial organisms are mainly subjected to day/night alternation, coastal and estuarine marine species inhabit an even more cycling biotope. They are indeed also submitted to tides. Nevertheless, biological rhythms in marine species are still unrecognized and molecular mechanisms of the underlying oscillator(s) are to date not determined. At the same time, toxic algae blooms are increasing since the 1970s and represent a major ecological concern, both at local and international levels. An objective of the present work was the characterization of biological rhythms in the oyster Crassotrea gigas and of their origin (molecular mechanism, zeitgebers). Furthermore, the work was designed to study the potential disruption of these rhythms by the toxic algal of worldwide distribution Alexandrium minutum, which produces paralytic toxins. The present results show the existence of a weak and dual circadian rhythm of valve activity in the oyster, and do not provide evidence for the existence of any circatidal clock. We suggested that, in the oyster C. gigas, the tidal rhythm could either be generated exogenously or endogenously by the tidally-synchronized circadian clock. Molecular analyses performed on the circadian gene cryptochrome in the adductor muscle of oyster, the effector of valve movements, revealed that Cgcry oscillates at tidal frequency in the striated muscle. This result supports our second hypothesis. Furthermore, A. minutum represses the expression of genes involved in key metabolic pathways: struggle against oxidative stress (cat, gpx), mitochondrial respiration (cox1), immunity (ilk), detoxification (mdr). Moreover, A. minutum impacts C. gigas at DNA level, being thus genotoxic.