New insights into ion regulation of cephalopod molluscs: a role of epidermal ionocytes in acid-base regulation during embryogenesis

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues: as in teleost fish epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a pCO2 of 0.2-0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization and vital dye staining techniques. We found one group of cells that is recognized by Concavalin A and MitoTracker, which also expresses Na+/H+ exchangers (NHE) and Na+/K+-ATPase. Similar to findings obtained in teleosts these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa which were identified as powerful acid-base regulators during hypercapnic challenges already exhibit strong acid-base regulatory abilities during embryogenesis

Osmoregulation and salinity tolerance in zoeae and juveniles of the snow crab

Osmoregulation and salinity tolerance were studied in zoeae 1 and instar IX juveniles of Chionoecetes opilio. In zoeae 1, the lower and upper lethal salinities for 50% of the animals (LS 50) at 14 °C were about 10 and 42‰ at 24 h, 18 and 41‰ at 48 h, 25 and 38‰ at 96 h. In juveniles, the approximate 48 h LS 50 s at 6 °C were 13.5 and 46‰. Both developmental stages are able to withstand relatively ample but brief variations of salinity. Their short-term euryhalinity is discussed in relation to the salinity of their habitat. Zoeae were hyper-osmoconformers. Juveniles were osmoconformers and isoionic to the external medium except for Mg++ which was hypo-regulated. Isosmotic and isoionic regulation in post-metamorphic stages are presumably an attribute of the family Majidae. The pattern of osmoregulation in zoeae 1 and juveniles relate C. opilio to the first of three groups of species previously characterized by their pattern of ontogeny of osmoregulation

Embryonic occurrence of ionocytes in the european sea bass dicentrarchus labrax emergence of the osmoregulatory function and early adaptation to salinity variations

Le Loup ou Bar Dicentrarchus labrax est une espèce euryhaline dont les femelles pondent généralement en eau de mer. Pendant son cycle de vie, des migrations vers les estuaires et les lagunes, peuvent exposer très tôt les jeunes stades à des variations de salinité. Les mécanismes de l'osmorégulation sont bien connus chez les prélarves, les larves et les adultes de D. labrax en eau de mer (EM, 38) et en eau de mer diluée (EMD, 5), cependant les possibilités d'osmorégulation et leurs mécanismes sont inconnus chez les embryons. Le but de cette étude a été d'évaluer la mise en place de la fonction osmorégulatrice chez les embryons de D. labrax.Tout d'abord le développement embryonnaire des différents sites osmorégulateurs a été décrit, en se focalisant sur le tube digestif, en incluant le pharynx et les premières fentes branchiales. La formation de ces structures débute au stade 12 somites (S) et a été décrite jusqu'à l'ouverture de la bouche, 5 jours après l'éclosion.En second lieu, le lieu et la cinétique d'apparition des premières cellules osmorégulatrices, les ionocytes ont été recherchés. Ces cellules ont été identifiées au stade 12S sur la membrane de la vésicule vitelline et au niveau des premières fentes branchiales et du tube digestif primitif au stade 14S. La fonctionnalité de ces cellules a été étudiée grâce à des immunomarquages des principales protéines transmembranaires impliqués dans l'osmorégulation [l'ATPase Na+/K+ (NKA), le cotransporteur Na+/K+/2Cl- (NKCC) et le canal à chlore (CFTR)], et avec une étude ultrastructurale. Des ionocytes potentiellement fonctionnels sont présents à partir du stade 25S au niveau de la membrane de la vésicule vitelline et du tube digestif primitif, mais les ionocytes des premières fentes branchiales ne sont pas totalement fonctionnels à l'éclosion. L'existence d'un phénomène de boisson passive qui permettrait la régulation hydrique chez D. labrax est envisagé.Finalement, l'osmorégulation embryonnaire existant en EM et en EMD a été étudiée. Des mesures nanoosmométriques des fluides embryonnaires indiquent une capacité à hyper- et hypo-osmoréguler. Cependant, en EMD, des analyses en qRT-PCR et des immunomarquages de NKA, NKCC et CFTR révèlent que les mécanismes de l'hyper-osmorégulation peuvent limiter les pertes ioniques mais ne sont pas suffisamment efficaces pour permettre une acclimatation totale à l'EMD à ce stade très précoce.The European sea bass Dicentrarchus labrax is a euryhaline species which usually spawns in seawater. Due to its life cycle that includes migrations to lagoon and estuaries, young stages can be exposed early to salinity variations. Osmoregulatory patterns are well known in prelarvae, larvae and adults D. labrax in seawater (SW, 38) and in dilute seawater (DSW, 5), but the possibility and mechanisms of embryonic osmoregulation are still unknown. The goal of this study was to investigate the occurence of the omoregulatory function in the embryos of D. Labrax.First, the embryonic development of the different osmoregulatory sites was described, focusing on the digestive system including the pharynx and the first gill slits. The formation of these structures is initialized at stage 12 somites (S) and was described throughout the opening of the mouth five days after hatching.Secondarily, the time and the location of the occurrence of the first osmoregulatory cells, the ionocytes were followed. These cells were identified at stage 12S on the yolk sac membrane and at stage 14S in the first gill slits and in the posterior primitive gut. The functionality of these cells was studied, using immunostaining of the main ionic transporters involved in osmoregulation [the Na+/K+ ATPase (NKA), the Na+/K+/2Cl- cotransporter (NKCC) and the chloride channel (CFTR)], and through ultrastructural investigations. Potentially functional ionocytes are present from stage 25S in the yolk sac membrane and in the gut, but gill slits ionocytes are not fully functional at hatching. Passive drinking is suspected to regulate water balance in D. labrax.Finally, the embryonic osmoregulation in SW and DSW was investigated. Nanoosmometry measurements of the embryonic fluids demonstrated some capabilities of hyper- and hypo-osmoregulation. However, in DSW, qRT-PCR and imunostaining of NKA, NKCC and CFTR, reveal that hyper-osmoregulatory mechanisms can only limit ion loss but are not efficient enough to allow a full acclimation at this early life stage.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Ontogénèse des glandes antennaires chez les crustacés (Etude comparative de leur implication dans l'osmorégulation au cours du développement chez l'écrevisse Astacus leptodactylus et le hormard Homarus gammarus)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Adaptations morpho-fonctionnelles des crustacés caridés et brachyoures à la salinité du milieu hydrothermal profond

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Crabs conquer freshwater and land: Ontogeny of osmoregulation and life-history strategies

Ontogeny of osmoregulation and salinity tolerance were investigated throughout the larval development of two congeneric species of sesarmid crab, Armases ricordi (H. Milne Edwards) and A. roberti (H. Milne Edwards), and compared with previous observations from two further congeners, A. miersii (Rathbun) and A. angustipes (Dana). In the semiterrestrial coastal species A. ricordi, the zoeal stages were only at moderately reduced salinities (17-25.5) capable of hyper-osmoregulation, being osmoconformers at higher concentrations. The megalopa was the first ontogenetic stage of this species, which exhibited significant hyper-osmoregulation at further reduced salinities (≥5), as well as a moderately developed function of hypo-regulation at high concentrations (32-44). The riverine species A. roberti showed similar overall patterns in the ontogeny of osmoregulation, however, also some striking differences. In particular, its first zoeal stage showed already at hatching a strong capability of hyper-osmoregulation in salinities down to 5. Interestingly, this early expressed function became significantly weaker in the subsequent zoeal stages, where survival and capabilities of hyper-osmoregulation were observed only at salinities down to 10. The function of hyper-regulation in strongly dilute media re-appeared later, in the megalopa stage, which tolerated even an exposure to freshwater (0.2). Differential species- and stage-specific patterns of osmoregulation were compared with contrasting life styles, reproductive behaviours, and life-history strategies. In A. ricordi, the larvae are released into coastal marine waters, where salinities are high, and thus, no strong hyper-osmoregulation is needed throughout the zoeal phase. The megalopa stage of this species, by contrast, may invade brackish mangrove habitats, where osmoregulatory capabilities are required. Strong hyper-osmoregulation occurring in both the initial and final larval stages (but not in the intermediate zoeal stages) of A. roberti correspond to patterns of ontogenetic migration in this species, including hatching in freshwater, larval downstream transport, later zoeal development in estuarine waters, and final re-immigration of megalopae and juvenile crabs into limnic habitats, where the conspecific adults live. Similar developmental changes in the ecology and physiology of early life-history stages seem to occur also in A. angustipes. A. miersii differs from all other species, showing an early expression and a gradual subsequent increase of the function of hyper-osmoregulation. This ontogenetic pattern corresponds with an unusual reproductive biology of this species, which breeds in supratidal (i.e. land-locked) rock pools, where variations in salinity are high and unpredictable. Matching patterns in the ontogeny of osmoregulation and life-history strategies indicate a crucial adaptive role of osmoregulation for invasions of (by origin marine) crabs into brackish, limnic and terrestrial environments

Tolérance à la salinité et osmorégulation chez les post-larves de

Chez deux espèces de crevettes pénéides, Penaeus japonicus et P. chinensis, les conditions de survie en fonction de la température et de la salinité ont été déterminées sur les post-larves P20, P40 et P60. Les SL50, 96 heures moyennes pour les trois stades sont respectivement de 19,3 ‰ à 10 °C, 12,1 ‰ à 14 °C, 6,4 ‰ à 18 °C et 5,4 ‰ à 25 °C pour P. japonicus et de 11,3 ‰ à 14 °C, 7,2 ‰ à 18 °C et 11,1 ‰ à 25 °C pour P. chinensis. L'abaissement de la température de 18-25 °C à 10-14 °C diminue fortement la tolérance des post-larves de P. japonicus aux faibles salinités alors que pour P. chinensis ce phénomène est beaucoup moins marqué. Quelle que soit la température, la mortalité est minimum dans les milieux à peu près isosmotiques à l'hémolymphe. Des essais de survie à long terme (35 jours) ont été effectués sur les post-larves des deux espèces. La régulation osmotique des post-larves et des adultes des deux espèces est hyper-osmotique en milieu dilué et hypo-osmotique en eau de mer et 900 mosm.kg−1. Chez P. japonicus il n'y a pas de différence significative entre les courbes de régulation osmotique des P20, P40 et P60 en milieu dilué. Par contre les capacités osmorégulatrices des adultes sont inférieures à celles des post-larves. Chez P. chinensis l'intensité de la régulation en milieu dilué augmente avec l'âge des post-larves et les adultes présentent une capacité hyper-régulatrice supérieure à celle des P60. L'étude de l'action de la température sur l'osmorégulation des P40 de P. japonicus a montré que l'abaissement de la température de 18 à 10 °C pendant 8 jours modifie fortement la régulation qui devient isosmotique en eau de mer. Les différents résultats obtenus permettent de définir les conditions optimales de température et de salinité pour les élevages de post-larves et les ensemencements en bassins ou lagunes

Impact of cadmium on the structure of gills and epipodites of the shrimp

The objective of this study was to investigate the impact of cadmium on the structure of gills and epipodites in late juvenile Penaeus japonicus. Cadmium titrations were performed by atomic absorption flame spectrophotometry, and structural observations were performed through light- and electron-microscopy. The cadmium concentration increased in different tissues (mainly gills, epipodites and hepatopancreas) according to the external cadmium concentration and the exposure time. The structural changes which occurred in the gill and epipodite cells appeared to be a function of cadmium accumulation in these tissues. Gill cells of shrimps exposed to 2 and 200 μg Cd·L−1 for 15 d did not display discernible structural changes. An increased number of nephrocytes was noted in gill filaments of shrimps exposed to 200 μg Cd·L−1. Exposure to 2 000 and 4 000 μg Cd·L−1 during 4 d resulted in profound structural changes. The epithelial cells were necrosed, disorganized and vacuolized. Exposure to 2 μg Cd·L−1 for 15 d did not result in discernible structural changes of epitodite cells. Exposure to 200 μg Cd·L−1 for 15 d, 2 000 and 4 000 μg Cd·L−1 during 4 d induced profound alterations of the epipodites: increased thickness, decreased number of apical microvilli, basal infoldings and mitochondria, occurrence of pseudomyelinic structures. These alterations are discussed in relation to the respiratory and osmoregulatory functions of gills and epipodites