Mireille Charmantier-Daures

Ontogeny of Osmoregulation and Salinity Tolerance in Two Decapod Crustaceans: Homarus americanus and Penaeus japonicus

Osmoregulation and salinity tolerance were studied in larvae and post-larvae of two species of crusta ceans, Homarus americanus and Penaeus japonicus, that have different types of embryonic development. In both species, salinity tolerance decreased through the larval stages, was at a minimum at metamorphosis, and increased in post-larval stages. In H. americanus, the lethal salinity for 50%of the animals (24 h LS50) at 20°C was about 17%» at metamorphosis, and about 10.5-12%« in stages IV and V. In P. japonicus, the 24 h LS50 at 25°C was about 25%« at metamorphosis, and about 7-10%o from the sixth post-larval stage onwards. In both species, larvae were hyper-osmoconformers and the osmoregulatory pattern changed after metamor phosis to the juvenile/adult type. In H. americanus, stages IV and V slightly hyper-osmoregulated in low sa linities. In P. japonicus, post-larvae hyper-hypo-regu lated, and their regulatory capacity increased up to the fifth post-larval stage. In young stages of H. americanus and P. japonicus, Osmoregulation and salinity tolerance appear correlated, and are modified at metamorphosis. These results are discussed with regard to their ecological and physiologi cal implications and to previous studies on other species.

Effect of copper on survival and osmoregulation of various developmental stages of the shrimp Penaeus japonicus bate (Crustacea, Decapoda)

The object of this study was to evaluate the acute toxicity of cadmium in different post-embryonic stages of the penaeid shrimp Penaeus japonicus (Bate, 1888) and to determine the effect of sublethal cadmium on the osmoregulatory capacity used as an indicator of physiological condition. Tolerance to cadmium increases with the developmental stage. The least tolerant stages are the nauplii (48 h LC50: 124 μg Cd l-1) and the zoeae (96 h LC50: ≃10 to 30 μg Cd l-1). The most tolerant stages are the postlarvae (96 h LC50: ≃200 to 3500 Cd l-1) and juveniles (96 h LC50: 5500 μg Cd l-1). In juvenile shrimp, 2000 μg Cd l-1 significantly reduce hypo- and hyper-OC. The effect of cadmium on hypo- and hyper-osmoregulatory capacity illustrates a dose- and time-dependent response. Surviving shrimp recover their hypo-osmoregulatory capacity after 6 d of readaptation in cadmium-free seawater.

Impact of cadmium on the structure of gills and epipodites of the shrimp Penaeus japonicus (Crustacea: Decapoda)

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 ug 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 epipodite cells. Exposure to 200 ·g 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.Abstract 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 ug 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 epipodite cells. Exposure to 200 ·g 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.

Ontogeny of osmoregulatory structures and functions in the green crab Carcinus maenas (Crustacea, Decapoda)

SUMMARY The ontogeny of osmoregulation, the development of branchial transporting epithelia and the expression of the enzyme Na+/K+-ATPase were studied in Carcinus maenas (L.) obtained from the North Sea, Germany. Laboratory-reared zoea larvae, megalopae and young crabs were exposed to a wide range of salinities, and hemolymph osmolality was measured after 24 h exposure time (72 h in juveniles). Zoea I larvae slightly hyper-regulated in dilute media (10.2‰ and 17.0‰) and osmoconformed at> 17‰. All later zoeal stages (II-IV) osmoconformed in salinities from 10.2‰ to 44.3‰. The megalopa hyper-regulated at salinities from 10.2 to 25.5‰. Young crabs hyperregulated at salinities from 5.3‰ to 25.5‰, showing an increase in their osmoregulatory capacity. The development of transporting epithelia and the expression of Na+/K+-ATPase were investigated by means of transmission electron microscopy and immunofluorescence microscopy. In the zoea IV, only a very light fluorescence staining was observed in gill buds. Epithelial cells were rather undifferentiated, without showing any features of ionocytes. Gills were present in the megalopa, where Na+/K+-ATPase was located in basal filaments of the posterior gills. In crab I juveniles and adults, Na+/K+-ATPase was noted in the three most posterior pairs of gills, but lacking in anterior gills. Ionocytes could first be recognized in filaments of megalopal posterior gills, persisting through subsequent stages at the same location. Thus, the development of the gills and the expression of Na+/K+-ATPase are closely correlated with the ontogeny of osmoregulatory abilities. The morphological two-step metamorphosis of C. maenas can also be regarded as an osmo-physiological metamorphosis, (i) from the osmoconforming zoeal stages to the weakly regulating megalopa, and (ii) to the effectively hyper-regulating juvenile and adult crabs.

METAMORPHOSIS IN THE LOBSTER HOMARUS (DECAPODA): A REVIEW

ABSTRACT A homarid lobster hatches as a prelarva and then molts successively through three larval instars (stages I, II, and III), one postlarval stage (IV), and numerous juvenile stages. Between the larval and the postlarval stages their anatomy undergoes major changes as the anatomical features characteristic of larval forms are replaced by those found on juveniles and adults. Physiology, ecology, and ethology also change progressively through stages IV and V, during which the lobster shifts from the planktonic behavior of the larva to the benthic existence of the juvenile and adult. This transition from larva to postlarva, specifically the molt from stage III to stage IV, is a true metamorphosis in which the eyestalk neuroendocrine tissue seems to be involved.

Ovarian cycle and embryonic development in Gammarus fossarum: Application for reproductive toxicity assessment

Among freshwater invertebrates, Gammarus fossarum is an important test organism and is currently used in ecotoxicology for acute and chronic assays; nevertheless, reproductive toxicity test methods are not yet available for these species. In the present study, the reproductive cycle in Gammarus fossarum was characterized in order to propose a reproductive toxicity test encompassing molting, follicle growth, and embryonic development that will provide a better understanding of the mode of action of chemicals disrupting these hormone-regulated processes. A detailed description of the reproductive cycle in Gammarus fossarum was obtained. As in some amphipods, molt and reproductive cycles of G. fossarum females occur concurrently, lasting 30 d at 12°C. Each molt stage is characterized by a specific marsupial embryonic development stage and the size of developing follicles visible on the ovarian membrane. Based on these results, a 21-d reproductive toxicity test is proposed for this species. This new bioassay was applied to identify the specific impact of different stressors: cadmium, methomyl, nonylphenol, and a starvation diet. Good reproducibility was obtained for different endpoints under control conditions and throughout the experiments. Preliminary robust reference values or benchmarks were proposed for these endpoints. Cadmium was found to specially inhibit secondary vitellogenesis. Nonylphenol had a specific concentration-dependent effect on embryonic development, with an increase in the percent abnormality from a concentration of 0.05 µg/L. A restricted food diet led to a significant delay in the molt cycle, which in turn induced inhibition of secondary vitellogenesis.

Ontogeny of Osmoregulatory Structures in the Shrimp Penaeus japonicus (Crustacea, Decapoda)

The ontogeny of differentiated osmoregulatory epithelia in the branchial chamber (gills, branchiostegite, pleura, epipodite) was studied by transmission electron microscopy throughout the postembryonic development of Penaeus japonicus. These epithelia are characterized by typical cytological features, including apical microvilli and numerous basal infoldings associated with mitochondria. Differentiated osmoregulatory structures are not observed in the early larval stages: nauplii and zoea 1. In the next larval stages, zoeas and mysis, gills and epipodites are developed as buds only, but osmoregulatory epithelia are observed in the branchiostegites and pleurae. The differentiated structures of the branchiostegites and pleurae are still present in postlarvae but disappear in juveniles and adults. Gills and epipodites develop progressively in the postlarval stages, with early differentiation of osmoregulatory epithelia in the epipodites. In juveniles and adults, the gill epithelium is poorly differentiated; in contrast, abundant differentiated osmoregulatory structures are observed in the epipodites. Ontogenetical comparisons of these observations with previous studies in the same species reveal strong correlations between the development of osmoregulatory epithelia, the ability to osmoregulate, the activity of Na+-K+ ATPase, and salinity tolerance during the postembryonic development of Penaeus japonicus.

Localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk ofHomarus gammarus larvae by immunocytochemistry and in situ hybridization

This study deals with the localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk of larvae and postlarvae ofHomarus gammarus, by immunocytochemistry and in situ hybridization. The CHH and GIH neuropeptides are located in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis, in their tract joining the sinus gland, and in the neurohemal organ itself, at larval stages I, II and III and at the first postlarval stage (stage IV). In all the investigated stages, the mRNA encoding the aforementioned neuropeptides could only be detected in the perikarya of these neuroendocrine cells. In stage I, approximately 19 CHH-immunopositive and 20 GIH-immunopositive cells are present, both with a mean diameter of 7±1 μm. GIH cells are preferably localized at the periphery of the X-organ surrounding the CHH cells that are centrally situated. Colocalization of CHH and GIH immunoreactions can be observed in some cells. The cell system producing CHH and GIH in the larval and postlarval eyestalk is thus functional and is morphologically comparable to the corresponding neuroendocrine center in the adult lobster.

Osmoregulation, Immunolocalization of Na/K-ATPase, and Ultrastructure of Branchial Epithelia in the Developing Brown Shrimp, Crangon crangon (Decapoda, Caridea)

Aspects of osmoregulation including salinity tolerance, osmoregulatory capacity, location of transporting epithelia, and the expression of the enzyme Na+/K+‐ATPase were investigated in the developing brown shrimp, Crangon crangon (L.), from the North Sea. Early developmental stages and large juveniles were exposed to a wide range of salinities for measurement of hemolymph osmolality and survival rates. In media ranging from 17.0‰ to 32.2‰, salinity tolerance was generally high (survival rates: 70%–100%) in all developmental stages, but it decreased in media <10.2‰. Zoeal stages and decapodids slightly hyperregulated at 17.0‰ and osmoconformed in media ≥25.5‰. At 10.2‰, these stages showed high mortality, and only juveniles survived at 5.3‰. Juveniles hyperregulated at 10.2‰ and 17.0‰, osmoconformed at 25.5‰, and hyporegulated in media ≥32.2‰. Large juveniles hyperregulated also at 5.3‰. Expression of the Na+/K+‐ATPase and ion‐transporting cells was located through immunofluorescence microscopy and transmission electron microscopy. In zoeae I and VI, a strong immunoreactivity was observed in cells of the inner epithelia of the branchiostegites and in epithelial cells lining the pleurae. Their ultrastructure showed typical features of ion‐transporting cells. In decapodids and juveniles, ionocytes and expression of Na+/K+‐ATPase remained located in the branchiostegite epithelium, but they disappeared from the pleurae and appeared in the epipodites. In large juveniles, the cells of the gill shaft showed positive immunolabeling and ultrastructural features of ionocytes. In summary, the adult pattern of osmoregulation in C. crangon is accomplished after metamorphosis from a moderately hyperosmoconforming decapodid to an effectively hyper‐/hyporegulating juvenile stage. Salinity tolerance and osmoregulatory capacity are closely correlated with the development of ion‐transporting cells and the expression of Na+/K+‐ATPase.

Immunolocalization of Na+,K(+)-ATPase in the organs of the branchial cavity of the European lobster Homarus gammarus (Crustacea, Decapoda).

Abstract The localization of Na+,K+-ATPase in epithelia of the organs of the branchial cavity of Homarus gammarus exposed to seawater and dilute seawater was examined by immunofluorescence microscopy and immunogold electron microscopy with a monoclonal antibody IgGα5 raised against the avian α-subunit of the Na+,K+-ATPase. In juveniles held in seawater, fluorescent staining was observed only in the epithelial cells of epipodites. In juveniles held in dilute seawater, heavier immunoreactivity was observed in the epithelial cells of epipodites, and positive immunostaining was also observed along the inner-side epithelial layer of the branchiostegites. No fluorescent staining was observed in the gill epithelia. At the ultrastructural level, the Na+,K+-ATPase was localized in the basolateral infolding systems of the epipodite and inner-side branchiostegite epithelia of juveniles held in dilute seawater, mostly along the basal lamina. The expression of Na+,K+-ATPase therefore differs within tissues of the branchial cavity and according to the external salinity. These and previous ultrastructural observations suggest that the epipodites, and to a lesser extent the inner-side epithelium of the branchiostegites, are involved in the slight hyper-regulation displayed by lobsters at low salinity. Enhanced Na+,K+-ATPase activity and de novo synthesis of Na+,K+-ATPase within the epipodite and branchiostegite epithelia may be key points enabling lobsters to adapt to low salinity environments.