Guy Charmantier

Osmoregulatory capacity as a tool in monitoring the physiological condition and the effect of stress in crustaceans

Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustacean capable of osmoregulation usually results in a decrease of its Na+ and Cl− regulation and/or of its osmoregulatory capacity (OC: difference between the osmotic pressures of the hemolymph and of the external medium, at a given salinity). The partial or complete loss of osmoregulatory and ionoregulatory capacity is generally linked to disruptions of the osmotic and ionic regulations. Different causes of these variations include alterations in the structure and ultrastructure of the branchial and excretory organs and changes in Na+,K+-ATPase activity, ionic fluxes and surface permeability. OC appears therefore as a nonspecific bioindicator. Hyper-OC and/or hypo-OC can be measured according to the type of osmoregulation of the species, respectively, in low-salinity media and in seawater or high-salinity media. Provided several precautions are taken (control of salinity and temperature, size, nutritional status, developmental stage, molt stage, use of controls), measurement of OC variations is proposed as a convenient and reliable way to monitor the physiological condition and the effect of stressors in crustaceans.

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.

Physiological and behavioural responses of Gammarus pulex (Crustacea: Amphipoda) exposed to cadmium.

The aim of this study was to investigate the effects of cadmium on physiological and behavioural responses in Gammarus pulex. In a first experiment, cadmium LC50s for different times were evaluated in 264 h experiment under continuous mode of exposure (LC50(96 h)=82.1 microgL(-1), LC50(120 h)=37.1 microgL(-1), LC50(168 h)=21.6 microgL(-1), LC50(264 h)=10.5 microgL(-1)). In a second experiment, the physiological and behavioural responses of the amphipod exposed to cadmium (0, 7.5 and 15 microgL(-1)) were investigated under laboratory conditions. The mortality and the whole body cadmium concentration of organisms exposed to cadmium were significantly higher than in controls. Concerning physiological responses, cadmium exposure exerted a significant decrease on osmolality and haemolymph Ca(2+) concentration, but not on haemolymph Na(+) and Cl(-) concentrations, whereas the Na(+)/K(+)-ATPase activity was significantly increased. Behavioural responses, such as feeding rate, locomotor and ventilatory activities, were significantly reduced in Cd exposed organisms. Mechanism of cadmium action and consequent energetic reallocation in favour of maintenance functions (i.e., osmoregulation) are discussed. The results of this study indicate that osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystem and to assess the health of organisms.

Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus.

SUMMARY This study investigates the involvement of eyestalk neuroendocrine factors on osmoregulation in the crayfish Astacus leptodactylus maintained in freshwater. Eyestalk removal was followed by a significant decrease in hemolymph osmolality and Na+ concentration and by a 50% increase in mass after one molting cycle. Several neurohormones have been isolated from the sinus gland through high-performance liquid chromatography (HPLC), and different crustacean hyperglycemic hormone (CHH)-related peptides, including stereoisomers (L-CHH and D-Phe3 CHH), have been identified by direct enzyme-linked immunosorbent assay (ELISA). A glucose quantification bioassay demonstrated a strong hyperglycemic activity following injection of the immunoreactive chromatographic fractions and showed that the D-Phe3 CHH was the most efficient. Destalked crayfish were then injected with purified CHH HPLC fractions. The D-Phe3 CHH fraction significantly increased the hemolymph osmolality and Na+ content 24 h after injection. Two other CHH-related peptides caused a smaller increase in Na+ concentration. No significant variation was observed in hemolymph Cl- concentration following injection of any of the CHH isoforms. These results constitute the first observation of the effects of a CHH isoform, specifically the D-Phe3 CHH, on osmoregulatory parameters in a freshwater crustacean. The effects of eyestalk ablation and CHH injection on osmoregulation and the identification of different CHH-related peptides and isoforms in crustaceans are discussed.

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.

The Na + /K + /2Cl - cotransporter in the sea bass Dicentrarchus labrax during ontogeny: involvement in osmoregulation

SUMMARY This study combines a cellular and molecular analysis of the Na+/K+/2Cl- cotransporter (NKCC) to determine the osmoregulatory role of this protein in different tissues during the ontogeny of the sea bass. We have characterized the complete sequence of the NKCC1 isoform isolated from the sea bass gills and have identified, by immunofluorescence, NKCC1, and other isoforms, within the epithelium of the major osmoregulatory organs. Different (absorptive and secretory) functions have been attributed to this protein according to the tissue and salinity. The effects of short- (1-4 days), medium- (7-21 days) and long (6 months)-term freshwater (FW) adaptations were investigated, in comparison with seawater (SW)-maintained sea bass. In adult sea bass after long-term adaptation to FW and SW, the gills had the highest expression of NKCC mRNA compared with the median/posterior kidney and to the posterior intestine. Expression of NKCC mRNA in the kidney was 95% (SW) and 63% (FW) lower, and in the intestine 98% (SW) and 77% (FW) lower. Compared to SW-maintained sea bass, long-term FW adaptation induced a significant 5.6-fold decrease in the branchial NKCC gene expression whereas the intestinal and renal expressions did not vary significantly. The cells of the intestine and collecting ducts as well as a part of the epithelium lining the urinary bladder expressed NKCC apically. Within the gill chloride cells, NKCC was found basolaterally in SW-acclimated fish; some apically stained cells were detected after 7 days of FW exposure and their relative number increased progressively following FW acclimation. The appearance of FW-type chloride cells induces a functional shift of the gills from a secretory to an absorptive epithelium, which was only completed after long-term exposure to FW. Short- and medium-term exposure to FW induced a progressive decrease in total NKCC content and an increase in functionally different branchial chloride cells. During development, the cotransporter was already expressed in tegumentary ionocytes and along the digestive tract of late embryos. NKCC was recorded in the branchial chamber and along the renal collecting ducts in prelarvae and also in the dorsal part of the urinary bladder in larvae. The expression of NKCC along the osmoregulatory epithelial cells and the presence of Na+/K+-ATPase within these cells contribute to the increase of the osmoregulatory capacity during sea bass ontogeny.

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.

Effect of molt stage and hypoxia on osmoregulatory capacity in the peneid shrimp Penaeus vannamei

The osmoregulatory capacity (OC) was used to study the effects of hypoxia in Penaeus vannamei. Since OC varied with molt stages with a tendency for animals to show a reduced OC before and after ecdysis, only shrimps at stages C-D0 were consequently used. Hyper-OC and hypo-OC, respectively, measured at low salinity and in seawater, were both depressed after 1–2 days exposure to low oxygen tension (PO2) ranging from 4 to 8 kPa. Low PO2 effect was time-dependent. OC recovered fully after 24 h in an O2 saturated medium. OC measurement is confirmed as a convenient tool to monitor the physiological condition and the effect of stress in crustaceans.

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.