Marine mammals as indicators of Anthropocene Ocean Health.

The current state of marine mammal populations reflects increasing anthropogenic impacts on the global Ocean. Adopting a holistic approach towards marine mammal health, incorporating healthy individuals and healthy populations, these taxa present indicators of the health of the overall Ocean system. Their present deterioration at the animal, population and ecosystem level has implications for human health and the global system. In the Anthropocene, multiple planetary boundaries have already been exceeded, and quiet tipping points in the Ocean may present further uncertainties. Long and short-term monitoring of marine mammal health in the holistic sense is urgently required to assist in evaluating and reversing the impact on Ocean Health and aid in climate change mitigation.

Ingestion of microplastics by fish and other prey organisms of cetaceans, exemplified for two large baleen whale species.

Knowledge on microplastic (MP) ingestion by cetaceans is difficult to obtain. We infer the potential for MP uptake by cetaceans from the occurrence of MP in prey species. First, we reviewed information on whale prey species, focussing on common minke (Balaenoptera acutorostrata) and sei whale (B. borealis), for which the most comprehensive quantitative datasets exist. Second, evidence of MP ingestion by their prey species was reviewed. We found common minke whales forage opportunistically on fish from various families: Ammodytidae, Clupeidae, Gadidae, Engraulidae and Osmeridae. Sei whales mostly feed on copepods, Engraulidae, Clupeidae and Scombridae. High levels of MP contamination are reported for Scombridae in the Atlantic and Engraulidae in the Northwest Pacific Ocean. Copepods exhibit low levels of MP ingestion in the Northeast Pacific Ocean. Species-specific prey preferences and feeding strategies imply different cetaceans have varied potential for MP uptake, even if they feed in similar geographic areas.

Marine debris in harbour porpoises and seals from German waters.

Records of marine debris in and attached to stranded harbour porpoises (Phocoena phocoena), harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) were studied comprising information on 6587 carcasses collected along the German coast between 1990 and 2014, the decomposition state allowed for necropsy in 1622 cases. Marine debris items were recorded in 31 carcasses including 14 entanglements (5 harbour porpoises, 6 harbour seals, 3 grey seals) and 17 cases of ingestion (4 harbour porpoises, 10 harbour seals, 3 grey seals). Objects comprised general debris (35.1%) and fishing related debris (64.9%). Injuries associated with marine debris included lesions, suppurative ulcerative dermatitis, perforation of the digestive tract, abscessation, suppurative peritonitis and septicaemia. This study is the first investigation of marine debris findings in all three marine mammal species from German waters. It demonstrates the health impacts marine debris can have, including severe suffering and death. The results provide needed information on debris burdens in the North and Baltic Seas for implementing management directives, such as the Marine Strategy Framework Directive (MSFD).

The invasive bighead goby Ponticola kessleri displays large-scale genetic similarities and small-scale genetic differentiation in relation to shipping patterns.

Colonization events, range expansions and species invasions leave genetic signatures in the genomes of invasive organisms and produce intricate special patterns. Predictions have been made as to how those patterns arise, but only very rarely, genetic processes can be monitored in real time during range expansions. In an attempt to change that, we track a very recently established invasive population of a fish species, the bighead goby Ponticola kessleri, with high temporal and spatial resolution through 2 years to identify patterns over time. We then compare Swiss and German samples of bighead goby along the river Rhine using microsatellites, mitochondrial D-loop sequences and geometric morphometrics to investigate geographic patterns. We detect weak temporal and strong geographic patterns in the data, which are inconsistent with isolation by distance and indicate long range transport. In search of an explanation for our observations, we analyse the vector properties and travel patterns of commercial vessels on the river Rhine. We present evidence that freshwater cargo ships and tankers are plausible vectors for larvae of invasive goby species. We also present indications that cargo ships and tankers act as differential vectors for this species. In summary, we present genetic data at unique temporal resolution from a vertebrate invasion front and substantiate the paramount role of commercial shipping in freshwater fish translocations.

Molecular and cellular effects of chemicals disrupting steroidogenesis during early ovarian development of brown trout (Salmo trutta fario).

A range of chemicals found in the aquatic environment have the potential to influence endocrine function and affect sexual development by mimicking or antagonizing the effects of hormones, or by altering the synthesis and metabolism of hormones. The aim of this study was to evaluate whether the effects of chemicals interfering with sex hormone synthesis may affect the regulation of early ovarian development via the modulation of sex steroid and insulin-like growth factor (IGF) systems. To this end, ex vivo ovary cultures of juvenile brown trout (Salmo trutta fario) were exposed for 2 days to either 1,4,6-androstatriene-3,17-dione (ATD, a specific aromatase inhibitor), prochloraz (an imidazole fungicide), or tributyltin (TBT, a persistent organic pollutant). Further, juvenile female brown trout were exposed in vivo for 2 days to prochloraz or TBT. The ex vivo and in vivo ovarian gene expression of the aromatase (CYP19), responsible for estrogen production, and of IGF1 and 2 were compared. Moreover, 17ß-estradiol (E2) and testosterone (T) production from ex vivo ovary cultures was assessed. Ex vivo exposure to ATD inhibited ovarian E2 synthesis, while T levels accumulated. However, ATD did not affect ex vivo expression of cyp19, igf1, or igf2. Ex vivo exposure to prochloraz inhibited ovarian E2 production, but did not affect T levels. Further prochloraz up-regulated igf1 expression in both ex vivo and in vivo exposures. TBT exposure did not modify ex vivo synthesis of either E2 or T. However, in vivo exposure to TBT down-regulated igf2 expression. The results indicate that ovarian inhibition of E2 production in juvenile brown trout might not directly affect cyp19 and igf gene expression. Thus, we suggest that the test chemicals may interfere with both sex steroid and IGF systems in an independent manner, and based on published literature, potentially lead to endocrine dysfunction and altered sexual development.

Effects of river morphology, hydraulic gradients, and sediment deposition on water exchange and oxygen dynamics in salmonid redds.

Fine sediment decreasing gravel permeability and oxygen supply to incubating salmonid embryos, is often considered the main contributing factor for the observed decline of salmonid populations. However, oxygen supply to salmonid embryos also depends on hydraulic conditions driving water flow through the redd. A more generalized perspective is needed to better understand the constraints on successful salmonid incubation in the many heavily modified fluvial ecosystems of the Northern Hemisphere. The effects of hydraulic gradients, riverbed and redd morphology as well as fine sediment deposition on dissolved oxygen (DO) and water exchange was studied in 18 artificial redds at three sites along a modified river. Fifty percent of the redds in the two downstream sites were lost during high flow events, while redd loss at the upstream site was substantially lower (8%). This pattern was likely related to increasing flood heights from up- to downstream. Specific water infiltration rates (q) and DO were highly dynamic and driven on multiple temporal and spatial scales. Temporally, the high permeability of the redd gravel and the typical pit-tail structure of the new built redds, leading to high DO, disappeared within a month, when fine sediment had infiltrated and the redd structure was leveled. On the scale of hours to days, DO concentrations and q increased during high flows, but decreased during the falling limb of the water level, most likely related to exfiltration of oxygen depleted groundwater or hyporheic water. DO concentrations also decreased under prolonged base flow conditions, when increased infiltration of silt and clay particles clogged the riverbed and reduced q. Spatially, artificial log steps affected fine sediment infiltration, q and interstitial DO in the redds. The results demonstrate that multiple factors have to be considered for successful river management in salmonid streams, including riverbed structure and local and regional hydrogeological conditions.

Development of an ex vivo brown trout (Salmo trutta fario) gonad culture for assessing chemical effects on steroidogenesis.

A variety of natural and synthetic environmental substances have been shown to disrupt vertebrate reproduction through mimicking or modifying the regulation of the endocrine system. Tests to screen for any such chemicals that directly interact with the steroid hormone receptors are widely available; however, few tests have been developed to identify chemicals that affect endocrine function through non-receptor mediated mechanisms. The aim of this study was, therefore, to develop an assay for the identification of substances that disrupt the activity of enzymes involved in the sex steroid biosynthesis cascade, in particular the aromatase enzyme, CYP19, that catalyses the final conversion of androgens to estrogens. A gonad ex vivo assay was developed using gonad explants harvested from juvenile brown trout and cultured in a modified Leibovitz medium. Effects on sex steroid biosynthesis were quantified through measurement of 17ß-estradiol (E2) and testosterone (T) concentrations in the medium after 2 days incubation. Exposure of ovary explants to 100 ng/mL 1,4,6-androstatriene-3,17-dione (ATD), a potent pharmaceutical aromatase inhibitor, reduced E2 concentrations and elevated T concentrations confirming that CYP19 activity could be inhibited in the assay. Exposure of ovary explants to 250 ng/mL prochloraz, an imidazole fungicide, also reduced E2 concentrations but did not affect T levels, consistent with reports that in addition to inhibiting CYP19 activity, prochloraz also inhibits enzymes in the steroidogenic pathway upstream of the CYP19 enzyme. Exposure to a third chemical, tributyltin (TBT), did not affect T or E2 concentrations, further supporting previous evidence that the CYP19 modulating effects of this chemical are not mediated through direct inhibition of CYP19 activity. These results demonstrate that the gonad ex vivo assay developed here can be successfully used to identify substances that disrupt sex steroid biosynthesis and further that it has the potential to inform on their specific mode of action.

Morphological organ alterations and infectious diseases in brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss exposed to polluted river water.

Poor water quality is discussed as a major factor causing a decline of brown trout populations in Swiss rivers. For our study we have chosen a river in the Swiss midlands, where the brown trout population has decreased dramatically during the last 10 yr and where feral fish have shown distinctive pathological alterations. The objective of our study was to investigate whether river water may be responsible for impaired fish health leading to an increased mortality in the river. In an active monitoring program, groups of brown and rainbow trout were exposed to polluted river water for 24 mo. Fish held in tap water served as a reference. Mortality, macroscopic and histopathologic changes, and infectious agents were investigated. Compared with the reference group, high mortality rates and severe pathological alterations of the inner organs were observed in fish held in river water. Especially gills, liver and kidney of these fish showed significantly higher changes than fish from tap water. These changes were dominated by degenerative and inflammatory reactions. Additionally, several infectious agents were diagnosed in fish exposed to river water. The most important findings were furunculosis and proliferative kidney disease. Brown trout seemed to be more sensitive than rainbow trout to environmental stress and infectious agents.

Effluent from a sewage treatment works causes changes in serum chemistry of brown trout (Salmo trutta L.).

To evaluate the impact of effluent from a sewage treatment works on fish health, serum chemistry variables were investigated in brown trout (Salmo trutta L.) held in cages (active monitoring) and wild brown trout (passive monitoring). Means of the measured serum parameters of the different treatment groups were close or within normal ranges. However, the results of the active monitoring demonstrated that the serum variables of reference trout held in tap water were clearly different from those of the river treatment groups. In the active monitoring, fish exposed to effluent from the sewage treatment works had significantly different blood urea nitrogen and bilirubin values than fish kept in river water. In the passive monitoring, total protein, blood urea nitrogen, and alkaline phosphatase were significantly different between the two groups. Of the numerous correlations between serum chemistry parameters and histological lesions, blood urea nitrogen and alkaline phosphatase were found to most strongly indicate gill and liver lesions, respectively. In the passive monitoring correlations between serum chemistry variables and histopathological lesions were restricted to bilirubin and liver lesions. This indicates that the application of serum chemistry variables as indicators of histological lesions in case of chronic exposure is questionable. A multivariate discriminant analysis was used to consider relationships between the single serum variables concurrently.

Nonylphenol provokes a vesiculation of the Golgi apparatus in three fish epidermis cultures.

The aim of this work was to study the effects of nonylphenol and waste water on the cell ultrastructure of fish skin. Therefore, besides a recently established primary cell culture and a cell line, an epidermal tissue culture of fish was developed and tested. In all three systems a prominent vesiculation of the Golgi apparatus was observed after exposure to nonylphenol, which has not been described before and therefore strongly suggests an effect that might indicate exposure to nonylphenol and/or related substances. The Epithelial papulosum cyprini cell line was the most sensitive to nonylphenol, followed by the primary cell culture of epidermis cells and then the explant tissue culture. The vesiculation of the Golgi apparatus was accompanied by degenerative changes in the two cell cultures only. The lack of degenerative changes in the cells of the tissue culture was discussed with respect to the presence of differentiated cells that might better be able to protect themselves by mucous or by an activated xenobiotic metabolism. In a second type experiment, a waste water sample containing small concentrations of nonylphenol was applied to the cultures. It did not lead to a vesiculation of the Golgi apparatus, probably because the nonylphenol concentrations in the waste water were too low to induce the vesiculation. The cultures exposed to waste water revealed unspecific degenerative cellular changes. Additionally, explant cultures were prepared from fish that had survived a 6-month exposure to polluted river water. In these cultures a higher number of mitochondria containing myelin bodies were observed when compared to control cultures. Consequently, exposure to polluted water containing a mixture of substances in vitro and in vivo was found to lead to degenerative alterations in the ultrastructure of the cells.

Exposure of rainbow trout (Oncorhynchus mykiss) to nonylphenol is associated with an increased chloride cell fractional surface area.

Nonylphenol is a biodegradation product of a widely used group of non-ionic detergents. Because of its ubiquitous distribution and persistence, nonylphenol is present in surface waters as a pollutant. Little is known about its biological effects at environmentally relevant concentrations other than its action as a xenoestrogen. The goal of the present paper was to study the trout gill surface epithelium as the major interface between fish and water in view of possible morphological alterations due to exposure to nonylphenol. Rainbow trout were intermittently exposed to 10 micrograms/l nonylphenol and gill samples from experimental and control animals were investigated by scanning electron microscopy. Gill surface epithelium was scrutinised for changes in chloride cell density and their status regarding cell surface modifications. In addition, chloride cell fractional surface area (CCFA) was determined by morphometrical methods. Statistical analysis revealed a highly significant increase of CCFA in animals exposed to nonylphenol as compared to control animals (P = 0.0001). Semi-quantitative assessment of the other parameters suggested a higher chloride cell density and a larger proportion of chloride cells bearing microvilli. Taken together, these results provide evidence that exposure of trout to nonylphenol is associated with a substantial increase in the active interface of chloride cells with water. We interpret these findings as being a means to further the fish's capacity for calcium exchange.

Nonylphenol affects the granulation pattern of epidermal mucous cells in rainbow trout, Oncorhynchus mykiss.

Nonylphenol is a biodegradation product of nonionic surfactants and has recently attracted considerable attention due to its estrogenic potential. Sexually mature male rainbow trout were repeatedly exposed (one to four periods of 10 days each) to environmentally relevant concentrations of nonylphenol (1 microg/L, 10 microg/L) and for comparison, trout were injected with estradiol. Since estrogens are known to induce structural changes within the fish skin, a similar effect of xenobiotics with estrogen-like activity was assumed. Samples of skin were evaluated by means of light and electron microscopy and histochemistry. In trout exposed to nonylphenol and to estradiol, the structure of the epidermis was altered: an irregular overall architecture was often accompanied by detached pavement cells, vacuolation of the cytoplasm, and severely deformed cell nuclei. However, the granulation pattern of the mucous cells was influenced exclusively after exposition to nonylphenol. The number of large and irregularly shaped mucosomes depended more on the exposure period than on the concentration of nonylphenol. Furthermore, this alteration has not yet been reported for any other pollutant or stressor and, thus, can be classified as an effect that would strongly indicate exposure to nonylphenol.

Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol.

Three types of epidermal cultures of fish were used for toxicological investigations, a primary cell culture and a tissue culture prepared from the rainbow trout Oncorhynchus mykiss Walbaum and the cell line EPC, derived from a skin tumour of the carp Cyprinus carpio L. Two studies were carried out to compare the different culture systems. In the first cultures were incubated with nonylphenol and in the second set of experiments the cell cultures were exposed to a wastewater sample containing low concentrations of nonylphenol (NP). Both cell cultures were similarly sensitive to nonylphenol with respect to the endpoints cell viability (LC50 (24 h) 47.1 µM NP (primary cell culture) and 44.2 µM NP (EPC)) values and apoptotic rate (significantly increased apoptotic rate after exposure to 50 µM NP for 24 h, p < 0.001 (primary cell culture), p = 0.008 (EPC)). The explant culture was slightly less sensitive (increased apoptotic rate after exposure to 50 µM NP for 24 h, but not significant: p = 0.385), which could be due to the capabilities of a differentiated tissue, providing more protective repair mechanisms, compared with single cells. All cultures revealed a concentration-response relationship for the endpoint apoptotic rate after the application of nonylphenol for 24 h. After wastewater exposure, a significant decrease in the apoptotic rate was measured in the primary cell culture (dilution wastewater : medium 1:1:p = 0.018; dilution wastewater : medium 1:2:p = 0.003), whereas the cell line EPC did not reveal any effects. Our results show that the endpoint apoptotic rate is more sensitive than the parameter cell viability for detecting adverse effects of a wastewater sample.

Increase of metallothionein-immunopositive chloride cells in the gills of brown trout and rainbow trout after exposure to sewage treatment plant effluents.

Metallothionein, a biomarker of exposure and toxicity of heavy metals, has been detected in the gills of brown trout (Salmo trutta fario L.) and rainbow trout (Oncorhynchus mykiss Richardson) by means of immunohistochemistry. A very prominent labelling of chloride cells was found after exposure to diluted sewage plant effluents. No significant increase was observed in either the number of labelled cells or their labelling intensity after exposure to water of a polluted river compared to fish kept in tap water. These results do not correlate with findings of a histopathological study, suggesting that the metal levels at the sewage treatment plant were too low to produce gross histopathology. A comparison between the species indicated that the rainbow trout showed a generally higher metallothionein expression than the brown trout.

Toxicity of 4-chloroaniline in early life stages of zebrafish (Danio rerio): II. Cytopathology and regeneration of liver and gills after prolonged exposure to waterborne 4-chloroaniline.

Ultrastructural alterations in liver and gills of embryonic and larval zebrafish (Danio rerio) following prolonged exposure to waterborne 0.05, 0.5, and 5 mg/L 4-chloroaniline for up to 31 days as well as after a 14-day regeneration period were investigated by means of light and electron microscopy. Acute toxicity was also tested at 25 and 50 mg/L. Survival of zebrafish embryos and larvae was only impaired from 25 mg/L 4-chloroaniline, but-after a transient stimulation following exposure to 0.5 mg/L-4-chloroaniline hatching was retarded after exposure to >/=5 mg/L, and fish displayed increasing rates of abnormal development and pigmentation. In contrast, hepatocytes displayed a time- and dose-dependent response from 0.05 mg/L 4-chloroaniline, including changes in nuclei, mitochondria, peroxisomes, endoplasmic reticulum, Golgi fields, lysosomes, and hepatic glycogen and lipid stores, as well as invasion of macrophages. In gills, dose-dependent effects were evident from 0.5 mg/L 4-chloroaniline and included deformation of secondary lamellae due to vacuolization and desquamation of respiratory epithelial cells in conjunction with dilation of intercellular spaces. Respiratory epithelial cells displayed progressive mitochondrial changes, induction of cytoplasmic myelinated structures, augmentation of lysosomes, and modifications of Golgi fields. Erythrocytes were severely deformed. A 14-day regeneration period was sufficient for almost complete recovery of pathological symptoms in both liver and gills. Only minor volumetric changes in hepatocellular organelles and a limited number of myelinated bodies, lysosomes, and cytoplasmic vacuoles were reminiscent of prior 4-chloroaniline exposure. In both qualitative and quantitative terms, most effects in hepatocytes after exposure of embryonic and larval zebrafish to waterborne 4-chloroaniline are comparable to the reaction of hepatocytes in adult zebrafish liver after prolonged sublethal exposure as well as in larval zebrafish after microinjection. Morphological changes in erythrocytes indicate disturbance of respiration as an additional mode of action of 4-chloroaniline.

Transient increase in chloride cell number and heat shock protein expression (hsp70) in brown trout (Salmo trutta fario) exposed to sudden temperature elevation.

The native cold-adapted brown trout (Salmo trutta fario) is often the subject of biomonitoring field studies. Groups of trout were exposed to a sudden temperature rise, from 8 degrees C to 19 degrees C for two hours, and thereafter set back to 8 degrees C. Gill samples of control animals, of fish after the exposure period, and after 24 and 48 hours of recovery at a temperature of 8 degrees C were examined histologically, immunohistochemically, electron microscopically, and by Western blot analysis. By means of immunohistochemistry and electron microscopy, an increase of chloride cells was observed after the temperature elevation. During the recovery period the number of chloride cells decreased. Western blot analysis for stress proteins (hsp70), widely used as a biomarker for environmental stress, was performed from skin and gill. Whereas in the gill both isoforms, the constitutive and the heat inducible form, of hsp70 were detected in all groups, in the skin the control animals only showed the constitutive form. After two hours of exposure both isoforms were visible. An increased expression of hsp70 could be demonstrated in both organs after the exposure. Comparison of the hsp70 values between gill and skin showed tissue-specific differences during the recovery period. In the gill hsp70 rapidly decreased, while in the skin the level remained elevated over the whole observation period. When hsp70 is used as a biomarker in field studies, the fast and organ-specific reaction in the gill and skin of brown trout has to be taken into consideration.

Heat shock protein (hsp70) in brown trout epidermis after sudden temperature rise.

So far, hsp70 has not yet been studied in the fish skin. This organ has a potential as an indicator organ and we investigate whether hsp70 could be used as a biomarker. In this study, we examined whether and how the epidermis reacts to a temperature rise. Brown trout, Salmo trutta fario, were exposed to higher temperature for 2 h and were allowed to recover subsequently. Samples were taken from controls, after heat shock, as well as after 24 and 48 h of recovery. The occurrence of hsp70 in trout skin was examined by Western blot. The amount of hsp70 was higher after 2-h heat shock and was rising until the end of the experiment. Immunocytochemically, hsp70 was detected in epidermal filament cells. After 2-h heat shock, hsp70 was predominantly located in the nucleus. At this time, light and electron microscopy revealed several features known to occur under a variety of stressors. Ultrastructurally, the appearance of compact filament aggregates in pavement cells was remarkable. After 24 h of recovery, filament compaction was lacking and after 48 h aspects of regeneration were obvious. However, an increased amount of apoptotic cells in the epidermis was prominent at this time only.

Primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum.

This is the first report on a primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum. These primary cells revealed a low seeding efficiency after 3 days (11.6 +/- 4.6%), whereas subcultured cells had a higher seeding efficiency at the same time point (75.5 +/- 34.0%) and increased in cell number (150-200% of seeded cells after 20 to 30 days). The cells were characterized applying histological, immunocytochemical and ultrastructural methods. The culture consisted of undifferentiated keratinocytes. Mucous cells as well as differentiated epithelial cells were absent. To date the cells were cultured for maximally 9 passages and 402 days and therefore provide the possibility for long-term studies.

Immunohistochemical detection of vitellogenin in male brown trout from Swiss rivers.

The detection of vitellogenin, a yolk precursor protein, may serve as a biomarker for exposure to environmental oestrogens as its induction by xenobiotic oestrogens in the immature and male fish has been reported repeatedly. In the present work, juvenile brown trout were injected with oestradiol (5 microg g(-1) body weight oestradiol benzoate) in order to assess the induction and organ distribution of vitellogenin by means of immunohistochemistry. In addition, brown trout collected from Swiss rivers were analysed. Vitellogenin was detected in the oestradiol-injected juvenile trout but not in uninjected controls. The presence of vitellogenin was also demonstrated in a male and an immature feral brown trout from one of two locations downstream of three sewage treatment plants. In contrast, no positive staining was found in livers of trout upstream of the respective plants. The results demonstrate the suitability of immunohistochemistry for monitoring feral fish for the presence of vitellogenin production.

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin.

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na+/K+ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions.