Serotonin-like immunoreactivity in the epidermal club cells of teleost fishes

The present immunocytochemical study concerns the distribution of serotonin in the epidermis of three species of teleost fish. Serotonin-like immunoreactivity was found in the club cells of Heteropneustes fossilis and Carapus acus but not in those from the sea eel Conger conger. This study is the first immunocytochemical identification of serotonin in the club cells of teleost epidermis. By comparing data from the literature (Zaccone et al. 1986, 1987, 1988) regarding the occurrence of serotonin and GRP/bombesin in the exocrine sacciform gland cells of piscine skin, it is worthy mentioning here that the serotonin contained in the club cells of the species studied may have the ability to affect the pheromonal or other possible functions of these cells. The presence of serotonin in these systems has been correlated with the capacity of the exocrine glands of fish skin to secrete, ectopically, amine messengers in contrast to those produced eutopically i.e. in the neuron-paraneuron system in some vertebrates (Fujita et al. 1988).     

Distribution patterns of the paraneuronal endocrine cells in the skin, gills and the airways of fishes as determined by immunohistochemical and histological methods

Summary The neuro-endocrine cells of fish skin and respiratory surfaces, and their bioactive secretion as far as is known, are reviewed, and compared with similar elements in tetrapods, particularly amphibians. In the skin of teleost fish, immunohistochemistry has shown that Merkel cells react for serotonin, neuron-specific enolase and enkephalins. The pharmacology is not established in dipnoans or lampreys. In some teleosts, neuromasts react for substance P and leu-enkephalins; substance P is also reported from some ampullary organs (electroreceptors). Taste buds of teleosts may react for enkephalin and substance P. Basal cells of taste buds react for serotonin and neuron-specific enolase. Some unicellular skin glands of teleosts express bioactive compounds, including serotonin and some peptides; this ectopic expression is paralleled in amphibian skin glands. The dipnoan Protopterus has innervated pulmonary neuro-endocrine cells in the pneumatic duct region with dense-cored vesicles. In Polypterus and Amia the lungs have serotonin-positive neuro-endocrine cells that are apparently not innervated. In fish gills, a closed type of neuro-endocrine cell reacts for serotonin, an open type for enkephalins and some calcium-binding proteins (calbindin, calmodulin and S-100 protein). The functions of neuro-endocrine cells in fishes await investigation, but it is assumed they are regulatory.     

Epidermal club cells and the innate immune system of minnows

Thousands of fish species belonging to the Superorder Ostariophysi possess specialized club cells in their epidermis. Damage to the cells, as would occur during a predator attack, releases chemical substances that evoke antipredator responses in nearby shoalmates. These chemical substances have often been referred to as alarm substances and the cells that release them as alarm cells. Understanding the evolution of the cells in an alarm context has been difficult. The fish needs to be captured prior to the chemicals being released, hence the benefit to the receiver is unclear. Recent studies have suggested that the club cells are part of the immune system and are maintained by natural selection owing to the benefits that they confer against pathogens, parasites, and general injury to the epidermis. In the present study, we gave fathead minnows intraperitoneal injections of cortisol, a known immunosuppressant, or injections of a control substance (corn oil). We found that fish exposed to cortisol had suppressed immune systems (as measured by a respiratory burst assay) and that they also reduced their investment in club cells. This is the best evidence to date indicating that the club cells of Ostariophysan fishes are part of the innate immune system and that the alarm function of the cells evolved secondarily. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 891–897.     

The effects of ultraviolet radiation on a freshwater prey fish: physiological stress response,club cell investment,and alarm cue production

Recent anthropogenic activities have caused deleterious effects to the stratospheric ozone layer, resulting in a global increase in the level of ultraviolet radiation (UVR). Understanding the way that organisms respond to such stressors is key to predicting the effects of anthropogenic activities on aquatic ecosystems and the species that inhabit them. The epidermal layer of the skin of fishes is not keratinized and acts as the primary interface between the fish and its environment. The skin of many species of fishes contains large epidermal club cells (ECCs) that are known to release chemicals (alarm cues) serving to warn other fishes of danger. However, the alarm role of the cells is likely secondary to their role in the immune system. Recent research suggests that ECCs in the epidermis may play a role in protecting the fish from damage caused by UVR. In the present study, we examined the effects of in vivo exposure to UVR on fathead minnows (Pimephales promelas), specifically investigating ECC investment, physiological stress responses, and alarm cue production. We found that fish exposed to UVR showed an increase in cortisol levels and a substantive decrease in ECC investment compared to non‐exposed controls. Unexpectedly, our subsequent analysis of the behavioural response of fish to alarm cues revealed no difference in the potency of the cues prepared from the skin of UV‐exposed or non‐exposed minnows. Our results indicate that, although nonlethal, UVR exposure may lead to secondary mortality by altering the fish immune system, although this same exposure may have little influence on chemically‐mediated predator–prey interactions. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 832–841.     

Conspecific and heterospecific alarm substance induces behavioral responses in piau fish Leporinus piau

In ostariophysan fish, the detection of alarm substance released from the skin of a conspecific or a sympatric heterospecific may elicit alarm reactions or antipredator behavioral responses. In this study, experiments were performed to characterize and quantify the behavioral response threshold of Leporinus piau, both individually and in schools, to growing dilutions of conspecific (CAS) and heterospecific skin extract (HAS). The predominant behavioral response to CAS stock stimulation was biphasic for fish held individually, with a brief initial period of rapid swimming followed by a longer period of immobility or reduced swimming activity. As the dilution of skin extract was increased, the occurrence and magnitude of the biphasic alarm response tended to decrease, replaced by a slowing of locomotion. Slowing was the most common antipredator behavior, observed in 62.5% of animals submitted to HAS stimulation. School cohesion, measured as proximity of fish to the center of the school, and swimming activity near the water surface significantly increased after exposure to CAS when compared with the control group exposed to distilled water. Histological analysis of the epidermis revealed the presence of Ostariophysi-like club cells. The presence of these cells and the behavioral responses to conspecific and heterospecific skin extract stimulation suggest the existence of a pheromone alarm system in L. piau similar to that in Ostariophysi, lending further support for the neural processing of chemosensory information in tropical freshwater fish.     

Epidermal club cells do not protect fathead minnows against trematode cercariae: a test of the anti‐parasite hypothesis

Epidermal club cells of fishes in the superorder ostariophysi have puzzled evolutionary biologists because they were historically linked to chemical alarm signalling and relied on group selectionist explanations. Alternative hypotheses to explain the existence of these cells include the possibility of an anti‐pathogenic or anti‐parasitic function. If this is so, individual fish should invest in increased numbers of club cells after exposure to parasites, and club cells should contain components that reduce the infectivity of skin‐penetrating larvae. Infectivity of cercariae of the trematode Ornithodiplostomum sp. was significantly reduced when exposed to the skin extract of fathead minnows (an ostariophysan), but also to skin extract of mollies (a non‐ostariophysan that lacks club cells), respectively, compared to controls. Moreover, club cell density was not affected by exposure to cercariae. Taken together, these results are inconsistent with an anti‐parasite function for these cells and instead suggest a generic role in response to injury. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 884–890.     

A comparative study of the epidermis of the common carp and the three Indian major carp

A comparative study has been made of the mucogenic epidermis of the common carp, Cyprinus carpio var. communis, and the three Indian major carps, Catla catla, Labeo rohita and Cirrhina mrigala: on the basis of epidermis structural organization, these species are easily differentiated. The epithelial cells in the superficial layer, as in most fishes, show secretory activity, evidenced by positive histochemical reactions, which is high in C. carpio var. communis, moderate in C. catla and low in L. rohita and C. mrigala. The epithelial cells in the underlying two or three layers also give positive reactions, though their intensity is relatively weak. The mucous cells in C. carpio var. communis are distributed in large numbers arranged in several superimposed layers in the outer regions of the epidermis, whereas in C. catla they are fewer in number and are widely separated in the surface layers as well as in the deeper layers of the epidermis; in both species the mucous cells appear rounded, large, and open on the surface by wide pores. In contrast, in L. rohita and C. mrigala the mucous cells are smaller, restricted mainly to the superficial layer, close together in a single row, and open on the surface by narrow pores. The overall density of mucous cells in L. rohita and C. mrigala, as in C. catla, is much lower than in C. carpio var. communis. In the epidermis of C. carpio var. communis there are a large number of mucous cells, and the few club cells are restricted to the deeper layers. In contrast, in the epidermis of the three Indian major carp the overall density of the mucous cells is much lower and the club cells are very numerous. It is suggested that the high density of club cells compensates an overall low density of mucous cells as an adaptation for an effective defence mechanism. Increased mucus production in the epidermis of C. carpio var. communis, as evidenced by a large number of mucous cells in outer regions and high secretory activity of superficial layer epithelial cells, is associated with increased precipitation of mud held in suspension, needed as an adaptation to the species’peculiar bottom-scooping habits. The varied density of the taste buds in the epidermis of the four carp is associated with their feeding habits.     

An examination of surface epithelium structures of the embryo across the genus Poeciliopsis (Poeciliidae)

In viviparous, teleost fish, with postfertilization maternal nutrient provisioning, embryonic structures that facilitate maternal‐fetal nutrient transfer are predicted to be present. For the family Poeciliidae, only a handful of morphological studies have explored these embryonic specializations. Here, we present a comparative morphological study in the viviparous poeciliid genus, Poeciliopsis . Using microscopy techniques, we examine the embryonic surface epidermis of Poeciliopsis species that vary in their level of postfertilization maternal nutrient provisioning and placentation across two phylogenetic clades and three independent evolutionary origins of placentation. We focus on surface features of the embryo that may facilitate maternal‐fetal nutrient transfer. Specifically, we studied cell apical‐surface morphology associated with the superficial epithelium that covers the body and sac (yolk and pericardial) of embryos at different developmental stages. Scanning electron microscopy revealed common surface epithelial cells across species, including pavement cells with apical‐surface microridges or microvilli and presumed ionocytes and/or mucus‐secreting cells. For three species, in the mid‐stage embryos, the surface of the body and sac were covered in microvillus epithelium. The remaining species did not display microvillus epithelium at any of the stages examined. Instead, their epithelium of the body and sac were composed of cells with apical‐surface microridges. For all species, in the late stage embryos, the surface of the body proper was composed of apical‐surface microridges in a “fingerprint‐like arrangement.” Despite the differences in the surface epithelium of embryos across Poeciliopsis species and embryonic developmental stages, this variation was not associated with the level of postfertilization maternal nutrient provisioning. We discuss these results in light of previous morphological studies of matrotrophic, teleost fish, phylogenetic relationships of Poeciliopsis species, and our earlier comparative microscopy work on the maternal tissue of the Poeciliopsis placenta.     

Quantitative studies on the skin of the paired species of lampreys,Lampetra fluviatilis (L.) and Lampetra planeri (BLOCH)

The number of mucous, club, and granular cells in the epidermis, and the number of rows of subcutaneous adipose cells, as well as the thickness of the epidermis and the dermal collagen layer, have been recorded for the larval and metamorphosing stages of the anadromous parasitic lamprey, Lampetra fluviatilis, and for the larval, metamorphosing, and adult stages of the nonparasitic lamprey, Lampetra planeri. In L. fluviatilis, the mucous cells predominated in all stages but were more abundant in fully metamorphosed individuals than in larvae. During metamorphosis, the number of granular cells increased continuously, whereas the club cells showed little change. Although lampreys do not feed during metamorphosis, there was an increase in the thickness of the epidermis and in the dermal collagen sheath; the latter increase probably foreshadows the increase in activity by the adults. Simultaneously, there is a reduction in the subcutaneous fat layer, which can be attributed to mobilization of lipid as an energy source. Changes similar to those just described for L. fluviatilis were also found in metamorphosing L. planeri. However, the pattern altered markedly during adult stages in this nonparasitic species. There were marked declines in the number of cells, in the thickness of the epidermis, in the width of the collagen sheath, and in the quantity of subcutaneous fat.     

Quantitative scanning electron microscopy of solitary chemoreceptor cells in cyprinids and other teleosts

Synopsis Solitary chemosensory cells (SCC) occur in the epidermis of many lower, aquatic vertebrates. By scanning electron microscopy, SCC apices were counted and density distributions estimated along various transects at the head and body of 12 species of teleost fishes, 7 cyprinids, 2 perciforms, 2 catfish and 1 characinid. In contrast to taste buds (TB), the distribution of SCCs is relatively even, with slightly higher densities at the forehead and along the dorsal trunk. In most species 1000 to 1500 SCC apices per mm² of skin were counted. Considerably higher densities occur in halos around free neuromasts. Depending on fish size and apex density, the epidermis of individuals may contain millions of SCCs. SCCs are considerably more abundant in individual fish than TB sensory cells. Highest average SCC densities (2000–4000 per mm²) were found in the cyprinids, roach, nase, chub and bream. Lowest densities (250 per mm²) occurred in the neon tetra. No correlations could be found between SCC densities and TB densities or relative size of the brain stem facial lobe, supporting the view of different functions and biological roles of the SCC and the TB systems. Whether teleost SCCs generally respond to mucoid substances, as in the case of the rocklings, remains an open question.     

Morphology of the Epidermis of the Neotropical Catfish Pimelodella lateristriga (Lichtenstein, 1823) with Emphasis in Club Cells

The epidermis of Ostariophysi fish is composed of 4 main cell types: epidermal cells (or filament containing cells), mucous cells, granular cells and club cells. The morphological analysis of the epidermis of the catfish Pimelodella lateristriga revealed the presence of only two types of cells: epidermal and club cells. The latter were evident in the middle layer of the epidermis, being the largest cells within the epithelium. Few organelles were located in the perinuclear region, while the rest of the cytoplasm was filled with a non-vesicular fibrillar substance. Club cells contained two irregular nuclei with evident nucleoli and high compacted peripheral chromatin. Histochemical analysis detected prevalence of protein within the cytoplasm other than carbohydrates, which were absent. These characteristics are similar to those described to most Ostariophysi studied so far. On the other hand, the epidermal cells differ from what is found in the literature. The present study described three distinct types, as follows: superficial, abundant and dense cells. Differences among them were restricted to their cytoplasm and nucleus morphology. Mucous cells were found in all Ostariophysi studied so far, although they were absent in P. lateristriga, along with granular cells, also typical of other catfish epidermis. The preset study corroborates the observations on club cells'' morphology in Siluriformes specimens, and shows important differences in epidermis composition and cell structure of P. lateristriga regarding the literature data.     

Structure and Histochemistry of the Skin of a Torrent Catfish, Liobagrus mediadiposalis

The epidermis of the torrent catfish, Liobagrus mediadiposalis, consists of three layers: the outermost layer, middle layer and stratum germinativum. The epidermis consists of two types of skin glands, small mucus cell and voluminous club cell. The unicellular mucus cell contains acid sulfomucins (some sialomucins) and the club cell, sometimes binucleate, is proteinaceous. Well-developed vascularization is one of the characteristics of epidermis of L. mediadiposalis. Well-developed lymphatic spaces contain lymphocytes in the epidermis. The dermis lacks scales and consists mostly of a thick, dense connective tissue; its superficial region just below the basal membrane is supplied with fine blood capillaries. These histological features of the skin in L. mediadiposalis are consistent with that required for cutaneous respiration.     

Cellular responses of the skin and changes in plasma cortisol levels of trout (Oncorhynchus mykiss) exposed to acidified water

The skin structure and the plasma cortisol levels of trout, Oncorhynchus mykiss, were examined during 7 days of exposure to water of pH 5. By day-4 and-7, the thickness of the epidermis was significantly (P<0.05) less in acid exposed fish than in controls, and degenerative cells were common in the upper epidermal layers. Many epidermal cells exhibited signs of necrosis, and by day-7 many apoptotic cells were also present. Secretory vesicles of high electron density were abundant in the filament cells of the 3–4 outermost layers of epidermis, and intercellular spaces had increased. Mitotic figures occureed throughout the epidermis, with the exception of the outermost cell layer. Mucous cells became elongated after day-1, and later, newly differentiating mucous cells could be seen close to the skin surface, and many mucocytes contained mucosomes of high electron density. Rodlet cells were occasionally seen. Chloride cells appeared similar to those of control fish. Many leucocytes, mainly macrophages and lymphocytes, had penetrated the epidermis via the highly undulating basal lamina, and at day-7, numerous apoptotic lymphocytes were found. In the dermis, melanosomes became dispersed in the cytoplasmic extensions of melanocytes which were present in the epidermis of all acid-exposed fish. Iridocytes were rate after day-4, while fibroblasts were abundant and secreted large amounts of collagen. After 1 day of exposure to acidified water, a significant (P<0.05) elevation of the plasma cortisol level had occurred, but this subsequently declined, and had returned to control values by day-7. The changes in skin structure, however, remained throughout the whole exposure period.     

An immunocytochemical study of intrapancreatic ganglia, nerve fibres and neuroglandular junctions in Brockmann bodies of the tompot blenny (Blennius gattoruggine), a marine teleost

The innervation of the Brockmann bodies in the teleost fish, Blennius gattoruggine, was studied using immunocytochemical techniques at both the light and electron microscopy levels. Islet innervation consisted of intrapancreatic ganglia, generally localized inside the rim of the exocrine tissue of the Brockmann bodies, in proximity to the islet, nerve fibres and nerve terminals with synaptic complexes. The intrapancreatic ganglia were of variable size, with different numbers of ganglionic cells, that appeared unipolar in section. The cell bodies showed immunoreactivity to galanin, oxytocin, peptide tyrosine tyrosine and glucagon. The extrinsic and intrinsic nerve fibres passed through the exocrine parenchyma and crossed the connectival septa and islet connectival sheath, penetrating into the islets, where they became increasingly thinner. They terminated on the endocrine cells with dilated nerve terminals. At least three types of terminals were detected, depending on the different vesicle content: peptidergic, cholinergic or adrenergic. They presented specialized synaptic structures, the neuroglandular junctions, some of which contained neurosecretory granules immunogold labelled by galanin antiserum. This new finding confirms the role of galanin as a neurotransmitter. This rich supply of innervation may be important in the regulation and integration of islet secretion.     

Surface folds in superficial epidermal cells of three species of teleost fish

Scanning electron microscope studies of fish epidermis demonstrate whorled arrangements of microfolds on the surface of superficial epidermal cells in the following species of teleost fish: the guppyfish Poecilia reticulata (Peters), the goldfish Carassius auratus auratus (L.), and the clown anemone fish Amphiprion percula (Lacépède). The pattern is most pronounced in epidermal cells covering the scales, but is also seen in the epidermal cells of the tail and body fins. The whorled pattern was not present on the gill epithelium or the surface of the eyes. These observations differ from transmission electron microscope studies which have described the skin surface as being composed of microvilli.     

Interactions between developmental processes,growth, and food selection in the larvae and juveniles of Rutilus rutilus (L.) (Cyprinidae)

Summary The aim of this study was to assess the effects of developmental events, occurring in fish during the first weeks after hatching, on the quantity and quality of the ingested food and on growth. The investigation was carried out with the larvae and juveniles of Rutilus rutilus, the single cyprinid species occurring in an oligotrophic subalpine lake in Tirol, Austria. Comparison between availability of prey in the water and gut contents suggests that the selection of food by the young fish is strongly influenced by developmental processes. For example, the prevalence of indigestible phytoplankton in the gut of young larvae can be taken as a sign of the not yet fully developed sensory and locomotory capacities of the young fish (El-Fiky et al. 1987). Furthermore, quantitative and qualitative changes in the gut contents correlate strongly with changes in the form and relative length of the gut, but reflect only weakly the availability of prey in the water. In the Seefelder See population of R. rutilus the switch from a phytoplankton to a cladoceran dominated diet is accompanied by an increase in relative growth rate by nearly one order of magnitude (Wieser et al. 1988).     

On the occurrence of merkel cells in the epidermis of teleost fishes

Summary The ultrastructure of a differentiated cell type in the epidermis of two species of teleost fish, Ictalurus melas and Phoxinus phoxinus, is described. This cell type has a synaptic association with nerve fibres, microvillus-like peripheral processes, and membrane-bounded inclusions, which together are the diagnostic features of the Merkel cells of tetrapod vertebrates. Other cytoplasmic features are shared with the epithelial cells. The appearance of the membrane-bounded granules depends on the fixative used; after fixation with glutaraldehyde the granules are of a size and electron-density comparable to that found in tetrapod Merkel cells, but after fixing in osmium tetroxide the granules are inconspicuous.Our thanks are due to Mr. A.C. Wheeler of the British Museum (Natural History) for help with the identification of the species of Ictalurus, and to Mr. E. Perry for technical assistance. One author (EBL) was supported by a SRC research studentship     

Structure and histochemistry of the epithelia of lips and associated structures of a catfishRita rita

InRita rita the upper lip is associated with the rostral cap and the lower lip with a skin fold. The epithelia of the lips, which are modified with respect to the peculiar feeding behaviour of the fish, are mucogenic. The superficial layer of epithelial cells, in addition to the mucous cells, are involved in active secretion of mucopolysaccharides. Mucus, apart from its diverse functions, as in fish epidermis, lubricates the epithelia giving protection against possible mechanical injury during the searching and catching of prey from bottom debris. Furthermore, the epithelia are thick, an adaptation to their mucogenic nature, so as to provide additional protection. The contents of the club cells are proteinaceous, which correlates with the helical filaments reported in similar cells in fish epidermis. Characteristic ridges on the surface of the lips are considered to be an adaptation to assist in manipulation during the procuring of food. Mucus secreted in the grooves between them reduces friction and prevents the deposition of foreign matter. The skin fold facilitates an increase in gape. The taste buds and ampullary organs, which on the lips and the rostral cap are associated with the localization of prey and triggering of a pick-up reflex, are not located on the skin fold, as the latter often remains concealed between the lower lip and the ventral head skin. The importance of histochemical analysis as a supplement to histological studies is emphasised.     

Relationship between urotensin II- and somatostatin-immunoreactive spinal cord neurons of Catostomus commersoni and Oncorhynchus kisutch (Teleostei)

Summary This immunocytochemical study describes the presence of separate immunoreactive (IR)-urotensin II (UII) and IR-somatostatin (SOM) systems in the spinal cord of two species of teleost fish. Both systems are arranged in a close spatial interrelationship in which IR-SOM fibres apparently innervate cerebrospinal fluid (CSF)-contacting IR-UII neurons. Specimens of Oncorhynchus kisutch also display CSF-contacting IR-SOM neurons located in the lateral ependymal walls of the central canal, in addition to CSF-contacting IR-UII neurons located ventrally. It is suggested that, in this species, CSF-contacting IR-SOM and IR-UII neurons perceive different stimuli from the CSF and are integrated in such a way that one peptidergic system may modulate the function of the other.     

A study on the epidermal structure of <Emphasis Type="Italic">Periophthalmodon</Emphasis> and <Emphasis Type="Italic">Periophthalmus</Emphasis> mudskippers with reference to their terrestrial adaptation

Epidermal structures of three species of Periophthalmus (Ps.) and two species of Periophthalmodon (Pn.) were investigated in relation to their lifestyle. All species of both genera lack a dermal bulge, which species of other two oxudercine genera, Boleophthalmus and Scartelaos, have in their epidermis. In Periophthalmus and Periophthalmodon species, which are highly terrestrial, the middle cells are well developed in the epidermis and the capillaries are distributed in the surface of the epidermis on the head and dorsal body. In Periophthalmus species and Pn. septemradiatus, the capillaries and blood vessels are also distributed in the epidermis of the abdomen, superficially in Ps. modestus and deeply in other species. In Ps. modestus, the capillaries are also densely distributed on the surface of the epidermis in the caudal area, whereas in other species, the epidermal capillaries and blood vessels of this area are located deep with a very low density. In Pn. schlosseri, the epidermal capillaries are not found in either the abdominal area or caudal area. A comparison of the distribution of epidermal capillaries among Boleophthalmus, Periophthalmodon, Periophthalmus, and Scartelaos species revealed that the skin makes a larger contribution to respiration in the species having a more terrestrial lifestyle. Goblet mucous cells are completely lacking in Periophthalmus species, whereas slimelike materials were often found on the skin surface of Periophthalmus species. This finding suggests that Periophthalmus species have some unknown mechanism for producing mucus. In Pn. schlosseri, exposure of the dense capillary net on the surface of the head is likely to increase cutaneous respiration, but it also makes the fish an attractive target of bloodsucking insects.Supplementary material to this paper is available in electronic format at http://dx.doi.org/10.1007/s10228-003-00173-7