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.     

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.     

Die Feinstruktur der Kolbenzellen (Schreckstoffzellen) in der Epidermis von Astyanax mexicanus Filippi 1853 (Characinidae,Pisces) und seinen Höhlenderivaten “Anoptichthys”

Fine Structure of the Club Cells (Alarm Substance Cells) in the Epidermis of Astyanax mexicanus Filippi 1853 (Characinidae, Pisces) and its Cave Forms “Anoptichthys” The club cells in the epidermis of Astyanax mexicanus are of the same ultrastructure as those in other Ostariophysan fish. There are also no essential differences to be found between the club cells of epigean Astyanax mexicanus and its cave forms “Anoptichthys”. Our findings correspond to former statements in that the function of the club cells producing and releasing alarm substances has been maintained in the cave forms.     

The ontogeny of chemically mediated antipredator responses of fathead minnows Pimephales promelas

The antipredator responses of adult and larval fathead minnows Pimephales promelas to chemical alarm cues prepared throughout ontogeny were tested using various behavioural assays. Larval epidermis was also examined during ontogeny using standard haematoxylin and eosin staining techniques. Adults elicited an antipredator response to chemical alarm cue made from larvae as young as 8–17 days post‐hatch. Interestingly, larvae did not possess visible club cells until 28–37 days post‐hatch and did not respond to conspecific chemical alarm cue until 48–57 days post‐hatch. These results suggest that chemical alarm cue may not be contained within club cells and that the components of larval and adult chemical alarm cue may be similar throughout ontogeny.     

Anti‐predator response of naïve and experienced common bully to chemical alarm cues

Histological analysis of the skin of common bully Gobiomorphus cotidianus , a New Zealand native eleotrid fish, revealed the presence of club cells in the epidermis. Epidermal club cells are frequently associated with the production of alarm substance (Schreckstoff). The behavioural responses of perch‐naïve and perch‐experienced common bullies to either conspecific skin extract or chemical cues from an introduced predator, perch Perca fluviatilis , were then examined. Both perch‐naïve and perch‐experienced common bullies exhibited a behavioural response when exposed to conspecific skin extract, indicating the probable presence of an alarm substance. In contrast, only perch‐experienced common bullies recognized and exhibited a subsequent behavioural response to the odour of perch. This study is the first to document the presence of epidermal club cells and a behavioural response to a conspecific chemical alarm signal for fishes in the Eleotridae. The results indicate that common bully can learn to recognize perch odour as a threat, and that this ability may be a result of previous predator labelling involving a conspecific alarm substance.     

Within and between Population Variation in Epidermal Club Cell Investment in a Freshwater Prey Fish: A Cautionary Tale for Evolutionary Ecologists

Many prey fishes possess large club cells in their epidermis. The role of these cells has garnered considerable attention from evolutionary ecologists. These cells likely form part of the innate immune system of fishes, however, they also have an alarm function, releasing chemical cues that serve to warn nearby conspecifics of danger. Experiments aimed at understanding the selection pressures leading to the evolution of these cells have been hampered by a surprisingly large intraspecific variation in epidermal club cell (ECC) investment. The goal of our current work was to explore the magnitude and nature of this variation in ECC investment. In a field survey, we documented large differences in ECC investment both within and between several populations of minnows. We then tested whether we could experimentally reduce variation in mean ECC number by raising fish under standard laboratory conditions for 4 weeks. Fish from different populations responded very differently to being held under standard laboratory conditions; some populations showed an increase in ECC investment while others remained unchanged. More importantly, we found some evidence that we could reduce within population variation in ECC investment through time, but could not reduce among-population variation in mean ECC investment. Given the large variation we observed in wild fish and our limited ability to converge mean cell number by holding the fish under standard conditions, we caution that future studies may be hard pressed to find subtle effects of various experimental manipulations; this will make elucidating the selection pressures leading to the evolution of the cells challenging.     

Socializing makes thick-skinned individuals: on the density of epidermal alarm substance cells in cyprinid fish, the crucian carp (Carassius carassius)

In cyprinid fish, density of epidermal club cells (i.e. alarm substance cells) has been found to vary between lakes with different predator fauna. Because predators can be labelled with chemical cues from prey, we questioned if club cell density could be controlled indirectly by predators releasing prey cues. In particular, we suspected a possible feedback mechanism between chemical alarm signals and their cellular source. We raised crucian carp singly and in groups of four. For both rearing types, fish were exposed to skin extracts of either conspecifics or brown trout (without club cells), and provided either low or high food rations. Independent of rearing type, condition factor and club cell density increased with food ration size, but no change was found in club cell density following exposure to conspecific alarm signals. However, the density of club cells was found significantly higher for fish raised in groups than for fish raised alone. We conclude that an increased condition factor results in more club cells, but crucian carp may also possess an awareness of conspecific presence, given by higher club cell densities when raised in groups. This increase in club cell density may be induced by unknown chemical factors released by conspecifics.     

The role of olfaction in the behavioural and physiological responses to conspecific skin extract in Brycon cephalus

A behavioural alarm reaction was shown by 78% of the non-operated juvenile matrinxã Brycon cephalus exposed to a conspecific skin extract, whereas the other 22% did not change their behaviour. The main reaction (72% of those reacting) consisted of a brief initial phase (2 min) of dashing or very rapid swimming followed by a long-lasting period of immobility (up to 30 min). The second most frequent behaviour (22% of the reacting fish) was immobility, with the animals becoming motionless immediately after exposure to the conspecific skin extract, for periods ranging from 5 to 30 min. Only one animal slowed its swimming activity. The responses, evaluated by the number of times the animal crossed the grid in the rear of the aquarium, were significantly lower when compared to the baseline swimming activity. Histological examination of matrinxã epidermis revealed the existence of club cells. Complete sectioning of the olfactory tracts as well as bilateral sections of the olfactory sub-tracts (medial or lateral), stopped the alarm reaction, confirming the importance of olfaction in the ability to recognize the chemical stimulus. Despite the behavioural responses, no variation was detected in blood glucose or cortisol levels, a result that is discussed in terms of the intensity of the disturbance caused by the alarm substance.     

Cortisol influences the antipredator behavior induced by chemical alarm cues in the Frillfin goby

We evaluated the effect of increased plasma cortisol levels on fish antipredator behavior induced by conspecific chemical alarm cues. The experimental model for the study was the Frillfin goby Bathygobius soporator. We first confirmed that the alarm substance induces typical defensive antipredator responses in Frillfin gobies and described their alarm substance cells (epidermal ‘club’ cells). Second, we confirmed that intraperitoneal cortisol implants increase plasma cortisol levels in this species. We then demonstrated that exogenous cortisol administration and subsequent exposure to an alarm substance decreased swimming activity to a greater extent than the activity prompted by either stimulus alone. In addition, cortisol did not abolish the sheltering response to the alarm chemical cue even though it decreased activity. As predators use prey movements to guide their first contact with the prey, a factor that decreases swimming activity clearly increases the probability of survival. Consequently, this observation indicates that cortisol helps improve the antipredator response in fish.     

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.     

Response of club cells in the skin of the carp Cyprinus carpio to exogenous stressors

The ultrastructure of club cells and neighbouring filament cells and leucocytes in the epidermis of carp, was studied under normal conditions and after exposure to several stressors: acid water, heavy metals, organic manure, brackish water and wounding. The effects of the stressors were remarkably similar. The club cells increased in size and contained more endoplasmic reticulum and Golgi areas. In both control and stressed fish, most mitotic figures of the filament cells were found adjacent to club cells, as was demonstrated after colchicine injection. Whereas in the controls apoptosis of filament cells was scarce and limited to the upper layer of the epithelium, in the stressed fish it was commonly seen in close proximity to the club cells but not in other mid-epidermal parts of the epithelium. This indicates that club cells influence the cellular kinetics of the filament cells. Under stress conditions leucocytes infiltrated the epidermis. Some were seen inside club cells. Apparently these leucocytes were taken up in phagosomes and subsequently they showed signs of necrotic degeneration. Leucocyte incorporation and degeneration in club cells were not observed in control fish. Control of the cellular turnover of filament cells and the elimination of leucocytes may represent new functions for club cells, which have mainly been associated with the production of pheromones.     

Alarm reaction and alert state inGambusia Affinis (Pisces,Poeciliidae) in response to chemical stimuli from injured conspecifics

We studied the behavior of the Poeciliid fishGambusia affinis after the introduction of 3 substances into their tank: a homogenization ofGambusia affinis, a homogenization of the Anabantid fishBetta splendens, and a blank made of distilled water. The response of the fish was measured as a change in their spatial distribution in the tank after the introduction of the substance. Two sizes of fish were used, and theGambusia homogenization produced clear alarm reactions in both, the fish fleeing to the bottom of the tank. This is one of a few examples available of recognition of alarm substances in non-ostariophysian fish. In addition, we found that the small fishes that had recently been exposed to the alarm substance stayed in an ‘alert state’, in which they had an increased sensitivity to mechanical and visual fright stimuli.     

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

Summary 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).     

Death-associated odors induce stress in zebrafish

Living animals exploit information released from dead animals to conduct adaptive biological responses. For instance, a recently published study has shown that avoidance behavior is triggered by death-associated odors in zebrafish. Stress can clearly act as an adaptive response that allows an organism to deal with an imminent threat. However, it has not been demonstrated whether these chemical cues are stressful for fish. Here, we confirmed that dead zebrafish scents induce defensive behavior in live conspecifics. Additionally, we show for the first time in fish that these scents increase cortisol in conspecifics. To reach this conclusion, firstly, we exposed zebrafish to multi-sensorial cues (e.g., visual, tactile, chemical cues) from dead conspecifics that displayed defensive behaviors and increased cortisol. Also, when we limited zebrafish to chemical cues from dead conspecifics, similar responses arose. These responses coincide with the decaying destruction of epidermal cells, indicating that defensive and stress responses could take place as an effect of substances emanating from decaying flesh, as well as alarm substance released due to rupture of epidermal cells. Taken together, these results illustrate that living zebrafish utilize cues from dead conspecific to avoid or to cope with danger and ensure survival.     

The ‘club’ cell and behavioural and physiological responses to chemical alarm cues in the Nile tilapia

The alarm response to skin extract has been well documented in fish. In response to skin extract, there is a decline in both locomotion activity and aggressive interactions. Our observation herein of these responses in the cichlid Nile tilapia, Oreochromis niloticus, confirmed the existence of the alarm response in this species. However, so far there has been a paucity of information on the autonomic correlates of this response. In this study, the ventilatory change in response to the chemical alarm cue was evaluated. This parameter was measured 4 min before and 4 min after exposure to 1 mL of either conspecific skin extract or distilled water (extract vehicle). Skin extract induced an increase in the ventilation rate, which suggested an anticipatory adjustment to potentially harmful stimuli. The chemical cue (alarm substance) also interfered with the prioritisation of responses to different environmental stimuli (stimuli filtering); this was suggested by the observation that the Nile tilapia declined to fight after exposure to a cue that indicates a risk of predation. Furthermore, histological analysis of the Nile tilapia skin revealed the presence of putative alarm substance-producing (club) cells.     

Embryonic substances induce alarm response in adult zebrafish (Danio rerio)

Many aquatic animals rely on chemicals released by injured individuals of the same species to assess predation risk. Among these chemical cues, alarm substances released from the injured skin of ostariophysan fishes have been extensively examined. In most fish species examined, these cues appear to be released by all injured individuals (including larvae, juveniles and adults) and elicit alarm responses in conspecifics. Adult alarm cues also affect development and physiology of embryos. Nonetheless, whether embryos produce alarm cues that affect adults is not known. This study reports that extracts of zebrafish (Danio rerio) embryos at 36 h post-fertilization or later induce antipredator behaviours reminiscent of those induced by skin alarm substances. At an equivalent of 10⁻⁶ g embryo per millilitre, the extract induced bottom-dwelling and freezing in adults. These behaviours are consistent with those induced by adult alarm substances. This study concludes that zebrafish embryos produce alarm substances.     

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.     

Olfactorily-mediated cortisol response to chemical alarm cues in zebrafish Danio rerio

Chemical cues released as a by-product of predation mediate antipredator behaviour, but little is known about the physiological responses to olfactory detection of predation risk. In this study, zebrafish Danio rerio were exposed to either chemical alarm cues from conspecifics, or water (control). Compared with water controls, D. rerio exposed to alarm cues responded behaviourally with antipredator behaviours such as erratic dashing and an increase in time spent near the bottom of the test aquarium. Danio rerio were sacrificed 5 min after exposure to test cues (alarm cues or water). Enzyme-linked immunosorbent assay (ELISA) revealed whole-body levels of cortisol that were significantly higher for fish exposed to alarm cues (mean ± SE, 11.9 ± 3.4 ng g⁻¹) than control fish (1.5 ± 0.7 ng g⁻¹). These data provide a benchmark for future studies of the proximate mechanisms of olfactorily mediated antipredator responses, modelling effects on aquatic life in a changing climate and, as a model organism, Danio rerio can further our understanding of anxiety in humans.     

繁殖期4种淡水鱼类化学预警行为及其与性腺指数的关联

化学预警信息（Chemical alarm cue,CAC）在鱼类捕食者-猎物关系中发挥重要作用,并与二者生存适合度密切关联。繁殖期鱼类的繁殖生理投入和化学预警响应均具有较高的能量需求,因而可能存在某种程度上的生存-繁殖的权衡（Trade-off）。选取高体鳑鲏（Rhodeus ocellatus,卵生,繁殖投入较低）、斑马鱼（Danio rerio,卵生,繁殖投入较高）、孔雀鱼（Poecilia reticulata,卵胎生,繁殖投入较高）和皮球鱼（Poecilia latipinna,卵胎生,繁殖投入很高）等繁殖方式各异、能量投入不同的4种淡水鱼类为实验对象,测定了繁殖IV期实验鱼对不同浓度的化学预警信息（Damage-released chemical alarm cues,DCAC）的行为响应模式,并探究了4种实验鱼的化学预警响应与其性腺指数（Gonadosomatic index,GSI）的关联。结果表明：（1）DCAC对4种实验鱼的空间分布、活跃状态、逃逸行为等大部分化学预警响应参数均有不同程度的影响（P<0.05）;（2）4种实验鱼GSI与其化学预警响应参数的变化无显著相关性（P>0.05）（除高浓度DCAC处理组皮球鱼GSI与底栖时间的变化负相关外）。研究回答了长期以来有关繁殖期鱼类化学预警通讯是否缺失的问题,证实了繁殖期不同繁殖生理投入/繁殖对策的4种淡水鱼类均有显著的化学预警响应,提示鱼类GSI与其化学预警响应程度通常不相关（仅高风险环境下繁殖生理投入很高的物种中可能呈现负相关）。     

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.