Cichlid Fish Oreochromis Mossambicus Research Articles

The opioid peptide leucine enkephalin modulates hypothalamic-hypophysial axis in the cichlid fish Oreochromis mossambicus

In vertebrates, opioid peptides are thought to be involved in the regulation of reproduction; however, the significance of enkephalins in testicular function remains unclear. We examined the influence of δ-opioid receptor agonist leucine enkephalin (L-ENK) on the hypophysial-testicular axis of the cichlid fish Oreochromis mossambicus. Treatment with a low dose of L-ENK (60 µg) caused a significant increase in the numbers of primary and secondary spermatocytes and early and late spermatids, concomitant with intense immunolabelling of testicular androgen receptors, but did not significantly alter serum luteinizing hormone (LH) and 11-ketotestosterone (11-KT) levels compared to those of controls. Nevertheless, treatment with a high dose of L-ENK (200 µg) caused a significant reduction in the numbers of secondary spermatocytes as well as late spermatids associated with marginal immunolabelling of androgen receptors and significantly lower concentrations of serum 11-KT and LH compared to controls. In addition, the serum cortisol level was not affected in low-dose L-ENK-treated fish, but its level was significantly increased in the high-dose L-ENK-treated group. Together, these findings indicate that a low dose of L-ENK stimulates the germ cells at the meiosis stage and promotes further stages of spermatogenesis, whereas a high concentration of L-ENK inhibits spermatogenesis at the advanced stages. This effect appears to be mediated through the suppression of testicular steroidogenesis and the reduction of LH release in the pituitary gland of tilapia. The findings also suggest that elevated L-ENK levels in teleosts may exert their inhibitory influence on the hypophysial-testicular axis via glucocorticoids.

Serotonin-immunoreactivity in the brain of the cichlid fish Oreochromis mossambicus.

Serotonin (5-HT) is an evolutionarily conserved monoaminergic neurotransmitter found in the central nervous system and peripheral nervous system across invertebrates and vertebrates. Although the distribution of 5-HT-immunoreactive (5-HT-ir) neurons is investigated in various fish species, the organization of these neurons in cichlid fishes is poorly understood. These fish are known for their adaptability to diverse environments, food habits, and complex mating and breeding behaviors, including parental care. In this paper, we describe the organization of 5-HT-ir neurons in the brain of the cichlid fish Oreochromis mossambicus. Aggregations of 5-HT-ir neurons were spotted in the granule cell layer of the olfactory bulb and near the ventricular border in the preoptic area and magnocellular subdivisions of the nucleus preopticus. Although the presence of 5-HT-ir cells and fibers in the hypothalamic and thalamic regions, cerebellum, and raphe nuclei was comparable to that of other teleosts, the current study reveals the occurrence of 5-HT-ir cells and fibers for the first time in some areas, such as the nucleus posterior tuberis, nucleus oculomotorius, and nucleus paracommissuralis in the tilapia. While the presence of 5-HT-ir cells and fibers in gustatory centers suggests a role for serotonin in the processing of gustatory signals, distinctive pattern of 5-HT immunoreactivity was seen in the telencephalon, pretectal areas, mesencephalic, and rhombencephalic regions, suggesting a cichlid fish specific organization of the serotonergic system. In conclusion, the 5-HT system in the tilapia brain may serve several neuroendocrine and neuromodulatory roles, including regulation of reproduction and sensorimotor processes.

Organization of the Melanin concentrating hormone secreting neuronal system in the brain of the cichlid fish Oreochromis mossambicus

Melanin concentrating hormone (MCH) is a highly conserved cyclic peptide present in vertebrates. In this study, we describe the organization of MCH-immunoreactive (MCH-ir) cells and fibres in different regions of the brain in the cichlid fish Oreochromis mossambicus. Only MCH-ir fibres were observed in dorsal and ventral subdivisions of the telencephalon, the preoptic area and magnocellular and parvocellular divisions of the nucleus preopticus, and in hypothalamic areas such as the suprachiasmatic nucleus and tuberal area. Distinctly labelled MCH-ir perikarya were observed in the paraventricular organ, lateral and medial subdivisions of the nucleus lateralis tuberis, nucleus recessus lateralis and in the nucleus posterior tuberis. The pituitary gland showed MCH-ir fibres in the proximal pars distalis, neurohypophyseal ramifications and in pars intermedia where the dark accumulations of MCH-ir content corresponded to enlarged axon terminals. In the diencephalon, MCH-ir fibres were also labelled in the pretectal area, thalamic nuclei and preglomerular complex. In the midbrain tegmentum, a cluster of MCH-ir neurons was detected in the dorsal tegmental nucleus, whereas MCH-ir fibres were distributed in the torus semicircularis and optic tectum. In the rhombencephalon, MCH-ir fibres were located in the nucleus lateralis valvulae, cerebellum and secondary gustatory nucleus. Overall, the widespread distribution of MCH-ir cells and fibres in the brain suggests diverse roles for MCH such as regulation of sensorimotor and neuroendocrine functions in the tilapia.

Chronic exposure to aquacultural stressors affects pituitary-testis axis in the Mozambique tilapia Oreochromis mossambicus.

Reproduction in fish is modulated by several factors that include environmental and endocrine components. The aim of this study was to elucidate the effect of aquacultural stressors along the pituitary-testis axis in a continuously breeding cichlid fish Oreochromis mossambicus. The fish (35.05 ± 2.20g) were divided into three groups (n = 10 in each group, n = 5 in each replicate), namely initial controls (euthanized on the day of initiation of experiment), time-matched controls (kept undisturbed), and stressed fish, which were subjected to different kinds of randomized aquacultural stressors such as handling, chasing, frequent netting, and low water levels, daily for a period of 21days. Although the gonadosomatic index and the mean numbers of spermatogonia-A and spermatogonia-B did not differ significantly among different experimental groups, significant decrease was observed in the mean numbers of primary spermatocytes, secondary spermatocytes, early spermatids, and late spermatids in fish exposed to stressors compared to those of initial controls and time-matched controls. While the diameter of the seminiferous lobule was significantly lower, the size of the lumen and the serum levels of cortisol were significantly increased in stressed fish compared with initial controls and time-matched controls. Furthermore, weak androgen receptor immunoreactivity was observed in the Sertoli cells of the testis in contrast to the strongly immunoreactive androgen receptors in initial controls and time-matched controls. Concomitant with this, there was a significant decrease in the percent area and the intensity of luteinizing hormone (LH) immunoreactive content in the proximal pars distalis (PPD) region of the pituitary gland in stressed fish compared with initial controls and time-matched controls. Overall, these results suggest that exposure to chronic aquacultural stressors causes suppression of LH synthesis in the pituitary gland concomitant with decreased androgen receptor expression and blockade of recruitment of germline cells at the meiosis stage. This inhibition appears to be mediated through the hypothalamic-pituitary-interrenal axis in the tilapia O. mossambicus.

The effect of high or low temperature on testicular activity in the cichlid fish Oreochromis mossambicus

The effect of high or low temperature on testicular activity in the cichlid fish Oreochromis mossambicus

The effect of α-MSH treatment on the hypothalamic-pituitary-gonad axis in the cichlid fish Oreochromis mossambicus.

In this investigation, we examined the influence of alpha-melanocyte stimulating hormone (α-MSH), a proopiomelanocortin-derived peptide, along the hypothalamic-pituitary-gonad axis in a cichlid fish Oreochromis mossambicus. Administration of α-MSH (40µg/0.1ml saline) for 22days did not affect the number of stage I (previtellogenic) follicles but caused significant reduction in the mean numbers of previtellogenic (stages II and III), vitellogenic (stage IV) and preovulatory (stage V) follicles compared to those of controls. While the gonadosomatic index was significantly lower, the rate of follicular atresia in stages II, III and IV remained significantly higher in α-MSH-treated fish compared to the controls. Furthermore, the mean percent area of gonadotropin-releasing hormone-immunoreactive (GnRH-ir) fibres and luteinizing hormone-immunoreactive (LH-ir) cells were significantly reduced in the proximal pars distalis of the pituitary gland in α-MSH-treated fish compared with the controls. Together, our findings suggest for the first time that the treatment of α-MSH blocks the follicular developmental process during the ovarian cycle, possibly through the inhibition of GnRH-LH pathway in teleosts.

Neuroanatomical organization of methionine-enkephalinergic system in the brain of the Mozambique tilapia Oreochromis mossambicus

Enkephalins are a class of opioid peptides implicated in several physiological and neuroendocrine responses in vertebrates. In this study, using immunocytochemical or immunofluorescence technique, we examined the neuroanatomical distribution of methionine enkephalin (M-ENK) immunoreactivity in the central nervous system (CNS) of the cichlid fish Oreochromis mossambicus. In the telencephalon, no M-ENK-like-immunoreactive (M-ENK-L-ir) perikarya, but sparsely distributed fibres were detected in the glomerular layer and the granule cell layer of the olfactory bulb. Although intensely labeled M-ENK-L-ir cells and fibres were found in the pallium, no M-ENK immunoreactivity was observed in the subpallium. The preoptic area showed a few M-ENK-L-ir somata and dense innervations of fibres. In the hypothalamic area, M-ENK-L-ir cells and fibres were located in magnocellular and parvocellular subdivisions of the nucleus preopticus, and medial and lateral subdivisions of the nucleus lateralis tuberis. Surrounding the recessus lateralis of the third ventricle, several intensely stained and packed M-ENK-L-ir cells and fibres were seen in dorsal, lateral and ventral subdivisions of the nucleus recessus lateralis. In the diencephalon, M-ENK immunoreactivity was restricted to the habenula, the thalamus, the pretectal area and the nucleus posterior tuberis. Dense aggregations of M-ENK-L-ir fibres were seen in the mesencephalic subdivisions, the optic tectum and the torus semicircularis, whereas a few fusiform M-ENK-L-ir cells and fibres were scattered in the midbrain tegmentum. In the rhombencephalon, different populations of ovoid or spindle shaped M-ENK-L-ir cells were observed in the secondary gustatory nucleus, the sensory trigeminal nerve nucleus, the nucleus reticularis medialis and the vagal motor nucleus, whereas bands of fibres were seen in the rostral spinal cord. Collectively, the widespread distribution of M-ENK immunoreactivity in the CNS suggests a role for this opioid peptide in regulation of neuroendocrine control of reproduction and modulation of sensorimotor functions in fish.

Alpha-melanocyte stimulating hormone immunoreactivity in the brain of the cichlid fish Oreochromis mossambicus

This study reports the distribution of a pro-opiomelanocortin-derived neuropeptide α–MSH in the brain of the cichlid fish Oreochromis mossambicus. α–MSH-ir fibres were found in the granule cell layer of the olfactory bulb, the medial olfactory tract, the pallium and the subpallium, whereas in the preoptic area of the telencephalon, few large α–MSH-ir perikarya along with extensively labeled fibres were observed close to the ventricular border. Dense network of α–MSH-ir fibres were seen in the hypothalamic areas such as the nucleus preopticus pars magnocellularis, the nucleus preopticus pars parvocellularis, the suprachiasmatic nucleus, the nucleus anterior tuberis, the paraventricular organ, the subdivisions of the nucleus recessus lateralis and the nucleus recessus posterioris. In the nucleus lateralis pars medialis, some α–MSH-ir perikarya and fibres were found along the ventricular margin. In the diencephalon, numerous α–MSH-ir fibres were detected in the nucleus posterior tuberis, the nucleus of the fasciculus longitudinalis medialis and the nucleus preglomerulosus medialis, whereas in the mesencephalon, α–MSH-ir fibres were located in the optic tectum, the torus semicircularis and the tegmentum. In the rhombencephalon, α–MSH-ir fibres were confined to the medial octavolateralis nucleus and the descending octaval nucleus. In the pituitary gland, densely packed α–MSH-ir cells were observed in the pars intermedia region. The widespread distribution of α–MSH-immunoreactivity throughout the brain and the pituitary gland suggests a role for α–MSH peptide in regulation of several neuroendocrine and sensorimotor functions as well as darkening of pigmentation in the tilapia.

Leucine-enkephalin-immunoreactive neurons in the brain of the cichlid fish Oreochromis mossambicus

Enkephalins are the pentapeptides involved in pain relief and neuroendocrine responses with high affinity for delta opioid receptors in vertebrates. In the present investigation, we studied the distribution of leucine-enkephalin-immunoreactive (L-ENK-ir) neurons in the brain of the cichlid fish Oreochromis mossambicus. Application of the antisera against L-ENK revealed the presence of numerous L-ENK-ir perikarya and fibres in subdivisions of the dorsal and the ventral telencephalon, the medial olfactory tract and the nucleus entopeduncularis, whereas intensely labelled L-ENK-ir fibres were noticed in the olfactory bulb. Furthermore, the presence of L-ENK-ir cells and dense accumulations of fibres in the preoptic area and its subdivisions, the nucleus preopticus pars magnocellularis and the nucleus preopticus pars parvocellularis suggested a role for this peptide in regulation of reproduction. While intensely labelled cells and fibres were found in the nucleus lateralis tuberis pars lateralis as well as the nucleus lateralis tuberis pars medialis, some L-ENK-ir fibres were seen at the hypothalamo-hypophyseal tract indicating the possible hypophysiotrophic role for this peptide. Numerous L-ENK-ir cells and dense network of fibres were observed in the subdivisions of the nucleus recess lateralis and the pretectal area, whereas intensely labelled thick network of L-ENK- fibres were found in the ventromedial thalamic nucleus, the sub-layers of the optic tectum and the rostral spinal cord. The widespread distribution of L-ENK-immunoreactivity in the olfactory bulb, the telencephalon, the diencephalon and the mesencephalon regions of the brain as well as the spinal cord suggests the possible involvement of this peptide in the regulation of diverse functions such as neuroendocrine, antinociceptive, visual and olfactory responses in O. mossambicus.

Effect of food deprivation on neuropeptide Y immunoreactivity and concurrent biochemical and gonadal responses in tilapia Oreochromis mossambicus (Peters, 1852)

The effect of short- and long-term fasting on neuropeptide Y (NPY) secreting neurons in the brain and concurrent biochemical and gonadal responses were determined in the cichlid fish Oreochromis mossambicus (Peters, 1852). Exposure of fish to fasting for 6 or 12 days resulted in intensely stained NPY-immunoreactive neurons in the nucleus entopeduncularis and the nucleus preopticus regions of the brain in contrast to moderately stained neurons in control fishes, whereas food supplementation after 6 days starvation restored NPY-immunoreactive profile to the same state as in control groups in both male and female fish. The gonadosomatic index remained significantly lower in female fishes subjected to fasting alone as well as fasting followed by refeedings compared to controls. While the hepatosomatic index (HSI) also showed a significant decrease in fasting male and female fish compared to controls, it increased subsequently in fishes exposed to refeeding after starvation. In addition, a significant decrease in the serum levels of glucose and triglycerides as well as the hepatic tissue protein content in fasting fish compared to those of controls in both sexes suggested gluconeogenesis. Although the serum levels of glucose and triglycerides were reinstated in both males and female fishes subjected to starvation and refeeding, the hepatic tissue protein levels were not completely restored in females. Collectively, these results suggest that food-deprivation, either for short or long term duration, significantly increases the secretory activity of NPY neurons in the brain concomitant with a significant decrease in the serum levels of glucose and triglycerides, the hepatic tissue protein content and the GSI in both sexes. Although males recovered from the effects of starvation following refeeding, females appeared to be more vulnerable to the effects of fasting.

Distribution of endomorphin-like-immunoreactive neurones in the brain of the cichlid fish Oreochromis mossambicus.

Endomorphins (EMs) are tetrapeptides involved in pain and neuroendocrine responses with a high affinity for μ-opioid receptors in mammals. In the present study, we investigated the distribution of EM-like-immunoreactive (EM-L-IR) neurones in the brain of the cichlid fish Oreochromis mossambicus. Application of antisera against EM-1 and 2 (EM-1-2) revealed the presence of EM-L-IR somata and fibres throughout the different subdivisions of the olfactory bulb, such as the olfactory nerve layer and the granule cell layer. Although the extensions of EM-L-IR fibres were seen along the medial olfactory tract, intensely labelled EM-L-IR somata were found in different subdivisions of the telencephalon. In the diencephalon, intensely stained EM-L-IR neurones were noted in the preoptic area, the nucleus preopticus pars magnocellularis, the suprachiasmatic nucleus, the nucleus lateralis tuberis pars lateralis and the nucleus lateralis tuberis pars medialis regions, whereas projections of EM-L-IR fibres were also seen along the hypothalamic-hypophyseal tract, suggesting a possible hypophysiotrophic role for these neurones. Intense to moderately stained EM-L-IR neurones were noted in different subdivisions of thalamic nucleus, such as the dorsal posterior thalamic nucleus, commissura posterior, ventromedial thalamic nucleus, nucleus posterior tuberis, ventrolateral thalamic nucleus and medial preglomerular nucleus. Numerous intensely stained perikarya and axonal fibres were also noted throughout the inferior lobe, along the periventricular margin of the reccessus lateralis and in the nucleus recesus lateralis regions. In addition, numerous moderately labelled EM-like neuronal populations were found in the secondary gustatory nucleus and rostral spinal cord. The widespread distribution of EM-L-IR neurones throughout the brain and spinal cord indicates the diverse roles for these cells in neuroendocrine and neuromodulatory responses for the first time in fish. The present study provides further insights into the possible existence of EM-like peptides in early vertebrate lines and suggests that these peptides might have been well-conserved during the course of evolution.

Formation of the inner ear during embryonic and larval development of the cichlid fish (Oreochromis mossambicus)

ABSTRACTBackground: The vertebrate inner ear comprises mineralized elements, namely the otoliths (fishes) or the otoconia (mammals). These elements serve vestibular and auditory functions. The formation of otoconia and otoliths is described as a stepwise process, and in fish, it is generally divided into an aggregation of the otolith primordia from precursor particles and then a growth process that continues throughout life. Results: This study was undertaken to investigate the complex transition between these two steps. Therefore, we investigated the developmental profiles of several inner ear structural and calcium-binding proteins during the complete embryonic and larval development of the cichlid fish Oreochromis mossambicus in parallel with the morphology of inner ear and especially otoliths. We show that the formation of otoliths is a highly regulated temporal and spatial process which takes place throughout embryonic and larval development. Conclusions: Based on our data we defined eight phases of otolith differentiation from the primordia to the mature otolith.

A case of complete loss of gill parasites in the invasive cichlid Oreochromis mossambicus.

This study investigates the recent evolution of a rich parasite community associated with one of the world's most invasive species, the cichlid fish Oreochromis mossambicus. Populations from the species' native range (Mozambique) are compared to a population from New Caledonia (Wester Pacific), an island where the species was introduced in 1954. The results support the complete local extinction of the gill parasite community in the course of the invasion process. Up to six gill parasite species per locality were documented in the O. mossambicus native range, and previous surveys consistently reported at least one parasite species introduced along African cichlid species established out of Africa. The absence of parasites in New Caledonia is therefore exceptional. This can be attributed to local factors, such as a strong initial population bottleneck, the likely absence of multiple host introductions, and the frequent occurrence of brackish watersheds that might enhance the probability for natural deparasitation.

Expression of SPARC and the osteopontin-like protein during skeletal development in the cichlid fish Oreochromis mossambicus.

Bones are mainly composed of calcium hydroxyapatite and a proteinous matrix. In this study, we focus on the bone matrix proteins, the fish osteopontin orthologous protein (osteopontin-like protein; OP-L) and SPARC, because the current knowledge regarding their expression is fragmentary or contradictory. We first provide a comprehensive and detailed description of skeletal development in the cichlid fish Oreochromis mossambicus. Following this, we analyzed the expression pattern of OP-L and SPARC in detail during development. OP-L expression was only found in tissues that undergo ossification (i.e., developing bones and teeth). Furthermore, we show that there is a fundamental difference in cartilage formation of the splanchnocranium and all other cartilages, concerning SPARC expression. Significantly, we show that the initial calcification of cranial bones occurs simultaneously with the expression of OP-L and SPARC in the osteoblast-like cells, which appear early in development. The difference in SPARC expression during chondrogenesis of the splanchnocranium is likely based on its different evolutionary history compared with the dermatocranium and chondrocranium. Moreover, our results suggest a co-occurrence of the initial calcium deposition and bone matrix protein expression during osteogenesis. Overall, this study enhances our understanding of fish skeletal development and evolution.

Evidence for the involvement of dopamine in stress-induced suppression of reproduction in the cichlid fish Oreochromis mossambicus.

In the present study, we examined whether stress-induced suppression of reproduction is mediated through the catecholaminergic neurotransmitter dopamine (DA) in the female cichlid fish Oreochromis mossambicus. In the first experiment, application of antibody against tyrosine hydroxylase (TH; a marker for DA) in brain sections revealed the presence of intensely stained TH immunoreactive cells in the preoptic area (POA) and nucleus preopticus (NPO) during the previtellogenic phase. These cells showed weak immunoreactivity during the vitellogenic and prespawning phases concomitant with darkly stained luteinising hormone (LH) immunoreactive content in the proximal pars distalis (PPD) of the pituitary gland and fully ripened follicles (stage V) in the ovary of control fish. However, in fish exposed to aquacultural stressors, TH secreting cells remained intensely stained in POA and NPO regions during the prespawning phase, indicating increased synthetic and secretory activity, which was reflected by a significantly higher DA content compared to controls. Increased DA activity as a result of stress was associated with a decrease in the LH immunoreactive content in the PPD and an absence of stage V follicles in the ovary. In the second experiment, administration of DA caused effects similar to those in stressed fish, whereas DA receptor antagonist domperidone (DOM) treatment significantly increased the LH content in the PPD and the number of stage V follicles in unstressed fish. On the other hand, treatment of stressed fish with DOM resulted in dark accumulations of LH immunoreactive content in the PPD accompanied by the presence of stage V follicles in the ovary. Taken together, these results suggest an additional pathway for the inhibitory effects of stress through dopaminergic neurones along the reproductive axis.

Naltrexone attenuates stress-induced suppression of LH secretion in the pituitary gland in the Cichlid fish Oreochromis mossambicus: evidence for the opioidergic mediation of reproductive stress response

Opioid peptide β-endorphin (β-EP) plays a modulatory role in vertebrate reproduction. However, the role of opioid peptides in reproductive stress response is least understood in fishes. The aim of the present study was to determine the effect of different doses of β-EP on luteinizing hormone (LH) secretion in normal and the opioid receptor antagonist naltrexone (NALT) in stressed female tilapia Oreochromis mossambicus. Administration of 4μg β-EP, but not 0.5 or 1.5μg β-EP, daily for 22days caused suppression of LH-secreting cells at the proximal pars distalis of the pituitary gland, concomitant with a significant reduction in the mean GSI and HSI in 4μg β-EP-treated fish compared to controls. On the other hand, exposure of the fish to mild acute stressors for 22days caused changes in the LH-secreting cells similar to that of high dose of β-EP, whereas administration of NALT attenuated these effects. Taken together, the results indicate that increased concentration of β-EP as may occur during stressful conditions can cause suppression of LH secretion, leading to the inhibition of spawning, and that treatment of NALT attenuates the stress-induced inhibition of LH secretion in fish.

Near-infrared orientation of Mozambique tilapia Oreochromis mossambicus

Light plays a pivotal role in animal orientation. Aquatic animals face the problem that penetration of light in water is restricted through high attenuation which limits the use of visual cues. In pure water, blue and green light penetrates considerably deeper than red and infrared spectral components. Submicroscopic particles and coloured dissolved organic matter, however, may cause increased scattering and absorption of short-wave components of the solar spectrum, resulting in a relative increase of red and infrared illumination. Here we investigated the potential of near-infrared (NIR) light as a cue for swimming orientation of the African cichlid fish (Cichlidae) Oreochromis mossambicus. A high-throughput semi-automated video tracking assay was used to analyse innate behavioural NIR-sensitivity. Fish revealed a strong preference to swim in the direction of NIR light of a spectral range of 850–950nm at an irradiance similar to values typical of natural surface waters. Our study demonstrates the ability of teleost fish to sense NIR and use it for phototactic swimming orientation.

The role of predictability in the stress response of a cichlid fish

In recent years there has been an increasing interest in the cognitive abilities of fish with implications for animal welfare and management of rearing operations. Although it is known that psychological factors can modulate the stress response in mammals, this aspect has seldom been investigated within stress in fish. In this study we investigate whether the perception (appraisal) that fish make of significant environmental events modifies their behavioural and physiological response. For this purpose we have used a predictable vs. unpredictable paradigm for positive (feeding) and negative (confinement) events using the cichlid fish Oreochromis mossambicus as a model species. Results show that there is a differential effect of predictability for the feeding and confinement events. In the confinement experiment, predictability involved more attention to the visual cue and lower cortisol. The feeding event triggered higher levels of anticipatory behaviour and a tendency for higher cortisol in the predictable group. Therefore, predictable negative events reduce the cortisol response. Predictable positive events may elicit an anticipatory response, and when there is a significant delay between the visual cue and the actual occurrence of the event, it may also contain elements that can be interpreted as a stress response. These findings demonstrate that fish can appraise relevant aspects of the environment, with welfare implications for housing, husbandry and experimental procedures.

Histochemical localisation of carbonic anhydrase in the inner ear of developing cichlid fish, Oreochromis mossambicus

Inner ear otolith growth in terms of mineralisation mainly depends on the enzyme carbonic anhydrase (CAH). CAH is located in specialised, mitochondria-rich macular cells (ionocytes), which are involved in the endolymphatic ion exchange, and the enzyme is responsible for the provision of the pH-value necessary for otolithic calcium carbonate deposition. In the present study, for the first time the localisation of histochemically demonstrated CAH was analysed during the early larval development of a teleost, the cichlid fish Oreochromis mossambicus. CAH-reactivity was observed already in stage 7 animals (onset of otocyst development; staging follows Anken et al. [Anken, R., Kappel, T., Slenzka, K., Rahmann, H. The early morphogenetic development of the cichlid fish, Oreochromis mossambicus (Perciformes, Teleostei). Zool. Anz. 231, 1–10, 1993]). Neuroblasts (from which sensory and supporting cells are derived) proved to be CAH-positive. Already at stage 12 (hatch), CAH-positive regions could be attributed to ionocyte containing regions both in the so-called meshwork and patches area of the macula (i.e., clearly before ionocytes can be identified on ultrastructural level or by employing immunocytochemistry). In contrast to the circumstances observed in mammalian species, sensory hair cells stained negative for CAH in the cichlid. With the onset of stage 16 (finray primordia in dorsal fin, yolk-sac being increasingly absorbed), CAH-reactivity was observed in the vestibular nerve. This indicates the onset of myelinisation and thus commencement of operation. The localisation of CAH in the inner ear of fish (especially the differences in comparison to mammals) is discussed on the basis of its role in otolith calcification. Since the vestibular system is a detector of acceleration and thus gravity, also aspects regarding effects of altered gravity on CAH and hence on the mineralisation of otoliths in an adaptive process are addressed.

Sites of calcium uptake of fish otoliths correspond with macular regions rich of carbonic anhydrase

Based on pharmacological data, it has been suggested that the enzyme carbonic anhydrase (CAH) plays a prominent role in the mineralization of fish otoliths. To directly test this proposal, the topographical distribution of CAH was histochemically analyzed in the utricular and saccular maculae of larval cichlid fish Oreochromis mossambicus. Further investigations were focussed on the sites of otolithic calcium uptake using the fluorescent calcium tracer alizarin-complexone (AC). Both in the utricle and the saccule, CAH-reactivity was prominent in regions on both sides of the sensory macula (centrifugal (cf) and centripetal (cp) areas), which reportedly contain ionocytes, specialized cells regulating the ionic composition of the endolymph. (The terms centrifugal and centripetal were chosen instead of lateral and medial, because the saccule is positioned perpendicular to the utricle; “lateral” and “medial” thus do not allow an unambiguous allocation of the respective regions.) In the saccule, the size of cf and cp did not differ from each other, whereas, in the utricle, cp was considerably larger as compared to cf (CAH-reactivity per μm 2 was nearly identical in both areas of both endorgans). AC-incubation resulted in a fluorescent band on the proximal surface of the otoliths (this surface lies next to the sensory epithelium). In saccular otoliths (sagittae), the area of the band did not differ between centrifugal and centripetal otolith regions, whereas in the utricular otoliths (lapilli), the area of the centripetal AC-band was larger in size as compared to the centrifugal one (AC-fluorescence per μm 2 did not differ between the areas analyzed in both types of otoliths). These results strongly suggest that calcium/carbonate uptake of otoliths takes place especially in those regions of their proximal face which are located adjacent to CAH-rich areas of the macular epithelium. It is thus concluded that CAH is directly involved in otolith calcification. The differences in CAH-localization/AC-uptake in the two endorgans analyzed are discussed on the basis of their different functional properties (saccule, hearing; utricle, graviperception).