Mercuric chloride induced toxicity responses in the olfactory epithelium of Labeo rohita (HAMILTON): a light and electron microscopy study

Bioaccumulation of mercury and histomorphological changes in the olfactory epithelium of Labeo rohita were investigated after exposing the fish to two sublethal concentrations of HgCl₂ (66 and 132 μg/L) for 15 and 30 days. Mercury deposition increased in the tissue significantly (p < 0.05) with dose- and duration-dependent manner. Severe damage to the olfactory epithelium was evident. When fish exposed to 66 μg/L for 15 days, the histology of olfactory epithelium exhibited that mucous cell proliferation was upregulated and cell size was significantly increased from the control. Similar trends were found in 30 days exposure in both treated groups. Histology showed that mercury induced degeneration of columnar sensory cells, supporting cells and ciliated non-sensory cells and induced basal cell proliferation. Basal cell hyperplasia led to form intraepithelial proliferative lesion, thickening of epithelium, basal lamina disruption and cyst formation. Scanning electron microscopy revealed that mercury exposure at 66 μg/L caused clumping and loss of cilia, erosion in microridges on the supporting cells and proliferation of mucous cell opening. Complete degeneration of ciliated cells and cyst formation was observed in the fish when exposed to 132 μg/L HgCl₂. This result suggests that prolonged exposure to mercury might cause irreversible damage to the olfactory epithelium and impair the olfactory function of fish.     

Molecular evolution of neurohypophysial hormones in relation to osmoregulation: the two fish options

In vertebrates, water and osmolyte homeostasis is controlled by at least three hormonal systems: the hypothalamo-neurohypophysial system, the renin-angiotensin-aldosterone system and the atrial natriuretic factor system. The first system implies neuroendocrine reflexes involving an afferent neural limb from osmo- and baroreceptors to hypothalamus and an efferent endocrine limb from secretory neurons to target cell receptors and transduction to intracellular effectors, namely water and sodium channels. Evolution can affect any level of this molecular cascade. Whereas virtually all vertebrate species have two neurohypophysial hormones, an oxytocin-like and a vasopressin-like peptides, the most primitive vertebrates, Cyclostomata (lampreys and hagfishes) possess a single peptide, vasotocin, so that an early gene duplication occurred before the emergence of fishes, about 400 million years ago. The remarkable evolutionary stability of neurohypophysial hormones in bony vertebrates allows us to trace two main lineages: isotocin-mesotocin-oxytocin and vasotocin-vasopressin. In contrast, in cartilaginous fishes, the oxytocin-like peptides display a great evolutionary diversity: we have identified glumitocin in rays, aspargtocin and valitocin in the spiny dogfish, asvatocin and phasvatocin in the spotted dogfish, and oxytocin in the chimaera. Whereas bony vertebrates regulate their blood osmotic pressure (about 250–450 mOsm kg^-1 H₂O) essentially through salts, cartilaginous fishes use urea as the main osmolyte for adjusting their osmotic pressure above the external medium (about 1050 mOsm kg^-1 H₂O for marine fishes). The hypothesis is made that the stability of neurohypophysial hormones in bony vertebrates is due to their implication in ion-based osmoregulation (selective evolution), whereas in Chondrichthyes the occurrence of urea-based osmoregulation has relieved the hormones from this function and therefore made them free to vary (neutral evolution).     

Gyrodactylus salmonis infection impairs the olfactory system of rainbow trout

Monogenean worms are ectoparasites that are known to be infectious to a wide variety of fish. Few species of monogenean parasites have been reported in the olfactory chamber of fish in current peer‐reviewed literature. However, the impacts of these parasites on the olfactory system are not well understood. In this study, the effects of Gyrodactylus salmonis on the olfactory system structure and performance were investigated in rainbow trout (Oncorhynchus mykiss). The olfactory performance of the infected fish was examined using an electro‐olfactography (EOG) technique, while the ultrastructure of the olfactory rosette was studied using scanning electron microscopy (SEM) and light microscopy (LM). The infected rainbow trout displayed reduced responses to two standard olfactory cues (L‐alanine and TCA). The SEM micrographs revealed that many regions of the olfactory epithelium in the infected fish were heavily pitted and the LM examination of the olfactory epithelium showed local proliferation of mucous cells in the sensory regions as compared to the control group. The results of this study demonstrated that G. salmonis causes physical damage to the olfactory system of fish that lead to olfactory impairment.     

Detection of thyroid hormone receptors in the olfactory system and brain of wild masu salmon,Oncorhynchus masou (Brevoort), during smolting by in vitro autoradiography

Thyroid hormone regulates a number of physiological functions during smolting in salmonids. However, the target sites and roles of thyroid hormone in the central nervous system (CNS) are not known in detail. We detected thyroid hormone-specific binding sites (i.e. thyroid hormone receptors) in the olfactory epithelium and the brain (the olfactory bulb, the telencephalon, the mid-brain and the cerebellum) of wild masu salmon, Oncor- hynchus masou (Brevoort), during smolting by means of in vitro autoradiography with frozen sections. A saturation experiment with the brain indicated the presence of a single class of binding sites of high affinity. T3-specific binding was detected in the olfactory epithelium and in all regions of the brain except the olfactory bulb. The T3-specific binding value in the olfactory epithelium was higher than in all other regions of the brain. This binding value in the olfactory epithelium increased at the full-smolt stage. The presence of thyroid hormone receptors in various regions of the CNS suggests that thyroid hormone plays an important role in the functional change in the brain and the olfactory epithelium during smolting.     

Cloning and expression of prostaglandin E2 receptor subtype 1 (ep 1 ) in Bostrichthys sinensis

Our previous studies suggested that prostaglandin E₂ (PGE₂) is a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. We found immunoreactivities of PGE₂ receptor subtypes (Ep_1–3) expressed in the olfactory sac, but only Ep₁ presented higher density of immunoreactivity in mature fish than that in immature fish in both sexes. To gain a better understanding of the underlying molecular mechanism for the detection of PGE₂ in the olfactory system, we cloned an ep ₁ cDNA from the adult olfactory sac. The open-reading frame of the ep ₁ consisted of 1,134-bp nucleotides that encoded a 378-amino acid-long protein with a seven-transmembrane domain, typical for the G protein-coupled receptors superfamily. Expression of ep ₁ mRNA was observed in all tissues examined, with higher levels obtained in the olfactory sacs and testes. The expression of ep ₁ mRNA in the olfactory sacs and gonads was significantly higher in both sexes of mature fish than in those of immature ones. Taken together, our results suggested that Ep₁, which is highly expressed in the olfactory sacs and gonads of mature fish, is important for the control of reproduction and may be involved in PGE₂-initiated spawning behavior in B. sinensis.     

Morphological and histochemical characteristics of the epithelium of ovarian lamellae of <Emphasis Type="Italic">Genypterus blacodes</Emphasis> (Schneider, 1801)

The physiological significance of the glycoproteins (GPs) secreted by the epithelium of ovarian lamellae is discussed in reference to the reproductive biology of G. blacodes. Histochemical procedures for localising and characterising GPs were used to determine the cytoplasmic components of cells of the epithelium that covers the ovarian lamellae of pink cuskeel, Genypterus blacodes (Schneider, 1801) (Pisces, Ophidiidae), during spawning. This species is one of the most valuable demersal fish resources in the Argentine Sea, mainly due its large size and flesh quality. GPs with oxidizable vicinal diol groups, sialic acid with or without O-acyl substituents, O-acyl sugars, neutral sugars and GPs with carboxyl and sulphate groups were detected. Light microscope examination showed morphological changes in the epithelium of ovarian lamellae during the spawning season, associated with a secretory activity of mucus. Optical density studies revealed the presence of polyploid cells encompassing those morphological changes. Results of the present study suggest that the epithelium of ovarian lamellae of G. blacodes performs a secretory role, which is intensified during ovarian maturity, suggesting that G. blacodes could release masses of eggs enveloped in mucus.     

Presence and ontogeny of intestinal and pancreatic phospholipase A2-like proteins in the Red Sea bream,Pagrus major. An immunocytochemical study

We have studied the location and the ontogeny of the digestive enzyme, phospholipase A₂ (PLA₂) immunohistochemically in the adult and larvae/juvenile of the red sea breamPagrus major by using an antiserum against theNaja naja venom PLA₂. The antiserum reacts with at least one enzyme among the PLA₂s purified from the fish hepatopancreas or intestine. Although the reactivities were comparatively low, it labelled zymogen granules of the pancreatic acinar cells and secretory materials of certain epithelial cells in the depths of epithelial crypts in the pyloric caeca of the adult. The immunoreactivities of PLA₂s were investigated in the viscera of larvae and juveniles of the 0 to 85^th day after hatch. In the larvae of the 13^th day, accumulation of PLA₂-positive zymogen granules in the pancreatic acinar cells were first recognized by the immunostaining. The intensity of the labelling subsequently became stronger and dramatically increased between the 20^th and 30^th day. This increase appeared to be one of the physiological changes associated with the transition to a new benthic life as juveniles. Lack of PLA₂ in the pancreas before the 13^th day may suggest the possibility that larvae utilized exogenous PLA₂, inherent in their prey, to digest the phospholipids. On the other hand, no reactivity was found in the intestine until the 85^th day.     

刀鲚MOR-2AK2的克隆、序列分析及组织表达

嗅觉是鱼类感知周围环境的重要工具,可能参与生殖洄游的过程。嗅觉由嗅觉受体(olfactory receptor)基因所编码的受体蛋白识别气味分子所引发,主嗅觉受体(MOR)基因是数量最大的一类嗅觉基因,可识别水溶性气味分子。为了弄清刀鲚定居型与洄游型种群的主嗅觉基因差异,本研究通过RACE技术获得了洄游型刀鲚MOR-2AK2基因,其开放阅读框长度972 bp,为单外显子结构,可编码323个氨基酸残基。预测表明,MOR-2AK2基因所编码的蛋白为7个疏水性的α-螺旋跨膜结构,属于G-蛋白偶联受体。对10种组织所作的定量分析表明,MOR–2AK2基因在嗅囊和性腺中的表达量远远高于其他组织器官,并且嗅囊中的表达量还高于性腺中的7~25倍。MOR–2AK2基因的表达量也存在性别差异,其中雌性嗅囊中的表达量约为雄性中的2倍,但精巢中的表达量却约是卵巢中的2倍。序列分析显示,MOR–2AK2基因的5′-UTR区域存在着一段微卫星序列(GT)5,其中定居型多出洄游型14个碱基(GTGTGTGTGTGTTT),这导致了二者所编码的氨基酸序列的相似度仅为84%。这些结果表明,MOR–2AK2基因不但与嗅觉功能有关,也可能参与了刀鲚的性腺发育或生殖洄游过程,同时也可能与定居型种群的形成相关。     

Gill denervation eliminates the barostatic reflex in a neotropical teleost,the tambaqui (<Emphasis Type="Italic">Colossoma macropomum</Emphasis>)

The baroreflex is one of the most important regulators of cardiovascular homeostasis in vertebrates. It begins with the monitoring of arterial pressure by baroreceptors, which constantly provide the central nervous system with afferent information about the status of this variable. Any change in arterial pressure relative to its normal state triggers autonomic responses, which are characterized by an inversely proportional change in heart rate and systemic vascular resistance and which tend to restore pressure normality. Although the baroreceptors have been located in mammals and other terrestrial vertebrates, their location in fish is still not completely clear and remains quite controversial. Thus, the objective of this study was to locate the baroreceptors in a teleost, the Colossoma macropomum. To do so, the occurrence and efficiency of the baroreflex were both analyzed when this mechanism was induced by pressure imbalancements in intact fish (IN), first-gill-denervated fish (G1), and total-gill-denervated fish (G4). The pressure imbalances were initiated through the administration of the α₁-adrenergic agonist phenylephrine (100 µg kg^?1) and the α₁-adrenergic antagonist prazosin (1 mg kg^?1). The baroreflex responses were then analyzed using an electrocardiogram that allowed for the measurement of the heart rate, the relationship between pre- and post-pharmacological manipulation heart rates, the time required for maximum chronotropic baroreflex response, and total heart rate variability. The results revealed that the barostatic reflex was attenuated in the G1 group and nonexistent in G4 group, findings which indicate that baroreceptors are exclusively located in the gill arches of C. macropomum.     

Biochemical properties of the sensitivity to GABA<Subscript>A</Subscript>ergic ligands,Cl<Superscript>−</Superscript>/HCO<Subscript>3</Subscript><Superscript>−</Superscript>-ATPase isolated from fish (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) olfactory mucosa and brain

This paper presents a comparative study of the roles of Cl^? and HCO₃ ^? in the functioning of the GABA_AR-associated Cl^?/HCO₃ ^?-ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl^?(15–30 mmol l^?1), whereas the enzyme from MBNs was not significantly affected by Cl^?. By contrast, HCO₃ ^?(15–30 mmol l^?1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl^?(15 mmol l^?1)/HCO₃ ^?(27 mmol l^?1) and these activities were inhibited in the presence of picrotoxin (100 μmol l^?1), bumetanide (150 μmol l^?1), and DIDS (1000 μmol l^?1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~?56 kDa that binds [³H]muscimol and [³H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-³²P and Mg²⁺, as well as Cl^?/HCO₃ ^? sensitive dephosphorylation, is also associated with this 56 kDa peptide. Both preparations also showed one subunit with molecular mass 56 kDa that was immunoreactive with GABA_AR β3 subunit. The use of a fluorescent dye for Cl^? demonstrated that HCO₃ ^?(27 mmol l^?1) causes a twofold increase in Cl^? influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO₃ ^? had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl^? and HCO₃ ^? in the regulation of the Cl^?/HCO₃ ^?-ATPases functioning in neurons with different specializations.     

Lipolytic activities in developing turbot larvae as influenced by diet

The development of neutral lipase and phospholipase activities was studied in larval turbot fed live prey. Activities of neutral lipase and phospholipase (activity larva⁻¹) increased significantly between days 6 and 24 after hatching in turbot larvae. The specific activities of both enzymes (activity μg protein⁻¹) decreased in older larvae. Feeding of a microdiet for 3 days (days 10–13) affected the lipolytic activity of neutral lipase and phospholipase negatively, compared to the larvae fed on rotifers. Since neutral lipase activities in whole larval homogenates and in the gut were significantly lower, it suggests a reduced synthesis rate and a reduced secretion of the enzyme in larvae fed the microdiet. A correlation between neutral lipase and phospholipase activities was found in larvae fed rotifers, but not in larvae fed the microdiet. This may indicate different regulating and stimulating mechanisms for these enzymes. The contribution of exogenous enzymes from ingested live prey to the total larval enzyme activity was about 6% for neutral lipase and 10% for phospholipase on day 6. The exogenous prey enzymes accounted for only 2% of the total activities in 12-day-old turbot larvae, suggesting that enzymes from prey did not contribute considerably to the digestion of lipids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enteroendocrine profile of α-transducin immunoreactive cells in the gastrointestinal tract of the European sea bass (Dicentrarchus labrax)

In vertebrates, chemosensitivity of nutrients occurs through the activation of taste receptors coupled with G-protein subunits, including α-transducin (G_αtran) and α-gustducin (G_αgust). This study was aimed at characterising the cells expressing G_αtran immunoreactivity throughout the mucosa of the sea bass gastrointestinal tract. G_αtran immunoreactive cells were mainly found in the stomach, and a lower number of immunopositive cells were detected in the intestine. Some G_αtran immunoreactive cells in the stomach contained G_αgust immunoreactivity. Gastric G_αtran immunoreactive cells co-expressed ghrelin, obestatin and 5-hydroxytryptamine immunoreactivity. In contrast, G_αtran immunopositive cells did not contain somatostatin, gastrin/cholecystokinin, glucagon-like peptide-1, substance P or calcitonin gene-related peptide immunoreactivity in any investigated segments of the sea bass gastrointestinal tract. Specificity of G_αtran and G_αgust antisera was determined by Western blot analysis, which identified two bands at the theoretical molecular weight of ~45 and ~40 kDa, respectively, in sea bass gut tissue as well as in positive tissue, and by immunoblocking with the respective peptide, which prevented immunostaining. The results of the present study provide a molecular and morphological basis for a role of taste-related molecules in chemosensing in the sea bass gastrointestinal tract.     

The effect of light on activity and growth of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae

The growth and activity of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae exposed to light of differing intensities and wavelengths were investigated every fifth day. The experiments were conducted at 6 ^oC from day 1 until day 34 post hatch. Four intensities of constant white light (2.0, 0.3, 0.03 and 0.005 μ Em^?2 s^?1, λ_max 590 nm), and constant coloured light of equal intensities (0.03μ Em^?2s^?1) in the blue, green and red spectrums (λ_max 450, 560 and 670 nm, respectively) were used. In addition to a control treatment in constant darkness, one treatment was incubated in a 12:12 h light: dark photoperiod. The light treatments did not have any discernible effect on the total length, myotome height, dry weights or yolk conversion efficiencies. The most intense white light resulted in an increased activity on days 24 and 30 post hatch, resulting in a temporarily reduced length and myotome height for the larvae in these groups compared to the other treatments. Larvae from all treatments were of the same size 34 days post hatch. The dry weights of the larvae and yolk-sacs were unaffected by light treatment. The activity increased independently of light treatment until 120 degree-days, and thereafter, the strongest white light resulted in an temporarily increased activity. The distribution of activity changed independently of light regime in the beakers during development.     

两种壳色福寿螺免疫功能的比较研究

为了解黑壳和黄壳福寿螺免疫功能的差异,本实验研究了氯硝柳胺对两种壳色福寿螺的急性毒性效应,并对四川地区黑壳福寿螺和黄壳福寿螺血细胞酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化物酶(POX)、中性红(NRD)、脂类(PAS)和糖原(SBB)细胞化学染色的观察及中性红试验(NRRT)的差异进行了比较。结果显示,黑壳福寿螺对氯硝柳胺24 h和48 h的半致死浓度分别为0.77 mg/L和0.46 mg/L,均高于黄壳福寿螺。透明细胞和颗粒细胞的细胞化学染色阳性主要分布在细胞质,且颗粒细胞中的颗粒物质呈强烈阳性;黑壳福寿螺ACP、AKP、POX阳性比例均大于黄壳福寿螺,且前者AKP和POX阳性细胞比例显著大于后者,NRD和SBB着染程度和阳性比例均无显著差异,黑壳福寿螺PAS着染程度更为强烈。NRRT实验发现,黑壳福寿螺血细胞的中性红滞留时间(9.15 min)是黄壳福寿螺的1.8倍。研究表明,黑壳福寿螺对氯硝柳胺死亡率及血细胞AKP、POX、糖原成分及中性红滞留时间是影响其与黄壳福寿螺免疫功能差异的重要因素。     

Histochemical identification of the black-brown pigment granules found in the alimentary canal of Siberian sturgeon (Acipenser baeri) during the lecitotrophic stage

The histochemical characteristics of the black-brown pigment granules accumulated during the lecitotrophic stage in the digestive tract of Siberian sturgeon, Acipenser baeri larvae, were studied in order to identify the nature of such pigments. Pigment granules appeared just after hatching in contact with the endodermal epithelium and in the periphery of the yolk-sac matrix, and increased in number with digestive system differentiation. At 3 days post hatch, the accumulation of pigment granules in the spiral valve formed a pigmented plug, which was ejected with first faeces after first feeding. Pigment granules were insoluble in all solubilising solutions tested (distilled water, H₂O₂, NH₄OH, KMnO₄, absolute methanol and different acid, basic and oxidising solutions). Pigment granules were completely bleached with the oxidising processes of Gomori and Casella (15 min) and with H₂O₂ (20%) during 48 h. Histochemical characteristics of bleached pigmented granules revealed that they were basophilic (Eosine negative), presented a moderate methachromasia (Toluidine Blue positive) and argentaffinity (Masson Fontana method positive), and contained carboxylated and sulphated (strongly and weakly ionised) glycoconjugated residues (Alcian Blue pH 0.5, 1.0, 2.5 positive). Bleached pigment granules also contained reductor groups (Ferric ferricyanide-Fe III positive), while they were negative for Bromophenol Blue, Perl's Prussian Blue, Red Oil O and Sudan Black B, indicating the absence of proteins, iron and lipidic substances respectively. According to such results, the black-brown pigment granules observed in the alimentary canal of Siberian sturgeon larvae during the lecitotrophic stage correspond to melanins.     

Effect of tank colour on larval performance of the Amazon River prawn Macrobrachium amazonicum

The aim of this study was to evaluate the effects of hatchery‐tank colours (white, yellow, red, blue, green and black) on the performance of larval culture of Macrobrachium amazonicum. The larvae were fed daily with newly hatched Artemia nauplii. The hatchery‐tank colours affected the light level inside the tanks, the consumption of Artemia nauplii (AN), larval development, survival, mass gain and productivity of postlarvae (PL). The overall consumption of Artemia nauplii per larva during the larval cycle was 30% and 45% higher in the green and red tanks respectively. The significant variation of AN consumption among tank colours (P = 0.0006) indicates that M. amazonicum larvae are visual predators. Survival was higher in the black, blue and green tanks, reaching more than 75%. However, the highest productivity was obtained in the black tanks (80.1 PL L^?1). Lighter coloured tanks and excess luminosity (more than 2 μmol s^?1 m² at tank bottom) appear to be important stress factors for larvae, contributing to reduce survival and productivity. The results indicate that rearing M. amazonicum in black tanks will improve larvae condition, ensure greater productivity of postlarvae and lower Artemia consumption, increasing technological and economic viability.     

Effects of stocking density,temperature, and salinity on larval survival and growth of the red race of the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Selenka)

The red race of the sea cucumber Apostichopus japonicus was introduced into China from Japan for large-scale seed production because of its economic value. This paper reports the effects of stocking density, temperature, and salinity on survival and growth of early larvae before and after feeding, in order to establish conditions for optimal larval growth and production. To maximize the yield per unit of space, densities of 0.5–1 larvae/ml are recommended for non-feeding larvae, while 0.1–0.2 larvae/ml are best for feeding larvae. Higher survival and growth values were obtained for both non-feeding and feeding larvae at temperature ranges from 21 to 24°C. Larvae reared at a salinity of 30‰ always showed maximum growth and survival. Based on results of this study, a temperature range from 21 to 24°C and a salinity of 30 are considered optimal for early development of the red A. japonicus.     

Role of thyroid hormones in tilapia larvae (Oreochromis mossambicus): I. Effects of the hormones and an antithyroid drug on yolk absorption,growth and development

Treatment of one-day old yolksac larvae of tilapia (Oreochromis mossambicus) by immersion in 0.05 ppm T₄ or 0.01 ppm T₃ significantly accelerated the differentiation and growth of all the fins, particularly pectoral and tail fins. Both the treatments also significantly accelerated yolk absorption and transition to free-swimming activity in the larvae. The treatments also significantly accelerated the growth of the larvae, with T₃ at 0.01 ppm having a greater effect than T₄ at 0.05 ppm. The yolk conversion efficiency was found not to be significantly affected by the hormone treatments but the treated larvae exhibited an increased heart beat, suggesting metabolic stimulation by the hormones.On the other hand, yolk absorption and free-swimming activity were significantly delayed in tilapia larvae immersed in 25 ppm solution of an antithyroid drug, phenylthiocarbamide (PTC). PTC also retarded the growth of the larvae. T₄ (0.05 and 0.10 ppm) or T₃ (0.01 and 0.02 ppm) therapy removed the PTC-inhibition,albeit not completely, suggesting that thyroid hormones are involved in the larval growth and development of tilapia.