The first appearance of Rodlet cells in carp (Cyprinus carpio L.) ontogeny and their possible roles during stress and parasite infection.

The origin and function of rodlet cells (RCs) are still a matter of discussion. Whereas the exogenous hypothesis considers them parasites, the endogenous hypothesis regards them as a genuine fish cell population with a secretory and/or leukocyte function. In order to shed more light on these questions we focused on the location and appearance of RCs during carp (Cyprinus carpio) ontogeny. Typical RCs were seen at 5days post fertilisation (dpf) between kidney and intestine, at 6dpf in the intestine and at 8dpf in both anterior and posterior kidney and in the abdominal cavity among the mesothelial cells. The RC number increased with age and after 14dpf they were also present in gills. The early appearance of the RCs during carp ontogeny support the endogenous hypothesis stating that RCs are genuine constituents of fish tissue and suggest that they are 'immune cells'. The fact that the RCs of the gills secrete their content into the surrounding water, combined with the strategic location around blood vessels in kidney and within intestinal epithelium, would also support an important role in host defense. To investigate whether RC numbers in gills and kidney are related to typical fluctuations in the physiology during stress and infection we counted their number in gills and kidney after parasite infection and stress. In the gills the number of RCs increased after infection but did not change after stress while in the kidney their number increased after stress and no significant changes were observed after infection.

Corticotropin-releasing hormone-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP) are expressed in the gills and skin of common carp Cyprinus carpio L. and respond to acute stress and infection.

We established that corticotropin-releasing hormone (CRH), CRH-binding protein (CRH-BP) and CRH-receptor 1 (CRH-R1) are expressed in the gills and skin of common carp Cyprinus carpio, an early vertebrate. Immunoreactive CRH was detected in macrophage-like cells in gills and skin, in fibroblasts in the skin and in endothelial cells in the gills. The involvement of the CRH system in gills and skin was investigated in response to infection and in an acute restraint stress paradigm. Carp were infected with the protozoan leech-transmitted blood flagellate Trypanoplasma borreli and subjected to acute restraint stress by netting for 24 h. The expression of CRH-BP and CRH-R1 genes in the gills and in the skin is downregulated after both infection and restraint. Thus the peripheral CRH system reacts to infection and stress. The gills and skin separate the internal from the external environment and are permanently exposed to stress and pathogens. Because of their pivotal role in maintaining the homeostatic equilibrium, these organs must act locally to respond to diverse stresses. Clearly, the CRH system is involved in the response of the integument to diverse stresses at the vulnerable interface of the internal and external milieu.

A short-term in vitro gill culture system to study the effects of toxic (copper) and non-toxic (cortisol) stressors on the rainbow trout, Oncorhynchus mykiss (Walbaum).

A short-term (24 h) method of gill filament culture system was developed to predict the effects of environmental contamination and stress in fish. Gill culture system containing two or three rainbow trout gill filaments in sterile glutamine supplemented Leibovitz 15 (L-15) media was submitted for 24 h to six different treatments: (i) CONT (control, medium only); (ii) CORT (cortisol, 0.28 microM cortisol); (iii) BLOCK (glucocorticoid receptor blocker, 14 microM RU 486); (iv) CORT+BLOCK (cortisol and blocker, 0.28 microM cortisol+14 microM RU 486); (v) CORT+CU (cortisol and copper, 100 microM CuSO4+0.28 microM cortisol); (vi) CU (copper, 100 microM CuSO4). After 24 h, the overall gill structure and cellular components resembled those of salmonids in vivo. Lactate dehydrogenase (LDH) activity in the culture media increased in the CORT+CU and CU groups but was significantly lower in the CORT+CU compared to CU group. Apoptotic cells increased in the CORT and CORT+BLOCK. The numbers of glucocorticoid (GR) receptor-positive cells were lower in the CU group. This short-term culture system seems to be suitable for studying the effects of both external and internal stress effectors (toxicants and hormones respectively), as it contains all cell types found in the gills and the cells give similar biological response as in vivo.

Physiological responses to sulfide toxicity by the air-breathing catfish, Hoplosternum littorale (Siluriformes, Callichthyidae).

Hemolytic anemia accompanied by changes in the immunology system is one of the sulfide intoxication harmful effects on Hoplosternum littorale. Hematological parameters are considered as effective indicators of stress caused by this hydrogen sulfide. During sulfide exposure, H. littorale neither alters the methemoglobin concentration nor forms sulfhemoglobin in the presence of high levels of dissolved sulfide in the water. Cytochrome c oxidase shows little activity in the gills and blood of H. littorale when exposed to sulfide. Alternative metabolic routes are suggested through which the accumulation of pyruvate leads to the formation of an end product other than lactate.

Hematological and physiological changes induced by short-term exposure to copper in the freshwater fish, Prochilodus scrofa

Hematological and physiological changes in the blood of juveniles of the freshwater fish, Prochilodus scrofa were determined after acute exposure to 20, 25, and 29 mugCu L-1 in water (pH 7.5; hardness 24.5 mg L-1 as CaCO3) for 96 h. Copper exposure to 25 and 29 mugCu L-1 caused significant increase in the hematocrit and red blood cell values. The increase in red blood cells was associated with increase in whole blood hemoglobin only in fish exposed to 29 mugCu L-1. Leukocytes increased following copper exposure and were significantly higher in fish exposed to 29 mugCu L-1. Differential leukocyte percentage displayed significant reduction in lymphocytes and an increase in neutrophils in fish exposed to 25 and 29 mugCu L-1. The percentage of monocytes remained unchanged after copper exposure. The thrombocytes did not change. There was a significant decrease in plasma [Na+] and [Cl-] and a significant drop in blood pH in fish exposed to 25 and 29 mugCu L-1 while [K+] showed significant increase in fish exposed to 29 mugCu L-1. Copper exposure led to ionoregulatory impairment, although chloride cell hypertrophy was induced. The changes in red blood cells suggest a compensatory response to respiratory surface reduction of gills (tissue damage and cell proliferation) in order to maintain oxygen transference from water to the tissues, allowing the fish to survive during the so-called shock phase of LC50 exposure, at least while at rest

Blood parameters and metabolites in the teleost fish Colossoma macropomum exposed to sulfide or hypoxia.

Juvenile tambaqui, Colossoma macropomum, were exposed to sulfide and hypoxia for 12, 24, 48 and 96 h. Hemoglobin concentrations, red blood cell counts, and mean cell hemoglobin, were higher at 12 h in fish exposed to hypoxia. However, control fish and those exposed to sulfide and hypoxia had lower red blood cell count, hemoglobin concentration and hematocrit at 96 h. Methemoglobin was higher than in the controls, probably due to the hypoxemia induced by these stressors. Sulfhemoglobin was not detected in significant amounts in the blood of fish exposed to sulfide (in vivo), yet hemoglobin converted into sulfhemoglobin at 1-15 mM sulfide in vitro. Anaerobic metabolism seemed to be an important mechanism for adapting to sulfide exposure and blood pH returned to control values after 24 h of sulfide, preventing acidosis. The high sulfide tolerance in tambaqui is associated with its high tolerance to hypoxia.

Gill cellular changes induced by copper exposure in the South American tropical freshwater fish Prochilodus scrofa.

The cellular changes in gill tissue induced by exposure to copper were studied in the tropical freshwater fish Prochilodus scrofa, with emphasis on chloride and pavement cells. Damage to gills included epithelial changes such as lifting, rupture, peeling of lamellar epithelium, lamellar fusion, hyperplasia, and cellular hypertrophy. Cell degeneration by necrosis and apoptosis was intense in fish exposed to 25 and 29 microg Cu L-1. Pavement cells showed microridge reduction on their surface. Chloride cells proliferated in the lamellar epithelia close to the onset of the lamellae. However, no changes in total chloride cell density in contact with the water were observed. The chloride cell apical area of fish exposed to copper increased, but only fish exposed to 25 microg Cu L-1 showed significant increase in the chloride cell fractional area. At this water copper concentration, almost 60% of the chloride cells were apoptotic. Necrotic chloride cells increased with copper in water, reaching 70% in fish exposed to 29 microg Cu L-1 (=LC50 calculated for this species). Pavement and chloride cell proliferation and hypertrophy on lamellar epithelia increased the thickness of the water-blood barrier. Our findings suggest severe impairment of ion regulation and gas transfer of fish exposed to copper.

Hematological and physiological changes induced by short-term exposure to copper in the freshwater fish, Prochilodus scrofa.

Hematological and physiological changes in the blood of juveniles of the freshwater fish, Prochilodus scrofa were determined after acute exposure to 20, 25, and 29 microgramsCu L-1 in water (pH 7.5; hardness 24.5 mg L-1 as CaCO3) for 96 h. Copper exposure to 25 and 29 microgramsCu L-1 caused significant increase in the hematocrit and red blood cell values. The increase in red blood cells was associated with increase in whole blood hemoglobin only in fish exposed to 29 microgramsCu L-1. Leukocytes increased following copper exposure and were significantly higher in fish exposed to 29 microgramsCu L-1. Differential leukocyte percentage displayed significant reduction in lymphocytes and an increase in neutrophils in fish exposed to 25 and 29 microgramsCu L-1. The percentage of monocytes remained unchanged after copper exposure. The thrombocytes did not change. There was a significant decrease in plasma [Na+] and [Cl-] and a significant drop in blood pH in fish exposed to 25 and 29 microgramsCu L-1 while [K+] showed significant increase in fish exposed to 29 microgramsCu L-1. Copper exposure led to ionoregulatory impairment, although chloride cell hypertrophy was induced. The changes in red blood cells suggest a compensatory response to respiratory surface reduction of gills (tissue damage and cell proliferation) in order to maintain oxygen transference from water to the tissues, allowing the fish to survive during the so-called shock phase of LC50 exposure, at least while at rest.