Morphological and Immunohistochemical Modifications in Zebrafish (Danio rerio) Gills After Short-Term Exposure to the Fungicide Tebuconazole.

In agroecosystems, a variety of pesticides may enter aquatic habitats exerting a number of toxic effects on living organisms. Due to their wide use and the repeated application, fungicides can be found at a much greater concentration than other agrochemicals in all environmental compartments. One of these fungicides, tebuconazole (TBZ), has been recognized as an endocrine-disrupting chemical, and the available literature focuses mainly on this mechanism of action. In the present study by subjecting the zebrafish model to an environmentally realistic concentration of TBZ, we clearly demonstrate that this fungicide has the potential to induce severe morphological and ultrastructural alterations in gills. The main morphological effects recorded in this study were as follows: the proliferation of the main epithelium, ectopia of chloride cells, lamellar shortening, necrosis, and apoptosis. TBZ exposure also resulted in the overtime decrease in the expression of both Na+/K+-ATPase and aquaporin 3 and in the induction of oxidative stress enzyme (superoxide dismutase-1) as revealed by immunohistochemical analysis. Our results clearly indicate that respiratory and osmoregulatory disorders represent an important mechanism of TBZ toxicity on Danio rerio gills. This is the first evidence of sublethal effects exerted by TBZ on fish gills and highlights the harmful properties of this fungicide, even at very low concentration.

Species-specific impacts of suspended sediments on gill structure and function in coral reef fishes.

Reduced water quality, in particular increases in suspended sediments, has been linked to declines in fish abundance on coral reefs. Changes in gill structure induced by suspended sediments have been hypothesized to impair gill function and may provide a mechanistic basis for the observed declines; yet, evidence for this is lacking. We exposed juveniles of three reef fish species (Amphiprion melanopus, Amphiprion percula and Acanthochromis polyacanthus) to suspended sediments (0-180 mg l-1) for 7 days and examined changes in gill structure and metabolic performance (i.e. oxygen consumption). Exposure to suspended sediments led to shorter gill lamellae in A. melanopus and A. polyacanthus and reduced oxygen diffusion distances in all three species. While A. melanopus exhibited impaired oxygen uptake after suspended sediment exposure, i.e. decreased maximum and increased resting oxygen consumption rates resulting in decreased aerobic scope, the oxygen consumption rates of the other two species remained unaffected. These findings imply that species sensitive to changes in gill structure such as A. melanopus may decline in abundance as reefs become more turbid, whereas species that are able to maintain metabolic performance despite suspended sediment exposure, such as A. polyacanthus or A. percula, may be able to persist or gain a competitive advantage.

The effects of hypoxia on active ionic transport processes in the gill epithelium of hyperregulating crab, Carcinus maneas.

Effects of hypoxia on the osmorespiratory functions of the posterior gills of the shore crab Carcinus maenas acclimated to 12ppt seawater (DSW) were studied. Short-circuit current (Isc) across the hemilamella (one epithelium layer supported by cuticle) was substantially reduced under exposure to 1.6, 2.0, or 2.5mg O2/L hypoxic saline (both sides of epithelium) and fully recovered after reoxygenation. Isc was reduced equally in the epithelium exposed to 1.6mg O2/L on both sides and when the apical side was oxygenated and the basolateral side solely exposed to hypoxia. Under 1.6mg O2/L, at the level of maximum inhibition of Isc, conductance was decreased from 40.0mScm-2 to 34.7mScm-2 and fully recovered after reoxygenation. Isc inhibition under hypoxia and reduced 86Rb+ (K+) fluxes across apically located K+ channels were caused preferentially by reversible inhibition of basolaterally located and ouabain sensitive Na+,K+-ATPase mediated electrogenic transport. Reversible inhibition of Isc is discussed as decline in active transport energy supply down regulating metabolic processes and saving energy during oxygen deprivation. In response to a 4day exposure of Carcinus to 2.0mg O2/L, hemolymph Na+ and Cl- concentration decreased, i.e. hyperosmoregulation was weakened. Variations of the oxygen concentration level and exposure time to hypoxia lead to an increase of the surface of mitochondria per epithelium area and might in part compensate for the decrease in oxygen availability under hypoxic conditions.

Jellyfish Stings Trigger Gill Disorders and Increased Mortality in Farmed Sparus aurata (Linnaeus, 1758) in the Mediterranean Sea.

Jellyfish are of particular concern for marine finfish aquaculture. In recent years repeated mass mortality episodes of farmed fish were caused by blooms of gelatinous cnidarian stingers, as a consequence of a wide range of hemolytic, cytotoxic, and neurotoxic properties of associated cnidocytes venoms. The mauve stinger jellyfish Pelagia noctiluca (Scyphozoa) has been identified as direct causative agent for several documented fish mortality events both in Northern Europe and the Mediterranean Sea aquaculture farms. We investigated the effects of P. noctiluca envenomations on the gilthead sea bream Sparus aurata by in vivo laboratory assays. Fish were incubated for 8 hours with jellyfish at 3 different densities in 300 l experimental tanks. Gill disorders were assessed by histological analyses and histopathological scoring of samples collected at time intervals from 3 hours to 4 weeks after initial exposure. Fish gills showed different extent and severity of gill lesions according to jellyfish density and incubation time, and long after the removal of jellyfish from tanks. Jellyfish envenomation elicits local and systemic inflammation reactions, histopathology and gill cell toxicity, with severe impacts on fish health. Altogether, these results shows P. noctiluca swarms may represent a high risk for Mediterranean finfish aquaculture farms, generating significant gill damage after only a few hours of contact with farmed S. aurata. Due to the growth of the aquaculture sector and the increased frequency of jellyfish blooms in the coastal waters, negative interactions between stinging jellyfish and farmed fish are likely to increase with the potential for significant economic losses.

Effects of Loma morhua (Microsporidia) infection on the cardiorespiratory performance of Atlantic cod Gadus morhua (L).

The microsporidian Loma morhua infects Atlantic cod (Gadus morhua) in the wild and in culture and results in the formation of xenomas within the gill filaments, heart and spleen. Given the importance of the two former organs to metabolic capacity and thermal tolerance, the cardiorespiratory performance of cod with a naturally acquired infection of Loma was measured during an acute temperature increase (2 °C h(-1)) from 10 °C to the fish's critical thermal maximum (CT(Max)). In addition, oxygen consumption and swimming performance were measured during two successive critical swimming speed (U(crit)) tests at 10 °C. While Loma infection had a negative impact on cod cardiac function at warm temperatures, and on metabolic capacity in both the CT(Max) and U(crit) tests (i.e. a reduction of 30-40%), it appears that the Atlantic cod can largely compensate for these Loma-induced cardiorespiratory limitations. For example, (i) CT(Max) (21.0 ± 0.3 °C) and U(crit) (~1.75 BL s(-1)) were very comparable to those reported in previous studies using uninfected fish from the same founder population; and (ii) our data suggest that tissue oxygen extraction, and potentially the capacity for anaerobic metabolism, is enhanced in fish infected with this microsporidian.

Uso de biomarcador histopatológico em brânquias de Centropomus undecimalis (Bloch, 1972) na avaliação da qualidade da água do Parque Ecológico Laguna da Jansen, São Luís - MA / Using histopathological biomarker in gills of Centropomus undecimalis (Bloch, 1972) in assessing the water quality at Ecological Park Laguna da Jansen, São Luís -MA

Com o objetivo de analisar a intensidade das alterações histológicas em brânquias de Centropomus undecimalis para avaliar a qualidade da água do Parque Ecológico Laguna da Jansen, em São Luís, Estado do Maranhão, coletaram-se exemplares de peixes no período chuvoso de 2012 e amostras de água nos períodos seco de 2011 e chuvoso de 2012 para análises físico-química e bacteriológica. Os resultados mostraram que a maioria dos parâmetros físico-químicos e bacteriológicos estava de acordo com os valores estabelecidos pela Resolução do CONAMA nº 357 de 2005. As alterações histológicas observadas foram: elevação do epitélio lamelar; proliferação excessiva de células dos epitélios filamentar e lamelar causando fusão total ou parcial das lamelas; alteração da estrutura das lamelas; hiperplasia das células mucosas; ruptura do epitélio lamelar; espessamento descontrolado do tecido proliferativo filamentar e lamelar e aneurismas de vários tamanhos. Também foi observada a presença de parasito em alguns exemplares. O Índice de Alteração Histológica (IAH) por indivíduo variou de 2 a 114. O valor médio do IAH obtido foi de 40,3, demonstrando que as brânquias dos indivíduos amostrados da espécie C. undecimalis apresentaram alterações de moderadas para severas no tecido. As alterações histológicas encontradas indicam que esses indivíduos desenvolveram mecanismos de defesa contra a ação de estressores presentes na água da laguna e a um provável desequilíbrio parasito-hospedeiro-ambiente associado à baixa qualidade do ambiente...

Aiming to analyze the intensity of the histological changes in the gills of Centropomus undecimalis to assess the water quality at Ecological Park of the Laguna da Jansen, in São Luís, State of Maranhão, specimens of fish were collected during the rainy season of 2012 and water samples during the dry season in 2011 and rainy season in 2012 for physical-chemical and bacteriological analyses. The results showed that most of the physical-chemical and bacteriological parameters analyzed agreed with the values established by CONAMA Resolution nº 357 of 2005. The histological changes were elevation of the lamellar epithelium, excessive cell proliferation of the filamentum and lamellar epithelium causing total or partial melting of the lamellar; changing the structure of the lamellar, hyperplasia of mucous cells, disruption of the lamellar epithelium, uncontrolled proliferative tissue filamentum thickening and lamellar and aneurysms in various sizes. We noted the presence of parasites in some samples. The Index Histological Amendment (IAH) per individual ranged from 2 to 114. The mean IAH obtained was 40.3, demonstrating that the gills of the individuals of the C. undecimalis species showed moderate to severe changes in the tissue. The histological findings indicate that these individuals have developed defense mechanisms against the action of stressors present in the water of the Laguna da Jansen and a probable parasite-host-environment imbalance associated with the environment's low quality...

In vivo changes in carbonic anhydrase activity and histopathology of gill and liver tissues after acute exposure to chlorpyrifos in rainbow trout.

Chlorpyrifos is an organophosphate pesticide widely used in agriculture and aquaculture. This study investigated its effects on carbonic anhydrase (CA) enzyme activity and histopathology of rainbow trout gill and liver. The fish were exposed to 2.25 (25% of 96 h LC50), 4.5 (50% of 96 h LC50), and 6.75 µg L⁻¹ (75% of 96 h LC50) of chlorpyrifos for 24, 48, 72, and 96 h. CA activity was measured in liver and gills and histopathological changes were examined by light microscopy. The most common liver changes at most of the chlorpyrifos concentrations were hyperaemia and degenerative changes. Gill tissues were characterised by lamellar hyperaemia, lamellar oedemas, clumping, cellular degeneration, hyperplasia, and lamellar atrophy. CA enzyme activity in the gills decreased at all concentrations at 48, 72, and 96 h after exposure to chlorpyrifos (p<0.05). Similarly, there was a time-dependent decrease in CA activity at all of the concentrations in liver tissues (p<0.05). The present study indicated that chlorpyrifos inhibits CA enzyme activity and causes histopathological damage in gill and liver tissues.

Stress in African catfish (Clarias gariepinus) following overland transportation.

Of the many stressors in aquaculture, transportation of fish has remained poorly studied. The objective of this study was therefore to assess the effects of a (simulated) commercial transportation on stress physiology of market-size African catfish (Clarias gariepinus). Catfish weighing approximately 1.25 kg were returned to the farm after 3 h of truck-transportation, and stress-related parameters were measured for up to 72 h following return. Recovery from transportation was assessed through blood samples measuring plasma cortisol, glucose and non-esterified fatty acids (NEFA) and gill histology. Also, the number of skin lesions was compared before and after transport. Pre-transport handling and sorting elevated plasma cortisol levels compared to unhandled animals (before fasting). Plasma cortisol levels were further increased due to transportation. In control fish, plasma cortisol levels returned to baseline values within 6 h, whereas it took 48 h to reach baseline values in transported catfish. Plasma glucose and NEFA levels remained stable and were similar across all groups. Transported catfish did not, on average, have more skin lesions than the handling group, but the number of skin lesions had increased compared to unhandled animals. The macroscopic condition of the gills was similar in control, transported and unhandled catfish; however, light microscopy and immunohistochemistry revealed atypical morphology and chloride cell migration normally associated with adverse water conditions. From our data, we conclude that transportation may be considered a strong stressor to catfish that may add to other stressors and thus inflict upon the welfare of the fish.

Branchial O(2) chemoreceptors in Nile tilapia Oreochromis niloticus: Control of cardiorespiratory function in response to hypoxia.

This study examined the distribution and orientation of gill O(2) chemoreceptors in Oreochromis niloticus and their role in cardiorespiratory responses to graded hypoxia. Intact fish, and a group with the first gill arch excised (operated), were submitted to graded hypoxia and their cardiorespiratory responses (oxygen uptake - V˙O(2) , breathing frequency - fR, ventilatory stroke volume - VT, gill ventilation - V˙G, O(2) extraction from the ventilatory current - EO(2) , and heart rate - fH) were compared. Their responses to bolus injections of NaCN into the bloodstream (internal) or ventilatory water stream (external) were also determined. The V˙O(2) of operated fish was significantly lower at the deepest levels of hypoxia. Neither reflex bradycardia nor ventilatory responses were completely abolished by bilateral excision of the first gill arch. EO(2) of the operated group was consistently lower than the intact group. The responses to internal and external NaCN included transient decreases in fH and increases in fR and Vamp (ventilation amplitude). These cardiorespiratory responses were attenuated but not abolished in the operated group, indicating that chemoreceptors are not restricted to the first gill arch, and are sensitive to oxygen levels in both blood and water.

Physiological consequences of gill remodeling in goldfish (Carassius auratus) during exposure to long-term hypoxia.

Goldfish (Carassius auratus) acclimated to 7 degrees C and exposed to hypoxia ( approximately 10 mmHg) for 7 days exhibited a pronounced remodeling of the gill consisting of the removal of an interlamellar cell mass (ILCM). Subsequent experiments were designed to assess the impact of gill remodeling and the associated increase in functional lamellar surface area on the distribution of branchial ionocytes and Cl(-) flux across the gill. Despite the increased functional lamellar surface area during hypoxia, there was no corresponding increase in Cl(-) loss or efflux of the extracellular marker polyethylene glycol (PEG 4000). However, when hypoxic fish were returned to normoxic water for 12 h, rates of Cl(-) and PEG efflux were markedly stimulated in keeping with an increased surface area for solute movement. Similarly, the rate of branchial Cl(-) uptake was reduced (105 +/- 22 vs. 45 +/- 8 micromol x kg(-1) x h(-1)) in normoxic and hypoxic fish, respectively, but then stimulated (345 micromol x kg(-1) x h(-1)) upon reestablishment of normoxic conditions. Hypoxia (7 days) was accompanied by a significant decrease in the total cross-sectional area of branchial ionocytes owing to a decrease in their numbers and individual sizes. Thus, despite experiencing an increase in functional lamellar surface area, hypoxic goldfish limit branchial Cl(-) loss likely by a hypoxia-mediated decrease in paracellular permeability. In normoxic fish, the ionocytes were largely confined to the outer edges of the ILCM. During hypoxia, preexisting ionocytes migrated with the shrinking ILCM, while a smaller proportion of newly differentiated cells appeared below the surface of the ILCM. The capacity to maintain a population of ionocytes in contact with the water is an appropriate strategy to retain ionoregulatory capabilities regardless of whether the lamellae are uncovered or covered.

Complete intracellular pH protection during extracellular pH depression is associated with hypercarbia tolerance in white sturgeon, Acipenser transmontanus.

Sturgeons are among the most CO2 tolerant of fishes investigated to date. However, the basis of this exceptional CO2 tolerance is unknown. Here, white sturgeon, Acipenser transmontanus, were exposed to elevated CO2 to investigate the mechanisms associated with short-term hypercarbia tolerance. During exposure to 1.5 kPa Pco2, transient blood pH [extracellular pH (pHe)] depression was compensated within 24 h and associated with net plasma HCO3- accumulation and equimolar Cl- loss, and changes in gill morphology, such as a decrease in apical surface area of mitochondrial-rich cells. These findings indicate that pHe recovery at this level of hypercarbia is accomplished in a manner similar to most freshwater teleost species studied to date, although branchial mechanisms involved may differ. White sturgeon exposed to more severe hypercarbia (3 and 6 kPa Pco2) for 48 h exhibited incomplete pH compensation in blood and red blood cells. Despite pHe depression, intracellular pH (pHi) of white muscle, heart, brain, and liver did not decrease during a transient (6 h of 1.5 kPa Pco2) or prolonged (48 h at 3 and 6 kPa Pco2 blood acidosis. This pHi protection was not due to high intrinsic buffering in tissues. Such tight active cellular regulation of pHi in the absence of pHe compensation represents a unique pattern for non-air-breathing fishes, and we hypothesize that it is the basis for the exceptional CO2 tolerance of white sturgeon and, likely, other CO2 tolerant fishes. Further research to elucidate the specific mechanisms responsible for this tremendous pH regulatory capacity in tissues of white sturgeon is warranted.

Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors.

O2 chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O2-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H2S) metabolism is involved in O2 sensing in vascular smooth muscle. Here, we examined the possibility that H2S is an O2 sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O2 sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H2S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H2S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H2S-synthesizing enzymes, cystathionine beta-synthase and cystathionine gamma-lyase, was present in branchial tissue. Homogenized gills produced H2S enzymatically, and H2S production was inhibited by O2, whereas mitochondrial H2S consumption was O2 dependent. Ambient hypoxia did not affect plasma H2S in unanesthetized trout, but produced a PO2-dependent increase in a sulfide moiety suggestive of increased H2S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H2S, produced a similar approximately 10-mV depolarization. These studies are consistent with H2S involvement in O2 sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H2S concentration ([H2S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to PO2 and may provide an exquisitely sensitive, yet simple, O2 sensor in a variety of tissues.

Histopathology of gills, kidney and liver of a Neotropical fish caged in an urban stream

Histological changes in gills, kidney and liver were used to evaluate the health of the Neotropical fish species Prochilodus lineatus, subjected to in situ tests for 7 days in a disturbed urban stream and in a reference site, during winter and summer. In fish caged in the urban stream the most common lesions were epithelial lifting, hyperplasia and hypertrophy of the respiratory epithelium, lamellar fusion, and aneurysms in the gills; enlargement of the glomerulus, reduction of Bowman's space, occlusion of the tubular lumen, cloudy swelling and hyaline droplet degeneration in the kidneys; hepatocytes with hypertrophy, cytoplasmic and nuclear degeneration, melanomacrophage aggregates, bile stagnation and one case of focal necrosis in the liver. The lesions were comparatively most severe in the liver. Histopathology showed to be a very suitable biomarker for use in conjugation with the in situ test, because the seasonal variation did not interfere in the results and it was possible to differentiate the sites in the urban stream from the reference site

Alterações histológicas nas brânquias, rim e fígado foram usadas para estudar a saúde da espécie neotropical de peixe Prochilodus lineatus, submetida a testes in situ por 7 dias em um ribeirão urbano contaminado e em um local de referência, durante o inverno e o verão. Nos peixes confinados no ribeirão urbano as lesões mais comumente encontradas foram elevação epitelial, hiperplasia e hipertrofia do epitélio respiratório, fusão lamelar e aneurismas, nas brânquias; aumento do glomérulo, redução do espaço de Bowman, oclusão da luz tubular, degeneração granular e degeneração hialina, no rim; hepatócitos hipertrofiados, degeneração citoplasmßtica e nuclear, agregados de melanomacrófagos, estagnação biliar e um caso de necrose focal, no fígado. As lesões foram comparativamente mais severas no fígado. A histopatologia mostrou ser um biomarcador muito apropriado quando usada conjuntamente com os testes in situ, porque parece não ter sofrido interferência da variação sazonal e permitiu diferenciar os locais estudados no ribeirão urbano do local de referência

Cell proliferation and gill morphology in anoxic crucian carp.

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius), by examining 5'-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G(0) cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells ( approximately 1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.

Induction of apoptosis in a flounder gill cell line by lymphocystis disease virus infection.

Lymphocystis disease virus (LCDV), a large icosahedral DNA virus classified to the iridovirus family, is the causative agent of lymphocystis, a disease which occurs in marine and freshwater fish species and is characterized by formation of papilloma-like lesions on the surface of the skin. In vitro, LCDV infection causes flounder gill cells, an adherent cell line, to exhibit an obvious cytopathic effect (CPE). In order to test whether apoptosis is responsible for the observed CPE, cells infected with LCDV at a multiplicity of infection (m.o.i.) of 5 PFU per cell were examined at various time intervals for the appearance of apoptotic signs. Nuclear fragmentation, DNA laddering and caspase activation were observed in the infected cells at the time (i.e. 10 days post-infection) when an intensive CPE was observed. These findings demonstrate that LCDV is capable of inducing apoptosis in vitro, which is different from the result of LCDV infection in vivo, and consequently suggest an intricate LCDV-host interaction.

Hormonal, morphological, and physiological responses of yellow perch (Perca flavescens) to chronic environmental metal exposures.

The effects of a chronic environmental exposure to metals on the hormonal, physiological, and reproductive status were assessed in yellow perch (Perca flavescens) sampled in six lakes situated along a contamination gradient of Cd, Zn, Cu, Pb, and Ni in the mining region of Rouyn-Noranda, Québec. Fish were captured in the summer and fall, and sampled before or after a confinement of one hour. Metal concentrations in the kidneys and the interrenal tissues (homologous to mammalian adrenals) were measured to compare tissue-specific metal accumulation. An exposure-related decrease of condition factor, gonadosomatic index (GSI), branchial Na+/K(+)-ATPase activity, plasma thyroxine (T4), triiodothyronine (T3), and 17 beta-estradiol and an impaired capacity to enhance cortisol levels after confinement were observed. Fish from the metal-contaminated lakes possessed gonads at less mature stages and exhibited structural alterations of their gills, interrenal cells, and thyroid follicle epithelium. A comparison of the morphological, biochemical, and physiological endpoints measured in the present study revealed that plasma concentrations of hormones and parameters of gill function were the most affected by metal contamination. The results of this study indicate that lifelong exposures to sublethal concentrations of metals alter the physiological functions of fish and delay reproduction.

Intermittent hypoxia and plasticity of respiratory chemoreflexes in metamorphic bullfrog tadpoles.

We tested the hypothesis that intermittent hypoxia elicits plasticity in respiratory chemoreflexes in bullfrog tadpoles. Metamorphic tadpoles (Taylor-Kollros stages XVI-XX) were subjected to intermittent hypoxia (PW(O(2))=45 Torr; 12 h/day) or constant normoxia (PW(O(2))=156 Torr) for 2 weeks before ventilatory responses to hypoxia and hypercarbia were measured. Buccal pressure changes were used to quantify the frequency and amplitude of movements associated with gill and lung ventilation. Morphometric assessment showed that intermittent hypoxia delayed development in comparison with controls. Oxygen consumption was enhanced in tadpoles subjected to intermittent hypoxia; however, this increase was not sufficient to affect basal ventilatory activity or the hypoxic ventilatory response. During acute hypercarbic exposure, tadpoles subjected to intermittent hypoxia showed (1) a greater decrease in gill ventilation frequency and (2) a greater increase in lung ventilation frequency than tadpoles maintained under control conditions. We conclude that intermittent hypoxia augments the responsiveness to hypercarbia, thereby promoting lung ventilation when animals face this stimulus. This manifestation of respiratory plasticity may reflect uncoupling between physiological and morphological development in the bi-modally breathing bullfrog tadpole.

Gill and lung ventilation responses to steady-state aquatic hypoxia and hyperoxia in the bullfrog tadpole.

Gill ventilation frequency (fG), the pressure amplitude (PBC) and stroke volume (VS) of buccal ventilation cycles, the frequency of air breaths (fL), water flow over the gills (VW), gill oxygen uptake (MGO2), oxygen utilization (U), and heart frequency (fH) have been measured in unanaesthetized, air breathing Rana catesbeiana tadpoles (stage XVI-XIX). The animals were unrestrained except for ECG leads or cannulae, and were able to surface voluntarily for air breathing. They were subjected to aquatic normoxia, hyperoxia and three levels of aquatic hypoxia, and their respiratory responses recorded in the steady state. The experiments were performed at 20 +/- 0.5 degrees C. In hyperoxia there was an absence of air breathing, and fG, PBC and VW fell from the normoxic values, while U increased, resulting in no significant change in MGO2. Animals in normoxia showed a very low fL which increased in progressively more hypoxic states. VW increased from the normoxic value in mild hypoxia (PO2 = 96 +/- 2 mm Hg), but fell, associated with a reduction in PBC, in moderate (PO2 = 41 +/- 1 mm Hg) and severe (PO2 = 21 +/- 3 mm Hg) hypoxia in the presence of lung ventilation. Gill MGO2 was not significantly different from the normoxic value in mild hypoxia but fell in moderate hypoxia, while in severe hypoxia oxygen was lost to the ventilating water from the blood perfusing the gills. There was no significant change in fH from the normoxic value in either hypoxia or hyperoxia. These data indicate, that in the bimodally breathing bullfrog tadpole, aquatic PO2 exerts a strong control over both gill and lung ventilation. Furthermore, there is an interaction between gill and lung ventilation such that the onset of a high frequency of lung ventilation in moderate and severe hypoxia promotes a suppression of gill ventilation cycles.

Ventilatory response to hypercapnia in the larger spotted dogfish Scyliorhinus stellaris.

Dogfish were exposed to sudden changes of Pco2 in inspired seawater. During hypercapnia breathing frequency remained constant, but gill ventilation was transiently increased to about 140% of control levels in the 1st h. O2 uptake was significantly increased also, but returned to the initial level before nomalization of gill ventilation. In contrast to the transient rise in gill ventilation and O2 uptake, arterial Po2 was increased for the whole period of hypercapnia. Hypercapnia results in a marked fall in pHa which returned to the initial value in 4-5 h even though hypercapnia is maintained. This rise in pHa with little change in PaCO2 was associated with an increase in plasma bicarbonate concentration. The increase of plasma bicarbonate was in part due to compensatory bicarbonate uptake from the seawater across the gills and in part was effected by transfer between intracellular tissue compartments and extracellular spaces. The compensatory bicarbonate exchange mechanism in the gills seems to have a delay both after onset and termination of hypercapnia.