Water quality requirements for first-feeding in marine fish larvae. II. pH,oxygen, and carbon dioxide

Previously unfed larvae representing eight species of marine teleosts (a soleid, Heteromycteris capensis Kaup; a cynoglossid. Trulla capensis Kaup; a gadid, Gaidropsarus capensis (Kaup); a congiopodid, Congiopodus spinifer (Smith); three sparids, Diplodus sargus (L.), Pachymetopon blochi (Val.), and an unidentified species; and a centracanthid, Pterosmaris axillaris (Boulenger)) were exposed for 24 h to various pH's and concentrations of dissolved oxygen and carbon dioxide and then tested for their ability to first-feed. Mortalities recorded during the 24-h exposure went into the fitting of response curves and the calculation of the LC₅₀ and LC₁₀. The incidence of successful first-feeding during the subsequent 4-h exposure to food yielded values for the EC₅₀ and EC₁₀. The exposure to sub-optimal conditions continued during the 4-h feeding test. The sensitivity of first-feeding larvae to high pH and dissolved oxygen may pose hazards for the marine fish culturist; low pH appears to be harmful (at least in the short term) only at levels below about pH 6.0; carbon dioxide poisoning is of doubtful importance in practical fish culture. There were only minor interspecific differences in calculated 24-h first-feeding EC₅₀'s, which suggests general applicability of the results to a wide variety of first-feeding marine fish larvae.     

Nitrate Reduction to Nitrite,Nitric Oxide and Ammonia by Gut Bacteria under Physiological Conditions

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.     

Morphological evidence of species differentiation within Lepus capensis Linnaeus, 1758 (Leporidae,Lagomorpha) in Cape Province,South Africa

A morphological study was carried out of the hares (Lepus) from Cape Province previously assigned to the subspecies L. capensis capensis, L. c. centralis, and L. c. grantii. The purpose of the study was to characterize the species L. capensis Linnaeus, 1758. In doing so, it was possible to distinguish two populations which we consider different species, as each shows homogeneous cranial, dental and pelage features. One of them, defined as L. capensis, is distributed near Cape Town not far from the coast, between Lambert's Bay and Cape Agulhas. The other species, defined as L. centralis, which includes L. grantii as a synonym, is distributed in central and western Cape Province. L. capensis and L. centralis have a parapatric distribution, with a small area of sympatry in a contact zone in Compagnies Drift area, near Lambert's Bay. With respect to cranial differences between the two species, L. capensis has a stronger maxilla and more robust dental series, while L. centralis has larger tympanic bullae. Among dental characters, L. centralis usually has a deeper groove and more abundant cement than L. capensis in the first upper incisor, and its internal lobe is squared, while in L. capensis is rounded. As for pelage color and pattern, L. capensis shows a more extended white ventral area than L. centralis. Our results are of interest for further research on taxonomic problems regarding Old World hare populations in which L. capensis is concerned.     

Zschokkella hildae Auerbach, 1910: Phylogenetic position,morphology, and location in cultured Atlantic cod

The myxozoan Zschokkella hildae Auerbach, 1910, was detected with a prevalence of 100% in cultured Atlantic cod, Gadus morhua L. aged 1+ from a culture facility on the west coast of Scotland. Sporogonic stages of Z. hildae, plasmodia producing 2–5 mature spores, were located predominantly in the collecting ducts and ureters of the kidney, and spores were present in the urine collected from the bladder. Less frequently, plasmodia were detected in the interstitial tissue of the kidney. The parasite prevalence in cultured fish was considerably higher than reported in wild fish but no obvious signs of pathology were detected. SSU rDNA sequencing and phylogenetic analysis showed that Z. hildae is closely related to a Sinuolinea sp. from the urinary system of turbot, Psetta maxima (L.), and that these two species, together with other myxozoans from the urinary system of marine fish cluster together in a sub-clade of the recognised marine clade of myxozoans. This sub-clade is characterised by a specific linear expansion segment, helix E23_15 in the secondary structure of variable region V4 of the SSU rDNA. Z. hildae and Sinuolinea sp. show extraordinary large linear expansion segment in both V4 and V7 and an important number of complementary base changes in the conservative regions of the SSU rDNA, indicating considerable evolutionary changes in the SSU rDNA of these species when compared with other myxozoans from the marine environment.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Effects of elevated ammonia concentrations on survival, metabolic rates, and glutamine synthetase activity in the Antarctic pteropod mollusk Clione limacina antarctica

Information on the effects of elevated ammonia on invertebrates in general, and polar Mollusks in particular, is scant. Questions of ammonia sensitivity are interesting for several reasons, particularly since predicted global change scenarios include increasing anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species are often lipid-rich, and authors have speculated that there is a linkage between elevated levels of lipids/trimethylamine oxide and enhanced ammonia tolerance. In the present study, we sought to examine ammonia tolerance and effects of elevated exogenous ammonia on several key aspects of the physiology and biochemistry of the pteropod mollusk, Clione limacina antarctica. We determined that the 96-h LC₅₀ value for this species is 7.465?mM total ammonia (Upper 95% CL?=?8.498?mM and Lower 95% CL?=?6.557?mM) or 0.51?mg/L as unionized ammonia (NH₃) (at a pH of 7.756). While comparative data for mollusks are limited, this value is at the lower end of reported values for other species. When the effects of lower ammonia concentrations (0.07?mM total ammonia) on oxygen consumption and ammonia excretion rates were examined, no effects were noted. However, total ammonia levels as low as 0.1?mM (or 0.007?mg/l NH₃) elevated the activity of the ammonia detoxification enzyme glutamine synthetase by approximately 1.5-fold. The values for LC₅₀ and observable effects on biochemistry for this one species are very close to permissible marine ammonia concentrations, indicating a need to more broadly determine the sensitivity of zooplankton to potential elevated ammonia levels in polar regions.     

Different sensitivity of mayflies (Insecta, Ephemeroptera) to ammonia, nitrite and nitrate: linkage between experimental and observational data

Complex toxic effects of ammonia, nitrite and nitrate to aquatic animals are not well investigated. In rivers of southwestern Siberia, Russia, elevated ammonia and nitrite concentrations corresponded to significant reduction in species diversity of mayflies. The objectives of the study were to evaluate the sensitivity of six mayfly species to the mixture of ammonia, nitrite and nitrate in acute laboratory tests and to compare the sensitivity found with results of the first river bioassessment in the region considered and with species saprobic indexes. The rank of the species sensitivity was: Baetis vernus < Potamanthus luteus < B. fuscatus=Cloeon bifidum < Ephemerella lenoki < Heptagenia sulphurea (p < 0.05). The experiments revealed variation in sensitivity among the species by a factor of 7.5. Comparison of the tests results and the available field data shows that species which exhibit higher tolerance in the tests inhabit comparatively greater amount of sites include contaminated places. Final conclusion is addressed in comparison of the results found with more spatially and temporally extensive observations. Saprobic indexes of the species and their acute tolerances (LC50s) tend to be positively correlated (r=0.93, p=0.02).     

Two novel myxosporean parasite species of Ceratomyxa Thélohan, 1892 from the banded cusk-eel Raneya brasiliensis (Kaup) (Ophidiiformes: Ophidiidae) off Patagonia,Argentina

We described two novel myxozoan parasite species Ceratomyxa argentina n. sp. and Ceratomyxa raneyae n. sp. from the gall bladder of Raneya brasiliensis (Kaup) from the Patagonian coast of Argentina. Both species can be distinguished from other ceratomyxids by myxospore and polar capsule (nematocyst) morphology and morphometry, fish host and geographic locality. Phylogenetic reconstruction using ssrDNA gene sequences showed that the two new species are placed in a long-branching ceratomyxid clade which also include Ceratomyxa appendiculata Thélohan, 1892, Ceratomyxa anko Freeman, Yokoyama and Ogawa, 2008, Ceratomyxa pantherini Gunter, Burger and Adlard, 2010 and Pseudoalataspora kovalevae Kalavati, MacKenzie, Collins, Hemmingsen and Brickle, 2013. This study documents additional biodiversity of marine myxozoans in the South Atlantic, a region still largely unexplored for this group of parasitic cnidarians.     

Differential effect of irradiance and nutrient nitrate on the relationship of in vivo and in vitro nitrate reductase assay in chlorophyllous tissues

Growth at increasing continuous irradiance (at high nutrient nitrate) and nutrient nitrate concentrations (at high continuous irradiance) furnished increases in the in vivo and in vitro nitrate reductase activities of corn (Zea mays L.), field peas (Pisum arvense L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and globe amaranth (Gomphrena globosa L.) leaves and of marrow (Cucurbita pepo L.) cotyledons. Ratios of in vivo to in vitro activity declined exponentially in all species with increasing nitrate reductase levels promoted by nutrient nitrate. The ratios were more nearly independent of nitrate reductase levels generated by adjusting the irradiance; major exceptions were marrow and wheat at low (1.5 klux and less) irradiances and peas throughout the irradiance range, where decreases in the ratio were accompanied by increases in in situ nitrate concentration. The ratio also increased at the highest irradiance (39.2 klux) in wheat and barley, associated with a decline of in vitro nitrate reductase. These differences in response to irradiance and nutrient nitrate indicate that the in vivo assay does not provide a simple measure of nitrate reductase but rather yields a more composite measure of nitrate reduction, possibly related both to nitrate reductase level and to the supply of reductant for in vivo activity.     

Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium <Emphasis Type="Italic">Marichromatium gracile</Emphasis>

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO?=?7.20–7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO?=?4.08–4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO?=?0.36–0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.     

Free nitrous acid selectively inhibits and eliminates nitrite oxidizers from nitrifying sequencing batch reactor

In a complete nitrification sequencing batch reactor (CNSBR), where ammonium containing wastewater (200–1,000 mg N/L) is completely oxidized to nitrate up to 2.4 kg NH₄ ⁺–N/m³ d, both ammonia oxidizers and nitrite oxidizers were enriched in the sludge granules. Quantitative fluorescence in situ hybridization analyses of the sludge granules of the CNSBR showed that ammonia oxidizers and nitrite oxidizers occupied 31 and 4.2% of total bacteria, respectively. Most of the nitrite oxidizers were Nitrobacter species (95% of the nitrite oxidizers) and the remainder was Nitrospira species. The population of nitrite oxidizers was significantly higher than that of partial nitrification SBR (PNSBR) where most of the ammonium was oxidized to nitrite. The PNSBR had 37% (ammonia oxidizers) and 0.4% (nitrite oxidizers) of total bacteria. Comparative study with CNSBR and PNSBR revealed that free nitrous acid, rather than free ammonia, played a critical inhibition role to wash out nitrite oxidizers from the reactor. The concentrations of free ammonia and nitrite as well as free nitrous acid in the CNSBR selected Nitrobacter as the dominant nitrite oxidizers rather than Nitrospira.     

Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation.     

Habitat partitioning in juvenile fishes associated with three vegetation types in selected warm temperate estuaries,South Africa

Three common vegetation types were studied to assess habitat partitioning in juvenile fishes in select warm temperate estuaries of South Africa. Vegetated habitat types are known as productive and important areas for predator avoidance and feeding and are often preferred by juvenile fishes. Habitat partitioning is not well understood, with previous studies mostly focusing on seagrass (Zostera capensis). This study aimed to assess three common vegetated fish nursery areas in estuaries to aid conservation planning. Fishes were collected by means of double-winged, six-hooped fyke nets, 1 mm mesh size secured in placed on the nocturnal flood tide at each inundated vegetation type (Phragmites australis, Zostera capensis, Spartina maritima) and at an adjacent unvegetated site for three consecutive months in 2014 and 2015 during the summer recruitment period for juvenile fishes. Higher catches were frequently recorded in vegetated areas for solely estuarine and marine estuarine dependent species. In general, the previously unstudied reed, P. australis showed the highest species richness and abundance of juvenile fishes overall, followed by, Z. capensis and the intertidal salt marsh species, S. maritima. Results from this study supports international trends on the value of vegetated areas as refugia for young fishes in estuaries.     

Detrimental effects of Ulva lactuca (L.) exudates and low oxygen on estuarine crab larvae

Conventional theory postulates that associations between marine macrophytes and animals are generally positive. This paper presents evidence, however, that a common species of green macroalga, Ulva lactuca (L.), is detrimental to estuarine invertebrates due to the production of toxic exudates and low oxygen tensions which occur in the seaweed beds at night. Bioassays of the responses of zoeae of five species of estuarine crabs (Callinectes sapidus Rathbun, Carcinus maenas L., Eurypanopeus depressus Smith, Neopanope texana savi Smith and Rhithropanopeus harissii Gould), using water in which Ulva lactuca was cultured for 24 h, produced 100% mortality after 22 days. No crabs survived the molt into megalopa. Hypoxic water, 0.5 ± 0.3 ppm oxygen, caused a decline in larval activity (movement), but there was no mortality over an 8-h period, Ulva-water purged to 0.4 ± 0.1 ppm oxygen caused 100% mortality in 13–40 min. These synergistic effects could be critical in estuaries where dense U. lactuca beds cause periods of low dissolved oxygen. We hypothesize larval recruitment may be limited in such systems, particularly in areas where flushing is poor.     

NITROGEN METABOLISM OF AQUATIC ORGANISMS. II. THE ASSIMILATION OF NITRATE,NITRITE, AND AMMONIA BY BIDDULPHIA AURITA1

Biddulphia aurita, a centric diatom, can grow on either nitrate, nitrite, or ammonia as its sole nitrogen, source. Cells remove ammonium nitrogen from the medium 2.3–2.4 times faster than either nitrate or nitrite nitrogen and, when grown for 24 hr in the ammonium medium, contain higher levels of non-protein nitrogen than cells grown in the nitrate or nitrite medium for the same period of time. The nitrogenous compounds in the nonprotein nitrogen fraction from cells grown in the nitrate, nitrite, or ammonium medium contain the same level of soluble-free amino nitrogen, combined amino nitrogen, and ammonium nitrogen. The high level of soluble nonprotein nitrogen in the medium of the cells grown in the ammonium medium is due to soluble amide nitrogen which represents 18% of the total soluble nitrogen present in these cells, whereas it represents only 2% in cells from the nitrite medium, and its level is negligible in cells from the nitrate medium. Cells grown in the nitrate medium have both nitrate- and nitrite-reductase activity. Cells grown in the nitrite medium have only nitrite-reductase activity in significant levels, while cells grown in the ammonium medium lack both enzymes.     

Intracellular localization of nitrate reductase, nitrite reductase, and glutamic Acid dehydrogenase in green leaf tissue

Greenhouse grown seedlings of corn (Zea mays L.) and foxtail (Setaria faberii Herrm.) were used as source material in determining the intracellular localization of nitrate reductase, nitrite reductase, and glutamic acid dehydrogenase, Nonaqueous and aqueous isolation techniques were used to establish that nitrite reductase is localized within the chloroplasts, but that nitrate reductase and glutamic acid dehydrogenase are not. Nonaqueous isolation gives distribution patterns of nitrite reductase which are the same as those observed for NADP-dependent 3-phosphoglyceraldehyde dehydrogenase but which differ drastically from the patterns observed for pyruvic acid kinase. The distribution patterns for nitrate reductase are the same as those of pyruvic acid kinase. The techniques used do not eliminate the possibility that nitrate reductase and pyruvic acid kinase are localized on the external chloroplast membrane.

The data obtained establish that glutamic acid dehydrogenase of green leaves is localized within the mitochondria.

     

Are Amazonian fish more sensitive to ammonia? Toxicity of ammonia to eleven native species

Little is known about the tolerance of Amazonian fish to ammonia. However, elevated ammonia of anthropogenic origin may now occur. As Amazonian fish evolved in waters which are generally acidic (i.e., low NH₃), we hypothesized that they would be more sensitive to ammonia than other freshwater fish. The acute (96-h) toxicity of NH₄Cl was tested in native ion-poor soft water (pH 7.0, ~28 °C) using semi-static tests with 11 species. Species sensitivity distributions (SSDs) for LC50_96 h and LC10_96 h and calculations of the hazardous concentrations to the most sensitive 5% (HC5 values) were tabulated. Values of LC50_96 h/LC10_96 h (in mM total ammonia) ranged from 2.24/0.78 for Paracheirodon axelrodi (most sensitive) to 19.53/16.07 for Corydoras schwartzi (most tolerant). These results confirm our hypothesis that Amazonian fish are more sensitive to ammonia than other freshwater species. High levels of ammonia may be associated with hypoxia, especially during dry periods. Simultaneous hypoxia (15–20% saturation) exacerbated ammonia toxicity in the most sensitive species (P. axelrodi), but not in Astronotus ocellatus or Corydoras schwartzi, a facultative air-breather where prevention of air access doubled ammonia toxicity. The present data are useful in generating regulatory guidelines in Amazonian waters and indicate that further studies incorporating hypoxia and air access/denial are needed.     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.