Production of ciguatoxin and maitotoxin by strains of Gambierdiscus australes,G. pacificus and G. polynesiensis (Dinophyceae) isolated from Rarotonga,Cook Islands

The isolation and culturing of toxic Gambierdiscus species is desirable for obtaining reliable sources of toxins responsible for Ciguatera Fish Poisoning (CFP) in order to provide reference materials and purified standards for chemical analyses and bioassays. Gambierdiscus polynesiensis CAWD212, G. pacificus CAWD213 and G. australes CAWD149 and 216 have been isolated from samples of sea water from the Cook Islands. G. polynesiensis produced ciguatoxins (CTX; 18.2 pg per cell) and G. australes CAWD149 produced maitotoxin (MTX; 8.3 pg per cell) and in both extracts putative MTX-3 was detected. G. pacificus also produced putative MTX-3. The toxicities of extracts of G. polynesiensis and G. pacificus were similar by i.p. injection (LD_50s of 1.0 mg/kg and 0.8 mg/kg respectively), but when administered by gavage, the MTX-3 analogue producing G. pacificus (LD₅₀ 251 mg/kg) was 30-times less toxic than the CTX-producing G. polynesiensis (LD₅₀ 7.9 mg/kg). Consumption of fish contaminated by CFP has caused illness and even death in the Cook Islands and throughout the Pacific region and is now considered a globally neglected disease. Large-scale cultures of these organisms are now available, and isolation of the toxins they produce is in progress.     

Stability of oxidative stress biomarkers in flathead mullet,Mugil cephalus,serum during short-term storage

The production of reactive oxygen species (ROS) by aquatic organisms in response to stressful factor is a common feature in aquatic environment. Thus, the evaluation of parameters related to oxidative stress in fish, in first instance those showing the redox potential, is notable to characterize the environmental conditions.Short-term storage of samples is essential when blood must be transported from collection sites to laboratories. Therefore, excessive delays in processing might compromise the reliability of results. The aim of the present study was to assess the effect of short-term storage time at +4 °C on total oxidant status (TOS), total antioxidant capacity (TAC) and oxidative stress index (OSI) in flathead mullet (Mugil cephalus) serum. After blood collection, all sample were analyzed and the assays were repeated after 24, 48 and 72 h from sampling. results showed a significant effect of short-term storage on TOS and TAC (P ≤ 0.0001), while the statistical significance of the linear regression study for these parameters was reduced as consequence of storage. These results highlight that the activities of oxidants and antioxidants in flathead mullet serum change during short-term storage at 4 °C and should be assesses as soon as possible from collection.     

Toxigenic Pfiesteria species—Updates on biology,ecology, toxins,and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 10² to 10³ (rarely, 10⁴) flagellate cells mL⁻¹. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL⁻¹), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.     

Gambierdiscus (Dinophyceae) species diversity in the Flower Garden Banks National Marine Sanctuary,Northern Gulf of Mexico,USA

Globally, ciguatera fish poisoning (CFP) is the principal cause of non-bacterial illness associated with seafood consumption. The toxins (ciguatoxins) responsible for CFP are produced by dinoflagellates in the genus Gambierdiscus, which are endemic to tropical and sub-tropical areas. Ciguatoxins are lipophilic and bioaccumulate in marine food webs, typically reaching their highest concentrations in fish. Following a CFP event in 2008, the U.S. Food and Drug Administration (USFDA) issued a ciguatera toxin alert that included fish harvested in the northern Gulf of Mexico in and near the Flower Garden Banks National Marine Sanctuary (FGBNMS). The East Flower Garden Bank (EFGB) and West Flower Garden Bank (WFGB) are characterized by thriving coral communities that support Gambierdiscus growth. This study was undertaken specifically to document the diversity of Gambierdiscus species present in the sanctuary that may be sources of ciguatoxins entering the food web. Samples collected from the FGBNMS over a three year period were screened using species-specific polymerase chain reaction assays. A diverse assemblage of Gambierdiscus species was distributed to depths of >45 m, a new depth record for Gambierdiscus. Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ribotype 2 were all found on both East and West FGB with Gambierdiscus ruetzleri also recorded from the WFGB. The most common species was G. carolinianus, originally identified from samples collected between 35 and 40 m off the coast of NC, USA. Our findings are consistent with recent physiological studies showing that some Gambierdiscus species can grow year round at the temperatures and salinities at the FGBNMS and at light levels as low as 10 μmol photons m⁻² s⁻¹. Such irradiances are estimated to occur in the FGBNMS at depths of ∼70–80 m. The consistent recovery of Gambierdiscus species from deep sampling sites in areas known to produce ciguatoxic fish signals a substantial change in our concept of suitable habitats for Gambierdiscus to include depths greater than 50 m.     

Characterization of a toxic Pseudo-nitzschia spp. bloom in the Northern Gulf of Mexico associated with domoic acid accumulation in fish

A toxic bloom of Pseudo-nitzschia spp. was observed in the Alabama coastal waters of the northern Gulf of Mexico (NGOM) in June 2009 that resulted in the accumulation of domoic acid (DA) in fish. The bloom initiated following a large storm event that likely caused increased groundwater discharge 16–20 days prior to peak densities. Eleven sites, located in littoral shoreline waters and inshore embayments spanning the entire Alabama NGOM coastline, were sampled during peak densities to assess Pseudo-nitzschia species composition and toxicity, and associated water-quality parameters. Small fish (0.27–11.9 g body weight) were collected at six of these sites for analysis of DA content. High Pseudo-nitzschia spp. densities (8.27 × 10⁴–5.05 × 10⁶ cell l⁻¹) were detected at eight sites located in the littoral shoreline and particulate DA was detected at six of these littoral sites (48.0–540 pg ml⁻¹). The bloom consisted primarily (>90%) of Pseudo-nitzschia subfraudulenta, a species previously characterized as forming only a minor component of Pseudo-nitzschia assemblages and not known to produce DA. Pseudo-nitzschia spp. were at low densities or not detected at the inshore sites and DA was detected at these sites. Pseudo-nitzschia spp. density varied along an estuarine gradient, with greater densities occurring in the most saline, clear, and nutrient-poor waters. Cell density was strongly and negatively correlated with silicate (Si) concentrations and the ratios of silicate to dissolved inorganic nitrogen and phosphate (Si:DIN and Si:PO₄). Cell toxin quota was negatively correlated with phosphate, and strongly and positively correlated with the ratio of total nitrogen to total phosphorus (TN:TP). These relationships are consistent with previous observations that indicate Pseudo-nitzschia spp. density and toxicity are likely to be greater in high salinity, high irradiance, and nutrient-poor waters. DA was detected in 128 of 131 (98%) of the fish collected, which included seven primary and secondary consumer species. This is the first demonstration of trophic transfer of DA in this region of the NGOM, indicating that toxic blooms of Pseudo-nitzschia spp. in Alabama coastal waters have the potential to transfer DA to recreationally and commercially important fish species.     

Particle retention in suspension-feeding fish after removal of filtration structures

The suspension-feeding cichlids Oreochromis aureus (blue tilapia) and Oreochromis esculentus (ngege tilapia) are able to selectively retain small food particles. The gill rakers and microbranchiospines of these species have been assumed to function as filters. However, surgical removal of these oral structures, which also removed associated mucus, did not significantly affect the total number of 11–200 μm particles ingested by the fish. This result supports the hypothesis that the branchial arch surfaces themselves play an important role in crossflow filtration. Both species selectively retained microspheres greater than 50 μm with gill rakers and microbranchiospines intact as well as removed, demonstrating that neither these structures nor mucus are necessary for size selectivity to occur during biological crossflow filtration. After removal of the gill rakers and microbranchiospines, O. esculentus retained significantly more microspheres 51–70 μm in diameter and fewer 91–130 μm microspheres compared to retention with intact structures, but the particle size selectivity of O. aureus was not affected significantly. These results support conclusions from previous computational fluid dynamics simulations indicating that particle size can have marked effects on particle trajectory and retention inside the fish oropharyngeal cavity during crossflow filtration. The substantial inter-individual variability in particle retention by suspension-feeding fish is an unexplored area of research with the potential to increase our understanding of the factors influencing particle retention during biological filtration.     

Determination of food sources and trophic position in Malaysian tropical highland streams using carbon and nitrogen stable isotopes

Stable isotope analysis has been extensively used as an effective tool in determination of trophic relationship in ecosystems. In freshwater ecosystem, aquatic invertebrates represent main component of a river food web. This study was carried out to determine potential food sources of freshwater organism together with pattern of trophic position along the river food web. In this study, rivers of Belum-Temengor Forest Complex (BTFC) has been selected as sampling site as it is a pristine area that contains high diversity and abundance of organisms and can be a benchmark for other rivers in Malaysia. Stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) were applied to estimate trophic position and food web paradigm. Analysis of stable isotopes based on organic material collected from the study area revealed that the highest δ¹³C value was reported from filamentous algae (? 22.68 ± 0.126⁰/₀₀) and the lowest δ¹³C was in allocthonous leaf packs (? 31.58 ± 0.187⁰/₀₀). Meanwhile the highest δ¹⁵N value was in fish (8.45 ± 0.177⁰/₀₀) and the lowest value of δ¹⁵N was in autochthonous aquatic macrophyte (2.00 ± 1.234⁰/₀₀). Based on the δ¹⁵N results, there are three trophic levels in the study river and it is suggested that the trophic chain begins with organic matter followed by group of insects and ends with fish (organic matter < insects < fish).     

From water to edible fish. Transfer of metals and metalloids in the San Roque Reservoir (Córdoba,Argentina). Implications associated with fish consumption

The concentration of Mn, Fe, Zn, Cu, Cd, Cr, Ni, Ag, Mo, Nd, Al, Ce, As, Sr, Pb, Pt and Hg was analysed in water, sediments, and aquatic organisms from the San Roque Reservoir (Córdoba-Argentina), sampled during the wet and dry season, to evaluate their transfer through the food web. Stable nitrogen (δ¹⁵N) isotopes were used to investigate trophic interactions. According to this, samples were divided into three trophic groups: plankton, shrimp (Palaemonetes argentinus) and fish (Silverside, Odontesthes bonariensis). Liver and gills are the main heavy metal storage tissues in fish. Hg and As concentrations in the muscle of O. bonariensis exceed the Oral Reference doses for metals established by USEPA (2009). Trophic magnification factors (TMFs) for each element were determined from the slope of the regression between trace element concentrations and δ¹⁵N. Calculated TMFs showed fundamental differences in the trophodynamics of the studied elements during the wet and dry season in the San Roque Reservoir. Concentrations of Ni, Cd, Cr, Al, Mn, Fe, Mo, Ce, Nd, Pt and Pb during both seasons, and Sr during the dry season, showed statistically significant decreases (TMF < 1) with increasing trophic levels. Thus these elements were trophically diluted in the San Roque food chain. Conversely, Cu, Ag and As (dry season) showed no significant relationships with trophic levels. Among the elements studied, Hg in the wet season, and Zn in the dry season were the only ones showing a statistically significant increase (TMF > 1) in concentration with trophic level. Current results trigger the need for further studies to establish differential behaviour with different species within the aquatic web, particularly when evaluating the transfer of toxic elements to edible organisms, which could pose health risks to humans.     

Improving estimates of trophic shift (Δδtrophic) for diet reconstruction studies using enzyme activities

For diet reconstruction studies using stable isotopes, accurate estimates of trophic shift (Δδ_trophic) are necessary to get reliable results. Several factors have been identified which affect the trophic shift. The goal of the present experiment was to test whether measurements of the activities of enzymes could improve the accuracy of estimation of trophic shift in fish. Forty-eight Nile tilapia (Oreochromis niloticus) were fed under controlled conditions with two diets differing in their protein content (21 and 41%) each at four different levels (4, 8, 12 and 16 g kg^? 0.8 d^? 1). At the end of the feeding experiment, proximate composition, whole body δ¹³C and δ¹⁵N as well as the activities of enzymes involved in anabolism and catabolism were measured. Step-wise regression specified contributing variables for Δδ¹⁵N (malic enzyme, aspartate aminotransferase and protein content) and Δδ¹³C_{lipid-free material} (aspartate aminotransferase and protein content). Explained variation by using the significant main effects was about 70% for Δδ¹⁵N and Δδ¹³C_{lipid-free material}, respectively. The results of the present study indicate that enzyme activities are suitable indicators to improve estimates of trophic shift.     

Occurrence of palytoxins in marine organisms from different trophic levels of the French Mediterranean coast harvested in 2009

Four sites located in Nice and Villefranche-sur-Mer, on the French Mediterranean coast, were monitored during the summer of 2009 for the presence of epiphytic and planktonic Ostreopsis cf. ovata, and that of palytoxin (PlTX) and 2 of its analogues (ovatoxin-a (OVTX-a) and ostreocin-D (OST-D)) in different marine organisms.Several of the 15 species that were sampled between June and September 2009 were found to be contaminated with OVTX-a as the major toxin (90% of the toxin profile) and PlTX; this included fish, echinoderms, gastropods, crustaceans and cephalopods. The contamination levels varied geographically and between species, with the herbivorous species generally having higher toxin levels than carnivorous ones.The determination of the toxin distribution between the digestive tube (DT) and the remaining tissue (RT) or roe in the case of the sea urchin Paracentrotus lividus showed that the toxins were sequestered in the DT. The highest toxin level ever recorded over the course of the study was of 392.2 μg for the sum of OVTX-a and PlTX per kg of DT of the flathead mullet Mugil cephalus. No quantifiable levels of toxins were found in the roe of the sea urchins or in the RT of the other marine products. However, in several cases, the toxin level in the whole flesh of the analysed organisms was above 30 μg OVTX-a + PlTX/kg, when knowing that the European food safety authority's opinion is that an adult should not ingest more than 30 μg PlTX + OST-D per kg of shellfish meat to avoid putting the consumer's health at risk. This was observed for the following four species: the sea urchin P. lividus, the red-mouthed rock shell Stramonita haemastoma, the warty crab Eriphia verrucosa and the flathead mullet M. cephalus.The collection of such data is of great importance to refine and complete the risk assessment of PlTX and its analogues and has to be encouraged in order to provide reliable information for setting up a regulatory level that would protect the consumers of edible marine organisms     

Microbiological and chemical quality of a traditional salted-fermented fish (Hout-Kasef) product of Jazan Region,Saudi Arabia

The Hout-Kasef is traditional salted fermented fish product of natural fermentation of salted mullet fish of coastal area of Jazan region of Saudi Arabia. The present study was carried out to investigate the microbiological and chemical characteristic of Hout-Kasef. A total of twenty-four salted fish samples were purchased from fish market in Jazan and Abu-Arish at different times of the year. The microbial studies of salted-fermented fish revealed a total bacterial count ranging from 2.81 to 4.72 Log₁₀ CFU/g, yeast and mold counts ranging from 0.48 to 3.14 Log₁₀ CFU/g, total staphylococci count 2.71–3.85 Log₁₀ CFU/g, halophile bacteria count 3.26–5.14 Log₁₀ CFU/g, and coliforms count <1 Log₁₀ CFU/g. However, pathogenic bacteria such as Listeria monocytogenes, Vibrio spp., Campylobacter spp. and Yersinia species were not detected. The major bacteria species isolated and identified from the salted fermented fish were Bacillus Subtilus, Bacillus mycoides, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus xylosus, Staphylococcus saprophticus and Staphylococcus cahnii subsp cahnii. The chemical analysis of salted fermented fish showed high content of moisture (47.96%), protein (25.71%), ash (19.6%) and salt (15.19%) but low contents of lipid (7.25%). The salted-fermented fish also showed high level of total volatile basic nitrogen (78.86 mg/100 gm sample) and thiobarbutric acid number (32.32 mg malonaldehyde/kg) with a pH value of pH 6.3. Finally, this study showed the presence of gram positive and gram negative bacteria in the fish product. The predominant microorganisms found were Bacillus and Staphylococcus spp. The fish product had high content of salt and TVB-N levels.     

A framework to assess the health of rocky reefs linking geomorphology,community assemblage,and fish biomass

The recovery of historic community assemblages on reefs is a primary objective for the management of marine ecosystems. Working under the overall hypothesis that, as fishing pressure increases, the abundance in upper trophic levels decreases followed by intermediate levels, we develop an index that characterizes the comparative health of rocky reefs. Using underwater visual transects to sample rocky reefs in the Gulf of California, Mexico, we sampled 147 reefs across 1200 km to test this reef health index (IRH). Five-indicators described 88% of the variation among the reefs along this fishing-intensity gradient: the biomass of piscivores and carnivores were positively associated with reef health; while the relative abundances of zooplanktivores, sea stars, and sea urchins, were negatively correlated with degraded reefs health. The average size of commercial macro-invertebrates and the absolute fish biomass increased significantly with increasing values of the IRH. Higher total fish biomass was found on reefs with complex geomorphology compared to reefs with simple geomorphology (r² = 0.14, F = 44.05, P < 0.0001) and the trophic biomass pyramid also changed, which supports the evidence of the inversion of biomass pyramids along the gradient of reefs’ health. Our findings introduce a novel approach to classify the health of rocky reefs under different fishing regimes and therefore resultant community structures. Additionally, our IRH provides insight regarding the potential gains in total fish biomass that may result from the conservation and protection of reefs with more complex geomorphology.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

Effects of exogenous enzymes on apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed diets with high inclusion of plant-based protein

Plant-based protein ingredients are increasingly used in feed for carnivorous fish as replacement for fish meal. This study investigated if supplementing diets with high inclusion levels of plant-based protein with three different enzymes could improve the apparent nutrient digestibility in juvenile rainbow trout (Oncorhynchus mykiss). Three diets with high inclusion levels of either de-hulled, solvent extracted soybean meal (344 g/kg), sunflower meal (246 g/kg), or rapeseed meal (264 g/kg) were produced and feed batches were coated post-extrusion with the different enzymes: β-glucanase at 67 mg/kg, xylanase at 208 mg/kg, protease at 228 mg/kg, or a combination of the three enzymes at the same doses. Three consecutive digestibility trials were carried out using a flow-through, modified Guelph System. Each trial was designed to include five dietary treatment groups: a non-supplemented control diet and 4 diets supplemented with either β-glucanase, xylanase, protease or a combination of the three enzymes. Diets were fed to triplicate tanks and two faecal sampling periods for digestibility measurements for each tank were scheduled. Each experiment comprised a 10-day digestibility trial succeeded by a water sampling period for measuring the dissolved nitrogen (N) output. Each experiment lasted 17–19 days in total.Apparent digestibility coefficients (ADCs) of protein, fat, ash, phosphorus and dry matter (DM) were derived from the three digestibility trials, along with calculations of the specific growth rate (SGR, %/d) and feed conversion ratio (FCR). Nitrogen mass-balance and energy retention were evaluated for each dietary treatment group to elucidate on the utilization of digested nutrients and energy.Enzyme supplementation had only moderate effect on apparent nutrient digestibility in the sunflower and rapeseed experiments, while β-glucanase and protease improved the apparent digestibility of all dietary nutrients in the soybean experiment (P<0.05). The effect was more pronounced for lipid than for other nutrients. β-Glucanase had a positive effect on energy retention in the soybean experiment (P<0.05), while there were no effects on nitrogen retention or fish performance in any of the three experiments (P>0.05) during the short feeding periods. The study thus provides preliminary results on the potential of β-glucanase and protease to increase apparent nutrient digestibility of soybean meal in fish feed.     

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs (Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell? mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.     

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

Characterization of the toxicity of Cochlodinium polykrikoides isolates from Northeast US estuaries to finfish and shellfish

Harmful algal blooms caused by Cochlodinium polykrikoides are annual occurrences in coastal systems around the world. In New York (NY), USA, estuaries, bloom densities range from 10³ to 10⁵ mL^?1 with higher densities (≥10⁴ cells mL^?1) being acutely toxic to multiple fish and shellfish species. Here, we report on the toxicity of C. polykrikoides strains recently isolated from New York and Massachusetts (USA) estuaries to juvenile fish (Cyprinodon variegates) and bay scallops (Argopecten irradians), as well as on potential mechanisms of toxicity. Cultures of C. polykrikoides exhibited dramatically more potent ichthyotoxicity than raw bloom water with 100% fish mortality occurring within ～1 h at densities as low as 3.3 × 10² cells mL^?1. More potent toxicity in culture was also observed in bioassays using juvenile bay scallops, which experienced 100% mortality during 3 days exposure to cultures at cell densities an order of magnitude lower than raw bloom water (～3 × 10³ cells mL^?1). The toxic activity per C. polykrikoides cell was dependent on the growth stages of cultures with early exponential growth cultures being more potent than cultures in late-exponential or stationary phases. The ichthyotoxicity of cultures was also dependent on both cell density and fish size, as a hyperbolic relationship between the death time of fish and the ratio of algal cell density to length of fish was found (～10³ cells mL^?1 cm^?1 yielded 100% fish mortality in 24 h). Simultaneous exposure of fish to C. polykrikoides and a second algal species (Rhodomonas salina or Prorocentrum minimum) increased survival time of fish, and decreased the fish mortality suggesting additional cellular biomass mitigated the ichthyotoxicity. Frozen and thawed-, sonicated-, or heat-killed-, C. polykrikoides cultures did not cause fish mortality. In contrast, cell-free culture medium connected to an active culture through a 5 μm nylon membrane caused complete mortality in fish, although the time required to kill fish was significantly longer than direct exposure to the whole culture. These results indicate that ichthyotoxicity of C. polykrikoides isolates is dependent on viability of cells and that direct physical contact between fish and cells is not required to cause mortality. The ability of the enzymes peroxidase and catalase to significantly reduce the toxicity of live cultures and the inability of hydrogen peroxide to mimic the ichthyotoxicity of C. polykrikoides isolates suggests that the toxicity could be caused by non-hydrogen peroxide, highly reactive, labile toxins such as ROS-like chemicals.     

The effects of feeding on the swimming performance and metabolic response of juvenile southern catfish,Silurus meridionalis,acclimated at different temperatures

To test whether the effects of feeding on swimming performance vary with acclimation temperature in juvenile southern catfish (Silurus meridionalis), we investigated the specific dynamic action (SDA) and swimming performance of fasting and feeding fish at acclimation temperatures of 15, 21, 27, and 33 °C. Feeding had no effect on the critical swimming speeding (U_crit) of fish acclimated at 15 °C (p = 0.66), whereas it elicited a 12.04, 18.70, and 20.98% decrease in U_crit for fish acclimated at 21, 27 and 33 °C, respectively (p < 0.05). Both the maximal postprandial oxygen consumption rate (VO_2peak) and the active metabolic rate (VO_2active, maximal aerobic sustainable metabolic rate of fasting fish) increased significantly with temperature (p < 0.05). The postprandial maximum oxygen consumption rates during swimming (VO_2max) were higher than the VO_2active of fasting fish at all temperature groups (p < 0.05). The VO_2max increased with increasing temperature, but the relative residual metabolic scope (VO_2max? VO_2peak) during swimming decreased with increasing in temperature. The present study showed that the impairment of postprandial swimming performance increased with increasing temperature due to the unparalleled changes in the catfish's central cardio-respiratory, peripheral digestive and locomotory capacities. The different metabolic strategies of juvenile southern catfish at different temperatures may relate to changes in oxygen demand, imbalances in ion fluxes and dissolved oxygen levels with changes in temperature.