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Mass mortalities of juvenile Pacific oysters (Crassostrea gigas) in aquaculture operations have been linked to the ostreid herpes virus 1 (OsHV-1) or its variants. This oyster has heritable resilience to the virus which can provide a basis for selective breeding for higher OsHV-1 resistance, however, controlled challenge methods for improving selection response towards increased OsHV-1 resistance are required. The objective of this study was to compare the survival of sibling juvenile oysters exposed to either a laboratory virus challenge or a heat shock challenge (as proxy for the virus challenge) and the survival of the same families on two oyster farms where they were exposed to natural field conditions including the presence of OsHV-1. A strong correlation between the family rankings was observed for the two farm sites. Estimated narrow sense heritabilities (h2) on the underlying liability scale were 0.38 (on-farm survival), 0.45 (laboratory virus challenge survival) and 0.15 (heat shock challenge survival). Only on-farm survival and laboratory virus challenge had a high genetic correlation. The method for measuring heat shock resistance in juvenile oysters produced high variability in survival among families but low estimates of heritability. Overall, the results of this study indicate that the laboratory virus challenge is a simple and relatively effective tool for selective breeding specifically towards OsHV-1 resistance or as part of a controlled multi-trait program. Statement of relevance The OsHV-1 virus is a serious problem for commercial aquaculture of Pacific oysters in numerous countries. This paper tests two ways of evaluating oysters for resistance to OsHV-1 that can be incorporated into selective breeding programs.

A lack of understanding regarding the feeding biology of post-settlement mussels has considerably constrained the hatchery culture of many commercially important mussel species, including the Greenshell™ mussel, Perna canaliculus . Therefore, we evaluated how particles of different size (MySpat artificial diet full particle size range 2–60 μm, sieved MySpat diet with a size range of 2–15 μm; polystyrene beads, 3–15 μm, and microalgae, 4–6.5 μm) affected the retention and capture efficiency in post-settlement juveniles (PSJ). The response of PSJ of various sizes (PSJ-I: 0.3 mm shell length, PSJ-II: 1.0 mm, and PSJ-III: 7.0 mm) was different. Overall, smaller mussels (PSJ-I and PSJ-II) were unable to selectively capture particles by their size on all diets, conversely larger juveniles actively captured smaller particles between 2 and 8 μm. Independently of the PSJ size, the full particle size range MySpat artificial diet had a negative effect on the feeding activity of the mussels, with particles blocking the pallial organs in the non-selective PSJ-I and PSJ-II, and completely inhibiting the feeding activity in the more selective PSJ-III. These results provide useful information to understand the feeding biology and ecology of PSJ of this species that can be used for the improvement of hatchery feeding methods. Artificial and live food particle size can be chosen in order to avoid larger harmful particles (> 15 μm), and the exclusive use of smaller food particles should be considered for PSJ Statement of relevance This article provides information to better understand particle capture process in post-settlement juvenile mussel in order to improve hatchery/nursery feeding practices. The results of this research will allow making informed decisions regarding selection of food particle size in relation to the size of the juvenile mussels and their ability to deal with particles of different sizes. Furthermore will also help to decide the adequate size of transfer for grow-out as well as being useful for the development of suitable artificial diets.

Antibiotic management of aquacultured animals, such as Greenshell™ mussel (GSM) larvae, Perna canaliculus , is undesirable because of health concerns and political pressures; hence, alternatives are needed. Herein, two novel probiotic bacteria were identified and trialled in a GSM larval rearing hatchery. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately at the Glenhaven Aquaculture Centre hatchery facility during routine larval rearing and when the larvae were challenged with both a high (10 7 and 10 6 CFU ml −1 ) and low (10 6 and 10 5 CFU ml −1 ) pathogenic dose of Vibrio sp. DO1 and V. splendidus respectively. In all experiments, probiotic application improved larval survival significantly when administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. The administration of probiotics prior to pathogen challenge resulted in larval survival of 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This is the first report of A. macleodii and Neptunomonas sp. as probiotic bacteria in a large scale production facility.

This paper describes a simple method for cryopreserving sperm of the Pacific oyster ( Crassostrea gigas Thunberg) in quantities suitable for commercial spat production. Experiments to refine the cryoprotectant mixtures demonstrated the key role of trehalose. Trehalose alone (at 0.45 M final concentration) was an effective cryoprotectant. The addition of 2.5–15% dimethyl sulphoxide (DMSO) in combination with 0.45 M trehalose gave only modest improvement in fertility over trehalose alone ( p =0.056). There was no significant difference in fertility among DMSO concentrations ( p =0.611). Seawater (SW) without cryoprotectant gave very poor results, but yielded some fertilization at very high sperm concentrations (7±1% at 10 7 sperm mL −1 , 21±2% at 3.2×10 7 sperm mL −1 , mean±S.E., n =3). The fertility of unfrozen sperm was 30- to 100-fold higher than that of sperm cryopreserved with DMSO and/or trehalose. For sperm cryopreserved in 4.5-mL cryovials, two simplified freezing methods gave fertilization rates equivalent to sperm cryopreserved by controlled rate freezing ( p =0.386). These methods involved securing the cryovials to aluminium canes and then either placing them into a bath of methanol chilled with dry ice, or holding them on a floating rack 3 cm above liquid nitrogen. A third technique of plunging the cryovials directly into liquid nitrogen gave reduced and variable fertility relative to the methanol/dry ice bath method ( p =0.032). The commercial applicability of the protocols was demonstrated on a batch of 30 million eggs. Fertilization with cryopreserved sperm yielded 81% fertilization, and larval rearing by normal commercial practises yielded 3.7 million settled spat, which was comparable to the 2.5 million spat from a parallel batch fertilized with unfrozen sperm.

A new brevetoxin analog, brevetoxin B5 (BTX-B5) was isolated together with BTX-B1 and PbTx-3 from the New Zealand cockle Austrovenus stutchburyi harvested at an outbreak of neurotoxic shellfish poisoning early in 1993. The structure was elucidated by comparison of its spectral data (NMR and CAD FAB MS/MS) with those of BTX-B1 and PbTx-2, and confirmed by synthesis from PbTx-2 with SeO2 and H2O2. It was detected in the toxic greenshell mussel Perna canaliculus and Pacific oyster Crassostrea gigas collected at the same time.

Bacterial diseases remain a large problem in aquaculture hatcheries. The successful design and implementation of protective measures in the hatchery depends on an understanding of the dynamics of the infection process. Developing an in situ experimental protocol for pathogen challenge is therefore of paramount importance. Here, we demonstrated the minimum effective pathogenic dose (MEPD) of Vibrio splendidus (10(5) CFU ml(-1)) and a Vibrio coralliilyticus/neptunius-like isolate, Vibrio sp. DO1 (10(6) CFU ml(-1)), for New Zealand Greenshell mussel (GSM, Perna canaliculus) larvae during hatchery production. In a flow-through water hatchery system, larvae given 1 to 2 h of static water exposure to these pathogen doses showed respective average cumulative mortalities of 58 and 69% on the fourth day following pathogen exposure. After the 1 to 2 h static exposure, larvae were returned to flow-through water. Larvae exposed to a dosage one order of magnitude greater than the MEPD had higher mortalities of 73 and 96% for V. splendidus and Vibrio sp. DO1 respectively. These 4 levels of mortality were significantly greater than those of the non-exposed control larvae which respectively averaged 23 and 35% in experiments involving V. splendidus and Vibrio sp. DO1. Experiments were repeated 4 times to confirm reproducibility. After pathogen exposure, pathogens were detected in the larvae and tank water of treatments with dosages of > or =10(5) CFU ml(-1) (V. splendidus) and 10(6) CFU ml(-1) (Vibrio sp. DO1), but not in treatments with lower pathogen dosages. The challenge protocols are reproducible and provide an opportunity to assess measures for the protection of GSM larvae against infection in the hatchery environment.

A gram-negative, rod- to oval-shaped, aerotolerant anaerobic bacterium was isolated from an anaerobic enrichment inoculated with sediment taken from below the cyanobacterial mat of a high-salinity pond near Bratina Island on the McMurdo Ice Shelf, Antarctica. The organism was positive for terminal oxidase and catalase and was motile by means of a polar flagellum. Optimal growth of anaerobic cultures occurred at 12 degrees C, at pH 6.5, and at an NaCl concentration of 3% (w/v). Of a variety of polysaccharides tested, only starch and glycogen supported growth. No growth was observed on cellulosic substrates and xylan, and the organism was unable to attack esculin. Monosaccharides and disaccharides, including the cyanobacterial cell-wall constituent N-acetyl glucosamine, were fermented. Per 100 mol of hexose, the following products (in mol) were formed: acetate, 60; formate, 130; ethanol, 56; lactate, 73; CO2, 15; and butyrate, 2. Propionate, ethanol, n-propanol, n-butanol and succinate were not detectable in the culture medium (< 1 mol per 100 mol of monomer). Hydrogen was not detected in the head space (detection limit < 10(-5) atm). Growth yields in aerobic static liquid cultures were slightly higher than those in anaerobic culture, and fermentation favoured acetate at the expense of electron sink products. Growth was inhibited in aerobic shaking cultures, and the organism did not utilize nitrate or sulfate as electron acceptors. The G+C content of the DNA from the bacterium was 42.8 mol%. A phylogenetic analysis indicated that the organism is a member of the gamma-subgroup of Proteobacteria, but that it is distinct from other members of this group based on the sequence of its 16S rRNA gene, mol% G+C, morphology, and physiological and biochemical characteristics. It is designated as a new genus and species; the type strain is star-1 (DSM 10704).

A gram-positive, motile, rod-shaped, strictly anaerobic bacterium was isolated from an enrichment initiated with sediment taken from below the cyanobacterial mat of a low-salinity pond on the McMurdo Ice Shelf, Antarctica. The organism grew optimally at 12 degrees C, at pH 6.5, and at an NaCl concentration of0.5% (w/v). It survived freeze-thawing at low salt concentrations,but not exposure to temperatures over 25 degrees C for more than 20 h or short-term exposure to temperatures50 degrees C. Out of a variety of polysaccharides tested as growth substrates, only xylan supported growth. The organism also grew on a variety of mono- and disaccharides including the cyanobacterial cell wall constituent, N-acetyl glucosamine. Fermentation products on a mol product per 100 mol of hexose monomer fermented basis were: acetate, 72; formate, 72; butyrate, 55; hydrogen, 114; and CO2, 100. Not detectable in the culture medium(2 mol per 100 mol of monomer) were lactate, propionate, ethanol,n-propanol, n-butanol, and succinate. The G+C content of the DNA from the bacterium was 33 mol%, and a phylogenetic analysis indicated that it grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. It differed from other species of this genus with regard to growth temperature optimum, substrate range, and fermentation pattern, and is therefore designated as a new species of Clostridium for which the name Clostridium vincentii is proposed. The type strain is lac-1 (DSM 10228).

SUMMARY. 1 In sediment from Wintergreen Lake, Michigan, denitrification was not detectable by the acetylene inhibition method at in situ nitrate concentrations. When nitrate was added to sediment slurries, denitrification capacities up to 18.8μg N g-1 h-1 were measured. The denitrification capacities decreased with increasing sediment depth and distance from shore. 2 The high denitrification capacities in these sediments which under natural conditions had no supply of nitrate and oxygen suggested that denitrifies with alternative mechanisms for anaerobic energy conversion were present. Nitrous oxide was a significant portion of the N-gas produced immediately after the nitrate addition. Small amounts (4–5% of the total N-gas production) of nitric oxide accumulated in the early phase of nitrate reduction. Presumably after depletion of nitrate and nitrite both N2O and NO were further reduced to N2. 3 About 70%r of the added nitrate was denitrified, and the remainder was assumed to have been reduced to ammonium.

In January 1982, sediment microbial N transformations and inorganic N fluxes across the sediment/water interface were studied at nine sites off the South Island West Coast, New Zealand. The sediments showed a great variety in physical, chemical and biological properties. The sediment organic matter had a molar C N ratio of 5.9–10.9, and the total N P ratio was 1.2–4.0. The denitrification capacity in the top 7.5 cm of sediment was 0.1–77.2 mmol N m −2 day −1 and generally declined with increasing sediment depth. The in situ denitrification rate was 0.02–1.84 mmol N m −2 day −1 and highest activities were generally found in surface sediments and at 6–7.5 cm depth. Denitrification accounted for 82–100% of total nitrate reduction. Net N mineralization was indirectly estimated at 0.6–2.4 mmol N m −2 day −1 , and the experimental determination of this N transformation gave 0.6–3.2 mmol N m −2 day −1 . Denitrification accounted for 3–75% of net N mineralization. The diffusive flux of ammonium and nitrate across the sediment/water interface was 0.1–0.7 and 0.1–0.6 mmol N m −2 day −1 , respectively.

Characteristics of dissimilatory nitrate reduction by Propionibacterium acidi-propionici, P. freudenreichii, P. jensenii, P. shermanii and P. thoenii were studied. All strains reduced nitrate to nitrite and further to N2O. Recovery of added nitrite-N as N2O-N approached 100%, so that no other end product existed in a significant quantity. Specific rates of N2O production were 3 to 6 orders of magnitude lower than specific rates of N2 production by common denitrifiers. Oxygen but not acetylene inhibited N2O production in P. acidi-propionici and P. thoenii. Nitrite reduction rates were generally higher than nitrate reduction rates. The enzymes involved in nitrate and nitrite reduction were either constitutive or derepressed by anacrobiosis. Nitrate stimulated synthesis of nitrate reductase in P. acidi-propionici. Specific growth rates and growth yields were increased by nitrate. At 10 mM, nitrite was toxic to all strains, and at 1 mM its effect ranged from none to total inhibition. No distinction was obvious between incomplete forms of denitrification and dissimilatory nitrate reduction to ammonia. N2O production from nitrite by propionibacteria may represent a detoxication mechanism rather than a part of an energy transformation system.

Methanogenesis by a Syntrophomonas wolfei/ Methanospirillum hungatei coculture was inhibited in presence of ethylene and the hydrogenation catalyst Pd-BaSO4. However, butyrate oxidation by S. wolfei continued and ethylene was reduced to ethane. Per mol of butyrate oxidized, 2.4 mol acetate was produced and 0.8 mol ethylene was reduced. Acetylene, propylene and butene were less effective as H2 acceptors than ethylene, and addition of bromoethanesulfonic acid was necessary to inhibit methanogenesis in the presence of the two longer-chain olefins. Other hydrogenation catalysts were less effective in the order Pd-charcoal < PE-asbestos < Pd-PEI beads < Pt-Al2O3, Pd-CaCO3. Optimal ethylene hydrogenation was achieved with still incubation in presence of 7.2 mg Pd-BaSO4 and 0.7 g sand per ml medium. The higher catabolic rate of S. wolfei in presence of the methanogen indicated that the biological H2 removal mechanism was more efficient than the catalytic olefin reduction.

The acetylene block technique was used to assay denitrification in undisturbed sediment cores of an intertidal mud flat. Nitrogen loss due to this process was estimated at 1 to 41 kg of N/hectare (ha) per year. Anaerobic nitrate-saturated slurry of the same sediment had a denitrification capacity of 1,026 kg of N/ha per year. The acetylene block technique failed at low nitrate concentrations, so that denitrification at average in situ nitrate concentrations could not be determined. Denitrification followed zero-order kinetics at nitrate concentrations high enough to allow successful blockage of N 2 O reduction. Thus, an estimate of in situ rates based on kinetic parameters and in situ nitrate concentrations was impossible. No denitrification was observed in a slurry of the top 1.5 cm under aerated conditions and nitrate saturation. In undisturbed sediment, significant denitrification occurred in few discrete sites within a matrix of undetectable or low activity. Despite numerous errors contributing to the uncertainty of the estimate of in situ rates, the result obtained by this method was considered more valuable than the determination of denitrification capacities. Methods which include severe changes of physical and chemical parameters may frequently lead to overestimates of denitrification rates.

Among five hydrogenation catalysts, palladium on charcoal was the most reactive one when suspended in anaerobic culture medium, and Lindlar catalyst (Pd on CaCO 3 ) was the most reactive one when suspended in the gas phase of culture tubes. Palladium on charcoal in the culture medium (40 to 200 mg 10 ml −1 ) completely inhibited growth of Neocallimastix frontalis and partly inhibited Ruminococcus albus . Lindlar catalyst (40 to 200 mg per tube) suspended in a glass pouch above the culture medium did not affect the rate of cellulose degradation or the ratio of fermentation products by these organisms. Acetylene added to tubes containing Lindlar catalyst in pouches, and either of the two organisms in monoculture or coculture with Methanospirillum hungatei , was reduced to ethylene and then ethane, followed by hydrogen production. Similar results were obtained with 1-pentene. Neither acetylene nor 1-pentene affected cellulose degradation but both inhibited methanogenesis. In the presence of Lindlar catalyst and propylene or 1-butene, fermenter-methanogen cocultures continued to produce methane at the same rate as controls and no olefin reduction occurred. Upon addition of bromoethanesulfonic acid, methanogenesis stopped and olefin reduction took place followed by hydrogen evolution. In a gas mixture consisting of propylene, 1-butene, and 1-pentene, the olefins were reduced at rates which decreased with increasing molecular size. These results demonstrate the technical feasibility of combining in one reactor the volatile fatty acid production by anaerobic digestion with chemical catalyst-mediated reductions, using the valuable by-product hydrogen.