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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.

Chaetoceros calcitrans is a small, fast-growing diatom with a high nutritional value for filter feeders. It is often used in hatcheries, particularly in the production of bivalve spat. It is widely produced in batch culture systems ranging from carboys to plastic bags to high volume tanks. Continuous culture of C. calcitrans has generally not been successful due to frequent crashes of the culture. We report the continuous culture of C. calcitrans in a hanging plastic bag system that is suitable for use in commercial hatcheries. Runs of continuous production lasted up to 125 d, with an average bag life of 24.4 d. Crashes occurred at irregular intervals in individual bags and caused the cell concentration to drop by up to three orders of magnitude. Cultures recovered over a few days following a crash. The temporary reduction of production due to crashes in individual bags was compensated by a 30% higher production than required. At dilution rates of 1.0–1.3 volume exchanges d − 1 the system produced 7–13 × 10 6 cells mL − 1 d − 1 . The cell diameter mode as determined by a Coulter Counter was 2.8–3.0 μm. The cells had the typical flat rectangular box shape with well-developed setae. The culturable bacterial population of the algae culture was 10 5 –10 7 colony forming units mL − 1 . Genetic integrity of C. calcitrans and monospecific status of the continuous culture were maintained over 7 sequential bag-to-bag transfers during a 125-day run. The fatty acid profiles of continuous and batch cultures of C. calcitrans were similar. Growth and survival of Greenshell™ mussel ( Perna canaliculus ) larvae were identical when fed with C. calcitrans batch culture or continuous culture delivered directly from the harvest line. When continuous culture was first collected over 24 h, diluted and then delivered to the rearing tanks over 24 h, larval growth was markedly slower (24 d to reach settlement competency compared to 21 d for the other two treatments); however the subsequent settlement success of competent larvae was not statistically different between the three diets (‘Carboy’, ‘Continuous/Harvest line’, ‘Continuous/Feed-out bin’; 33.9–35.5% settled). Concomitant to the differences in larval performance we observed a difference in larval fatty acid profile: lower-performing larvae had a markedly lower proportion of mono-unsaturated fatty acids and a markedly higher proportion of poly-unsaturated fatty acids. Lower performance and different fatty acid profiles of larvae fed the ‘Continuous/Feed-out bin’ diet appeared to be caused by unidentified changes that occurred during storage of the C. calcitrans culture.

Earlier work identified two novel strains of probiotic bacteria, Alteromonas macleodii 0444 and Neptunomonas sp. 0536, for Greenshell™ mussel ( Perna canaliculus ) larvae. Herein, we investigated whether the combination of the two probiotics in a multi-strain mix provided a) enhanced larval production during routine rearing and b) improved larval protection during two separate pathogen-challenge tests ( Vibrio sp. DO1 and Vibrio splendidus ). The response of larvae to multi-strain or single-strain probiotic administration was compared to that of larvae without probiotics. Two concentrations of each probiotic were tested (10 7 and 10 8 CFU ml − 1 ). Addition of 10 8 CFU ml − 1 multi-strain mix in the routine rearing of larvae yielded smaller-sized larvae and a lower feeding rate when compared with the 10 7 CFU ml − 1 and control groups. During the challenge test against Vibrio sp. DO1 and V. splendidus , protection by single-strain probiotic administration was observed at both levels of probiotic administration (10 7 and 10 8 CFU ml − 1 ), with no apparent added protection from multi-strain probiotics. Although 10 8 CFU ml − 1 levels provided protection against pathogen attack, they were also potentially detrimental to normal larval rearing when administered in combination and, as such, where administration of A. macleodii 0444 and Neptunomonas sp. 0536 would be applied together, a multi-strain mix of probiotics at 10 7 CFU ml − 1 , is recommended as the best concentration of each probiotic.

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.

Bacterial pathogens of Greenshell mussel (GSM) larvae can cause batch losses during hatchery production. Twenty-two isolates were screened using a larval bioassay. Two strains were identified as potential pathogens. Phenotypic identification of these strains revealed two non-reactive Gram-negative, oxidase positive rods. Sequencing of the 16S rRNA gene identified Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate as pathogens of GSM larvae, with an ability to cause 83% and 75% larval mortality in vitro, respectively, at a concentration of 10(2) CFU mL(-1). Histopathology indicated that the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. This is the first report on pathogens of GSM larvae.

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.

A new brevetoxin B analog, brevetoxin B4 (BTXB4), was isolated as the major toxin in greenshell mussels, Perna canaliculus, collected in New Zealand at the time of the neurotoxic shellfish poisoning incident. The new analog accounted for nearly two-thirds of the mouse lethality of the shellfish and was determined to be a mixture of N-myristoyl-BTXB2 and N-palmitoyl-BTXB2. Copyright © 1999 John Wiley & Sons, Ltd.

A new brevetoxin analog, brevetoxin B2 (BTXB2), was isolated from greenshell mussels, Perna canaliculus, collected at the time of the neurotoxic shellfish poisoning incident in New Zealand. The structure was elucidated based on NMR, CAD FAB MS/MS, and chemical degradation experiments.

A new brevetoxin analog, brevetoxm B3 (BTXB3 1 ). was isolated from the greenshell mussel, Perna canaliculus from New Zealand. In BTXB3 the brevetoxin B skeleton is modified by cleavage of ring D. esterification of the resulting alcohol, and oxidation of the aldehydic terminus.

A new marine toxin, gymnodimine, was isolated from New Zealand oysters, Tiostrea chilensis, and the dinoflagellate Gymnodinium cf. mikimotoi. Its unique structure was elucidated by spectroscopic methods.

A new marine toxin, gymnodimine, was isolated from New Zealand oysters, Tiostrea chilensis, and the dinoflagellate Gymnodinium cf. mikimotoi. Its unique structure was elucidated by spectroscopic methods.

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 bioassay was developed to screen and select bacterial strains as probiotics for an alternative to antibiotic usage in the management of bacterial pathogens of Greenshell™ mussel (GSM) larvae, Perna canaliculus. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate, Vibrio sp. DO1, were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 h revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. © 2009 Elsevier B.V. All rights reserved.

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).

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.