Pathogenic threats and probiotic use in larviculture of the scallop,Pecten maximus

During a series of experiments, bacteriological elements in scallop larval rearing were investigated: larvae susceptibility to pathogens as a function of their age, and the use of probiotic bacteria during larviculture. Younger larvae (d5 PF) were highly more susceptible to pathogenic‐challenge than their older siblings, which were challenged at an older age (d15 PF). A challenge with 10⁴ CFU mL^?1 of V. pectenicida killed 100% of d5 PF larvae 7 days following challenge, yet killed only 9% of d15 PF larvae 9 days following challenge. Use of the probiotics Phaeobacter gallaeciensis, Alteromonas macleodii 0444 and Neptunomonas sp. 0536, provided for larger larvae, a high yield of competent larvae and, perhaps more importantly, protection against pathogen‐challenge similar to levels achieved from antibiotic use. When challenged with V. pectenicida, d29 survivals were 20.3%, 85.1% and 75.0% respectively for control (no probiotic), antibiotic treated, and ‘probiotic mix’ administered larvae. Use of potential probiotic Pseudoalteromonas sp. D41 appeared to hinder scallop larvae. Future use of probiotics in scallop larval rearing would benefit from combined use of P. gallaeciensis, A. macleodii 0444 and Neptunomonas sp. 0536.     

On the relationship between low winter temperatures and mortality of juvenile scallops, Pecten maximus L., cultured in western Norway

The mortality of juvenile scallops (Pecten maximus L.) cultured at two densities at 8‐ and 15‐m depth was investigated at 17 trial locations along the western Norwegian coast between latitudes 58 and 65°N. During the winter of 1995/1996, scallops suffered high mortality at many sites. The results show a clear relationship between minimum temperature recorded on the sites and scallop mortality. All the scallops died at sites where minimum temperatures were lower than 2°C, while a mortality rate below 30% was only obtained at locations where the minimum temperature was 4–5°C. At temperatures between 2 and 4°C, mortality was intermediate, ranging from 52% to 100%. There were no mortality differences between culture densities, while a significantly higher mortality at 8‐ than at 15‐m depth was shown for two of the four sites with a total mean mortality lower than 30%. The results are discussed in relation to low‐temperature conditions along the Norwegian coast to suggest risk assessments for site selection in scallop culture.     

Study of the distribution of active caspase‐3‐positive cells in turbot,Scophthalmus maximus (L.), enteromyxosis

Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria‐submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti‐(active caspase‐3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi‐infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi‐infected turbot compared with non‐infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase‐3‐positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.     

Digestion of lipids and carbohydrates from microalgae (Chaetoceros muelleri Lemmermann and Isochrysis aff. galbana clone T‐ISO) in juvenile scallops (Pecten maximus L.)

The digestion of laminaran and neutral lipids from the microalgae Chaetoceros muelleri or mixtures of C. muelleri and Isochrysis galbana clone T‐ISO in 4‐mm scallops Pecten maximus were studied. The microalgae were cultivated at fixed low and high growth rate (5% and 60% of μ_max). The cultivation method clearly affected the lipid and carbohydrate contents, as well as their enzyme activity. Chaetoceros muelleri and T‐ISO grown at low growth rate exhibited a considerably higher content of lipids and carbohydrates than those algae grown at high growth rate. The specific activity of laminaranase and neutral lipase was the highest in C. muelleri grown at the low growth rate. Growth rate of scallops was best when they were fed a mono‐species diet of C. muelleri grown at low growth rate. The growth rate of the scallops was positively correlated to the dietary carbohydrate content as well as to the specific activity of neutral lipase and laminaranase in the diet. The capacity to digest carbohydrates (laminaran) might be limiting for the young scallops, and the content of carbohydrates and enzymatic activities of laminaran in the diet should be considered when formulating an optimal diet, both when the diet consist of live microalgae and formulated diets.     

Effect of size‐fractionated fish protein hydrolysate on growth and feed utilization of turbot (Scophthalmus maximus L.)

Four experimental diets were fed to turbot to examine the effect of fish hydrolysate and ultra‐filtered fish hydrolysate on growth performance, feed utilization and non‐specific immune response. Fish hydrolysate was produced by enzymatic treatment and size fractionated using ultra‐filtration (UF). The permeate (molecular weight <1000 Da) after UF and the non‐ultra‐filtered fish hydrolysate (NUF) were tested as feed ingredients. Diets UF1, UF2 contained 3.7%, 1.2% ultra‐filtered fish hydrolysate to replace fish meal protein respectively. The diets UF1, NUF were identical in composition except that the molecular weight of fish hydrolysate in the diet. Fish meal was used in the control diet. All diets were made equal in protein, lipid and energy. Each experimental diet was fed to juvenile turbot (27.87 ± 0.04 g) in triplicate for 8 weeks. Results of this study indicate that the best overall growth and feed utilization of turbot juveniles were obtained with a diet containing higher dose of the small molecular weight compounds in fish hydrolysate. Acid phosphatase, alkaline phosphatase, lysozyme and superoxide dismutase activity in serum were not affected by diet. Total antioxidant capacity was improved with increasing level of low molecule weight fish hydrolysate (UF1).     

Evaluating the most suitable nonlinear growth model for turbot (Scophthalmus maximus) in aquaculture 2 (weight application): Multi‐criteria model selection and growth prediction

Seeking the most suitable model to describe the growth of turbot, we analysed growth data of two different turbot (Scophthalmus maximus) strains reared communally in a recirculating aquaculture system. We fitted 10 different nonlinear growth models to individual weight gain data (n = 2,010) during the grow‐out phase. Analyses were carried out for each strain, for sexes within strains and for a pooled data set containing both strains and sexes. To assess the model performance, three different criteria are used. Further, a growth‐simulation was performed to evaluate the shape of the generated curve. This way we could assess the capability of the models to predict future growth. The 3‐parametric Gompertz model achieved the best fit in 42.9% of all cases tested and the lowest Bayesian information criterion in 100% of cases. The model produced realistically shaped curves and asymptotic values matching the biological attributes of the species. In contrast, 5‐parametric functions projected unrealistically shaped curves and predicted improbable mature sizes. Our results show that increasing number of parameters do not lead to increasing goodness of fit, but tend to result in overfitting, and demonstrate the advantages of the 3‐parametric Gompertz model for describing the growth of turbot.     

Effectiveness of dietary vitamin supplementation to the performance of common carp (Cyprinus carpio L.) larvae in intensive rearing condition

Study of enrichment of different vitamins (C, B₁, B₆ and E) in common carp larvae through 4 weeks feeding in recirculation system was carried out. For this purpose, procedure for enrichment of Artemia nauplii with different vitamins was developed and investigated the effectiveness in larvae nutrition. The habituation of larvae to dry feeds was done using supplemented dry diets with the same vitamins. To study the effect of vitamins in young fish, the most common stress situation was planned for carp nursed fries, which occurs during transportation. Levels of vitamins and parameters of immune response in mucus were monitored before and after stress situation. At the end of the feeding trials using enriched Artemia nauplii, level of vitamin B₁, B₆ and C were increased in the body of fish, but remained on similar level or decreased after dry feeding period. Therefore, we concluded that Artemia is a good delivery vector of these vitamins. While handling stress, vitamin C and E concentration was decreased and sparing effect of antioxidant vitamins was confirmed in groups supplemented with both vitamins. Vitamin B₁ was consumed intensively during the stress, vigorously in groups without supplementation. Contrary to this, vitamin B₆ level increased in stress condition in group without supplementations. The levels of immunoglobulins in mucus were increased in all treated groups independently of treatment. Our results confirm that carp larvae reared in tank condition needs sufficient amount of vitamins C, B₁ and B₆ supplements to protect them from the mild handling stress.     

The differences in postprandial free amino acid concentrations and the gene expression of PepT1 and amino acid transporters after fishmeal partial replacement by meat and bone meal in juvenile turbot (Scophthalmus maximus L.)

Meat and bone meal (MBM) is a high‐quality alternative protein source used to replace fishmeal (FM). However, the molecular mechanisms of over‐substituted FM by MBM resulted in growth reduction are still not clear. The objective of the study was to evaluate the effect of FM replacement by MBM on the concentration of postprandial free amino acid (FAA) and mRNA abundance of peptide and amino acid transporters in juvenile turbot (Scophthalmus maximus L.). Fish were fed with FM diet (60% FM), MBM diet (33% FM + 34.2% MBM) and MBM + AA diet (MBM diet with essential amino acid (EAA) added to match the AA profile of FM) for 30 days. Results showed that compared with the FM diet, MBM diet led to a reduction in FAA concentration peak values in plasma and muscle. MBM + AA diet significantly elevated the peak values of FAA concentrations to FM diet level in plasma, but not in muscle. Furthermore, compared with FM diet, MBM diet significantly increased gene expression of PepT1 and major amino acid transporters in intestine, whereas MBM diet greatly downregulated gene expression of T‐type amino acid transporter‐1, system ASC amino acid transporter‐2 and cationic amino acid transporter‐2 in muscle. Supplemented EAA did not ameliorate these different effects in intestine and muscle. Overall, this study provided a comprehensive explanation for the relationship between diet, FAA concentrations and AA transportations, which provides a molecular basis for further using MBM to replace FM in aquafeeds.     

Fertilizing capacity and motility of tench Tinca tinca (L., 1758) sperm following cryopreservation

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar‐based Grayling and ion‐based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post‐thaw storage (4°C). Sperm prepared with the sugar‐based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post‐thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar‐based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.     

A cost‐effectiveness analysis of live feeds in juvenile turbot Scophthalmus maximus (Linnaeus, 1758) farming: copepods versus Artemia

The biological benefits of copepods as live feed for marine finfish larvae have already been well established in the literature. Copepods have better biochemical compositions that improve growth, reduce malpigmentations and allow successful farming of ‘new’ marine finfish species. However, their current usage is quite limited. One of the reasons has been lack of economic knowledge concerning the cost‐effectiveness of copepod application compared to other commonly used feed items such as the brine shrimp Artemia. In this study, a cost‐effectiveness analysis is made on two alternative live feed items (copepods and Artemia) in juvenile turbot farming. Unit cost of production and profit are compared between the two feeding regimes using a unique data set from an existing turbot fry production facility in Denmark. The result reveals that copepods are not only biochemically superior but they are also economically a cost‐effective alternative. Thus, a commercial use of copepods will significantly reduce the production costs for turbot. Furthermore, the unexploited economic potential can be utilized for the successful farming of other high‐valued marine finfish species such as tuna, flounders, cod, sole and halibut. Generally, the biochemical superiority coupled with economic benefits can lead to the commercial utilization of copepods as complementary live feed in the short run and in some situations as a substitute in the long run.     

Effect of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on the growth,immunity and muscle composition of turbot,Scophthalmus maximus (Linnaeus, 1758)

We evaluated the effect of different concentrations of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on the growth, immunity and muscle composition of turbot Scophthalmus maximus. Eight diets (containing no IMP or GMP, or 0.5 g/kg IMP, 1.0 g/kg IMP, 2.0 g/kg IMP, 0.5 g/kg GMP, 1.0 g/kg GMP, 2.0 g/kg GMP, or 0.5 g/kg IMP plus 0.5 g/kg GMP) were prepared. A total of 360 fish (average body weight of 105 g) were randomly selected and placed in groups into 24 plastic aquaria (8 treatments × 3 replicates × 15 individuals per plastic aquaria). The tanks were maintained at the temperature of 15 ± 2°C. The experimental diets were fed for 60 days. The specific growth rate (SGR) was significantly higher in S. maximus fed with IMP or GMP compared with fish fed neither IMP nor GMP. The highest SGR was observed in fish fed with 1.0 g/kg IMP. Supplementation with these dietary nucleotides had a positive, but not significant effect on the activity of superoxide dismutase, alkaline phosphatase and acid phosphatase. There was a significant difference in the moisture and crude lipid content of muscle from S. maximus fed the different diets compared with control fish. The highest moisture content was 83.44 for a diet of 0.5 g/kg IMP plus 0.5 g/kg GMP, which was also significantly higher when compared to fish fed alternative diets. The crude lipid content of S. maximus fed diets containing either IMP or GMP was significantly higher than those fed diets without IMP or GMP. Thus, according to these results, the optimal level of dietary IMP is 1.0 g/kg, which correlates with the largest increase in growth performance of S. maximus.     

Non‐invasive fast real‐time PCR assay for detection of the enteric parasite Enteromyxum scophthalmi in cultured turbot (Scophthalmus maximus L.)

Enteromyxum scophthalmi is a myxozoan parasite that causes severe parasitic diseases in cultured turbot affecting mainly the intestine of the host. It is characterized by producing acute enteritis, starvation and eventually death. Current diagnosis of E. scopthalmi use traditional techniques, based on the identification of the morphology of the parasite. These techniques take extended time to be carried out and do not favour the adoption of control measure at turbot farms and require the sacrifice of fish. This study develops a fast real‐time PCR molecular tool for the detection of E. scophthalmi in infected farmed turbot. This methodology is applicable for routine controls on the farm at every stage of the parasite infection. Results of the study demonstrate the robustness, specificity, efficiency and reliability of the technique. In addition, this study also provides a non‐invasive procedure of sampling through swaps. This allows control, prevention and diagnosis of the parasite infection at turbot farms while maintaining the welfare of the cultivated fish and avoiding sacrifice of the fish sampled.     

Harvesting,post‐harvest handling and live‐transport of freshwater prawns in the United States

Freshwater prawn Macrobrachium rosenbergii aquaculture continues to expand in the United States though is still a relatively small industry. Innovations continue to improve efforts in harvesting with novel harvest basin designs and concrete internal pond aprons. Purging and holding strategies are more defined. Some producers are now investing in water re‐use systems to extend holding times for many days, making prawns more readily available for market. The lucrative live‐market, which historically has had many challenges, seems poised for success when following new protocols which address handling and oxygen stress.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

Underwater evaluation of habitat partitioning in a European river between a non‐native invader,the racer goby and a threatened native fish,the European bullhead

We conducted underwater surveys using SCUBA gear to examine habitat and microhabitat competition between the Ponto‐Caspian racer goby Babka gymnotrachelus and native European bullhead Cottus gobio to assess the potential for competitive displacement of the native species by the invading species. In summer, 88 surveys were made in a tributary of the River Vistula within defined benthic areas across the entire width of the river bed. The occurrence of fish by total length class (small: <6 cm; large: >6 cm) and environmental conditions (depth, water velocity, substratum type, plant cover, shelter type) was recorded. We found a substantial separation between the species and size classes in relation to substratum, shelter type and water velocity. European bullheads were limited to lotic areas with stony bottoms, whereas racer gobies also occupied lentic areas over sand or mud. European bullheads usually took refuge under stones in contrast to a wider range of shelters used by racer goby, including tree roots and rubbish. In general, the breadth of habitat used by the racer goby was wider than that of the European bullhead, although habitat overlap between the species was not statistically significant except for the type of shelter occupied by small fish, selecting smaller stones. An inverse relationship was observed between small European bullheads and all racer gobies in areas where they co‐occurred, suggesting that invader may be having an adverse effect on the distribution and habitat use of small native bullheads, particularly in areas of moderate water velocities over small stones and gravel.     

Detection of Herpesvirus anguillae (AngHV‐1) in European eel Anguilla anguilla (L.) originating from northern Poland—assessment of suitability of selected diagnostic methods

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV‐1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false‐negative results, the main aim of the study was to optimize diagnostic methods for AngHV‐1 detection using conventional PCR, nested PCR and in situ hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV‐1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals.     

Hatchery cultivation of the common cockle (Cerastoderma edule L.): from conditioning to grow‐out

This study describes for the first time the cultivation of Cerastoderma edule on a commercial scale. A protocol to grow F₂ generation cockles was developed, which led to fine‐tuning experiments for broodstock conditioning and spat growth. Broodstock animals were conditioned with diets of Isochrysis galbana (T‐Iso) or Tetraselmis suecica, whereas a third group was not fed. The best diet, T. suecica, induced 12 females out of 100 animals to spawn a total of 3 380 000 eggs. The non‐fed group did not spawn. Cockle spat (4.9 ± 1.0 mm) grew best when given a mixed diet of C. muelleri, T‐Iso and Sceletonema costatum, or a mixture of P. tricornutum and S. costatum at a concentration of 240 cells μl^?1 day^?1, resulting in a tripling of their wet weight after 14 days. The impact of density, burrowing substrate and food availability on cockle spat growth (41 days old, 5.6 ± 1.2 mm) was studied for 11 weeks. Best results were obtained by culturing spat at ad libitum food conditions at 500 ind m^?2, resulting in an average growth rate of 168 μm day^?1, an average final size of 19.0 ± 1.9 mm and a total final biomass of 1040 g m^?2.     

Time‐series response of immune enzymes and catecholamines in juvenile sea cucumber Apostichopus japonicus during long‐term live transport

Sea cucumbers are free of protective exoskeleton and capable of autolysis under stress conditions. The live transport of small‐size juveniles to grown‐out sites induces hypoxia and congesting stress. The transport process is essential to production of outdoor healthy seedlings. Here, we evaluated the effects of damp transport and water transport on Apostichopus japonicus, an economic species inhabiting along the Asian coast. The survival after transport and immune performance of small‐size juveniles (0.05–0.1 g of immersed weight) were monitored during distinct duration of transport (1, 3, 5, 8 and 12 hr). The results revealed that there were no significant differences between the two transport systems within 5 hr. Given the survival for recovery of 48 hr and responses of immune enzymes and catecholamines during transport, water transport was less detrimental to sea cucumbers than damp transport when the transport duration was less than 8 hr. Once the duration prolonged to 12 hr, these immune parameters changed irregularly and the survival declined drastically. Water transport with a notably higher survival for juveniles was the more appropriate manner for long‐time transport. These findings provide insights into long‐term transport of small‐size A.  japonicus juveniles and might be beneficial for production of ecologically healthy seedlings.     

Recovery of Hafnia alvei from diseased brown trout,Salmo trutta L., and healthy noble crayfish,Astacus astacus (L.), in Bulgaria

Hafnia alvei was isolated in Bulgaria from healthy noble crayfish, Astacus astacus (L.), and then from farmed diseased brown trout, Salmo trutta L., with signs of haemorrhagic septicaemia. The isolates were identified initially with conventional phenotyping and commercial Merlin Micronaut and API 20E rapid identification systems, followed by sequencing of the 16S rRNA gene. Hafnia alvei Bt1, Bt2 and Aa4 were of low virulence to rainbow trout and brown trout, although cytotoxicity was demonstrated by Bt1 and Bt2, but not by Aa4.     

Co‐Infection with Pseudomonas anguilliseptica and Delftia acidovorans in the European eel,Anguilla anguilla (L.): a case history of an illegally trafficked protected species

Inspections by customs agents at Barcelona airport discovered 420 kg of contraband glass eels prepared for shipment to Hong Kong. After confiscation of these animals by police, they were transported to holding facilities to be maintained until after a judicial hearing. Upon arrival, they were separated into two groups and held under ambient flow‐through conditions in fresh water. During their captivity period, several peaks in mortality occurred and multiple bacterial strains were isolated from moribund animals. Sequencing of 16S rDNA was used to determine specific identity of the isolates. An initial isolation of Pseudomonas anguilliseptica was treated with oxytetracycline. A subsequent isolation of Delftia acidovorans proved resistant to oxytetracycline and was treated with gentamicin in combination with sulphadiazine–trimethoprim. Once the health condition of the animals was stabilized, they were partitioned into groups and subsequently released as part of a restocking effort for the species following the guidelines of Regulation (EC) 1100/2007 (Anon 2007). This represents the first record for both bacterial species in the host Anguilla anguilla in the Spanish Mediterranean.