Effect of cultivated copepods (Acartia tonsa) in first‐feeding of Atlantic cod (Gadus morhua) and ballan wrasse (Labrus bergylta) larvae

The aim of this study was to compare the nutritional composition and effects of short periods with cultivated copepod nauplii versus rotifers in first‐feeding. Atlantic cod (Gadus morhua) and ballan wrasse (Labrus bergylta) larvae were given four different dietary regimes in the earliest start‐feeding period. One group was fed the copepod Acartia tonsa nauplii (Cop), a second fed enriched rotifers (RotMG), a third fed unenriched rotifers (RotChl) and a fourth copepods for the seven first days of feeding and enriched rotifers the rest of the period (Cop7). Cod larvae were fed Artemia sp. between 20 and 40 dph (days posthatching), and ballan wrasse between 36 and 40 dph, with weaning to a formulated diet thereafter. In addition to assessing growth and survival, response to handling stress was measured. This study showed that even short periods of feeding with cultivated copepod nauplii (7 days) had positive long‐term effects on the growth and viability of the fish larvae. At the end of both studies (60 days posthatching), fish larvae fed copepods showed higher survival, better growth and viability than larvae fed rotifers. This underlines the importance of early larval nutrition.     

Fishmeal quality and ethoxyquin effects on the weaning performance of ballan wrasse (Labrus bergylta) larvae

Fishmeal diets are shown to be unfavourable for successful weaning of ballan wrasse (Labrus bergylta) larvae. Variable fishmeal quality, and/or a possible negative effect of ethoxyquin (EQ) and/or palatability could be the reason for the reduced growth and increased mortality observed. This study weaned wrasse larvae on either a control diet of freeze–dried shrimp and cod fillet, or fishmeal‐based diets with 0, 11 or 44 ppm EQ, for 1 month. Lower final body weight and higher mortality were related to increased inclusion of EQ. Furthermore, fishmeal diets had increased lipid oxidation values with decreased EQ content, whereas the control diet had the lowest lipid oxidation levels. A combined negative effect of oxidation and EQ supplementation could have lowered the palatability of the feeds and be the reason for the unsuccessful larval weaning. Use of EQ is inevitable in large‐scale production of feed ingredients; however, the use of high‐quality ingredients with low EQ inclusion, or its replacement with other antioxidants, may be necessary for successful intensive culture of some marine fishes.     

Genetic population structure of goldsinny wrasse, Ctenolabrus rupestris (L.), in Norway: implications for future management of parasite cleaners in the salmon farming industry

The application of goldsinny wrasse, Ctenolabrus rupestris (L.), as parasite cleaners in the salmon farming industry has led to transport of live wrasse between regions in Norway. Owing to the operation of salmon farms, some of the transferred wrasse escape from the salmon net pens and can therefore mix and potentially interbreed with the local populations. The genetic effects of such interbreeding are unknown and information about the genetic population structure of this species in Norwegian inshore waters is needed to evaluate the potential effects. A genetic study based on polymorphic tissue enzymes was therefore carried out. Four of the 35 protein coding loci screened were polymorphic at the 99% level, and only two at the 95% level. The allele frequencies of the IDDH-1*; PGM-1*; GPI-1* and GPI-2* loci were compared in wrasse samples (1442 specimens) collected at 12 localities. Significant differences were found for PGM-1* among the samples collected at two inner fjord localities. Significant differences at the IDDH-1*, PGM-1* and GPI-2* loci were found when data from a sample of transferred wrasse caught in southern Norway were compared with local wrasse in the recipient areas.     

Effects of continuous and alternate administration of β‐glucan and mannan‐oligosaccharide on the growth,immunity and resistance against Vibrio splendidus of sea cucumber Apostichopus japonicus (Selenka)

A 4‐week growth trial was conducted to compare the effects of different feeding strategies of dietary immunostimulants on the growth, immunity and resistance against Vibrio splendidus of sea cucumbers Apostichopus japonicus (Selenka). Six feeding strategies were set, including feeding immunostimulants‐free diet continuously (control), feeding dietary β‐glucan (1.25 g kg^?1 diet) continuously, feeding dietary mannan‐oligosaccharides (MOS; 2.00 g kg^?1 diet) continuously, feeding β‐glucan 2 days followed by MOS 5 days alternately, feeding β‐glucan 5 days followed by MOS 2 days alternately and feeding β‐glucan 7 days followed by MOS 7 days alternately. The sea cucumbers fed immunostimulants showed higher specific growth rate (SGR) and lower cumulative mortality than control (P < 0.05). When sea cucumbers were fed with β‐glucan continuously, total coelomocytes counts and superoxide anion were significantly higher than control on the 4th day (P < 0.05). However, these two immune parameters were not significantly higher than those in control after the 18th day (P > 0.05). While sea cucumbers continuously fed MOS, these two immune parameters were not significantly higher than control until the 15th day. All immune parameters of the sea cucumbers fed with β‐glucan and MOS alternately were significantly higher than those in control during the experiment (P < 0.05). The sea cucumbers fed with β‐glucan 7 days followed MOS 7 days alternately showed the highest SGR and second lowest cumulative mortality. It was suggested that this feeding strategy is most suitable for sea cucumbers.     

An aerolysin-like enterotoxin from Vibrio splendidus may be involved in intestinal tract damage and mortalities in turbot, Scophthalmus maximus (L.), and cod, Gadus morhua L., larvae

Vibrio splendidus is a pathogen that can cause major losses during the early stages of larval turbot rearing when live feed (rotifers or Artemia) is used. As haemolytic bacteria have often been associated with larval rearing losses, we studied the role of the V. splendidus haemolysin in infection of larvae. From a bank of over 10,000 transposon mutants of V. splendidus, two different types of haemolysin-negative mutants were obtained. Both had lost virulence for larval fish, and immunohistochemistry showed that the transposon mutant studied colonized the turbot larval intestinal tract at a similar level to the wild-type organism but did not cause damage or signs of enteritis found with the wild-type organism. One transposon insertion site was located within a gene with high homology to aerolysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we have termed vibrioaerolysin, had properties similar to aerolysin and osmotic protection studies showed that it formed pores in the membranes of erythrocytes of similar diameter to those of aerolysin. The Tn10 insertion site of the second transposon mutant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaerolysin. The gastroenteritis caused by Aeromonas spp. in humans is considered to be due to production of aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine fish larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial flora of the larval fish gut can secrete aerolysin-like toxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on blood agar plates of live feed is recommended with measures to reduce the concentrations of haemolytic bacteria in rearing systems.     

Applying the ontogeny of digestive enzyme activity to guide early weaning of pigfish,Orthopristis chrysoptera (L.)

Weaning marine fish larvae from live prey to a dry microdiet is an important step towards optimizing the commercial production, but early weaning is constrained by the lack of sufficient digestive enzymes at first feeding. This study quantified the activity of five digestive enzymes throughout the larval period of pigfish (Orthopristis chrysoptera [L.]) to assess ontogenetic changes in digestive abilities, and then trials were conducted that determined the optimal time for weaning. The activity of all digestive enzymes was low or undetectable at first feeding (3 days post hatching, dph; 2.5 mm standard length, SL). A substantial increase in activity occurred at 5.7 mm SL (17 dph), 6.9 mm SL (21 dph), 7.7 mm SL (23 dph), 8.4 mm SL (25 dph) and 11.2 mm SL (30 dph) for bile salt‐dependent lipase, trypsin, chymotrypsin, amylase and acid protease respectively. During the weaning experiment, larvae were co‐fed live prey and microdiet beginning 15 dph (4.8 mm SL). Live prey was withdrawn from the diet at 24, 28, 32 or 36 dph, with the control receiving live prey and microdiet throughout (to 43 dph). There were no significant differences in mean final SL among treatments, but survival was significantly lower when larvae were weaned at 24 dph compared to 32–43 dph. Based on the digestive enzyme activity and survival, weaning larval pigfish at 32 dph (11.7 mm SL) when reared at 24°C is recommended.     

Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non‐host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non‐ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.     

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

Embryonic and larval development of serpae tetra Hyphessobrycon eques (Steindachner, 1882)

In this study, the embryonic and larval development stages of one of the most important ornamental fish serpae tetra (Hyphessobrycon eques) are described. The early life stage is documented from fertilization until the beginning of the juvenile period. The fertilized eggs (the average diameter = 938.55 ± 35.20 µm) were incubated at a water temperature of 26 ± 0.5°C. The cleavage finished in 1:10 hr (=h) and the early blastula stage occurred at 1:26 hr post fertilization (hpf). The gastrulation started at 3:05 hpf, and 50% epiboly was observed at 3:25 hpf. Segmentation stage was monitored at 7:26 hpf. Embryonic developmental stage was completed and hatching occurred 20–21 hpf. The total length (TL) of newly hatched larvae was 2.64 ± 0.21 mm. The larval development of serpae tetra was divided into four different periods: Yolk‐sac larva (1–4 DAH, TL = 2.77 ± 0.09 mm ‐ 3.85 ± 0.11 mm), preflexion larva (5–12 DAH), flexion larva (13–15 DAH, TL = 5.78 ± 0.46 mm on the 15th day) and post‐flexion larva (16–30 DAH, TL = 10.7 ± 0.27 mm on the 28th–30th days). The mouth and anus are closed at 1 DAH. The mouth and anus opened at 4 DAH. Exogenous feeding started on the 4th day. The first gulping of the swim bladder was on days 3. The larva begins to swim freely, and the yolk sac was completely consumed at 4 DAH. Histological structures of the eye and brain of new hatched larva were clearly identified at 1 day after hatching (DAH). According to histological findings, the digestive system (stomach, intestine) started to develop and the liver could be seen on the ventral side of the swim bladder at 5 DAH. No histological difference was observed between the anterior intestine and the posterior intestine at 15–16 DAH. The larval metamorphosis was completed, and the larvae transformed into juveniles at 28–30 DAH.     

The use of preserved copepods in sea bream small‐scale culture: biometric,biochemical and molecular implications

Considering the well‐known problems arising from the use of rotifers and Artemia as live prey in larval rearing in terms of fatty acid deficiencies, the aim of this study was to evaluate a partial or complete replacement of traditional live prey with preserved copepods during the larviculture of gilthead sea bream (Sparus aurata). Sea bream larvae were randomly divided into 4 experimental groups in triplicates: group A larvae (control) fed rotifers followed by Artemia nauplii; group B fed a combined diet (50%) of rotifers–Artemia and preserved copepods; group C fed rotifers followed by preserved copepods; and group D fed preserved copepods solely. Survival and biometric data were analysed together with major molecular biomarkers involved in growth, lipid metabolism and appetite. Moreover, fatty acid content of prey and larvae was also analysed. At the end of 40 days treatment, a stress test, on the remaining larvae, was performed to evaluate the effects of different diets on stress response. Data obtained evidenced a positive effect of cofeeding preserved copepods during sea bream larviculture. Higher survival and growth were achieved in group B (fed combined diet) larvae respect to control. In addition, preserved copepods cofeeding was able to positively modulate genes involved in fish growth, lipid metabolism, stress response and appetite regulation.     

Susceptibility of corkwing wrasse Symphodus melops, goldsinny wrasse Ctenolabrus rupestis, and Atlantic salmon Salmo salar smolt, to experimental challenge with Vibrio tapetis and Vibrio splendidus isolated from corkwing wrasse

The virulence of two Vibrio strains, previously isolated from diseased corkwing wrasse Symphodus melops and identified as V. tapetis and V. splendidus, to corkwing and goldsinny wrasse Ctenolabrus rupestris and to Atlantic salmon Salmo salar, was studied under laboratory conditions. Both bacteria were shown to be opportunistically pathogenic to corkwing wrasse, causing significantly higher mortality in the challenged groups than in the controls. Bacterial cultivation of kidney samples and re-isolation of V. tapetis and V. splendidus from most mortalities confirmed the two strains as the probable cause of mortality in the challenged groups. The control group also suffered relatively high mortality, but no specific pathogens that were suspected to be the main cause of death were isolated, other than a mixture of Vibrio spp. and, in the case of one individual, atypical Aeromonas salmonicida. Following injection challenge with both bacterial strains, no mortality was recorded in Atlantic salmon. In bath challenge trials with goldsinny wrasse, only slight mortality was observed in the challenged groups and the unchallenged control group. Bacterial examination showed that atypical Aeromonas salmonicida was the probable cause of death in both bath challenged and control groups of goldsinny wrasse, and no indication of infection by any Vibrio sp. was found.     

Efficacy and toxicity of praziquantel in helminth‐infected barbel (Barbus barbus L.)

This study evaluated efficacy and toxicity of the pyrazinoisoquinoline anthelmintic praziquantel (PZQ) in barbel infected with metacercariae of Diplostomum spathaceum and adult Pomphorhynchus laevis, and assessed antioxidant biomarkers and the lipid peroxidation response in juvenile barbel post‐treatment. The estimated 96‐hr LC50 of PZQ was 28.6 mg/L. For evaluation of efficacy, barbel naturally infected with D. spathaceum were exposed to a 10 and 20 mg/L PZQ 4‐day bath treatment. Both concentrations were 100% effective against D. spathaceum and significantly (p < .01) affected the activity of catalase, superoxide dismutase, glutathione reductase and glutathione‐S‐transferase as well as levels of reduced glutathione in liver and muscle. The efficacy of orally administered PZQ was assessed in adult barbel naturally infected with P. laevis. Fish were administered 10, 30 and 50 mg/kg of body weight and examined via gut dissection after 6 days. The 50 mg/kg dose significantly decreased the intensity of infection. Praziquantel is a feasible bath treatment for barbel infected with D. spathaceum and has potential for oral treatment of broodfish infected with P. laevis.     

Studies on the broodstock production and larval rearing of Coral demoiselle Neopomacentrus nemurus (Bleeker 1857)

Steps taken for broodstock development of Neopomacentrus nemurus, Coral demoiselle (Bleeker, 1857) in a hatchery, its spawning and development of egg to the juvenile stage are described. Among the three treatments tried, only the trial using Parvocalanus crassirostris nauplii as the first diet helped in the development of larvae. Larval and post‐larval growth was studied for a period of 65 days post hatch. The larva measured 2–2.4 mm in total length at the time of hatching. The mouth size of larva at the time of hatching measured 237.92 µm. By the 5th day, the width of the larval body had significantly increased. All the fins were conjoined, except for the caudal fin which had begun its appearance on the 2nd day itself. The digestive system was functional by the 5th day and the copepod remains were seen in the digestive tract. The larva metamorphosed by the 15th day with fully developed fins and fin rays. On the 20th day, the larval body had begun to display signs of fin pigmentation. The larva assumed adult pigmentation by the 35th day. By the 65th day, the larva grew to a total length of about 27 mm and weighed about 0.56 g.     

Genotyping of Streptococcus dysgalactiae strains isolated from Nile tilapia,Oreochromis niloticus (L.)

Streptococcus dysgalactiae is an emerging fish pathogen that is responsible for outbreaks of disease on fish farms around the world. Recently, this bacterium was associated with an outbreak at a Nile tilapia, Oreochromis niloticus (L.), farm in Brazil. The aim of this study was to evaluate the genetic diversity, best genotyping method and aspects of molecular epidemiology of S. dysgalactiae infections in Nile tilapia farms in Brazil. Twenty‐one isolates from four farms located in different Brazilian states were characterized genetically using pulsed‐field gel electrophoresis (PFGE), ERIC‐PCR, REP‐PCR and sodA gene sequencing. The discriminatory power of the different typing methods was compared using Simpson's index of diversity. Identical sodA gene sequences were obtained from all isolates, and ERIC‐PCR and REP‐PCR were unable to discriminate among the isolates. PFGE typing detected three different genetic patterns between the 21 strains evaluated; thus, it was the best genotyping method for use with this pathogen. The strains from Ceará State were genetically divergent from those from Alagoas State. The S. dysgalactiae isolates analysed in this study constituted a genetically diverse population with a clear association between geographical origin and genotype.     

Effects of various sodium adsorption ratio (SAR) mediums on larval performance of the freshwater prawn Macrobrachium rosenbergii (de Man)

The sodium adsorption ratio (SAR) is used to describe the ratio of sodium to calcium and magnesium in water that is very important in both aquaculture and agriculture. SAR treatments including of 20, 30, 40, 50 and 60 were adjusted in 10‐L containers. Initial larval density was fixed at 100 larvae L^?1 and during the culture period, various larval quality parameters were evaluated. Our findings illustrated that at initial stages of larval development, different SAR treatments affected the larval condition index (LCI), but no influences on larval stage index (LSI) were apparent. Importantly, this process at the final larval stages became reversed. In addition, survival at SAR 30 was 12.4% greater than among any other treatments. The highest larval resistance to stress (191 ± 5 ppm), as measured by the 24‐h LC₅₀ formalin test also was observed for SAR 30, at the 11th larval stage. Analysis indicated that calcium absorption during larval developmental stages is affected by SAR levels in the environment and also showed that the optimal SAR treatment was 30 for Macrobrachium rosenbergii larviculture in inland hatchery brackish water. According to our findings and desired SAR for the environment and agricultural soil (<15), effluent of M. rosenbergii inland hatcheries must be treated at least two times by dilution and adding calcium and magnesium salts.     

The effect of photoperiod and feeding frequency on larval of the Amazonian ornamental fish Pyrrhulina brevis (Steindachner, 1876)

This study aimed to evaluate the performance of Pyrrhulina larvae, Pyrrhulina brevis, under different photoperiods and feeding frequencies. A completely randomized design was used, with four replicates in a 5 × 2 factorial, with five photoperiods (6L:18D, 12L:12D, 16L:8D, 20L:4D, 24L:0D) and two feeding frequencies (two or four times a day). Four hundred larvae with an initial length of 4.6 ± 0.09 mm were randomly distributed in 40, 1‐L containers at a density of 10 larvae/L. Feeding was performed with Artemia nauplii in the ratio of 150 Artemia nauplii/larva/feeding. After 15 days, all larvae were euthanized for measurements of length (mm), final weight (mg), survival rate (%) and larvae uniformity in weight an length (%). The data were subjected to analysis of variance and subsequently, in case of significance, a Tukey test was performed to compare means. There was no interaction between photoperiod and feeding frequency on studied variables (P > 0.05). Likewise, there was no influence of feed rate on the same variables (P > 0.05). There was only influence of photoperiod, where the larvae subjected to longest photoperiods (24L:0D and 20L:4D) showed improved weight and length (P < 0.05). Therefore, a photoperiod of 20L:4E, with feeding twice a day is recommended.