Inter‐individual variation in the dominant intestinal microbiota of reared Atlantic cod (Gadus morhua L.) larvae

The diversity of the microbiota in 15 Atlantic cod (Gadus morhua L.) larvae from three different hatcheries with variable food type and water treatment was characterized with culture dependent and independent approaches. The culture‐dependent approach was phenotypic characterization of bacterial isolates based on 16 tests, and the culture‐independent approach was terminal restriction fragment length polymorphism (T‐RFLP) of PCR amplified 16S rRNA. Both approaches revealed large inter‐individual differences in the microbiota of cod larvae. Data from real time PCR indicate substantially lower variation in bacterial number larva^?1 both within and between hatcheries than the data on colony forming units, resulting in an estimated four orders of magnitude variation in culturability between larvae. Considerably higher richness and diversity were observed with T‐RFLP than with phenotypic characterization, and no correlation between the two approaches was found for diversity indexes calculated at the individual level. Dissimilarity in the composition of the microbiota between individuals from the same rearing environment was comparable to the dissimilarities between hatcheries. The large inter‐individual variation is important to consider when designing experiments, as pooling of individuals will completely mask this variability. We conclude that colonization of individual cod larvae by bacteria seems to be a stochastic and not a deterministic process, which is not well understood.     

Microbial communities associated with early stages of intensively reared orange‐spotted grouper (Epinephelus coioides)

The gut microbiota plays key roles in the health and general welfare of fish larvae, the present study characterized the bacterial communities associated with grouper Epinephelus coioides larvae during a period of 22 days post hatch (DPH) in an intensive hatchery using both cultivation‐based and cultivation‐independent approaches. Both approaches confirmed that bacteria were present in the gut of larvae before and after the onset of exogenous feeding, and the number of cultiviable bacteria increased gradually from 2 DPH to 22 DPH. A more complex bacterial profile was present in larvae fed fertilizer oyster eggs for 4 days (8 DPH), probably as a result of the onset of exogenous feeding. Interestingly, similar internal microbiota were observed in larvae fed fertilized oyster eggs for 4 days (8 DPH) and rotifers for 2 weeks (22 DPH), although different microbial communities were present in the two feeds. This might suggest that the gut environment of E. coioides larvae selects for a common microbiota, which is more closely related with the rearing water than the two feeds. Therefore, bacterial community of the rearing water may play a critical role in the establishment of gut microbiota of fish larvae and more attention should be paid to its practical modulation by using probiotics. In addition, some potentially beneficial bacteria, such as Lactococcus spp., were the major components of the microbiota associated with fertilized oyster eggs, while these bacteria were not detected in larvae samples.     

Growth,survival rate and fatty acid composition of sterlet (Acipenser ruthenus) larvae fed fatty acid‐enriched Artemia nauplii

We evaluated the growth and survival rate of sterlet (Acipenser ruthenus) larvae fed Artemia nauplii enriched with Olioω3 or Red Pepper commercial emulsions (BernAqua NV, Belgium). Sterlet larvae, 0.022 ± 0.002 g body weight, were randomly assigned to one of three feeding regimes with two different feeding durations. After administering live feed for 7 or 14 days, larvae were weaned onto commercial food and reared to 36 days posthatching (28 days of feeding). There were no significant differences in body weight among groups at the end of the trial. A significantly higher survival rate (p < 0.05) was observed in larvae fed Artemia enriched with Red Pepper for 14 days compared to other feeding regimes. Based on the analysis of growth parameters, we can conclude that 7 days of live feeding to be sufficient for efficient rearing of sterlet larvae. And longer duration of live feeding with use of special enrichment can be recommended for a higher survival rate.     

Effect of florfenicol and oxytetracycline treatments on the intensive larval culture of the Chilean scallop Argopecten purpuratus (Lamarck, 1819)

The administration of antimicrobials to control bacterial pathologies in Chilean scallop hatcheries is a frequent practice, but their effects on these cultures remained unknown. This study was undertaken to obtain information on the effect of the administration of florfenicol and oxytetracycline on the growth, survival and bacterial content of scallop larvae under farming conditions. Florfenicol‐treated cultures exhibited high survival rates (44% after 17 days of culture), whereas cultures not treated or treated with oxytetracycline collapsed after 11 days of culture. Surprisingly, no significant differences in the heterotrophic (Tukey test; P = 0.226) and Vibrio (Tukey test; P = 0.666) concentrations between the oxytetracycline‐treated and untreated larval cultures were observed. Otherwise, florfenicol administered directly into rearing tanks produced significantly higher larval growth (Tukey test; P = 0.0001) and survival (Tukey test; P = 0.011) than bath treatment. When 2 and 4 mg L^?1 of florfenicol were compared, no significant differences in growth (t‐test; P = 0.4596) and survival (Tukey test; P = 0.057) were observed, suggesting that a concentration of 2 mg L^?1 is sufficient to ensure larval production. The present results demonstrate the efficacy of florfenicol‐based therapy to increase larval survival and growth at commercial scale and prompt the necessity to standardize its use in Chilean scallop hatcheries.     

Reduction of skeletal anomalies in meagre (Argyrosomus regius,Asso, 1801) through early introduction of inert diet

The consolidation of meagre (Argyrosomus regius) in aquaculture requires an understanding and optimization of larval rearing and nutritional conditions. The aim of this study was to analyse the effects of an early introduction of inert diets during larval rearing, on growth performance, digestive enzymes activity and development of skeletal anomalies. This study evaluated the effects of three different timings for the introduction of inert diet during larval rearing: a control group (CTRL) where inert diet was initiated at 14 days after hatching (DAH) and two treatment groups that had an earlier introduction of inert diet at 8 DAH (T1) and 11 DAH (T2). Meagre larvae exhibited similar pancreatic and intestinal enzymatic activities among the different dietary treatments. No differences in the overall prevalence of anomalies were observed between treatments at 25 or 50 DAH, however, a significant reduction was observed in all groups with the transition from larval to juvenile stage. The precocious introduction of inert diet shifted the distribution of vertebral anomalies to a more anterior vertebral column region. Altogether, this study shows that earlier introduction of inert diets in meagre hatcheries can be beneficial for meagre production in aquaculture.     

An investigation on the gamete quality of Black Sea trout (Salmo trutta labrax) broodstock fed with mealworm (Tenebrio molitor)

In this study, the effect of combined feeding Black Sea trout (Salmo trutta labrax) broodstock with commercial feed and mealworm larvae (Tenebrio molitor) for 50 days before the breeding period in order to evaluate the gamete quality. While the control group was fed with commercial feed, mealworm larvae were given as an additional protein source 2 days a week and 3 days a week to the experimental groups formed from female and male individuals. In addition to the growth parameters, the number of eggs (number/individual) and the egg diameter (mm), sperm volume (ml), density of spermatozoa (×10⁹ cell/ml), total motility (%), progressive motility (%) and average curvilinear velocity (VCL, µm/sec) values were determined. At the end of the study, the highest live weight gain was found as 46.2 g in the control group of male. The egg diameters were 4.3 ± 1.8 mm and 4.5 ± 1.4 mm in the worm treatment groups fed twice and three times with mealworm respectively. As a result, it was determined that feeding fish with mealworm larvae as an addition to the commercial diet in female individuals did not affect the amount of eggs (p > .05), but the egg diameters were significantly smaller in the control group than the mealworm groups (p < .05). In spermatological characteristics, only the amount of sperm was different between the groups (p < .05); all other parameters were found to be similar to each other with no statistical differences (p > .05).     

Microbial communities in various waters used for fish larval rearing

A major concern in larvae production is a mass mortality caused by fish diseases. In larvae production, pumped‐up natural seawater filtered through a sand filter system is used for fish rearing, and microalgae and rotifer cultures. Here, we investigated the community structures of eukaryotic microbes, as well as total bacteria and vibrios, in various processed ‘waters’ used in a larvae production site. We observed that ultraviolet irradiation of seawater was effective to reduce not only total bacteria and vibrios but also eukaryotic microbes. Moreover, the community structures of total bacteria and vibrios in rearing waters for fish larvae were different from those in rotifer cultures fed with Chlorella, but rather similar to those in natural seawater and microalgae cultures. These results suggest that the bacterial community in rearing waters may originate mainly from natural seawater and then be selected by microalgae in rearing water. Overall, this study provides useful information for avoiding the risk of fish disease outbreaks in a larvae production site.     

Use of Nannochloropsis sp. isolated from the East China Sea in larval rearing of sea cucumber (Apostichopus japonicus)

The supply of microalgae to hatcheries is a limiting factor for the mass larval production of sea cucumber (Apostichopus japonicus) in Fujian Province, China. In this study, Nannochloropsis sp. isolated from the East China Sea was tested as food for A. japonicus larvae. The first trial compared the effect of mono‐, bi‐ and trialgal diets comprising three microalgae (Chaetoceros muelleri, Dunaliella tertiolecta and Nannochloropsis sp.) on A. japonicus larval growth, survival, settlement and juvenile growth. The results showed that there were no significant differences in survival and settlement between larvae fed with Nannochloropsis sp. and other diets. All diet treatments yielded similar juvenile sea cucumber output. In the second trial, A. japonicus larvae were fed equally four times daily at three different rations (5000, 20 000 and 40 000 cells mL^?1 day^?1). Larvae fed 20 000 cells mL^?1 day^?1 were significantly larger than larvae in other groups and experienced the highest survival rate. In the third trial, A. japonicus larvae were fed 20 000 cells mL^?1 day^?1 in three different frequency (2, 3 and 4 meals day^?1). The greatest body length was observed in larvae that received 3 meals day^?1. Survival and settlement of larvae fed 2 meals day^?1 were significantly lower than other two groups. These results suggest that Nannochloropsis sp. can be used as a diet for the large‐scale production of A. japonicus seed, and larvae fed three times daily at a ration of 20 000 cell mL^?1 day^?1 are recommended for hatchery production of A. japonicus.     

Larval rearing of an Asian catfish Pangasius hypophthalmus (Siluroidei,Pangasiidae): Analysis of precocious mortality and proposition of appropriate treatments

Both in Indonesia and Vietnam, larval rearing of Pangasius hypophthalmus remains problematic due to the variable and generally low survival rates obtained. The first week represents the most critical period for these larvae and up to now, cannibalistic behaviour was considered as the main cause of mortality. In the present study, two experiments were carried out in order to better understand the evolution and causes of mortality of P. hypophthalmus larvae from hatching up to 8 d of age and to find measures to improve survival rates. The first experiment was designed to evaluate the importance of cannibalism and differences in mortality when larvae from two different females were reared either in groups of 30 individuals or in isolated condition (30 larvae reared separately). In both cases, the culture was carried out either with or without antibiotic. The aim of the second experiment was to test oxytetracycline and different disinfectants (chloramine-T, formalin and ‘formalin + malachite green oxalate’) at different dosages in order to prevent bacterial outbreaks in the culture. The results indicated that the survival rates of P. hypophthalmus larvae was dependent on the initial quality of larvae or eggs and that larval mortality was more a consequence of pathogenic infection than a direct effect of cannibalism. The present study demonstrated that the survival rates of larvae were systematically improved when rearing was carried out in water containing antibiotic (oxytetracycline at a dose of 5 to 20 mg·L¹). Survival rate and final mean body weight of larvae as high as those obtained using antibiotic were also reached with applications of disinfectants such as chloramine-T and formalin. The use of these disinfectants is recommended for commercial P. hypophthalmus hatcheries.     

Larval rearing of African carp,Labeo parvus Boulenger, 1902 (Pisces: Cyprinidae), using live food and artificial diet under controlled conditions

This study aimed at (1) evaluating the efficacy of live food organisms (Artemia and natural zooplankton) and an artificial diet in the larval rearing of African carp Labeo parvus and (2) determining appropriate rearing conditions. After yolk sac resorption, the larvae were separated into five different feeding trials with two replicates. At the end of the larval rearing period (28 days post‐hatching), the highest (94.6%) and the lowest (53.7%) cumulative survival rates were found when larvae were fed with natural zooplankton for 7 days followed by Nippai food for 21 days, and when larvae were fed from the beginning of exogenous feeding with Nippai food only respectively. The significant highest body weight (351.6 mg), total length (34.4 mm) and specific growth rates (15.5%day^?1) were recorded when the larvae were fed with Artemia nauplii for 14 days followed by Nippai food for 14 days. The lowest growth performance (body weight and specific growth rates) were obtained when larvae were fed exclusively Nippai food. These results indicate that L. parvus can be successfully cultured in indoor nursery systems from hatching to the early juvenile stage.     

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.     

The effect of insect meal as a feed ingredient on survival,growth, and metabolic and antioxidant response of juvenile prawn Palaemon adspersus (Rathke, 1837)

For successful prawn aquaculture, feeds should be based on readily consumed ingredients that promote survival and optimal growth performance. This pilot study investigates the rearing of juvenile Baltic prawn Palaemon adspersus. Two feeding trials were carried out for 60 days; both incorporated insect meals, the first one in fishmeal‐based diets, whereas the second one in plant meal ones. Insect meals derived from larvae of Tenebrio molitor (TM), Hermetia illucens (HI) and Musca domestica (MD) were tested as feed ingredients. This study indicated that the inclusion of HI in fishmeal diets resulted in significantly higher growth performance and survival of the prawns, whereas the MD diet led to similarly high growth performance reducing significantly their survival. Growth performance was not affected by the insect inclusion in the plant‐based diets, but survival was higher in the TM and HI inclusion diets. The inclusion of TM and HI resulted in higher protein and energy content of the prawns’ muscle when incorporated in fishmeal and plant meal diets respectively. No significant differences were observed in the activities of hepatopancreas’ amino acid‐catabolizing enzymes. Concluding, the combinations fishmeal–HI and plant meal–TM can be used for the successful rearing of Baltic prawn.     

Effect of temperature on survival,growth and development of Mytilus galloprovincialis larvae

Mussel aquaculture is widely prevalent worldwide, but generally relies on natural seed collection, which does not always meet the needs of the producers. Thus, development of mussel hatcheries is of economic interest in some parts of the world, such as Europe; it provides opportunities not only on annual reliability of seed but also on genetic improvements. To broaden knowledge on mussel larval physiology, we carried out temperature treatments (17, 20 and 24 °C) on Mytilus galloprovincialis larvae under laboratory conditions. The trials ended when 30% of the larval population was in the post‐larval stage. The temperature coefficient Q₁₀ indicated a strong relationship between temperature and increase in growth from 17 to 20 °C, but not between 20 and 24 °C. Exposure of M. galloprovincialis larvae to 17 °C resulted in poor growth, low survival and a delayed development and was considered to be inadequate for M. galloprovincialis larval culture. Rearing the larvae at 20 or 24 °C produced better growth, higher survival rates and faster metamorphosis as compared with 17 °C. The temperature region within 20 and 24 °C was suggested as adequate for the mussel M. galloprovincialis larval culture, and implications of these results on the development of commercial hatcheries were discussed.     

Larval growth of turbot, Scophthalmus maximus (L.) produced with fresh and cryopreserved sperm

The present paper assesses the fertilization and hatching rates, as well as the growth, of larvae obtained from four artificial fertilizations (AF) using fresh and cryopreserved sperm of the turbot Scophthalmus maximus (L.). Larvae growth in both sperm groups, measured in terms of length and weight at culture days 0, 7, 14 and 31, are compared, as well as their growth rates. The two groups' fertilization and hatching rates were not significantly different. Likewise, no significant differences in length and wet weight of 7‐ and 14‐day‐old larvae were found using fresh and cryopreserved sperm; however, significant differences were found in 31‐day‐old larvae, which were more attributable to the variability inherent in larval turbot culture, and to variability in the reproductive specimens used in our study, than to the type of sperm employed. These results indicate that the type of sperm used in artificial fertilization, i.e. fresh or cryopreserved, is not a determining factor, either for fertilization and hatching, or for subsequent larval development. Our results also confirm once again the high quality of cryopreserved turbot sperm, and its usefulness in commercial hatcheries.     

The Effects of light intensity on growth and survival of cuttlefish (sepia officinalis) hatchlings and Juveniles

The effects of three light intensities (100, 350 and 1200 lux) on cuttlefish hatchling rearing performance was studied in black tanks. A total of 270 cuttlefish with a mean wet weight (MWW) of 0.089 ± 0.012 g were used in the experiment, which was undertaken during the first 50 days after hatching (hatchling stage plus the transition to the juvenile stage). According to results of the present study, light intensity is an important factor for growth and survival consistency in cuttlefish rearing. All three light intensity groups displayed exponential growth. The effect of days, light intensity and their interaction only displayed differences (P < 0.05) between light groups in terms of mean wet weight. The 100 lux light intensity promoted the best absolute values of total biomass and total mortality. We believe that the higher mortality observed in 1200 lux reared cuttlefish during the first 10 days after hatching (DAH) was due to light intensity and individual adaptation to light conditions. Therefore, the 100 lux light intensity, obtained with daylight spectrum bulbs, is recommended for cuttlefish rearing during the first 50 DAH. This light setup promotes higher growth and survival rates and lower energetic costs, which are key aspects to consider in a cuttlefish hatchery.     

Combined effect of temperature and live feed period on growth and survival of Coregonus maraena (Bloch, 1779) larvae

Despite intensive stocking programmes, wild Maraena whitefish Coregonus maraena (Bloch, 1779)‐stocks are in danger of extinction in the Baltic sea region. Current aquaculture rearing methods in recirculating aquaculture systems (RAS) are lacking efficient protocols for transitioning larvae from endogenous to exogenous feeding. In this 34‐day‐experiment the offspring of wild fish was used. Maraena whitefish larvae were weaned at three different temperatures (15.9°C, 17.9°C, 19.7°C) and three feeding regimes, resulting in nine treatments. The first group received pure live feed (freshly hatched Artemia sp. nauplii) for 10 days and a 1:1‐mixture of live and dry feed for 3 days. The second group received the live and dry feed mixture for 13 days and the third group directly received a commercial dry feed. All nine treatments were stocked in triplicate with 500 larvae per tank (19 ind. L^?1). Feeding started 4 days post hatch. Survival was highest in the pure dry feed groups and lowest in the pure live feed groups. In contrast to growth, which was highest in the live feed groups and lowest in the dry feed group. Higher temperature increased growth in the live feed groups but had no effect on survival in all groups. These results will enhance the weaning of C. maraena in recirculation aquaculture.     

Flavobacteria colonizing the early life stages of hatchery‐incubated Chinook salmon Oncorhynchus tshawytscha (Walbaum 1792) are markedly diverse

Flavobacterial diseases are significant impediments to hatchery‐based fishery conservation and aquaculture productivity worldwide. Recent studies revealed a multitude of novel flavobacteria within the reproductive fluids and unfertilized eggs of feral Chinook salmon Oncorhynchus tshawytscha broodstock, some of which were associated with systemic disease. Herein, embryonated eggs/fry from these broodstock were assayed for flavobacteria while in incubator stacks in three hatcheries over 2 years, as was the water entering hatchery incubators. Overall, >65% of sampled eggs and 38% of fry were colonized by flavobacteria. One hundred and ninety‐one egg and fry‐associated flavobacterial isolates were characterized phenotypically and via 16S rRNA gene sequencing and phylogenetic analyses, revealing that the majority fell into 22 clades (i.e., 15 Flavobacterium spp. groups and seven Chryseobacterium spp. groups) that varied in presence by facility. Although some matched previously described fish‐pathogenic species, the majority were distinct from all described flavobacteria and likely represent novel species. Of concern, iodophor disinfection at the commonly utilized dose/duration for egg‐surface disinfection did not eliminate flavobacteria. Results also implicated maternal routes of infection and source water for some flavobacteria. In total, study findings underscore the complexity of flavobacterial ecology within hatchery environments and highlight the need for improved hatchery biosecurity practices.     

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.     

Mortality event involving larvae of the carpet shell clam Ruditapes decussatus in a hatchery: isolation of the pathogen Vibrio tubiashii subsp. europaeus

Diseases caused by bacteria belonging to the genus Vibrio are a common, as yet unresolved, cause of mortality in shellfish hatcheries. In this study, we report the results of routine microbiological monitoring of larval cultures of the carpet shell clam Ruditapes decussatus in a hatchery in Galicia (NW Spain). Previous episodes of mortality with signs similar to those of vibriosis affecting other species in the installation indicated the possibility of bacterial infection and led to division of the culture at the early D‐veliger larval stage. One batch was cultured under routine conditions, and the other was experimentally treated with antibiotic (chloramphenicol). Differences in larval survival were assessed, and culturable bacterial population in clams and sea water was evaluated, with particular attention given to vibrios. Severe mortalities were recorded from the first stages of culture onwards. The pathogen Vibrio tubiashii subsp. europaeus was detected in both batches, mainly associated with larvae. Moreover, initial detection of the pathogen in the eggs suggested the vertical transmission from broodstock as a possible source. Experimental use of antibiotic reduced the presence and diversity of vibrios in sea water, but proved inefficient in controlling vibrios associated with larvae from early stages and it did not stop mortalities.     

Spawning success and early life history of longnose suckers in Great Lakes tributaries

Fish eggs and larvae are often subject to very high mortality, and variation in early life survival can be important for population dynamics. Although longnose suckers (Catostomus catostomus) are widespread in northern North America, little is known about their early life history. We examined fecundity and early larval survivorship during sucker spawning events in three small Lake Michigan tributaries. Although egg deposition varied 25% among spawning events, estimated larval export to the lake varied over 25,000‐fold from around 1000 to 26 million. Based on variation in environmental conditions across years, it appears that spring flow and temperature may be important determinants of egg survival to larval outmigration. Larval age data suggest that most individuals that survived to outmigration hatched during a 2‐day period despite adult spawning across at least 10 days. Most larvae spent <2 weeks in the stream and emigrated around the time of transition from endogenous to exogenous feeding before substantial growth occurred. In two of three cases, larvae drifted exclusively at night; however, high drift rates occurred during both day and night in the case where larvae were very abundant, suggesting density‐dependent drift behaviour. Our results indicate that survival in tributary streams from egg deposition to larval export is highly variable in longnose suckers. These large differences in early life survival may translate into variability in recruitment, thereby influencing population structure and dynamics.