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Using a circular economy concept, the present study investigated the use of crude glycerol, a primary by-product of biodiesel production, as a low-priced nutrient source for heterotrophic cultivation of the fungus-like protist Schizochytrium limacinum SR21 strain. The whole biomass of this oleaginous microorganism, rich in docosahexaenoic acid (DHA) and high-quality proteins, was then paired with a vegetable oil (VO) source and used to replace fish oil (FO) in European sea bass (Dicentrarchus labrax) feeds. Four nutritionally balanced diets were formulated: diet FO (a FO-based diet), diet VO + 0 (a VO-based diet without S. limacinum), and diets VO + 5 and VO + 10 that were VO-based feeds supplemented with 5 and 10% of S. limacinum, respectively. After a 3-month feeding trial, fish of all dietary groups tripled their initial weight, but growth and feeding efficiencies of D. labrax were not significantly different among treatments. Although the formulated diets were balanced for polyunsaturated fatty acids (PUFAs), fish fed with feeds containing either VO or VO plus 5 and 10% of S. limacinum biomass had significantly higher levels of PUFAs in the flesh than fish fed the FO-based diet. Values of health-related lipid indexes, such as atherogenicity index, thrombogenicity index, and flesh lipid quality as well as n-6/n-3 and PUFAs/SFAs ratios confirmed the high nutritional value of sea bass filet, thus representing a healthy product for human consumption. Although the PUFAs/SFAs ratio showed a significantly higher value in fish fed with VO-based diets supplemented with S. limacinum than in those fed with FO diet, suggesting a better filet quality, the n-6/n-3 ratio clearly indicated that filet quality of dietary group FO was best (value of 0.55) and that of group VO + 10 second best (value of 0.98). We also evaluated the nutritional regulation of Δ6-desaturase (or fads2) gene expression in European sea bass liver. European sea bass fed the VO + 0 diet had the highest number of mRNA copies for Δ6-desaturase (or fads2), fish fed with diet VO + 10 the lowest. Our study adds to the growing body of literature concerning the use of thraustochytrid biomass as a valid alternative to marine-derived raw materials for European sea bass feeds.

© 2021 Moroni, Naya-Català, Piazzon, Rimoldi, Calduch-Giner, Giardini, Martínez, Brambilla, Pérez-Sánchez and Terova., The present research tested the effects of dietary nisin-producing Lactococcus lactis on growth performance, feed utilization, intestinal morphology, transcriptional response, and microbiota in gilthead sea bream (Sparus aurata). A feeding trial was conducted with fish weighting 70–90 g. Fish were tagged with passive, integrated transponders and distributed in nine 500 L tanks with 40 fish each. Fish were fed for 12 weeks with either a control (diet A) or experimental diets (diets B and C) in triplicate (3 tanks/diet). Extruded pellets of diets B and C were supplemented with a low (2 × 109 CFU/kg) and a high (5 × 109 CFU/kg) dose of probiotic, respectively. No significant differences were found between groups for the feed conversion ratio or specific growth rates. However, the final body weight of fish fed diet C was significantly higher than the control group with intermediate values for fish fed diet B. Histological analysis conducted using a semi-quantitative scoring system showed that probiotic did not alter the morphology of the intestine and did not trigger inflammation. With regard to the transcriptomic response, a customized PCR array layout was designed to simultaneously profile a panel of 44 selected genes. Significant differences in the expression of key genes involved in innate and acquired immunity were detected between fish fed probiotic and control diets. To analyze the microbiota associated to the feeds and the gut autochthonous microbial communities, we used the Illumina MiSeq platform for sequencing the 16S rRNA gene and a metagenomics pipeline based on VSEARCH and RDP databases. The analysis of gut microbiota revealed a lack of colonization of the probiotic in the host’s intestinal mucosa. However, probiotic did modulate the fish gut microbiota, confirming that colonization is not always necessary to induce host modification. In fact, diets B and C were enriched with Actinomycetales, as compared to diet A, which instead showed a higher percentage of Pseudomonas, Sphyngomonas, and Lactobacillus genera. These results were confirmed by the clear separation of gut bacterial community of fish fed with the probiotic from the bacterial community of control fish group in the beta-diversity and PLS-DA (supervised partial least-squares discriminant analysis) analyses., This work was supported by the EU Horizon 2020 Research Innovation Program under the TNA program (project AE130011) at IATS-CSIC research infrastructure within AQUAEXCEL2020 project (652831). This work was also co-funded by the EU Horizon 2020 AquaIMPACT project (Genomic and nutritional innovations for genetically superior farmed fish to improve efficiency in European aquaculture; 818367). This output reflects only the author’s view and the European Union cannot be held responsible for any use that may be made of the information contained herein. MP was funded by a Ram n y Cajal Postdoctoral Research Fellowship [RYC2018-024049-I/AEI/10.13039/501100011033 co-funded by the European Social Fund (ESF) & ACOND/2020 Generalitat Valenciana].

Background Aquaculture must continue to reduce dependence on fishmeal (FM) and fishoil in feeds to ensure sustainable sector growth. Therefore, the use of novel aquaculture feed ingredients is growing. In this regard, insects can represent a new world of sustainable and protein-rich ingredients for farmed fish feeds. Accordingly, we investigated the effects of full replacement of FM with Tenebrio molitor (TM) larvae meal in the diet of rainbow trout (Oncorhynchus mykiss) on fish gut and skin microbiota. Methods A feeding trial was conducted with 126 trout of about 80 g mean initial weight that were fed for 22 weeks with two isonitrogenous, isolipidic, and isoenergetic extruded experimental diets. Partially defatted TM meal was included in one of the diets to replace 100% (TM 100) of FM, whereas the other diet (TM 0) was without TM. To analyse the microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and Qiime pipeline were used to identify bacteria in the gut and skin mucosa, and in the diets. Results The data showed no major effects of full FM substitution with TM meal on bacterial species richness and diversity in both, gut mucosa- and skin mucus-associated microbiome. Skin microbiome was dominated by phylum Proteobacteria and especially by Gammaproteobacteria class that constituted approximately half of the bacterial taxa found. The two dietary fish groups did not display distinctive features, except for a decrease in the relative abundance of Deefgea genus (family Neisseriaceae) in trout fed with insect meal. The metagenomic analysis of the gut mucosa indicated that Tenericutes was the most abundant phylum, regardless of the diet. Specifically, within this phylum, the Mollicutes, mainly represented by Mycoplasmataceae family, were the dominant class. However, we observed only a weak dietary modulation of intestinal bacterial communities. The only changes due to full FM replacement with TM meal were a decreased number of Proteobacteria and a reduced number of taxa assigned to Ruminococcaceae and Neisseriaceae families. Conclusions The data demonstrated that TM larvae meal is a valid alternative animal protein to replace FM in the aquafeeds. Only slight gut and skin microbiota changes occurred in rainbow trout after total FM replacement with insect meal. The mapping of the trout skin microbiota represents a novel contribution of the present study. Indeed, in contrast to the increasing knowledge on gut microbiota, the skin microbiota of major farmed fish species remains largely unmapped but it deserves thorough consideration.

Alternative nutrient sources to fishmeal for fish feed, such as insect meals, represent a promising sustainable supply. However, the consequences for fish digestive function have not been exhaustively investigated. In the present study we evaluated the effect of partial fishmeal substitution with 10% Hermetia illucens (Hi10) larvae meal on the neuromuscular function of proximal and distal intestine in gilthead sea bream. In animals fed with insect meal, weight and growth parameters were similar to controls fed with conventional fishmeal. In addition, no anomalies in intestinal gross morphology and no overt signs of inflammation were observed. The gastrointestinal transit was significantly reduced in Hi10 fed animals. In the proximal and distal intestine longitudinal muscle, Hi10 feeding downregulated the excitatory cholinergic and serotoninergic transmission. Sodium nitroprusside-induced inhibitory relaxations increased in the proximal intestine and decreased in the distal intestine after Hi10 meal. Changes in the excitatory and inhibitory components of peristalsis were associated with adaptive changes in the chemical coding of both proximal and distal intestine myenteric plexus. However, these neuromuscular function alterations were not associated with considerable variations in morphometric growth parameters, suggesting that 10% Hi meal may represent a tolerable alternative protein source for gilthead sea bream diets.

The diffusion of novel SARS-CoV-2 coronavirus over the world generated COVID-19 pandemic event as reported by World Health Organization on March 2020. The huge issue is the high infectivity and the absence of vaccine and customised drugs allowing for hard management of this outbreak, thus a rapid and on site analysis is a need to contain the spread of COVID-19. Herein, we developed an electrochemical immunoassay for rapid and smart detection of SARS-CoV-2 coronavirus in saliva. The electrochemical assay was conceived for Spike (S) protein or Nucleocapsid (N) protein detection using magnetic beads as support of immunological chain and secondary antibody with alkaline phosphatase as immunological label. The enzymatic by-product 1-naphtol was detected using screen-printed electrodes modified with carbon black nanomaterial. The analytical features of the electrochemical immunoassay were evaluated using the standard solution of S and N protein in buffer solution and untreated saliva with a detection limit equal to 19 ng/mL and 8 ng/mL in untreated saliva, respectively for S and N protein. Its effectiveness was assessed using cultured virus in biosafety level 3 and in saliva clinical samples comparing the data using the nasopharyngeal swab specimens tested with Real-Time PCR. The agreement of the data, the low detection limit achieved, the rapid analysis (30 min), the miniaturization, and portability of the instrument combined with the easiness to use and no-invasive sampling, confer to this analytical tool high potentiality for market entry as the first highly sensitive electrochemical immunoassay for SARS-CoV-2 detection in untreated saliva., Highlights • Carbon black-modified screen-printed for smart immunosensor able to detect SARS-CoV-2. • Magnetic beads as support for rapid and reliable SARS-CoV-2 detection in untreated saliva. • Saliva clinical samples and cultured SARS-CoV-2 virus analysis. • Selectivity when tested with influenza pandemic/seasonal H1N1 viruses.

Background In the last two decades, research has focused on testing cheaper and sustainable alternatives to fish oil (FO), such as vegetable oils (VO), in aquafeeds. However, FO cannot be entirely replaced by VOs due to their lack of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids. The oilseed plant, Camelina sativa, may have a higher potential to replace FO since it can contains up to 40% of the omega-3 precursors α-linolenic acid (ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6). Methods A 90-day feeding trial was conducted with 600 gilthead sea bream (Sparus aurata) of 32.92 ± 0.31 g mean initial weight fed three diets that replaced 20%, 40% and 60% of FO with CO and a control diet of FO. Fish were distributed into triplicate tanks per diet and with 50 fish each in a flow-through open marine system. Growth performance and fatty acid profiles of the fillet were analysed. The Illumina MiSeq platform for sequencing of 16S rRNA gene and Mothur pipeline were used to identify bacteria in the faeces, gut mucosa and diets in addition to metagenomic analysis by PICRUSt. Results and Conclusions The feed conversion rate and specific growth rate were not affected by diet, although final weight was significantly lower for fish fed the 60% CO diet. Reduced final weight was attributed to lower levels of EPA and DHA in the CO ingredient. The lipid profile of fillets were similar between the dietary groups in regards to total saturated, monounsaturated, PUFA (n-3 and n-6), and the ratio of n-3/n-6. Levels of EPA and DHA in the fillet reflected the progressive replacement of FO by CO in the diet and the EPA was significantly lower in fish fed the 60% CO diet, while ALA was increased. Alpha and beta-diversities of gut bacteria in both the faeces and mucosa were not affected by any dietary treatment, although a few indicator bacteria, such as Corynebacterium and Rhodospirillales, were associated with the 60% CO diet. However, lower abundance of lactic acid bacteria, specifically Lactobacillus, in the gut of fish fed the 60% CO diet may indicate a potential negative effect on gut microbiota. PICRUSt analysis revealed similar predictive functions of bacteria in the faeces and mucosa, although a higher abundance of Corynebacterium in the mucosa of fish fed 60% CO diet increased the KEGG pathway of fatty acid synthesis and may act to compensate for the lack of fatty acids in the diet. In summary, this study demonstrated that up to 40% of FO can be replaced with CO without negative effects on growth performance, fillet composition and gut microbiota of gilthead sea bream.

This study evaluated the effects of partial substitution of dietary fishmeal (FM) with either fish protein hydrolysate (FPH) or autolysed dried yeast (HiCell®, Biorigin, Brazil) on intestinal microbiota of gilthead sea bream (Sparus aurata). A total number of 720 fish of 122.18 ± 6.22 g were fed for 92 days with three different diets in triplicate (3 tanks/diet). A diet based on FM/vegetable meal was used as control. The other two diets were formulated by replacing FM with 5% of either FPH or HiCell®. To analyze the gut microbiota associated to autochthonous and allochthonous microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME pipeline were used.A total number of 102 OTUs (operational taxonomic units) at 97% identity were identified in fish gut samples collected at the end of feeding trial. Fourteen OTUs constituted the core gut microbiota, i.e. those OTUs found in at least nine out of fifteen samples per group and shared regardless of the diet. Eight OTUs were assigned to Firmicutes represented by Lactobacillus, Staphylococcus, and Bacillus genera, and six to Proteobacteria phylum. Dietary dried yeast autolysate modulated the intestinal microbiota by promoting the growth of some beneficial bacteria. At order level, fish fed yeast showed an enrichment in Bacillales and Clostridiales as compared to the control group, whereas fish fed FPH showed a significantly lower amount of bacteria belonging to Alteromonadales and Enterobacteriales than the other two feeding groups. Although we did not observe any effect of 5% FM replacement with alternative nitrogen sources at phylum level, at lower taxonomical levels, the composition of gut microbiota, in terms of relative abundance of specific taxa, was significantly influenced by the dietary treatment.The metabarcoding analysis revealed a clearly intestinal microbiota modulation in response to dietary autolyzed yeast. The abundance of some beneficial bacteria, i.e. indigestible carbohydrate degrading- and SCFA producing bacteria, was positively affected. Autolysed dried yeast obtained by the fermentation of a strain of Saccharomyces cerevisiae could be a valid alternative protein source to FM as well as a valid functional ingredient for aquafeed production [corrected].

There is an increasing interest from the aquafeed industry in functional feeds containing selected additives that improve fish growth performance and health status. Functional feed additives include probiotics, prebiotics, organic acids, and phytogenics (substances derived from plants and their extracts). This study evaluated the effects of dietary inclusion of a mucilage extract rich in galactomannan oligosaccharides (GMOS), a mixture of garlic and labiatae-plants oils (PHYTO), and a combination of them (GMOSPHYTO), on gut microbiota composition of European sea bass (Dicentrarchus labrax) fed with a low fishmeal (FM) and fish oil (FO) diet. Three experimental diets and a control diet (plant-based formulation with 10% FM and 6% FO) were tested in a 63-days feeding trial. To analyze the microbiota associated to feeds and the intestinal autochthonous (mucosa-adhered) and allochthonous (transient) microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME2 pipeline were used. Metabarcoding analysis of feed-associated bacteria showed that the microbial communities of control (CTRL) feed deeply differed from those of experimental diets. The number of reads was significantly lower in CTRL feed than in other feeds. The OTU (operational taxonomic unit) number was instead similar between the feeds, ranging from 42 to 50 OTUs. The variation of resident gut microbiota induced by diet was lower than the variation of transient intestinal microbiota, because feedstuffs are a major source of allochthonous bacteria, which can temporarily integrate into the gut transient microbiome. However, the composition of transient bacterial communities was not simply a mirror of feed-borne bacteria. Indeed, the microbial profile of feeds was different from both faecal and mucosa profiles. Our findings suggest that the dietary inclusion of GMOS (0.5%) and PHYTO (0.02%) in a low FM and FO diet induces changes in gut microbiota composition of European sea bass. However, if on allochthonous microbiota the combined inclusion of GMOS and PHYTO showed an antagonistic effect on bactericidal activity against Vibrionales, at mucosa level, only GMOSPHYTO diet increased the relative abundance of Bacteroidales, Lactobacillales, and Clostridiales resident bacterial orders. The main beneficial effects of GMOS and PHYTO on gut microbiota are the reduction of coliforms and Vibrionales bacteria, which include several potentially pathogenic species for fish, and the enrichment of gut microbiota composition with butyrate producer taxa. Therefore, these functional ingredients have a great potential to be used as health-promoting agents in the farming of European sea bass and other marine fish.

Background This study evaluated the effects of partial substitution of dietary fishmeal (FM) with either fish protein hydrolysate (FPH) or autolysed dried yeast (HiCell®, Biorigin, Brazil) on intestinal microbiota of gilthead sea bream (Sparus aurata). A total number of 720 fish of 122.18 ± 6.22 g were fed for 92 days with three different diets in triplicate (3 tanks/diet). A diet based on FM/vegetable meal was used as control. The other two diets were formulated by replacing FM with 5% of either FPH or HiCell®. To analyze the gut microbiota associated to autochthonous and allochthonous microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME pipeline were used. Results A total number of 102 OTUs (operational taxonomic units) at 97% identity were identified in fish gut samples collected at the end of feeding trial. Fourteen OTUs constituted the core gut microbiota, i.e. those OTUs found in at least nine out of fifteen samples per group and shared regardless of the diet. Eight OTUs were assigned to Firmicutes represented by Lactobacillus, Staphylococcus, and Bacillus genera, and six to Proteobacteria phylum. Dietary dried yeast autolysate modulated the intestinal microbiota by promoting the growth of some beneficial bacteria. At order level, fish fed yeast showed an enrichment in Bacillales and Clostridiales as compared to the control group, whereas fish fed FPH showed a significantly lower amount of bacteria belonging to Alteromonadales and Enterobacteriales than the other two feeding groups. Although we did not observe any effect of 5% FM replacement with alternative nitrogen sources at phylum level, at lower taxonomical levels, the composition of gut microbiota, in terms of relative abundance of specific taxa, was significantly influenced by the dietary treatment. Conclusions The metabarcoding analysis revealed a clearly intestinal microbiota modulation in response to dietary autolyzed yeast. The abundance of some beneficial bacteria, i.e. indigestible carbohydrate degrading- and SCFA producing bacteria, was positively affected. Brewer’s yeast autolysate could be a valid alternative protein source to FM as well as a valid functional ingredient for aquafeed production.

Nutritional Programming (NP) has been shown to counteract the negative effects of dietary plant protein (PP) by introducing PP at an early age towards enhancement of PP utilization during later life stages. This study explored the effect of NP and its induction time on growth, expression of appetite-stimulating hormones, and any morphological changes in the gut possibly responsible for improved dietary PP utilization. At 3 days post-hatch (dph) zebrafish were distributed into 12 (3 L) tanks, 100 larvae per tank. This study included four groups: 1) The control (NP-FM) group received fishmeal (FM)-based diet from 13-36 dph and was challenged with PP-based diet during 36-66 dph; 2) The NP-PP group received NP with dietary PP in larval stage via live food enrichment during 3-13 dph followed by FM diet during 13-36 dph and PP diet during 36-66 dph; 3) The T-NP group received NP between 13-23 dph through PP diet followed by FM diet during 23-36 dph and PP diet during 36-66 dph; and 4) The PP group received PP diet from 13-66 dph. During the PP challenge the T-NP group achieved the highest weight gain compared to control and PP. Ghrelin expression in the brain was higher in T-NP compared to NP-FM and NP-PP, while in the gut it was reduced in both NP-PP and T-NP groups. Cholecystokinin expression showed an opposite trend to ghrelin. The brain neuropeptide Y expression was lower in NP-PP compared to PP but not different with NP-FM and T-NP groups. The highest villus length to width ratio in the middle intestine was found in T-NP compared to all other groups. The study suggests that NP induced during juveniles stages improves zebrafish growth and affects digestive hormone regulation and morphology of the intestinal lining – possible mechanisms behind the improved PP utilization in pre-adult zebrafish stages.

The expected future demand for highly nutrient animal food products will push the animal production system to search for new sources of high-quality protein feedstuffs. In this scenario, economic and environmental issues will have to be considered while reducing the competition with the plant-based human food chains. Legume grains and some oilseed cakes, by-products from the oil industry, are the main protein sources for ruminants and terrestrial monogastrics such as pigs and poultry. Their relevant role will hold in the next decades, but it is necessary to increase the diversification of sources that can be grown profitably throughout the world, including European countries. Microalgae are a promising source of protein and other nutrients for animal feeding. However, an amazing richness of biologically active substances makes these organisms very interesting as feed ingredients, as their role go far beyond the supply of nutrients. Due to the limited usage of microalgae as human foodstuffs or food ingredients, low competition between microalgae-based feed and food chains is predictable. This review aims to synthesise current knowledge on minor pulses and other protein-rich plant products and microalgae, as alternative ingredients to the conventional animal protein sources, focussing on their production, availability, and nutritional values. Points of strength, weakness, opportunity and threat related to the use of these protein sources in animal feeding are separately analysed through a SWOT approach to underlie future needs in terms of research and/or technological development that could help valorise these nutrient sources as feed ingredients.

The effects on the benthic ecosystem deriving from the activities of two land-based fish farms located in Italian coastal transitional ecosystems (CTEs) were investigated. Together with chemical and physical analysis, some biological analysis was performed. For each fish farm, three stations were sampled: two stations located inside the farm (ponds exit and farm exit) and one station located outside the fish farm, considered as reference site. The AZTI’s Marine Biotic Index (AMBI) and the BENTIX index were calculated, which are being used in assessing the ecological status of benthic communities within the European Water Framework Directive (WFD). Results were compared in order to evaluate the more suitable index for this study area. Both the indices gave similar results but AMBI resulted more sensitive than BENTIX, probably due to the small size of the collected samples. This study seems to suggest that AMBI, being not dependent from sample size, could be preferred as descriptor of benthic health status in CTEs.

Taurine (Tau) is an amino sulfonic acid, which is widely distributed in animal tissues, whereas it is almost lacking in plants with the exception of certain algae, seaweeds, and few others. In the aquafeed industry, Tau is mainly used as a feed additive to promote growth in marine fish species with limited cysteine sulfinate decarboxylase activity. In particular, Tau supplementation is required in feeds in which fishmeal (FM) is substituted with high percentages of plant-derived protein sources such as soybean meals (SBM) that have much lower levels of Tau than FM. In addition to being a growth promoter, Tau exert powerful antioxidant properties being a scavenger of the reactive oxygen species (ROS). Under sustained swimming conditions, an intracellular increase in ROS production can occur in fish red muscle where the abundance of mitochondria (the main site of ROS formation) is high. Accordingly, this study aimed at investigating the effects of dietary Tau on European seabass (Dicentrarchus labrax) growth and oxidative stress response induced by swimming exercise. Individually tagged fish of 92.57 &plusmn, 20.33 g mean initial weight were fed two experimental diets containing the same low percentage of FM and high percentage of SBM. One diet was supplemented with 1.5% of Tau. Tau supplemented in the diet had a positive effect on fish growth, and enhanced swimming performance and antioxidant status. Two swim endurance tests were performed during the feeding trial. Metabolic oxygen consumption (MO2) was measured during exercise at incremental swimming speeds (0.7, 1.4, 2.1, 2.8, 3.5, and then 4.2 BL (body length) s&minus, 1, until fatigue). Fish maximal sustainable swimming speed (Ucrit) was determined too. To investigate the antioxidant effect of dietary Tau, we also measured ROS production in fish blood by RBA (respiratory burst activity) assay and quantified the expression of genes coding for antioxidant enzymes by qPCR (quantitative polymerase chain reaction) , such as SOD (superoxide dismutase), GPX (glutathione peroxidase), and CAT (catalase) in red muscle and liver. There was a significant effect of Tau upon Ucrit during exercise. Additionally, ROS production was significantly lower in fish fed with Tau supplemented diet, supporting the role of Tau as ROS scavenger. The protective effect of Tau against oxidative stress induced by forced swimming was denoted also by a significant decrease in antioxidant enzymes gene expression in fish liver and muscle. Taken together these results demonstrate that Tau is beneficial in low FM-based diets for seabass.

The worldwide growing consumption of proteins to feed humans and animals has drawn a considerable amount of attention to insect rearing. Insects reared on organic wastes and used as feed for monogastric animals can reduce the environmental impact and increase the sustainability of meat/fish production. In this study, we designed an environmentally closed loop for food supply in which fruit and vegetable waste from markets became rearing substrate for Hermetia illucens (BSF&mdash, black soldier fly). A vegetable and fruit-based substrate was compared to a standard diet for Diptera in terms of larval growth, waste reduction index, and overall substrate degradation. Morphological analysis of insect organs was carried out to obtain indications about insect health. Processing steps such as drying and oil extraction from BSF were investigated. Nutritional and microbiological analyses confirmed the good quality of insects and meal. The meal was then used to produce fish feed and its suitability to this purpose was assessed using trout. Earthworms were grown on leftovers of BSF rearing in comparison to a standard substrate. Chemical analyses of vermicompost were performed. The present research demonstrates that insects can be used to reduce organic waste, increasing at the same time the sustainability of aquaculture and creating interesting by-products through the linked bio-system establishment.

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

European sea bass were fed four low FM/FO (10%/6%) diets containing galactomannan oligosaccharides (GMOS), a mixture of garlic oil and labiatae plants oils (PHYTO), or a combination of both functional products (GMOSPHYTO) for 63 days before exposing the fish to an intestinal Vibrio anguillarum infection combined with crowding stress. In order to evaluate functional diets efficacy in terms of gut health maintenance, structural, cellular, and immune intestinal status were evaluated by optical and electron microscopy and gene expression analyses. A semi-automated software was adapted to determine variations in goblet cell area and mucosal mucus coverage during the challenge test. Feeding with functional diets did not affect growth performance; however, PHYTO and GMOS dietary inclusion reduced European sea bass susceptibility to V. anguillarum after 7 days of challenge testing. Rectum (post-ileorectal valve) showed longer (p = 0.001) folds than posterior gut (pre-ileorectal valve), whereas posterior gut had thicker submucosa (p = 0.001) and higher mucus coverage as a result of an increased cell density than rectum. Functional diets did not affect mucosal fold length or the grade of granulocytes and lymphocytes infiltration in either intestinal segment. However, the posterior gut fold area covered by goblet cells was smaller in fish fed GMOS (F = 14.53; p = 0.001) and PHYTO (F = 5.52; p = 0.019) than for the other diets. PHYTO (F = 3.95; p = 0.049) reduced posterior gut goblet cell size and increased rodlet cell density (F = 3.604; p = 0.068). Dietary GMOS reduced submucosal thickness (F = 51.31; p = 0.001) and increased rodlet cell density (F = 3.604; p = 0.068) in rectum. Structural TEM analyses revealed a normal intestinal morphological pattern, but the use of GMOS increased rectum microvilli length, whereas the use of PHYTO increased (p≤0.10) Ocln, N-Cad and Cad-17 posterior gut gene expression. After bacterial intestinal inoculation, posterior gut of fish fed PHYTO responded in a more controlled and belated way in terms of goblet cell size and mucus coverage in comparison to other treatments. For rectum, the pattern of response was similar for all dietary treatments, however fish fed GMOS maintained goblet cell size along the challenge test.

Insects have been identified as an economically sustainable high-value, and safe protein-rich alternative to fishmeal in compound feeds for farmed fish. Accordingly, the present study aimed to evaluate the effects of substitution of fishmeal with insect meal from Hermetia illucens in the diet of rainbow trout (Oncorhynchus mykiss), on fish growth performance, and gut microbiota composition. For this purpose, three diets, with increasing levels of insect prepupae meal inclusion (10%, 20% and 30%) in partial substitution of fishmeal and a control diet without insect meal were tested in a 12-weeks feeding trial. Fish growth and feed conversion ratio were evaluated. The Illumina MiSeq platform for high-throughput amplicon sequencing of 16S rRNA gene and QIIME pipeline were used to analyse and characterize the whole microbiome associated to aquafeeds, and fish gut. The number of reads taxonomically classified according to the Greengenes database was 1,140,534. We identified 450 OTUs at 97% identity in trout fecal samples; 62 OTUs constituted the core gut microbiota. Actinobacteria, Firmicutes and Proteobacteria represented the dominant phyla in both experimental groups. Among them, the abundance of Actinobacteria and Proteobacteria was significantly influenced by including insect meal in the diet. In summary, our findings clearly indicated that insect meal positively modifies fish gut microbiota, increasing its richness and diversity and in particular, increasing the amount of beneficial lactic acid-and butyrate-producing bacteria, which contribute to the global health of the host. In addition, based on our present and previous studies, we believe that the prebiotic effect of insect meal is principally due to fermentable chitin.

Nutritional intervention with vegetable oils (VO) supplemented to fish broodstock diets enhances progeny ability to utilize low fish meal (FM) and fish oil (FO) diets. However, the intervention with vegetable meals (VM) in broodstock diets has been scarcely tested in marine fish. This study aims to determine the effect of FM replacement by VM alone or in combination with FO replacement by VO as a programming tool. Different broodstocks of gilthead sea bream were fed one of three diets: one contained 35% FM and 10% FO, another contained VM in replacement of FM (FM: 15%) or a third one had both VM and VO in replacement of FM and FO (15% FM and 2.7% FO). At the weight of 3 g, their offspring were challenged with a low FM (5%) and FO (3%) diet for 45 days. FM replacement by VM in broodstock diets did not affect fish reproductive performance but in offspring juveniles reduced growth and feed efficiency and in liver, altered fatty acids profiles and down-regulated fatty acyl desaturase 2 (fads2) expression. Combined supplementation of VM and VO in broodstock diets led to a poor reproductive performance, reducing female fecundity and the egg content in eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). At molecular level, the expression of fads2 and target of rapamycine (tor) genes in the eggs were upregulated. Similarly, in liver of juvenile progenies fads2 and elongase of very long chain fatty acids 6 (elovl6) expression were up-regulated, whereas fads2 expression was correlated to sterol regulatory element-binding protein transcription factor 1 (srebp1). However, methylation rates of CpG rich regions in fads2 promoter were not significantly different among juvenile groups. Thus, the replacement of FM by VM, alone or in combination with FO in diets for gilthead sea bream broodstock negatively affected growth performance of juvenile offspring, whereas the replacement of FO by VO enhanced the fatty acid desaturase and elongase activities in the offspring, especially when juveniles were fed a diet with a high replacement of FM and FO by VM and VO.

Levels of the oxidative stress-related minerals selenium (Se), zinc (Zn) and manganese (Mn) that should be supplied in microdiets for marine fish larvae depend on the availability of the molecular form of these minerals. The objective of this study was to determine how effectively Se, Zn and Mn in organic, inorganic and nanoparticle forms promote larval performance and bone development. Microdiets supplemented with Se, Zn and Mn were fed for 24 days to 20 dah seabream larvae. Microdiets without Se, Zn and Mn supplementation were associated with poor growth, low bone mineralization and a high incidence of anomalies in the branchial arches. Including Zn, Mn and Se in an amino acid chelate organic form promoted maximum larval growth, increased body lipid reserves, enhanced early mineralization and prevented branchial arches anomalies. In contrast, feeding with inorganic forms of these minerals was less effective than organic minerals in improving larval weight or bone mineralization in comparison to the non-supplemented diet. Moreover, the larvae were less resistant to stress, and fish showed higher bone anomalies in the pre-hemal region. Adding Zn, Mn and Se in the form of nanometals did not enhance growth, but improved stress resistance and bone mineralization. The study showed the need to supplement seabream with early weaning diets based on squid meal and krill oil with one or more of the antioxidant minerals, to promote larval growth, bone mineralization and prevention of skeleton anomalies, organic minerals being more effective than inorganic forms and nanometals in promoting mineralization and stress resistance.

We studied the molecular responses to different water oxygen levels in gills and swim bladder of spotted gar (Lepisosteus oculatus), a bimodal breather. Fish at swim-up stage were exposed for 71 days to normoxic, hypoxic, and hyperoxic water conditions. Then, all aquaria were switched to normoxic conditions for recovery until the end of the experiment (120 days). Fish were sampled at the beginning of the experiment, and then at 71 days of exposure and at 8 days of recovery. We first cloned three hypoxia-related genes, hypoxia-inducible factor 2α (HIF-2α), Na(+) /H(+) exchanger 1 (NHE-1), and NHE-3, and uploaded their cDNA sequences in the GeneBank database. We then used One Step Taqman® real-time PCR to quantify the mRNA copies of target genes in gills and swim bladder of fish exposed to different water O2 levels. We also determined the protein expression of HIF-2α and neuronal nitric oxide synthase (nNOS) in the swim bladder by using confocal immunofluorescence. Hypoxic stress for 71 days significantly increased the mRNA copies of HIF-2α and NHE-1 in gills and swim bladder, whereas normoxic recovery for 8 days decreased the HIF-2α mRNA copies to control values in both tissues. We did not found significant changes in mRNA copies of the NHE-3 gene in either gills or swim bladder in response to hypoxia and hyperoxia. Unlike in normoxic swim bladder, double immunohistochemical staining in hypoxic and hyperoxic swim bladder using nNOS/HIF-2α showed extensive bundles of HIF-2α-positive nerve fibers in the trabecular musculature associated with a few varicose nNOS immunoreactive nerve terminals.

In aquaculture research, one important aim of gut microbiota studies is to provide the scientific basis for developing effective strategies to manipulate gut microbial communities through the diet, promoting fish health and improving productivity. Currently, there is an increasing commercial and research interest towards the use of organic acids in aquafeeds, due to several beneficial effects they have on growth performance and intestinal tract’s health of farmed fish. Among organic acids, monoglycerides of short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) have attracted particular research attention also for their bacteriostatic and bactericidal properties. Accordingly, the present study aimed to evaluate the potential beneficial effects of SCFA and MCFA monoglycerides, used as a feed additive, on fish growth performance, and intestinal microbiota composition. For this purpose, a specific combination of short- and medium-chain 1-monoglycerides (SILOhealth 108Z) was tested in 600 juvenile gilthead sea bream (Sparus aurata) of about 60 g mean initial weight that were fed for 90 days with plant-based diets. Two isoproteic and isolipidic diets were formulated. The control fish group received a plant-based diet, whereas the other group received the same control feed, but supplemented with 0.5% of SILOhealth 108Z. The Illumina MiSeq platform for high-throughput amplicon sequencing of 16S rRNA gene and QIIME pipeline were used to analyse and characterize the whole microbiome associated both to feeds and S. aurata intestine. The number of reads taxonomically classified according to the Greengenes database was 394,611. We identified 259 OTUs at 97% identity in sea bream fecal samples; 90 OTUs constituted the core gut microbiota. Firmicutes, Proteobacteria and Actinobacteria represented the dominant phyla in both experimental groups. Among them, relative abundance of Firmicutes and Proteobacteria were positively and negatively affected by dietary SCFA monoglycerides supplementation, respectively. In summary, our findings clearly indicated that SILOhealth 108Z positively modulated the fish intestinal microbiota by increasing the number of beneficial lactic acid bacteria, namely, Lactobacillus, and reducing Gammaproteobacteria, which include several potential pathogenic bacteria. The specific composition of 1-monoglycerides of short- and medium-chain fatty acids contained in SILOhealth 108Z could thus have a great potential as a feed additive in aquaculture.

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2β, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.

Animal by-product meals from the rendering industry could provide a sustainable and commercially viable alternative to fishmeal (FM) in aquaculture, as they are rich in most essential amino acids and contain important amounts of water-soluble proteins that improve feed digestibility and palatability. Among them, poultry by-product meal (PBM) have given encouraging results in rainbow trout (Oncorhynchus mykiss). However, the introduction of new ingredients in the diet needs to be carefully evaluated since diet is one of the main factors affecting the gut microbiota, which is a complex community that contributes to host metabolism, nutrition, growth, and disease resistance. Accordingly, we investigated the effects of partial replacement of dietary FM with a mix of animal by-product meals and plant proteins on intestinal microbiota composition of rainbow trout in relation to growth and feeding efficiency parameters. We used 1540 trout with an initial mean body weight of 94.6 ± 14.2 g. Fish were fed for 12 weeks with 7 different feed formulations. The growth data showed that trout fed on diets rich in animal by-product meals grew as well as fish fed on control diet, which was rich in FM (37.3%) and PBM-free. High-throughput 16S rRNA gene amplicon sequencing (MiSeq platform, Illumina) was utilised to study the gut microbial community profile. After discarding Cyanobacteria (class Chloroplast) and mitochondria reads a total of 2,701,274 of reads taxonomically classified, corresponding to a mean of 96,474 ± 68,056 reads per sample, were obtained. Five thousand three hundred ninety-nine operational taxonomic units (OTUs) were identified, which predominantly mapped to the phyla of Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The ratio between vegetable and animal proteins proved to play a central role in determining microbiome profiles and Firmicutes and Proteobacteria phyla were particularly discriminatory for diet type in trout. Plant ingredients favoured a higher Firmicutes:Proteobacteria ratio than animal proteins. Acceptable abundance of Firmicutes was guaranteed by including at least 25% of vegetable proteins in the diet regardless of animal protein source and percentage. In summary animal by-product meals, as replacements to FM, gave good results in terms of growth performances and did not induce significant changes in gut microbial richness, thus proving to be a suitable protein source for use in rainbow trout aqua feed.

We evaluated the chemical coding of the myenteric plexus in the proximal and distal intestine of gilthead sea bream (Sparus aurata), which represents one of the most farmed fish in the Mediterranean area. The presence of nitric oxide (NO), acetylcholine (ACh), serotonin (5-HT), calcitonin-gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) containing neurons, was investigated in intestinal whole mount preparations of the longitudinal muscle with attached the myenteric plexus (LMMP) by means of immunohistochemical fluorescence staining. The main excitatory and inhibitory neurochemicals identified in intestinal smooth muscle were ACh, SP, 5HT, and NO, VIP, CGRP. Some neurons displayed morphological features of ascending and descending interneurons and of putative sensory neurons. The expression of these pathways in the two intestinal regions is largely superimposable, although some differences emerged, which may be relevant to the morphological properties of each region. The most important variances are the higher neuronal density and soma size in the proximal intestine, which may depend on the volume of the target tissue. Since in the fish gut the submucosal plexus is less developed, myenteric neurons substantially innervate also the submucosal and epithelial layers, which display a major thickness and surface in the proximal intestine. In addition, myenteric neurons containing ACh and SP, which mainly represent excitatory motor neurons and interneurons innervating the smooth muscle were more numerous in the distal intestine, possibly to sustain motility in the thicker smooth muscle coat. Overall, this study expands our knowledge of the intrinsic innervation that regulates intestinal secretion, absorption and motility in gilthead sea bream and provides useful background information for rational design of functional feeds aimed at improving fish gut health.

Currently available data show that shellfish and finfish production in Italy, derived both from fisheries and aquaculture activities, is on the order of 474,000 tons, each activity representing 50 % of the total amount. In this context, the finfish aquaculture industry contributes on average 31 % to the national aquaculture production and on average 59 % of its value, giving a total amount of 72,000 tons and a value of around 351 million € (2010). According to FEAP statistics, Italy is the fourth largest finfish producer in EU27, after the UK, Greece, and Spain, while it is also one of the six largest finfish producers among the non- EU and EU member countries, together with Norway, UK, Greece, Turkey, and Spain. Presently, fish culture activities are mainly focused on rainbow trout (Oncorhynchus mykiss, 55.5 %), followed by European sea bass (Dicentrarchus labrax, 13.6 %), gilthead sea bream (Sparus aurata, 12.2 %), gray mullet (Mugil cephalus, 5.3 %), sturgeon (Acipenser spp., 2 %), and European eel (Anguilla anguilla, 1.7 %). Over the last 20 years, freshwater fish production and aquaculture (trout, carp, and eel) have decreased in Italy, with the exception of sturgeon. In contrast, marine fish production has significantly increased during the same period, and the two leading species, European sea bass and gilthead sea bream, presently contribute 25.8 % of the finfish production. From 1,900 tons in 1990, production reached 19,000 tons in 2010, with a 900 % increase, at an average percentage of 4.5 %. In addition, new marine fish species were successfully cultured over the same period. This review outlines the past and present situation of finfish culture in Italy and discusses future developments and priorities, with particular emphasis on new, emerging aquaculture species.

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2)gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Znsod) and glutathione peroxidase 1 (gpx-1),suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.

The skin mucus of gilthead sea bream was mapped by one-dimensional gel electrophoresis followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2,000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, two-dimensional gel electrophoresis methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked daily farming activities. The major changes were associated with a higher abundance of cytokeratin 8 in the skin mucus proteome of stressed fish, which was confirmed by immunoblotting. Thus, the increased abundance of markers of skin epithelial turnover results in a promising indicator of chronic stress in fish., This study was funded by the European Union (AQUAEXCEL, FP7/2007/2013; grant agreement No. 262336, Aquaculture infrastructures for excellence in European fish research) project. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. Additional funding was obtained from the Spanish Ministerio de Economía y Competitividad (MI2-Fish, AGL2013-48560) and from Generalitat Valenciana (PROMETEO FASE II-2014/085). Proteomics study was done at Proteomics laboratory of University of Valencia, Spain (SCSIE). This laboratory is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+D+i 2013–2016, funded by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER).

E' ampiamente accettato che entro il 2050 il mondo ospiterà 9 miliardi di persone. Per accogliere questo numero, la produzione alimentare attuale dovrà quasi raddoppiare. La terra è insufficiente ed ampliare l'area dedicata all'agricoltura è difficilmente una soluzione valida o sostenibile. Gli oceani sono sovrasfruttati e il cambiamento climatico e la scarsità d'acqua correlata potrebbero avere profonde implicazioni nelle produzioni alimentari. Per soddisfare le sfide alimentari e nutrizionali attuali - nel mondo sono quasi 1 miliardo le persone che vivono una situazione di fame cronica - e future, ciò che mangiamo e come produciamo hanno bisogno di essere rivalutati. Le inefficienze devono essere corrette e gli sprechi ridotti. Dobbiamo trovare nuovi modi di produrre alimento. Gli insetti eduli sono sempre stati parte della dieta umana, ma in alcune società vi è un senso di disgusto all'idea del loro consumo. Sebbene la maggior parte degli insetti eduli siano raccolti da habitat forestali, molti paesi hanno iniziato ad implementare sistemi innovativi di allevamento di massa. Gli insetti offrono una opportunità significativa di fondere conoscenze tradizionali e scienza moderna sia nei Paesi sviluppati che in quelli in via di sviluppo. Questa pubblicazione ha le sue origini nel tentativo del Dipartimento Forestale della FAO di individuare le pratiche tradizionali di raccolta di insetti sia per cibo che per reddito, e di documentare i relativi impatti ecologici sugli habitat forestali. Successivamente, la FAO ha colto l'opportunità di collaborare con il Laboratorio di Entomologia dell'Università di Wageningen nei Paesi Bassi - un'istituzione in prima linea nella ricerca di base e applicata sugli insetti quali cibo e mangimi. Da allora, questo sforzo congiunto ha avuto nuovo impulso e ne è risultato un impegno di ampio respiro presso la FAO per esaminare le molteplici dimensioni della raccolta e dell'allevamento degli insetti quale valide alternative per alleviare la scarsa disponibilità alimentare. Questo libro si basa su numerose ricerche scientifiche sul contributo che gli insetti hanno sugli ecosistemi, le diete, la disponibilità alimentare e i mezzi di sussistenza sia nei Paesi sviluppati che in quelli in via di sviluppo. Ci auguriamo che possa contribuire a migliorare la percezione degli insetti come fonte di alimenti e mangimi presso le autorità alimentari nazionali ed internazionali. Speriamo inoltre che attiri l'attenzione degli agricoltori, dei media, del grande pubblico e dei governi, degli organi di finanziamento multilaterale e bilaterali, delle imprese di investimento, dei centri di ricerca, degli organismi di aiuto umanitario e delle industri alimentari e mangimistiche. Soprattutto, è nostra speranza che questa pubblicazione possa sensibilizzare sui molteplici ed importanti ruoli che gli insetti svolgono nel sostenere la natura e la vita umana e possa anche servire a documentare il ruolo che gli insetti già hanno nel diversificare le diete e migliorare la disponibilità alimentare.

Hypoxia can affect various fish populations, including yellow perch Perca flavescens, which is an economically and ecologically important species in Lake Erie, a freshwater system that often experiences hypoxia in the hypolimnetic part of the lake. Fish, similarly to mammals, possess molecular oxygen sensor—hypoxia-inducible factor-1 (HIF-1), a transcription factor that can affect expression of many downstream genes related to animal growth and locomotion, protein synthesis, as well as ATP and amino acid metabolism. HIF-1 is a heterodimer, which consists of two subunits: oxygen-sensitive and oxygen-insensitive subunits, α and β, respectively. In this study, we report first on the molecular cloning and sequencing of P. flavescens HIF-1α. The full-length complementary DNA (cDNA) was isolated and submitted to the GenBank with accession number KT783483. It consists of 3529 base pairs (bp) carrying a single open-reading frame that encompasses 2250 bp of the coding region, 247 bp of the 5′ untranslated region (UTR), and 1032 bp of the 3′ UTR. The “de novo” prediction of the 3D structure of HIF-1α protein, which consists of 749 amino acids, is presented, too. We then utilized One-Step Taqman® real-time RT-PCR technology to monitor changes in HIF-1α messenger RNA (mRNA) copies in response to chronic hypoxic stress. An experiment was conducted using 14-day post-swim-up stage yellow perch larvae with uninflated swim bladders. This experiment included three treatment groups: hypoxia, mid-hypoxia, and normoxia, in four replicates (four tanks per treatment) with the following dissolved oxygen levels: 3, 4, and >7 mg O2/L, respectively. At the end (2 weeks) and in the middle (1 week) of the experiment, fish from each tank were sampled for body measurements and molecular biology analysis. The results showed no differences in survival (∼90%) between treatment groups. Oxygen concentration was lowered to 3.02 ± 0.15 (mean ± SE) mg O2/L with no adverse effect on fish survival. The highest growth rate was observed in the normoxic group. A similar trend was observed with fish body length. The growth rate of fish declined with decreasing water-dissolved oxygen. The number of HIF-1α mRNA copies was not significantly different between hypoxic, mid-hypoxic, and normoxic conditions, and this was true for fish obtained in the middle and at the end of the experiment.

The objective of the present study was to investigate the effect of dietary methionine (Met) concentration and alternating feeding strategies in methionine delivery, on the mRNA transcript levels of genes involved in Met resynthesis (betaine-homocysteine methyltransferase, BHMT; S-adenosylhomocysteine hydrolase, SAHH) and net Met loss (taurine synthesis) (cystathionine beta-synthase, CBS) in Atlantic salmon ( Salmo salar ) liver. Salmon alevins (265 ± 3 mg) were distributed into 24 tanks (50 fish per tank; 3 replicates). The experimental diets were supplemented with l -methionine at 0, 1.9, 5.8, and 17.4 g/kg (M0, M1/3, M1, and M3, respectively). The M3 diet without glycine was prepared to examine Met toxicity (M3-G). These diets were provided via “mono-feeding strategy,” meaning fish were fed a designated single diet. This experiment also included alternative feeding groups with “duo-feeding” strategy: AF1 (fish fed M0 for 2 days followed by M1 for 1 day), AF2 (fish fed M0 for 2 days followed by M3 for 1 day), and AF3 (fish fed two meals of M0 followed by one meal of M3). Salmon fed M0 diet had smaller weight compared to all other groups. There was no effect of alternative feeding on the growth except with the M0 group. The highest expression of CBS gene was found in the M0, M1/3, and AF-1 groups compared to the M3 and M3-G groups. The expression of CBS gene in the M1 group was lower compared to the AF-1 group. The expression of SAHH gene was the highest in the M3 group compared to the M1, M1/3, and AF-3 groups. The highest expression of BHMT gene was found in the M0, M1/3, and AF-1 groups, indicating enhanced re-methylation of homocysteine by betaine to Met. The lowest BHMT expression in M1, M3, M3-G, AF-2, and AF-3 groups compared to other treatments can be indicative of downregulation of remethylation in the liver. The alternate provision of methionine in a form of combination of low and high methionine containing diets seemed to support its efficient utilization in the salmon liver.

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2) gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Zn sod) and glutathione peroxidase 1 (gpx-1), suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.

Simple Summary The composition of fish-gut microbial communities has been demonstrated to adapt when the host is fed different ingredients. Fishmeal and soy are the conventional protein sources used in aquafeeds. However, these feed options are not sustainable anymore due to the progressive depletion of wild marine fish stocks and the considerable environmental cost of protein-rich terrestrial plant cultivation. In this perspective, insects could be a promising source of protein and may help aquaculture to cope with the increasing global demand for new protein sources, representing the idea of “waste into feed” bioconversion. In this perspective, we evaluated the effects of dietary insect meal from Hermetia illucens (Hi) larvae on autochthonous gut microbiota of rainbow trout (Oncorhynchus mykiss). Hi larvae were grown on leftover fruit and vegetables provided by a local wholesale market. Three diets, with increasing levels of insect meal inclusion (10%, 20%, and 30%) and a control diet without insect meal were tested in a 3-month fish feeding trial. The data showed that feeding insects influences the intestinal bacterial communities, thus improving fish gut health. In our opinion, these findings represent a precious tool for future research on salmonid’s microbial communities and their interaction with diet and the host. Abstract This study evaluated the effects of dietary insect meal from Hermetia illucens larvae on autochthonous gut microbiota of rainbow trout (Oncorhynchus mykiss). Three diets, with increasing levels of insect meal inclusion (10%, 20%, and 30%) and a control diet without insect meal were tested in a 12-week feeding trial. To analyze the resident intestinal microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME pipeline were used. The number of reads taxonomically classified according to the Greengenes database was 1,514,155. Seventy-four Operational Taxonomic Units (OTUs) at 97% identity were identified. The core of adhered intestinal microbiota, i.e., OTUs present in at least 80% of mucosal samples and shared regardless of the diet, was constituted by three OTUs assigned to Propiobacterinae, Shewanella, and Mycoplasma genera, respectively. Fish fed the insect-based diets showed higher bacterial diversity with a reduction in Proteobacteria in comparison to fish fed the fishmeal diet. Insect-meal inclusion in the diet increased the gut abundance of Mycoplasma, which was attributed the ability to produce lactic and acetic acid as final products of its fermentation. We believe that the observed variations on the autochthonous intestinal microbiota composition of trout are principally due to the prebiotic properties of fermentable chitin.

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase.

Amino acids play an important role in the metabolism of all organisms. Their epithelial re-absorption is due to specific transport proteins, such as B(0)AT1, a Na(+)-coupled neutral amino acid symporter belonging to the solute carrier 6 family. Here, a recently cloned fish orthologue, from the intestine of Salmo salar, was electrophysiologically characterized with the two-electrode voltage clamp technique, in Xenopus laevis oocytes heterologously expressing the transporter. Substrate specificity, apparent affinities and the ionic dependence of the transport mechanism were determined in the presence of specific collectrin. Results demonstrated that like the human, but differently from sea bass (Dicentrarchus labrax) orthologue, salmon B(0)AT1 needs to be associated with partner proteins to be correctly expressed at the oocyte plasma membrane. Cloning of sea bass collectrin and comparison of membrane expression and functionality of the B(0)AT1 orthologue transporters allowed a deeper investigation on the role of their interactions. The parameters acquired by electrophysiological and immunolocalization experiments in the mammalian and fish transporters contributed to highlight the dynamic of relations and impacts on transport function of the ancillary proteins. The comparative characterization of the physiological parameters of amino acid transporters with auxiliary proteins can help the comprehension of the regulatory mechanism of essential nutrient absorption.

European sea bass (Dicentrarchus labrax) is one of the most important species for Mediterranean aquaculture. It is therefore crucial to establish appropriate feeding management regimens and to gain better knowledge of nutritional requirements for this species, exploring not only new feed ingredients and feeding strategies, but also understanding the molecular mechanisms that regulate the metabolism of nutrients. Accordingly, transcriptomic analysis represents a useful nutrigenomic discovery tool for identifying the molecular basis of biological responses to nutrition as well as nutritional biomarkers in fish. This study evaluated how the transcriptional activity of genes controlling lipid metabolism in European sea bass were modulated in a tissue-specific manner in response to fasting and refeeding. Such approach focused on a panel of 29 genes in which desaturases, elongases, triacylglycerol lipases, fatty acid-binding proteins, β-oxidation and oxidative phosphorylation enzymes, phospholipid-related enzymes, and transcription factors that regulate lipid homeostasis were represented. Fasting activated the lipolytic machinery in adipose tissue, liver and muscle of European sea bass, whereas markers of lipogenesis were downregulated in liver and adipose tissue. Genes involved in phospholipid and oxidative metabolism were differentially regulated in liver and skeletal muscle of fasted European sea bass. However, 12 days of refeeding were sufficient, for the most part, to reverse the expression of key genes. Overall, our data clearly showed a tissue-specific regulation of lipid-related genes according to the different metabolic capabilities of each tissue, being the brain the most refractory organ to changes in nutrient and energy availability and liver the most responsive tissue., This research was partly funded by AQUAEXCEL EU 7 FP Project (Grant Agreement 262336): Trans National Access Grant to S.R. for accessing to IATS-CSIC facilities. This work has been partly funded under the EU seventh Framework Program by the ARRAINA Project N288925: Advanced Research Initiatives for Nutrition & Aquaculture.

20 pages, 3 figures, 4 tables, supporting information https://dx.doi.org/10.1371/journal.pone.0160332, Bacteria that inhabit the epithelium of the animals’ digestive tract provide the essential biochemical pathways for fermenting otherwise indigestible dietary fibers, leading to the production of short-chain fatty acids (SCFAs). Of the major SCFAs, butyrate has received particular attention due to its numerous positive effects on the health of the intestinal tract and peripheral tissues. The mechanisms of action of this four-carbon chain organic acid are different; many of these are related to its potent regulatory effect on gene expression since butyrate is a histone deacetylase inhibitor that play a predominant role in the epigenetic regulation of gene expression and cell function. In the present work, we investigated in the European sea bass (Dicentrarchus labrax) the effects of butyrate used as a feed additive on fish epigenetics as well as its regulatory role in mucosal protection and immune homeostasis through impact on gene expression. Seven target genes related to inflammatory response and reinforcement of the epithelial defense barrier [tnfα (tumor necrosis factor alpha) il1β, (interleukin 1beta), il-6, il-8, il-10, and muc2 (mucin 2)] and five target genes related to epigenetic modifications [dicer1(double-stranded RNA-specific endoribonuclease), ehmt2 (euchromatic histone-lysine-N-methyltransferase 2), pcgf2 (polycomb group ring finger 2), hdac11 (histone deacetylase-11), and jarid2a (jumonji)] were analyzed in fish intestine and liver. We also investigated the effect of dietary butyrate supplementation on histone acetylation, by performing an immunoblotting analysis on liver core histone extracts. Results of the eight-week-long feeding trial showed no significant differences in weight gain or SGR (specific growth rate) of sea bass that received 0.2% sodium butyrate supplementation in the diet in comparison to control fish that received a diet without Na-butyrate. Dietary butyrate led to a twofold increase in the acetylation level of histone H4 at lysine 8, but showed no effect on the histone H3 at Lys9. Moreover, two different isoforms of histone H3 that might correspond to the H3.1 and H3.2 isoforms previously found in terrestrial animals were separated on the immunoblots. The expression of four (il1 β, il8, irf1, and tnfα) out of seven analyzed genes related to mucosal protection and inflammatory response was significantly different between the two analyzed tissues but only il10 showed differences in expression due to the interaction between tissue and butyrate treatment. In addition, butyrate caused significant changes in vivo in the expression of genes related to epigenetic regulatory mechanisms such as hdac11, ehmt2, and dicer1. Statistical analysis by two-way ANOVA for these genes showed not only significant differences due to the butyrate treatment, but also due to the interaction between tissue and treatment, This work has been funded under the seventh EU Framework Programme by the ARRAINA project N°288925: Advanced Research Initiatives for Nutrition & Aquaculture. Collaboration between the GT and FP labs was fostered by COST Action FA1201: Epigenetics and Periconception Environment (“Epiconcept”)

We studied the molecular responses to different water oxygen levels in gills and swim bladder of spotted gar (Lepisosteus oculatus), a bimodal breather. Fish at swim-up stage were exposed for 71 days to normoxic, hypoxic, and hyperoxic water conditions. Then, all aquaria were switched to normoxic conditions for recovery until the end of the experiment (120 days). Fish were sampled at the beginning of the experiment, and then at 71 days of exposure and at 8 days of recovery. We first cloned three hypoxia-related genes, hypoxia-inducible factor 2α (HIF-2α), Na(+) /H(+) exchanger 1 (NHE-1), and NHE-3, and uploaded their cDNA sequences in the GeneBank database. We then used One Step Taqman® real-time PCR to quantify the mRNA copies of target genes in gills and swim bladder of fish exposed to different water O2 levels. We also determined the protein expression of HIF-2α and neuronal nitric oxide synthase (nNOS) in the swim bladder by using confocal immunofluorescence. Hypoxic stress for 71 days significantly increased the mRNA copies of HIF-2α and NHE-1 in gills and swim bladder, whereas normoxic recovery for 8 days decreased the HIF-2α mRNA copies to control values in both tissues. We did not found significant changes in mRNA copies of the NHE-3 gene in either gills or swim bladder in response to hypoxia and hyperoxia. Unlike in normoxic swim bladder, double immunohistochemical staining in hypoxic and hyperoxic swim bladder using nNOS/HIF-2α showed extensive bundles of HIF-2α-positive nerve fibers in the trabecular musculature associated with a few varicose nNOS immunoreactive nerve terminals.

Although fish farming has been practiced for 4,000 years, aquaculture research dates back only to about 1,870, whereas molecular techniques, in addition to the more traditional methods of biotechnology, were introduced only recently. Contemporary genomic approaches (often adapted from human or medical research) such as cDNA cloning and sequencing, cDNA microarray/expression analysis, and functional genomics, combined with improvements in transgenic technologies, have enhanced possibilities for aquacultural biotechnologists for improving fish growth rates and increasing resistance to pathogens and stressors. Although genomic technologies in fish have been applied primarily to model organisms, such as zebrafish (Danio rerio), fugu or pufferfish (Tetraodon nigroviridis), and medaka (Oryzias latipes), many teleosts of interest for biological research and with potential application in aquaculture have unique physiological characteristics that cannot be directly investigated from the study of small laboratory fish models. As a consequence, large-scale genomic research studies are increasingly being applied to farmed species of economic relevance, such as farmed rainbow trout, Atlantic salmon, tilapia, catfish, and sea bass. Accordingly, we describe the utilization of molecular cloning and gene expression analysis in cultured European sea bass (Dicentrarchus labrax) to generate “transcriptome-focused” information which may enable a better understanding of the transcriptional programs that underlie fish respiratory physiology and immune response pathways.

DHA deficiency has been related to skeletal malformations in fish, but high DHA levels have produced controversial results that could relate to the oxidative status of fish tissues in the different reports. In the present study, gilthead seabream (Sparus aurata) larvae were fed deficient, adequate or high DHA levels, or high DHA levels supplemented with the antioxidant α-tocopherol. Larvae fed deficient DHA levels tended to be smaller, and showed the highest incidence of urinary bladder calculi, lordosis and kyphosis and the lowest number of mineralised vertebrae for any given size class. Elevation of dietary DHA increased larval growth and significantly enhanced the expression of the insulin-like growth factor 1 (IGF-1) gene. However, a DHA level increase up to 5 % raised the degree of lipid oxidation in larval tissues and deformities in cranial endochondral bones and in axial skeletal haemal and neural arches. The increase in dietary α-tocopherol supplementation in high-DHA feeds reduced again the occurrence of skeletal deformities. Moreover, the expression of genes coding for specific antioxidants such as catalase, superoxide dismutase or glutathione peroxidase, which neutralised reactive oxygen substances formed by increased dietary DHA, was significantly decreased in larvae fed high α-tocopherol levels. These results denoted the importance of DHA for early bone formation and mineralisation. Low dietary DHA levels delay early mineralisation and increase the risk of cranial and axial skeletal deformities. Excessive DHA levels, without an adequate balance of antioxidant nutrients, increase the production of free radicals damaging cartilaginous structures before bone formation.

The aim of the present study was to investigate by histological methods the impact of fasting and re-feeding induced compensatory growth, in the European sea bass ( Dicentrarchus labrax ) liver. In addition, the presence and distribution of leptin (LEP) and aquaglyceroporin 9 (AQP-9), two proteins involved in lipid storage and mobilization, were investigated in the liver in relation to fish nutritional status with the aim to verify their involvement in the compensatory growth. The histological results showed modifications in liver feature and in hepatic glycogen storage during fasting. Indeed, liver morphology was altered by food deprivation and was characterized by a large spectrum of vacuolization. During re-feeding, liver of the previously fasted fish showed a similar feature to that observed in the control fish, suggesting a recovery at the histomorphological level. The hepatic glycogen store was observed in the control, and in the re-fed fish, but not in the fasted group. Immunostaining showed an up-regulation in both LEP and AQP-9 during fasting and a down-regulation during re-feeding, whereas AQP-9 but not LEP immunoreactivity was higher than that of the controls. The study of fasting and re-feeding induced histological alterations provides additional knowledge on the fish cell and tissue condition, completing the information obtained with growth studies. We also demonstrated that although LEP and AQP-9 were both influenced by nutritional status in sea bass, only AQP-9 was involved in the compensatory growth of this species. Therefore, we suggest AQP-9 may play a role in the process of energy accumulation to lipid storage in sea bass liver during the phase of the compensatory growth.

The objective of the present study was to examine the effect of a wheat-gluten-based diet supplemented with the indispensable amino acid source in the form of free amino acid or dipeptide on growth, intestine oligopeptide transporter, PepT1, transcript levels using real-time RT-PCR, and muscle protein expression in yellow perch Perca flavescens juveniles. Yellow perch (initial size ~0.3 g) were randomly distributed into 12 glass aquaria, 60 fish per tank. Fish were fed 4 diets: wheat-gluten based diet supplemented with lysine–glycine dipeptide (LG), wheat-gluten based diet supplemented with free lysine (FL), diet not supplemented with lysine (NL; negative control) and a commercial diet (BO). Fish were fed at 90 % satiation level and the rate was re-adjusted to be equal across all treatments based on each day’s projected change in weight gain. It amounted to 3 % of the biomass per day for the first 14 days and 4–5 % until the end of the experiment. After 55 days of the experiment the mean weight of juvenile yellow perch fed the LG diet was larger compared to the NL diet fed group. There was no difference, however, between LG, FL, and BO groups (1.35 ± 0.11 g; 1.31 ± 0.03; 1.16 ± 0.10 g, respectively). Fish subjected to FL treatment showed an increase in the amount of PepT1 transcripts compared to the NL group value. The LG diet was associated with a significant increase in PepT1 mRNA transcript levels, compared with both FL and NL diets fed fish. We have also cloned and sequenced full-length cDNA representing yellow perch PepT1. The cDNA sequence (GeneBank: accession no. GQ906471), encompasses a total of 2,956 base pairs (bp) including a 5′-untranslated region of 94 bp, an open reading frame of 2,190 bp, and a 3′-untranslated region of 672 bp. The predicted 12 transmembrane domains and the 3D structure of the protein (729 amino acids) are presented. Proteomic fingerprinting showed that thirteen electrophoretically resolved protein/peptide bands from the muscle sarcoplasmic fraction were significantly different across treatments suggesting that muscle protein expression was influenced by dietary treatments.

The impact of an off-shore fish farm in Alghero Bay (northwest Sardinia, Italy) on the benthic ecosystem was investigated in 2007 and 2008. In addition to studying the chemical and physical characteristics of the area (i.e., currents and sediment analyses), some biological analyses were also performed. The AZTI's Marine Biotic Index (AMBI) and the multivariate AMBI (M-AMBI) were calculated, which are being used in assessing the ecological status of benthic communities within the European Water Framework Directive (WFD). Clear impact gradients were detected according to both methods; they are related to farm production, prevailing currents, and characteristics of the area (i.e., water depth and distance to the cages). The site affected most was detected within 84 m from the cages; the area that no longer showed effects was over 907 m from the cages. The gradient is shown by decreasing AMBI values and percentage of opportunistic species and increasing richness, diversity, and the presence of sensitive species. This study highlights the importance of setting reference conditions for different areas when calculating M-AMBI. These reference conditions correspond to those in undisturbed sites in the opposite direction of the prevailing currents within the area.

Microfluidic capillary electrophoresis and real-time PCR were successfully applied to investigate the postmortem alterations in RNA extracted from fish muscular tissue in relation to three parameters: slaughtering method, time, and storage temperature. Postmortem proteolytic degradation of fish filets, which leads to textural changes such as muscle softening and gaping, is a major problem for fish freshness. Endogenous proteases such as calpains and cathepsins are assumed to play the major role in this process, although the exact mechanisms underlying fish meat tenderization have yet to be determined. In the course of the present study we first identified the cDNA sequences coding for μ-calpain and cathepsin L in sea bass ( Dicentrarchus labrax ). Then we determined the total RNA and μ-calpain- and cathepsin L-specific mRNA integrity over 5 days of storage at two different temperatures (1 °C and 18 °C) in sea bass slaughtered by three different methods (asphyxia in air, hypothermia, and immediately severing the spinal cord). The results of this study show that, RNA degradation is a slow process under the conditions investigated and, although postmortem storage temperature negatively affects the integrity of total RNA (higher degradation at 18 °C), the transcripts of μ-calpain and cathepsin L are present for up to 5 days postmortem in the muscle of sea bass stored at either 1 °C or 18 °C.

Aquatic hypoxia is a frequent event and in fish a complex set of physiological and biochemical alterations are employed to cope with this environmental stress. Many of these adjustments depend to a large extent on changes in the expression of genes that encode for physiologically relevant proteins. Genes that are induced by hypoxia appear to share a common mode of transcriptional regulation. This induction depends upon activation of a transcription factor, the hypoxia-inducible factor-1 (HIF-1), which is composed of α and β subunits. In this study we report first on the molecular cloning and characterization of HIF-1α in sea bass ( Dicentrarchus labrax ). The full-length sea bass cDNA for HIF-1α was isolated and deposited in the GenBank with accession no. DQ171936 . It consists of 3317 base pairs (bp) carrying a single open-reading frame that encompasses 2265 bp of the coding region and 1052 bp of the 3′ UTR. The predicted sea bass HIF-1α protein (755 amino acids) shows a high level of conservation at the bHLH (basic helix–loop–helix domain), PAS A/B (Per-ARNT-Sim A/B domain), N-TAD (N-terminal transactivation domain), and C-TAD (C-terminal transactivation domain), whereas only few variations are found at the ODD (oxygen-dependent degradation domain) regions among vertebrates. We then utilized the real-time PCR technology to monitor dynamic changes in levels of HIF-1α mRNA in response to acute and chronic hypoxic stress. The number of HIF-1α mRNA copies was significantly increased in liver tissue in response to both acute (1.9 mg/L, dissolved oxygen for 4 h) and chronic (4.3 mg/l, DO for 15 days) hypoxia in sea bass whereas it remained unchanged in fish exposed to hyperoxic (DO 13.5 ± 1.2 mg/L, 155% saturation) conditions. This is the first study to investigate the behaviour of HIF-1α gene transcripts during hypoxia in a representative of marine, hypoxia-sensitive species.

Ghrelin is a novel appetite-inducing peptide hormone secreted by the stomach. The purpose of this study was first to identify the cDNA encoding sequence for ghrelin in sea bass (Dicentrarchus labrax). Using molecular cloning techniques we sequenced the cDNA corresponding to sea bass ghrelin mRNA. A total of 798 bases including a 5'-untranslated region (89 bp), an open reading frame (ORF) (324 bp), and a 3'-untranslated region (385 bp) were detected. Nucleotide sequence (ORF) encoded a 108 amino acid prepropeptide that demonstrated complete conservation of the N-terminal "biological active core" (GSSF) of the predicted mature ghrelin peptide. We also analyzed fasting-induced changes in the expression of ghrelin mRNA, using a one-tube two-temperature real-time RT-PCR with which the gene expression can be absolutely quantified using the standard curve method. Our results revealed that ghrelin was highly expressed in the stomach with much lower levels of expression in the proximal intestine and brain. Levels of ghrelin mRNA in the stomach were upregulated under conditions of negative energy balance, such as starvation, and downregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass ghrelin gene and support a role of this orexigenic hormone in the regulation of food intake in sea bass.

The exceptionally fast growth that fish experience after periods of fasting has been called ‘compensatory growth’ or ‘catch-up’ growth. This phenomenon, reported in a wide range of fish species, has been studied in intensive aquaculture as a means of enhancing feed conversion efficiency, but the mechanisms implicated are complex and not yet fully understood. In the present study, the authors describe the cloning and sequencing of the complete coding sequences of sea bass (Dicentrarchus labrax) insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II), which are potent mitogens known to play important roles in growth and development. Sea bass IGF-I has an open reading frame (ORF) of 561 bp that encodes a mature protein of 187 amino acids, whereas IGF-II has an ORF of 648 bp encoding a mature protein of 216 amino acids. At the amino acid level, sea bass IGF-I shares a 30% similarity with IGF-II. The authors then report the pattern of IGF-I and IGF-II gene expression in liver and myotomal muscle in response to prolonged fasting and refeeding. Nutritional status significantly influenced IGF-I messenger RNA copy number in both liver and muscle, inducing a down-regulation during fasting and an up-regulation during the recovery from fasting. The trend of IGF-II response was similar, but different feeding regimens did not affect the amounts of transcript as sharply as in IGF-I. Taken together these data indicate that IGF-I and IGF-II participate in promoting sea bass muscle compensatory growth induced by refeeding.

Perch are promising species for freshwater aquaculture and, differently from other fish, have not yet been domesticated through artificial selection; therefore, they show a wide genetic variability that is undesirable for aquaculture. In addition to the more traditional methods of aquatic biotechnology, the most recently developed molecular biological techniques can augment the overall efficiency of aquaculture. To help these new molecular techniques find their place in the everyday management of fish farming, we should make an effort to reduce the gap in genomic resources that separates farming species from "model organisms." We performed single-pass sequencing on 1237 randomly selected clones from a perch liver cDNA expression library, 350 clones of a brain-minus-liver, and 639 clones of a liver-minus-brain subtraction library. The sequences were deposited in the NCBI Expressed Sequence Tags database (www.ncbi.nlm.nih.gov/projects/dbEST). In the three libraries we identified 108, 46, and 104 genes, respectively. EST cataloguing and profiling of perch will provide a basis for functional genomic research in this species, but will also promote studies in comparative and environmental genomics, for identifying polymorphic markers that are useful, for example, to survey the disease resistance of fish and for discovering of new molecular markers of exposure. Using these genomic resources, micro- and macroarrays can be produced that will give immediate and practical benefits in the field of aquaculture, allowing early diagnosis of the fish conditions and helping in the generation of new mechanistic data on the nature of fish responses to different farming conditions.