Publications by authors named "Genciana Terova"

60 Publications

Dietary Phytogenics and Galactomannan Oligosaccharides in Low Fish Meal and Fish Oil-Based Diets for European Sea Bass () Juveniles: Effects on Gill Structure and Health and Implications on Oxidative Stress Status.

Front Immunol 2021 12;12:663106. Epub 2021 May 12.

Grupo de Investigación en Acuicultura (GIA), IU-ECOAQUA, Universidad de Las Palmas de Gran Canaria, Las Palmas, Spain.

An effective replacement for fish meal (FM) and fish oil (FO) based on plant-based raw materials in the feed of marine fish species is necessary for the sustainability of the aquaculture sector. However, the use of plant-based raw materials to replace FM and FO has been associated with several negative health effects, some of which are related to oxidative stress processes that can induce functional and morphological alterations in mucosal tissues. This study aimed to evaluate the effects of dietary oligosaccharides of plant origin (5,000 ppm; galactomannan oligosaccharides, GMOS) and a phytogenic feed additive (200 ppm; garlic oil and labiatae plant extract mixture, PHYTO) on the oxidative stress status and mucosal health of the gills of juvenile European sea bass (). The experimental diets, low FM and FO diets (10%FM/6%FO) were supplemented with GMOS from plant origin and PHYTO for 63 days. GMOS and PHYTO did not significantly affect feed utilization, fish growth, and survival. GMOS and PHYTO downregulated the expression of , , , , and in the gills of the fish compared with that in fish fed the control diet. The expression of and was upregulated and downregulated, respectively, in the GMOS group compared with that in the control group, whereas the expression of downregulated in the PHYTO group compared with that in the GMOS group. The morphological, histopathological, immunohistochemical, and biochemical parameters of the fish gills were mostly unaffected by GMOS and PHYTO. However, the PHYTO group had lower incidence of lamellar fusion than did the control group after 63 days. Although the tissular distribution of goblet cells was unaffected by GMOS and PHYTO, goblet cell size showed a decreasing trend (-11%) in the GMOS group. GMOS and PHYTO significantly reduced the concentration of PCNA+ in the epithelium of the gills. The above findings indicated that GMOS and PHYTO in low FM/FO-based diets protected the gill epithelia of . from oxidative stress by modulating the expression of oxidative enzyme-related genes and reducing the density of PCNA+ cells in the gills of the fish.
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http://dx.doi.org/10.3389/fimmu.2021.663106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8149968PMC
May 2021

Effects of full replacement of dietary fishmeal with insect meal from Tenebrio molitor on rainbow trout gut and skin microbiota.

J Anim Sci Biotechnol 2021 Feb 3;12(1):30. Epub 2021 Feb 3.

Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant, 3, 21100, Varese, Italy.

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.
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http://dx.doi.org/10.1186/s40104-021-00551-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860006PMC
February 2021

Intestinal microbial communities of rainbow trout (Oncorhynchus mykiss) may be improved by feeding a Hermetia illucens meal/low-fishmeal diet.

Fish Physiol Biochem 2021 Apr 3;47(2):365-380. Epub 2021 Jan 3.

Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant, 3, 21100, Varese, Italy.

With demands and reliance on aquaculture still growing, there are various challenges to allow sustainable growth and the shift from fishmeal (FM) to other protein sources in aquafeed formulations is one of the most important. In this regard, interest in the use of insect meal (IM) in aquafeeds has grown rapidly. Accordingly, the aim of the present study was to assess the effects of dietary IM from Hermetia illucens (Hi) larvae included in a low-FM diet on gut microbial communities of rainbow trout (Oncorhynchus mykiss), in terms of both composition and function of microbiome. A feeding trial was conducted using 192 trout of about 100-g mean initial weight. Fish were fed in quadruplicate (4 tanks/diet) for 131 days with two diets: the control (Ctrl) contained 20% of FM as well as other protein sources, whereas the Hi diet contained 15% of Hi larvae meal to replace 50% of the FM contained in the Ctrl diet. High-throughput sequencing of 16S rRNA gene was used to identify the major feed and gut bacterial taxa, whereas Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis was performed on gut bacterial genomes to identify the major active biological pathways. The inclusion of IM led to an increase in Firmicutes, mainly represented by Bacilli class and to a drastic reduction of Proteobacteria. Beneficial genera, such as Lactobacillus and Bacillus, were enriched in the gut of fish fed with the Hi diet, whereas the number of bacteria assigned to the pathogenic Aeromonas genus was drastically reduced in the same fish group. The metagenome functional data provided evidence that dietary IM inclusion can shape the metabolic activity of trout gut microbiota. In particular, intestinal microbiome of fish fed with IM may have the capacity to improve dietary carbohydrate utilization. Therefore, H. illucens meal is a promising protein source for trout nutrition, able to modulate gut microbial community by increasing the abundance of some bacteria taxa that are likely to play a key role in fish health.
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http://dx.doi.org/10.1007/s10695-020-00918-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026480PMC
April 2021

Effect of dietary oil from on the growth performance, fillet fatty acid profile and gut microbiome of gilthead Sea bream ().

PeerJ 2020 9;8:e10430. Epub 2020 Dec 9.

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.

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, , may have a higher potential to replace FO since it can contains up to 40% of the omega-3 precursors -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 () 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 and , were associated with the 60% CO diet. However, lower abundance of lactic acid bacteria, specifically , 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 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.
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http://dx.doi.org/10.7717/peerj.10430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733328PMC
December 2020

Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva.

Biosens Bioelectron 2021 Jan 3;171:112686. Epub 2020 Oct 3.

University of Rome "Tor Vergata", Department of Chemical Science and Technologies, Via della Ricerca Scientifica, 00133, Rome, Italy; SENSE4MED, Via Renato Rascel 30, 00128, Rome, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bios.2020.112686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833515PMC
January 2021

Effects of Partially Defatted Meal in Rainbow Trout Diet on Hepatic Methionine Metabolism.

Animals (Basel) 2020 Jun 19;10(6). Epub 2020 Jun 19.

Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant, 3, 21100 Varese, Italy.

This study investigated, for the first time, the effects of replacement of fishmeal (FM) with insect meal from (HI) on the transcript levels of three genes involved in methionine (Met) metabolism in rainbow trout () liver. Two target genes-betaine-homocysteine S-methyltransferase () and S-adenosylhomocysteine hydrolase ()-are involved in Met resynthesis and the third one-cystathionine β synthase ()-is involved in net Met loss (taurine synthesis). We also investigated the levels of two Met metabolites involved in the maintenance of methyl groups and homocysteine homeostasis in the hepatic tissue: S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Three diets were formulated, an FM-based diet (HI0) and two diets in which 25% (HI25) and 50% (HI50) of FM was replaced with HI larvae meal. A 78-day feeding trial involved 360 rainbow trout with 178.9 ± 9.81 g initial average weight. Dietary replacement of up to 50% of FM with HI larvae meal, without any Met supplementation, did not negatively affect rainbow trout growth parameters and hepatic Met metabolism. In particular, Met availability from the insect-based diets directly modulated the transcript levels of two out of three target genes (, ) to maintain an optimal level of one-carbon metabolic substrates, i.e., the SAM:SAH ratio in the hepatic tissue.
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http://dx.doi.org/10.3390/ani10061059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341315PMC
June 2020

Can intestinal absorption of dietary protein be improved through early exposure to plant-based diet?

PLoS One 2020 4;15(6):e0228758. Epub 2020 Jun 4.

Center for Fisheries, Aquaculture, and Aquatic Sciences, Southern Illinois University, Carbondale, Illinois, United States of America.

Nutritional Programming (NP) has been studied as a means of mitigating the negative effects of dietary plant protein (PP), but the optimal timing and mechanism behind NP are still unknown. The objectives of this study were: 1) To determine whether zebrafish (Danio rerio) can be programmed to soybean meal (SBM) through early feeding and broodstock exposure to improve SBM utilization; 2) To determine if NP in zebrafish affects expression of genes associated with intestinal nutrient uptake; 3) To determine if early stage NP and/or broodstock affects gene expression associated with intestinal inflammation or any morphological changes in the intestinal tract that might improve dietary SBM utilization. Two broodstocks were used to form the six experimental groups. One broodstock group received fishmeal (FM) diet (FMBS), while the other was fed ("programmed with") SBM diet (PPBS). The first ((+) Control) and the second group ((-) Control) received FM and SBM diet for the entire study, respectively, and were progeny of FMBS. The last four groups consisted of a non-programmed (FMBS-X-PP and PPBS-X-PP) and a programmed group (FMBS-NP-PP and PPBS-NP-PP) from each of the broodstocks. The programming occurred through feeding with SBM diet during 13-23 dph. The non-control groups underwent a PP-Challenge, receiving SBM diet during 36-60 dph. During the PP-Challenge, both PPBS groups experienced significantly lower weight gains than the (+) Control group. NP in early life stages significantly increased the expression of PepT1 in PPBS-NP-PP, compared to PPBS-X-PP. NP also tended to increase the expression of fabp2 in the programmed vs. non-programmed groups of both broodstocks. The highest distal villus length-to-width ratio was observed in the dual-programmed group, suggesting an increase in surface area for nutrient absorption within the intestine. The results of this study suggest that NP during early life stages may increase intestinal absorption of nutrients from PP-based feeds.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272038PMC
August 2020

Assessment of dietary supplementation with galactomannan oligosaccharides and phytogenics on gut microbiota of European sea bass (Dicentrarchus Labrax) fed low fishmeal and fish oil based diet.

PLoS One 2020 16;15(4):e0231494. Epub 2020 Apr 16.

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162502PMC
July 2020

Nutritional programming improves dietary plant protein utilization in zebrafish Danio rerio.

PLoS One 2020 6;15(3):e0225917. Epub 2020 Mar 6.

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.

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 juvenile 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225917PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059923PMC
May 2020

Dietary phytogenics and galactomannan oligosaccharides in low fish meal and fish oil-based diets for European sea bass (Dicentrarchus labrax) juveniles: Effects on gut health and implications on in vivo gut bacterial translocation.

PLoS One 2019 18;14(9):e0222063. Epub 2019 Sep 18.

Grupo de Investigación en Acuicultura (GIA), IU-ECOAQUA, Universidad de Las Palmas de Gran Canaria, Crta. Taliarte s/n, Telde, Las Palmas, Canary Islands, Spain.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222063PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750610PMC
March 2020

Protective Effect of Dietary Taurine from ROS Production in European Seabass under Conditions of Forced Swimming.

Animals (Basel) 2019 Aug 26;9(9). Epub 2019 Aug 26.

Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy.

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 () growth and oxidative stress response induced by swimming exercise. Individually tagged fish of 92.57 ± 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 (MO) 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, 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.
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http://dx.doi.org/10.3390/ani9090607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770007PMC
August 2019

A First Attempt to Produce Proteins from Insects by Means of a Circular Economy.

Animals (Basel) 2019 May 24;9(5). Epub 2019 May 24.

Dipartimento di Biotecnologie e Scienze della Vita, Università dell'Insubria, 21100 Varese, Italy.

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 (BSF- 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.
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http://dx.doi.org/10.3390/ani9050278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562786PMC
May 2019

The Effects of Dietary Insect Meal from Prepupae on Autochthonous Gut Microbiota of Rainbow Trout ().

Animals (Basel) 2019 Apr 2;9(4). Epub 2019 Apr 2.

Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21100 Varese, Italy.

This study evaluated the effects of dietary insect meal from larvae on autochthonous gut microbiota of rainbow trout (). 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.
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http://dx.doi.org/10.3390/ani9040143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523354PMC
April 2019

Antibiotic treatment-induced dysbiosis differently affects BDNF and TrkB expression in the brain and in the gut of juvenile mice.

PLoS One 2019 22;14(2):e0212856. Epub 2019 Feb 22.

Department of Medicine and Surgery, University of Insubria, Varese, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212856PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386304PMC
November 2019

Effect of a specific composition of short- and medium-chain fatty acid 1-Monoglycerides on growth performances and gut microbiota of gilthead sea bream ().

PeerJ 2018 31;6:e5355. Epub 2018 Jul 31.

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.

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.
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http://dx.doi.org/10.7717/peerj.5355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6074759PMC
July 2018

Neurochemical characterization of myenteric neurons in the juvenile gilthead sea bream (Sparus aurata) intestine.

PLoS One 2018 3;13(8):e0201760. Epub 2018 Aug 3.

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201760PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075763PMC
February 2019

Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification, and the expression of genes involved in intestinal transport of minerals, osteoblast differentiation, and oxidative stress response.

Fish Physiol Biochem 2018 Oct 17;44(5):1375-1391. Epub 2018 Jun 17.

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.

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.
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http://dx.doi.org/10.1007/s10695-018-0528-7DOI Listing
October 2018

Next generation sequencing for gut microbiome characterization in rainbow trout (Oncorhynchus mykiss) fed animal by-product meals as an alternative to fishmeal protein sources.

PLoS One 2018 6;13(3):e0193652. Epub 2018 Mar 6.

VRM S.r.l. Naturalleva, Cologna Veneta, Verona, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193652PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839548PMC
June 2018

Inorganic, organic, and encapsulated minerals in vegetable meal based diets for (Linnaeus, 1758).

PeerJ 2017 27;5:e3710. Epub 2017 Oct 27.

Grupo de Investigación en Acuicultura (GIA), University Institute Ecoaqua, University of Las Palmas de Gran Canaria, Telde, Las Palmas, Canary Islands, Spain.

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 ( 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 ) and glutathione peroxidase 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.
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http://dx.doi.org/10.7717/peerj.3710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661455PMC
October 2017

Skin Mucus of Gilthead Sea Bream ( L.). Protein Mapping and Regulation in Chronically Stressed Fish.

Front Physiol 2017 1;8:34. Epub 2017 Feb 1.

Fish Pathology Group Group, Biology, Culture and Pathology of Marine Species, Institute of Aquaculture Torre de la Sal (IATS-CSIC) Castellón, Spain.

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.
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http://dx.doi.org/10.3389/fphys.2017.00034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288811PMC
February 2017

The expression of hypoxia-inducible factor-1α gene is not affected by low-oxygen conditions in yellow perch (Perca flavescens) juveniles.

Fish Physiol Biochem 2017 Jun 18;43(3):849-862. Epub 2017 Jan 18.

Department of Biotechnology and Life Sciences, University of Insubria, Via Dunant, 3, 21100, Varese, Italy.

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 O/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 O/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. Graphical abstract.
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http://dx.doi.org/10.1007/s10695-017-0340-9DOI Listing
June 2017

Effects of Sodium Butyrate Treatment on Histone Modifications and the Expression of Genes Related to Epigenetic Regulatory Mechanisms and Immune Response in European Sea Bass (Dicentrarchus Labrax) Fed a Plant-Based Diet.

PLoS One 2016 29;11(7):e0160332. Epub 2016 Jul 29.

Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Passeig Marítim, 37-49, 08003, Barcelona, Spain.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160332PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966935PMC
August 2017

Amino acid transporter B(0)AT1 (slc6a19) and ancillary protein: impact on function.

Pflugers Arch 2016 08 2;468(8):1363-74. Epub 2016 Jun 2.

Department of Biotechnology and Life Science, University of Insubria, Via J.H. Dunant 3, 21100, Varese, Italy.

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.
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http://dx.doi.org/10.1007/s00424-016-1842-5DOI Listing
August 2016

The Effect of Hypoxia and Hyperoxia on Growth and Expression of Hypoxia-Related Genes and Proteins in Spotted Gar Lepisosteus oculatus Larvae and Juveniles.

J Exp Zool B Mol Dev Evol 2016 06 31;326(4):250-67. Epub 2016 May 31.

School of Environment and Natural Resources, Ohio State University, Columbus, Ohio.

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.
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http://dx.doi.org/10.1002/jez.b.22680DOI Listing
June 2016

Activity, Expression, and Substrate Preference of the Δ(6)-Desaturase in Slow- or Fast-Growing Rabbit Genotypes.

J Agric Food Chem 2016 Feb 20;64(4):792-800. Epub 2016 Jan 20.

Department of Biotechnology and Life Sciences (DBSV), University of Insubria , Via J. H. Dunant 3, 21100 Varese, Italy.

In the present paper liver fatty acid Δ(6) desaturation (fads2) activity was analyzed in two rabbit strains with slow- (S, 27.5 g/day) or fast-growing (F, 48.5 g/day) rate. The fatty acid profile of the liver showed a different PUFA profile in the two strains with a lower n-6/n-3 ratio in the S rabbits. The expression of fads2 was 2-fold higher in S than in F rabbits, whereas enzyme activity was higher in F and more oriented toward the desaturation of linoleic acid (90%). In contrast, S showed a higher preference for linolenic acid (38.9 vs 10%). This study identified a single difference in the fads2 amino acid sequence between these two strains. Such a difference consists in the substitution of Gly104 to Ser104 in the sequence of F fads2. These results indicate for the first time that genetic selection for performance may affect the preference for PUFA toward desaturation of linoleic/linolenic acid.
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http://dx.doi.org/10.1021/acs.jafc.5b05425DOI Listing
February 2016

Expression profile of six stress-related genes and productive performances of fast and slow growing broiler strains reared under heat stress conditions.

Meta Gene 2015 Dec 31;6:17-25. Epub 2015 Aug 31.

University of Insubria, Department of Biotechnology and Life Sciences, Varese, Italy ; Inter-University Centre for Research in Protein Biotechnologies "The Protein Factory" - Polytechnic University of Milan and University of Insubria, Varese, Italy.

High temperature is one of the prominent environmental factors causing economic losses to the poultry industry as it negatively affects growth and production performance in broiler chickens. We used One Step TaqMan real time RT-PCR (reverse transcription polymerase chain reaction) technology to study the effects of chronic heat stress on the expression of genes codifying for the antioxidative enzymes superoxide dismutase (SOD), and catalase (CAT), as well as for heat shock protein (HSP) 70, HSP90, glucocorticoid receptor (NR3C1), and caspase 6 (CASP6) in the liver of two different broiler genetic strains: Red JA Cou Nu Hubbard (CN) and Ross 508 Aviagen (RO). CN is a naked neck slow growing broiler intended for the free range and/or organic markets, whereas RO is selected for fast growing. We also analysed the effect of chronic heat stress on productive performances, and plasma corticosterone levels as well as the association between transcriptomic response and specific SNPs (single nucleotide polymorphisms) in each genetic strain of broiler chickens. RO and CN broilers, 4 weeks of age, were maintained for 4 weeks at either 34 °C or 22 °C. The results demonstrated that there was a genotype and a temperature main effect on the broilers' growth from the 4th to the 8th week of age, but the interaction effect between genotype and temperature resulted not statistically significant. By considering the genotype effect, fast growing broilers (RO) grew more than the slow growing ones (CN), whereas by considering the temperature effect, broilers in unheated conditions grew more than the heat stressed ones. Corticosterone levels increased significantly in the blood of heat stressed broilers, due to the activation of the HPA (hypothalamic-pituitary-adrenocortical axis). Carcass yield at slaughter was of similar values in the 4 cohorts (genotype/temperature combinations or treatment groups), ranging from 86.5 to 88.6%, whereas carcass weight was negatively influenced by heat stress in both broiler strains. Heat stress affected gene expression by downregulating CASP6 and upregulating CAT transcript levels. HSPs, SOD and NR3C1 mRNA levels remained unaffected by heat stress. The differences found in the mRNA copies of CASP6 gene could be partly explained by SNPs.
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http://dx.doi.org/10.1016/j.mgene.2015.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556841PMC
December 2015

Modulation of the Expression of Components of the Stress Response by Dietary Arachidonic Acid in European Sea Bass (Dicentrarchus labrax) Larvae.

Lipids 2015 Oct 2;50(10):1029-41. Epub 2015 Aug 2.

Grupo de Investigación en Acuicultura (GIA), Parque Científico Tecnológico Marino de Taliarte, Universidad de Las Palmas de Gran Canaria (ULPGC), Muelle de Taliarte s/n, Telde, 35214, Las Palmas, Canary Islands, Spain.

This study reports for the first time on European sea bass, Dicentrarchus labrax (L.), larvae, the effect of different levels of dietary arachidonic acid (ARA; 20:4n-6) on the expression of genes related to the fish stress response. Copies of mRNA from genes related to steroidogenesis [StAR (steroidogenic acute regulatory protein), c-Fos, and CYP11β (11β-hydroxylase gene)], glucocorticoid receptor complex [GR (glucocorticoid receptor) and HSP (heat shock proteins) 70 and 90) and antioxidative stress (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase] were quantified. Eighteen day-old larvae were fed for 14 days with three experimental diets with increasing levels of ARA (0.3, 0.6 and 1.2% d.w.) and similar levels of docosahexaenoic (22:6n-3) and eicosapentaenoic (20:5n-3) acids (5 and 3%, respectively). The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real time RT-PCR with the standard curve method (absolute quantification). Increase dietary levels of ARA induced a significantly (p < 0.05) down-regulation of genes related to cortisol synthesis, such as StAR and CYP11β and up-regulated genes related to glucocorticoid receptor complex, such as HSP70 and GR. No effects were observed on antioxidant enzymes gene expression. These results revealed the regulatory role of dietary ARA on the expression of stress-related genes in European sea bass larvae.
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http://dx.doi.org/10.1007/s11745-015-4057-1DOI Listing
October 2015

Intestinal B(0)AT1 (SLC6A19) and PEPT1 (SLC15A1) mRNA levels in European sea bass (Dicentrarchus labrax) reared in fresh water and fed fish and plant protein sources.

J Nutr Sci 2015 20;4:e21. Epub 2015 May 20.

Department of Biotechnology and Life Sciences , University of Insubria , 21100 Varese , Italy ; Inter-University Centre for Research in Protein Biotechnologies , "The Protein Factory" , Polytechnic University of Milan and University of Insubria , Varese , Italy.

The objective of the present study was to examine the effect of diets with descending fish meal (FM) inclusion levels and the addition of salt to the diet containing the lowest FM level on growth performances, feed conversion ratio, and intestinal solute carrier family 6 member 19 (SLC6A19) and oligopeptide transporter 1 (PEPT1) transcript levels, in freshwater-adapted European sea bass (Dicentrarchus labrax). We first isolated by molecular cloning and sequenced a full-length cDNA representing the neutral amino acid transporter SLC6A19 in sea bass. The cDNA sequence was deposited in GenBank database (accession no. KC812315). The twelve transmembrane domains and the 'de novo' prediction of the three-dimensional structure of SLC6A19 protein (634 amino acids) are presented. We then analysed diet-induced changes in the mRNA copies of SLC6A19 and PEPT1 genes in different portions of sea bass intestine using real-time RT-PCR. Sea bass were fed for 6 weeks on different diets, with ascending levels of fat or descending levels of FM, which was replaced with vegetable meal. The salt-enriched diet was prepared by adding 3 % NaCl to the diet containing 10 % FM. SLC6A19 mRNA in the anterior and posterior intestine of sea bass were not modulated by dietary protein sources and salt supplementation. Conversely, including salt in a diet containing a low FM percentage up-regulated the mRNA copies of PEPT1 in the hindgut. Fish growth correlated positively with the content of FM in the diets. Interestingly, the addition of salt to the diet containing 10 % FM improved feed intake, as well as specific growth rate and feed conversion ratio.
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http://dx.doi.org/10.1017/jns.2015.9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462763PMC
June 2015

Modulation of adrenocorticotrophin hormone (ACTH)-induced expression of stress-related genes by PUFA in inter-renal cells from European sea bass (Dicentrarchus labrax).

J Nutr Sci 2015 4;4:e16. Epub 2015 May 4.

Universidad de Las Palmas de Gran Canaria (ULPGC) , Grupo de Investigación en acuicultura (GIA) , Instituto Universitario de Sanidad Animal y Seguridad Alimentaria (IUSA) , c/ Transmontaña , s/n , 35413 , Arucas , Las Palmas , Canary Islands , Spain.

Dietary fatty acids have been shown to exert a clear effect on the stress response, modulating the release of cortisol. The role of fatty acids on the expression of steroidogenic genes has been described in mammals, but little is known in fish. The effect of different fatty acids on the release of cortisol and expression of stress-related genes of European sea bass (Dicentrarchus labrax) head kidney, induced by a pulse of adenocorticotrophin hormone (ACTH), was studied. Tissue was maintained in superfusion with 60 min of incubation with EPA, DHA, arachidonic acid (ARA), linoleic acid or α-linolenic acid (ALA) during 490 min. Cortisol was measured by RIA. The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real-time RT-PCR. There was an effect of the type of fatty acid on the ACTH-induced release of cortisol, values from ALA treatment being elevated within all of the experimental period. The expression of some steroidogenic genes, such as the steroidogenic acute regulatory protein (StAR) and c-fos, were affected by fatty acids, ALA increasing the expression of StAR after 1 h of ACTH stimulation whereas DHA, ARA and ALA increased the expression of c-fos after 20 min. ARA increased expression of the 11β-hydroxylase gene. Expression of heat shock protein 70 (HSP70) was increased in all the experimental treatments except for ARA. Results corroborate previous studies of the effect of different fatty acids on the release of cortisol in marine fish and demonstrate that those effects are mediated by alteration of the expression of steroidogenic genes.
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http://dx.doi.org/10.1017/jns.2015.6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4463938PMC
June 2015

Proteomic profiling of sea bass muscle by two-dimensional gel electrophoresis and tandem mass spectrometry.

Fish Physiol Biochem 2014 Feb 21;40(1):311-22. Epub 2013 Sep 21.

Department of Biotechnology and Life Sciences (DBSV), University of Insubria, Via J. H. Dunant, 3, 21100, Varese, Italy,

In this study, the proteome profile of European sea bass (Dicentrarchus labrax) muscle was analyzed using two-dimensional electrophoresis (2-DE) and tandem mass spectrometry with the aim of providing a more detailed characterization of its specific protein expression profile. A highly populated and well-resolved 2-DE map of the sea bass muscle tissue was generated, and the corresponding protein identity was provided for a total of 49 abundant protein spots. Upon Ingenuity Pathway Analysis, the proteins mapped in the sea bass muscle profile were mostly related to glycolysis and to the muscle myofibril structure, together with other biological activities crucial to fish muscle metabolism and contraction, and therefore to fish locomotor performance. The data presented in this work provide important and novel information on the sea bass muscle tissue-specific protein expression, which can be useful for future studies aimed to improve seafood traceability, food safety/risk management and authentication analysis. This work is also important for understanding the proteome map of the sea bass toward establishing the animal as a potential model for muscular studies.
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http://dx.doi.org/10.1007/s10695-013-9855-xDOI Listing
February 2014
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