The Replacement of Fish Meal with Poultry By-Product Meal and Insect Exuviae: Effects on Growth Performance, Gut Health and Microbiota of the European Seabass, Dicentrarchus labrax.

This study addressed the urgent need for sustainable protein sources in aquaculture due to the depletion of marine resources and rising costs. Animal protein sources, particularly poultry by-product meal (PBM) and insect exuviae meal, were investigated as viable alternatives to fishmeal (FM). The research study confirmed the successful replacement of FM with a combination of PBM and insect exuviae meal (up to 50%) in the diet of European seabass without compromising growth, feed conversion, gut health, and liver fat content. In particular, growth was robust with both PBM formulations, with the 25% PBM diet showing better results. Histological examinations showed good gut and liver health, contradicting the concerns of previous studies. This paper emphasizes the importance of holistic analyzes that go beyond growth parameters and include histomorphological investigations. The results show that PBM in combination with insect/exuviae meal is well tolerated by seabass, which is consistent with reports in the literature of it mitigating negative effects on gut health. A detailed analysis of the microbiota revealed a decrease in the Firmicutes/Proteobacteria ratio due to an increase in potentially pathogenic bacteria. However, the formulation containing insect exuviae partially counteracted this effect by preserving the beneficial Lactobacillus and promoting the synthesis of short-chain fatty acids (SCFAs), particularly butyrate. Chitin-rich components from insect exuviae were associated with improved gut health, which was supported by the increased production of SCFAs, which are known for their anti-inflammatory properties. This paper concludes that a combination of PBM and insect/exuviae meal can replace up to 50% of FM in the diet of seabass, supporting sustainable aquaculture practices. Despite some changes in the microbiota, the negative effects are mitigated by the addition of insect exuviae, highlighting their potential as a prebiotic to increase fish productivity and contribute to a circular economy in aquaculture.

The impact of diets containing Hermetia illucens meal on the growth, intestinal health, and microbiota of gilthead seabream (Sparus aurata).

The present study investigated the effect of replacing fishmeal (FM) with insect meal of Hermetia illucens (HI) in the diet of Sparus aurata farmed inshore on growth, gut health, and microbiota composition. Two isolipidic (18% as fed) and isoproteic (42% as fed) diets were tested at the farm scale: a control diet without HI meal and an experimental diet with 11% HI meal replacing FM. At the end of the 25-week feeding trial, final body weight, specific growth rate, feed conversion rate, and hepatosomatic index were not affected by the diet. Gross morphology of the gastrointestinal tract and the liver was unchanged and showed no obvious signs of inflammation. High-throughput sequencing of 16S rRNA gene amplicons (MiSeq platform, Illumina) used to characterize the gut microbial community profile showed that Proteobacteria, Fusobacteria, and Firmicutes were the dominant phyla of the gut microbiota of gilthead seabream, regardless of diet. Dietary inclusion of HI meal altered the gut microbiota by significantly decreasing the abundance of Cetobacterium and increasing the relative abundance of the Oceanobacillus and Paenibacillus genera. Our results clearly indicate that the inclusion of HI meal as an alternative animal protein source positively affects the gut microbiota of seabream by increasing the abundance of beneficial genera, thereby improving gut health and maintaining growth performance of S. aurata from coastal farms.

Polychaete (Alitta virens) meal inclusion as a dietary strategy for modulating gut microbiota of European seabass (Dicentrarchus labrax).

Recent research has revealed the significant impact of novel feed ingredients on fish gut microbiota, affecting both the immune status and digestive performance. As a result, analyzing the microbiota modulatory capabilities may be a useful method for assessing the potential functionality of novel ingredients. Therefore, this study aimed to evaluate the effects of dietary polychaete meal (PM) from Alitta virens on the autochthonous and allochthonous gut microbiota of European seabass (Dicentrarchus labrax). Two diets were compared: a control diet with 25% fishmeal (FM) and a diet replacing 40% of fishmeal with PM, in a 13-week feeding trial with juvenile fish (initial weight of 14.5 ± 1.0 g). The feed, digesta, and mucosa-associated microbial communities in fish intestines were analyzed using high-throughput sequencing of the 16S rRNA gene on the Illumina MiSeq platform. The results of feed microbiota analyses showed that the PM10 feed exhibited a higher microbial diversity than the FM diet. However, these feed-associated microbiota differences were not mirrored in the composition of digesta and mucosal communities. Regardless of the diet, the digesta samples consistently exhibited higher species richness and diversity than the mucosa samples. Overall, digesta samples were characterized by a higher abundance of Firmicutes in PM-fed fish. In contrast, at the gut mucosa level, the relative abundances of Mycobacterium, Taeseokella and Clostridium genera were lower in the group fed the PM10 diet. Significant differences in metabolic pathways were also observed between the FM and PM10 groups in both mucosa and digesta samples. In particular, the mucosal pathways of caffeine metabolism, phenylalanine metabolism, and sulfur relay system were significantly altered by PM inclusion. The same trend was observed in the digesta valine, leucine, and isoleucine degradation and secretion pathways. These findings highlight the potential of PM as an alternative functional ingredient in aquafeeds with microbiota modulatory properties that should be further explored in the future.

Aquaculture ecosystem microbiome at the water-fish interface: the case-study of rainbow trout fed with Tenebrio molitor novel diets.

BACKGROUND: Sustainable aquaculture relies on multiple factors, including water quality, fish diets, and farmed fish. Replacing fishmeal (FM) with alternative protein sources is key for improving sustainability in aquaculture and promoting fish health. Indeed, great research efforts have been made to evaluate novel feed formulations, focusing especially on the effects on the fish gut microbiome. Few studies have explored host-environment interactions. In the present study, we evaluated the influence of novel insect-based (Tenebrio molitor) fish diets on the microbiome at the water-fish interface in an engineered rainbow trout (Oncorhynchus mykiss) farming ecosystem. Using 16S rRNA gene metabarcoding, we comprehensively analyzed the microbiomes of water, tank biofilm, fish intestinal mucus, fish cutis, and feed samples. RESULTS: Core microbiome analysis revealed the presence of a highly reduced core shared by all sample sources, constituted by Aeromonas spp., in both the control and novel feed test groups. Network analysis showed that samples were clustered based on the sample source, with no significant differences related to the feed formulation tested. Thus, the different diets did not seem to affect the environment (water and tank biofilm) and fish (cutis and intestinal mucus) microbiomes. To disentangle the contribution of feed at a finer scale, we performed a differential abundance analysis and observed differential enrichment/impoverishment in specific taxa, comparing the samples belonging to the control diet group and the insect-based diet group. CONCLUSIONS: Omic exploration of the water-fish interface exposes patterns that are otherwise undetected. These data demonstrate a link between the environment and fish and show that subtle but significant differences are caused by feed composition. Thus, the research presented here is a step towards positively influencing the aquaculture environment and its microbiome.

Functional Additives in a Selected European Sea Bass (Dicentrarchus labrax) Genotype: Effects on the Stress Response and Gill Antioxidant Response to Hydrogen Peroxide (H2O2) Treatment.

Functional ingredients have profiled as suitable candidates for reinforcing the fish antioxidant response and stress tolerance. In addition, selective breeding strategies have also demonstrated a correlation between fish growth performance and susceptibility to stressful culture conditions as a key component in species domestication processes. The aim of the present study is to evaluate the ability of a selected high-growth genotype of 300 days post-hatch European sea bass (Dicentrarchus labrax) juveniles to use different functional additives as endogenous antioxidant capacity and stress resistance boosters when supplemented in low fish meal (FM) and fish oil (FO) diets. Three isoenergetic and isonitrogenous diets (10% FM/6% FO) were supplemented with 200 ppm of a blend of garlic and Labiatae plant oils (PHYTO0.02), 1000 ppm of a mixture of citrus flavonoids and Asteraceae and Labiatae plant essential oils (PHYTO0.1) or 5000 ppm of galactomannan-oligosaccharides (GMOS0.5). A reference diet was void of supplementation. The fish were fed the experimental diets for 72 days and subjected to a H2O2 exposure oxidative stress challenge. The fish stress response was evaluated through measuring the circulating plasma cortisol levels and the fish gill antioxidant response by the relative gene expression analysis of nfΚß2, il-1b, hif-1a, nd5, cyb, cox, sod, cat, gpx, tnf-1α and caspase 9. After the oxidative stress challenge, the genotype origin determined the capacity of the recovery of basal cortisol levels after an acute stress response, presenting GS fish with a better pattern of recovery. All functional diets induced a significant upregulation of cat gill gene expression levels compared to fish fed the control diet, regardless of the fish genotype. Altogether, suggesting an increased capacity of the growth selected European sea bass genotype to cope with the potential negative side-effects associated to an H2O2 bath exposure.

Sustainable Fish Feeds with Insects and Probiotics Positively Affect Freshwater and Marine Fish Gut Microbiota.

Aquaculture is the fastest-growing agricultural industry in the world. Fishmeal is an essential component of commercial fish diets, but its long-term sustainability is a concern. Therefore, it is important to find alternatives to fishmeal that have a similar nutritional value and, at the same time, are affordable and readily available. The search for high-quality alternatives to fishmeal and fish oil has interested researchers worldwide. Over the past 20 years, different insect meals have been studied as a potential alternate source of fishmeal in aquafeeds. On the other hand, probiotics-live microbial strains-are being used as dietary supplements and showing beneficial effects on fish growth and health status. Fish gut microbiota plays a significant role in nutrition metabolism, which affects a number of other physiological functions, including fish growth and development, immune regulation, and pathogen resistance. One of the key reasons for studying fish gut microbiota is the possibility to modify microbial communities that inhabit the intestine to benefit host growth and health. The development of DNA sequencing technologies and advanced bioinformatics tools has made metagenomic analysis a feasible method for researching gut microbes. In this review, we analyze and summarize the current knowledge provided by studies of our research group on using insect meal and probiotic supplements in aquafeed formulations and their effects on different fish gut microbiota. We also highlight future research directions to make insect meals a key source of proteins for sustainable aquaculture and explore the challenges associated with the use of probiotics. Insect meals and probiotics will undoubtedly have a positive effect on the long-term sustainability and profitability of aquaculture.

A Novel Approach in the Development of Larval Largemouth Bass Micropterus salmoides Diets Using Largemouth Bass Muscle Hydrolysates as the Protein Source.

This study's objectives were to determine the effect of Largemouth Bass (LMB) muscle hydrolysates obtained using same-species digestive enzymes and the degree of LMB muscle hydrolysis when included in the first feeds of growth performance and survival, skeletal development, intestinal peptide uptake, and muscle-free amino acid composition of larval LMB. LMB muscle was mixed with digestive enzymes from adult LMB, and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, the intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. Those diets were characterized by their level of each hydrolysate (presented as a ratio of 1.5, 3, and 6 Ts hydrolysates): 1:1:1, 1:3:6, 1:3:1, 6:3:1 for diets A, B, C, and D, respectively. To account for gut development, one group of larval LMB was fed a weekly series of diets B, C, and D to provide an increasing molecular weight profile throughout development. This group was compared against others that received either; (1) diets D, C, and B; (2) diet A; or (3) intact diet. The initial inclusion of the hydrolysates significantly improved the total length of the larval LMB; however, neither the hydrolysate inclusion nor the series of dietary molecular weight profiles improved the overall growth of larval LMB. The inclusion of hydrolysates significantly decreased the occurrence of skeletal deformities. The degree of hydrolysis did not have a significant effect on the parameters measured, except for intestinal peptide uptake, which was increased in the group that received the most hydrolyzed diet at the final time of sampling. The lack of overall growth improvement suggests that while the hydrolysates improve the initial growth performance, further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds for larval LMB.

Gill Oxidative Stress Protection through the Use of Phytogenics and Galactomannan Oligosaccharides as Functional Additives in Practical Diets for European Sea Bass (Dicentrarchus labrax) Juveniles.

The aim of the present study is to evaluate the potential of two functional additives as gill endogenous antioxidant capacity boosters in European sea-bass juveniles fed low-FM/FO diets when challenged against physical and biological stressors. For that purpose, two isoenergetic and isonitrogenous diets with low FM (10%) and FO (6%) contents were supplemented with 5000 ppm plant-derived galactomannan-oligosaccharides (GMOS) or 200 ppm of a mixture of garlic and labiate plant essential oils (PHYTO). A control diet was void from supplementation. Fish were fed the experimental diet for nine weeks and subjected to a confinement stress challenge (C challenge) or a confinement stress challenge combined with an exposure to the pathogen Vibrio anguillarum (CI challenge). Both GMOS and PHYTO diets attenuated fish stress response, inducing lower circulating plasma cortisol and down-regulating nfκß2 and gr relative gene-expression levels in the gill. This attenuated stress response was associated with a minor energetic metabolism response in relation to the down-regulation of nd5 and coxi gene expression.

How Different Dietary Methionine Sources Could Modulate the Hepatic Metabolism in Rainbow Trout?

In aquafeeds in which plant proteins are used to replace fishmeal, exogenous methionine (Met) sources are demanded to balance the amino acid composition of diets and meet the metabolic fish requirements. Nonetheless, since different synthetic Met sources are commercially available, it is important to determine their bioavailability and efficacy. To address this issue, we conducted a two-month feeding trial with rainbow trout (Oncorhynchus mykiss), which were fed diets supplemented with five different forms of Met: Met-Met, L-Met, HMTBa, DL-Met, and Co DL-Met. No differences in growth performance were found in trout fed with different Met forms, but changes in the whole-body composition were found. In particular, Met-Met and L-Met promoted a significant body lipid reduction, whereas the protein retention was significantly increased in fish fed with HMTBa and Co DL-Met. The latter affected the hepatic Met metabolism promoting the trans-sulfuration pathway through the upregulation of CBS gene expression. Similarly, the L-Met enhanced the remethylation pathway through an increase in BHMT gene expression to maintain the cellular demand for Met. Altogether, our findings suggest an optimal dietary intake of all tested Met sources with similar promoting effects on fish growth and hepatic Met metabolism. Nevertheless, the mechanisms underlying these effects warrant further investigation.

On the Compatibility of Fish Meal Replacements in Aquafeeds for Rainbow Trout. A Combined Metabolomic, Proteomic and Histological Study.

The sustainable development of modern aquaculture must rely on a significant reduction of the fish meal (FM) used in aquafeed formulations. However, FM substitution with alternative ingredients in diets for carnivorous fish species often showed reduced nutrient absorption, significantly perturbed metabolisms, and histological changes at both hepatic and intestinal levels. In the present study, rainbow trout (Oncorhynchus mykiss) were fed three different experimental aquafeeds. A control diet with higher FM content (27.3%) than two test formulations in which FM was substituted with two more sustainable and promising alternatives: insect meal (Hermetia illucens larvae = 10.1%, FM = 11.6%) and poultry by-products meal (PBM = 14.8%; FM = 11.7%). Combined metabolomics and proteomics analyses of fish liver, together with histological examination of liver and intestine demonstrated that a well-balanced formulation of nutrients in the three diets allowed high metabolic compatibility of either substitution, paving the way for a deeper understanding of the impact of novel raw materials for the fish feed industry. Results show that the main metabolic pathways of nutrient absorption and catabolism were essentially unaltered by alternative feed ingredients, and also histological alterations were negligible. It is demonstrated that the substitution of FM with sustainable alternatives does not have a negative impact on fish metabolism, as long as the nutritional requirements of rainbow trout are fulfilled.

Efficacy of Utilization of All-Plant-Based and Commercial Low-Fishmeal Feeds in Two Divergently Selected Strains of Rainbow Trout (Oncorhynchus mykiss): Focus on Growth Performance, Whole-Body Proximate Composition, and Intestinal Microbiome.

The present study aimed to investigate the growth performance, whole-body proximate composition, and intestinal microbiome of rainbow trout strains when selected and non-selected for weight gain on all-plant protein diets. A 2x2 factorial design was applied, where a selected (United States) and a non-selected (ITA) rainbow trout strain were fed using either an all-plant protein (PP) or a commercial low-FM diet (C). Diets were fed to five replicates of 20 (PP) or 25 (C) fish for 105 days. At the end of the trial, growth parameters were assessed, and whole fish (15 pools of three fish/diet) and gut samples (six fish/diet) were collected for whole-body proximate composition and gut microbiome analyses, respectively. Independent of the administered diet, the United States strain showed higher survival, final body weight, weight gain, and specific growth rate when compared to the ITA fish (p < 0.001). Furthermore, decreased whole-body ether extract content was identified in the PP-fed United States rainbow trout when compared to the ITA strain fed the same diet (p < 0.001). Gut microbiome analysis revealed the Cetobacterium probiotic-like genus as clearly associated with the United States rainbow trout, along with the up-regulation of the pathway involved in starch and sucrose metabolism. In summary, the overall improvement in growth performance and, to a lesser extent, whole-body proximate composition observed in the selected rainbow trout strain was accompanied by specific, positive modulation of the intestinal microbiome.

New value from food and industrial wastes - Bioaccumulation of omega-3 fatty acids from an oleaginous microbial biomass paired with a brewery by-product using black soldier fly (Hermetia illucens) larvae.

Research on bioconversion based on insects is intensifying as it addresses the problem of reducing and reusing food and industrial waste. To reach this goal, we need to find more means of pairing waste to insects. With this goal, brewers' spent grains (BSG) - a food waste of the brewing industry - paired with the oleaginous biomass of the thraustochytrid Schizochytrium limacinum cultivated on crude glycerol - a major waste of biodiesel production - were successfully used to grow Hermetia illucens larvae. Combining BSG and S. limacinum in the diet in an attempt to design the lipid profile of H. illucens larvae to contain a higher percentage of omega-3 fatty acids is novel. Insect larvae were grown on three different substrates: i) standard diet for Diptera (SD), ii) BSG, and iii) BSG + 10% S. limacinum biomass. The larvae and substrates were analyzed for fatty acid composition and larval growth was measured until 25% of insects reached the prepupal stage. Our data showed that including omega-3-rich S. limacinum biomass in the BSG substrate promoted an increase in larval weight compared to larvae fed on SD or BSG substrates. Furthermore, it was possible, albeit in a limited way, to incorporate omega-3 fatty acids, principally docosahexaenoic acid (DHA) from BSG + S. limacinum substrate containing 20% of DHA into the larval fat (7% DHA). However, H. illucens with this level of DHA may not be suitable if the aim is to get larvae with high omega-3 lipids to feed carnivorous fish.

Impact of polypropylene microplastics and chemical pollutants on European sea bass (Dicentrarchus labrax) gut microbiota and health.

Plastic pollution has become a global problem for marine ecosystems. Microplastics (MPs) are consumed by several marine organisms, including benthic and pelagic fish species that confuse them with food sources, thus contributing to bioaccumulation along the food chain. In addition to structural intestinal damage, ingestion of MPs represents a pathway for fish exposure to potentially hazardous chemicals, too. Most of them are endocrine disrupters, genotoxic or induce immune depression in fish. Accordingly, we assessed the combined toxicological effects of microplastics (MPs) and adsorbed pollutants by adding them to marine fish diet. European sea bass (Dicentrarchus labrax) juveniles were fed for 60 days with feeds containing polypropylene MPs, either virgin or contaminated with chemical pollutants (a blend of dichlorodiphenyldichloroethylene, chlorpyrifos, and benzophenone-3). The data demonstrated a synergic action of MPs and chemical pollutants to induce an inflammatory-like response in distal intestine of sea bass as shown by the up regulation of cytokine il-6 and tnf-α expression. Morphological analysis detected the presence of a focus of lymphocytes in anterior and posterior intestinal segments of fish fed with contaminants in the diet. With regard to microbiota, significant changes in bacterial species richness, beta diversity, and composition of gut microbiota were observed as a consequence of both pollutants and polluted MPs ingestion. These perturbations in gut microbial communities, including the reduction of beneficial lactic acid bacteria and the increase in potential pathogenic microorganism (Proteobacteria and Vibrionales), were undeniable signs of intestinal dysbiosis, which in turn confirmed the signs of inflammation caused by pollutants, especially when combined with MPs. The results obtained in this study provide, therefore, new insights into the potential risks of ingesting MPs as pollutant carriers in marine fish.

Effect of partial substitution of fishmeal with insect meal (Hermetia illucens) on gut neuromuscular function in Gilthead sea bream (Sparus aurata).

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.

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

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 (Dicentrarchus labrax). 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 ß-act, sod, gpx, cat, and gr in the gills of the fish compared with that in fish fed the control diet. The expression of hsp70 and ocln was upregulated and downregulated, respectively, in the GMOS group compared with that in the control group, whereas the expression of zo-1 was 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 D. labrax 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.

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

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.

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

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.

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.

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.

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

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.

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

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.