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.

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.

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.

HIF-1α mRNA levels in Eurasian perch (Perca fluviatilis) exposed to acute and chronic hypoxia.

The big advantage of using molecular biomarkers to monitor oxygen levels in aquatic systems is that responses at the molecular level tend to be more sensitive, and usually occur earlier than those at higher levels of biological organization Aquatic hypoxia is a frequent event, which can occur naturally in a variety of marine, estuarine and freshwater habitats. More often, however, hypoxia arises as a result of euthrophication of aquatic ecosystem and can lead to changes in community structure by eliminating hypoxia-sensitive species. Consequently fish have develop various physiological and biochemical mechanisms to cope with this environmental stress. Many of these adjustments depend to changes in expression of a wide range of genes. The transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor-1 (HIF-1), a heterodimer composed of an α and ß subunit. This study investigated if HIF-1α mRNA levels were regulated by hypoxia in Eurasian perch (Perca fluviatilis), a hypoxia-sensitive fresh water species. The real-time PCR was utilized to monitor dynamic changes in levels of HIF-1α mRNA in response to acute (DO 0.4 ± 0.1 mg/l for 1 h) and chronic (DO 2.8 ± 0.3 mg/l for 15 days) hypoxia. Our results indicated an up-regulation of HIF-1α in brain and liver, but not in muscle tissue after acute hypoxic treatment, whereas significant changes of HIF-1α mRNA levels were detected in muscle, but not in brain and liver after chronic hypoxia exposure. This study suggests that HIF-1α mRNA level in selected perch tissues could be an useful indicator of acute exposure to hypoxia.

Bio-Mos: an effective inducer of dicentracin gene expression in European sea bass (Dicentrarchus labrax).

Concern over the use of dietary antibiotics in aquaculture has encouraged the industry to search for alternatives that both enhance performance and afford protection from disease. Bio-Mos, derived from the outer cell wall of a specific strain of yeast Saccharomyces cerevisiae (Alltech Inc, USA) is a product that fits these criteria. Here, we present data on the impact of a Bio-Mos supplemented diet on the mRNA copy number of the antimicrobial peptide dicentracin, whose transcript regulation has not yet been explored in fish.We analyzed Bio-Mos-induced changes in the expression of sea bass (Dicentrarchus labrax) dicentracin,using a one-tube two-temperature real-time RT-PCR with which the gene expression can be absolutely quantified using the standard curve method. Our results revealed that 30 days of feeding fish with diets containing Bio-Mos supplemented at either 3 per thousand or 5 per thousand significantly increased the dicentracin mRNA copy number in the head kidney. Furthermore, the mRNA copy number in fish fed at 3 per thousand was significantly higher than that of the group fed at 5 per thousand for the same period of feeding Bio-Mos. A longer feeding period (60 days)did not further increase the dicentracin transcript levels as compared to the values recorded after 30 days of feeding either in the group fed at 3 per thousand or in the one fed at 5 per thousand diet. However, the transcript levels in fish fed at 3 per thousand proved to be significantly higher than those of the controls after 60 days of feeding. These findings offer new information about the response of antimicrobial peptides at the transcriptional level to diets supplemented with immune response modulators, and support a role of Bio-Mos in promoting sea bass nonspecific immune system.

Antibiotic susceptibility of coagulase-negative staphylococci isolated from goats' milk.

The antibiotic susceptibility of 70 strains of coagulase-negative staphylococci (CNS) isolated during the 2001 lactating period from the milk of dairy goats, was evaluated. The antibiotics tested were benzylpenicillin, cloxacillin, amoxicillin, amoxicillin plus clavulanic acid, cephalonium and cefoperazone, erythromycin and tylmicosin, kanamycin and tetracycline. Minimum inhibitory concentration (MIC) measurements showed that all beta-lactams (except cefoperazone) were effective against Staphylococcus epidermidis and Staphylococcus caprae, whereas the other antibiotics were either less effective or showed no activity. Other CNS species showed very variable sensitivity to the antibiotics; testing would be required before therapy for the clinical control of goat mammary infections.