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In the context of evaluating the impact of environmentally friendly and sustainably produced alternative protein sources in fish feed, the present study's aim was to examine the overall physiological stress response in one of the main fish species of European freshwater aquaculture, Oncorhynchus mykiss (rainbow trout), following the partial substitution of fish meal (FM) with a Tenebrio molitor (TM) (yellow mealworm) full-fat meal. In total, 222 rainbow trout individuals (115.2 ± 14.2 g) were allocated randomly into six tanks, three per dietary treatment, and were fed a formulated diet containing 60% yellow mealworm (TM60) compared to a control diet without insect meal (TM0). Both diets contained equal amounts of crude protein, dry matter and, lipid content, while the FM in TM60 was 100 g kg-1 corresponding to the one seventh of the TM0. Heat shock response (HSR), MAPK signalling, cell death pathways (apoptosis and autophagy), antioxidant defence mechanisms, and intermediate metabolism were evaluated. In general, HSR and MAPK signalling were activated in response to the inclusion of T. molitor. Moreover, triggering of apoptotic and autophagic processes and the onset of antioxidant defence mechanisms underlined the existence of physiological stress. Despite the apparent dietary-induced stress, rainbow trout in the present study exhibited no mortality and no significant effects regarding growth performance parameters. Specifically, TM60 dietary inclusion resulted in no changes in final body weight, weight gain, and specific growth rate. However, feed intake depicted a statistically significant decrease in TM60 fish compared to TM0 individuals. Nevertheless, nutrient stress should be considered a limiting factor regarding the utilization of T. molitor in O. mykiss diet due to the associated risks for health and welfare.
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The present study investigates the effects of water temperature under prolonged feed deprivation on meagre's (Argyrosomus regius) antioxidant defense system and apoptotic pathway. Therefore, triplicate groups of fish were subjected to 4 water temperatures (17, 20, 23, 26 °C) and deprived of feed for 60 days. Heat shock response (HSR) indicators (Hsp70 and Hsp90), activation of MAPK family members, uniquitination, antioxidant enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), and apoptotic indicators such as caspases levels and the ratio of Bax/Bcl-2 were determined in liver and muscle samples that were collected on days 0, 14, 41 and 60. Long term feed deprivation and temperature variations resulted in granulomatosis, HSR induction, MAPK activation, ubiquitination, and triggering of the antioxidant defense and apoptotic machinery of the meagre. Although these responses were differentially expressed at each different imposed temperatures, the most significant variations were mainly observed below 20 °C and above 23 °C. Correlation analysis showed a significant relation between the examined indicators. Thus, the obtained data herein indicated that long term food deprivation induces oxidative stress in fish.
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Antioxidantes , Perciformes , Animais , Antioxidantes/metabolismo , Peixes/metabolismo , Estresse Oxidativo , Perciformes/fisiologia , Superóxido Dismutase/metabolismo , Temperatura , ÁguaRESUMO
The present study addresses the effects of dietary Tenebrio molitor (TM) larvae meal inclusion on cytoprotective, cell death pathways, antioxidant defence, and intermediate metabolism in the heart, muscle, and digestive tract of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax). Three experimental diets were formulated to contain 0%, 25%, or 50% inclusion TM levels. Heat Shock Proteins (HSPs) induction was apparent in both species' muscle at 50% inclusion. Conversely, p44/42 Mitogen-Activated Protein Kinase (MAPK) activation was increased (p < 0.05) in both species' muscle and digestive tract at 25% inclusion. Regarding the apoptotic machinery, TM inclusion exerted no influence on gilthead seabream, while suppression through autophagy may have occurred in the muscle. However, significant apoptosis (p < 0.05) was evident in European sea bass muscle and digestive tract. Both fish species' heart seemed to additionally rely on lipids compared to muscle and digestive tract. In contrast to gilthead seabream, European sea bass exhibited increased (p < 0.05) antioxidant activity at 50% TM inclusion. The present findings highlight the dietary derived induction of cellular responses in a species- and tissue-specific manner, whereas European sea bass appears to be more susceptible to TM inclusion.
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Cell growth and differentiation signals of insulin-like growth factor-1 (IGF-1), a key regulator in embryonic and postnatal development, are mediated through the IGF-1 receptor (IGF-1R), which activates several downstream pathways. The present study aims to address crucial organogenesis and development pathways including Akt, MAPKs, heat shock response, apoptotic and autophagic machinery, and energy metabolism in relation to IGF-1R activation during five developmental stages of reared Seriola dumerili: 1 day prior to hatching fertilized eggs (D-1), hatching day (D0), 3 days post-hatching larvae (D3), 33 (D33) and 46 (D46) days post-hatching juveniles. During both the fertilized eggs stage and larval-to-juvenile transition, IGF-1R/Akt pathway activation may mediate the hypertrophic signaling, while p44/42 MAPK phosphorylation was apparent at S. dumerili post-hatching processes and juvenile organs completion. On the contrary, apoptosis was induced during embryogenesis and autophagy at hatching day indicating a potential involvement in morphogenetic rearrangements and yolk-sac reserves depletion. Larvae morphogenesis was accompanied by a metabolic turnover with increased substantial energy consumption. The findings of the present study demonstrate the developmental stages-specific shift in critical signaling pathways during the ontogeny of reared S. dumerili.
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Perciformes , Proteínas Proto-Oncogênicas c-akt , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Mitógenos , Transdução de Sinais , Perciformes/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1 , Larva/metabolismo , Morte CelularRESUMO
The present study examined the effects of dietary supplementation with extracts of pomegranate (Punica granatum) and onion (Allium cepa), either encapsulated in cyclodextrin (POMALCD group) or in an aqueous (POMALAQ group) form, on breast meat, thigh meat, and liver composition, oxidative stability, cellular signaling pathways, and the gene expression of certain hepatic genes. The results showed that breast and thigh meat contained significantly (p < 0.05) higher moisture content in the group with the aqueous extract, compared to the control and POMALCD groups. Moreover, the protein content was significantly (p < 0.05) higher in the thigh and liver samples of the treated groups in comparison to the control. The iron-induced challenge deteriorated (p < 0.001) the lipid and protein oxidative status of the control group, whereas both supplemented groups showed considerable tolerance in all tissues. The supplementation of pomegranate and onion extracts mitigated or maintained heat shock protein (HSP) levels and elevated (p < 0.05) the Bcl-2/Bad ratio in thigh and breast meat, whereas mitogen-activated protein kinase (MAPK) activation was modulated at a lower rate. After normalization to ß-actin expression, quantitative real-time PCR analysis revealed a significant (p < 0.05) induction in the expression of MTR and MSRB1 genes in the liver of the supplemented groups. No differences were observed for the TAT, SMS, and BHMT genes. In conclusion, dietary mixtures of herbal extracts with pomegranate and onion improved protein and lipid oxidative stability in meat, enhanced the hepatic energy status, and exerted ameliorative effects on stress-related proteins. The encapsulated extract of pomegranate and onion, using cyclodextrin as a carrier, appeared to reduce lipid oxidation to a greater extent than the aqueous extract. In contrast, the aqueous extract exhibited higher total antioxidant capacity (TAC) values and provided better protection against protein carbonyl formation.
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Metamorphosis entails hormonally regulated morphological and physiological changes requiring high energy levels. Probiotics as feed supplements generate ameliorative effects on host nutrient digestion and absorption. Thereby, the aim of the present research was to investigate the impact of the probiotic Phaeobacter inhibens as a water additive on cellular signaling pathways in the metamorphosis of greater amberjack (Seriola dumerili). Activation of insulin-like growth factor type 1 receptor (IGF-1R), protein kinase B (Akt), mitogen-activated protein kinases (MAPKs) and AMP-activated protein kinase (AMPK), induction of heat shock proteins (Hsps), and programmed cell death were assessed through SDS-Page/immunoblot analysis, while energy metabolism was determined through enzymatic activities. According to the results, greater amberjack reared in P. inhibens-enriched water entered the metamorphic phase with greater body length, while protein synthesis was triggered to facilitate the hypertrophic growth as indicated by IGF-1/Akt activation and AMPK inhibition. Contrarily, MAPKs levels were reduced, whereas variations in Hsps response were evident in the probiotic treatment. Apoptosis and autophagy were mobilized potentially for the structural remodeling processes. Furthermore, the elevated enzymatic activities of intermediary metabolism highlighted the excess energy demands of metamorphosis. Collectively, the present findings demonstrate that P. inhibens may reinforce nutrient utilization, thus leading greater amberjack to an advanced growth and developmental state.
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Insects have been proposed as a rich alternative source of protein for the partial or total replacement of fishmeal in aquaculture. For maximum safety and effectiveness of insect meals, control of the quality composition of these products is considered mandatory. The aim of this study was the genetic analysis of the composition of commercially available insect meals at the species level. Commercially available Hermetia illucens, Tenebrio molitor and Musca domestica individuals, as well as nine insect meals produced from these species, were analyzed. The genetic identification of insects at the species level was based on a COI fragment, and analysis of the insect meals' composition was performed with the processes of cloning and colony PCR. Genetic analysis indicated that the commercially available larvae morphologically identified as Musca domestica belonged to the species Muscina stabulans. In the commercially available insect meals, no other animal species was identified beyond the expected one. However, in the insect meal produced for research purposes, fungal growth was detected. The used methodology, herein, allows for the qualitative genetic identification of insect meals and could be included in the methods of traceability of products containing insects and other animal species.
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This work assesses the dietary use of two insect meals of Tenebrio molitor (TM) larvae reared in conventional (TM-10) or MAP-enriched substrates (MAP-TM-10) as fish meal replacements (10%) in the diets of gilthead seabream (Sparus aurata). Fish (n = 4500; 207.19 ± 1.47 g) were divided into three groups with triplicates: control (fed conventional diet), TM-10, and MAP-TM-10 groups. The fish were reared in floating cages for 12 weeks and the dietary effects on white blood cell activation, heat shock proteins, MAPKs, and apoptosis of the fish were evaluated. The MAP-TM-10 group exhibited the highest eosinophilic induction. Phosphorylated levels of p38 MAPK, p44/42 MAPK, HSP70, and HSP90 increased in the TM-10 and MAP-TM-10 groups. In terms of apoptosis, Bax levels were lower in the TM groups compared to the control, and the MAP-TM-10 group showed even lower levels than the TM-10 group. Bcl-2 levels increased in the TM-10 group compared to the control, and further increased in the MAP-TM-10 group. The Bax/Bcl-2 ratio, an apoptosis indicator, decreased in the TM groups, with the MAP-TM-10 group showing a further decrease compared to TM-10. These findings suggest that insects' breeding substrate being enriched with MAPs modulated the effect of TM on cellular stress and apoptosis.
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Herein, the effect of dietary inclusion of insect (Tenebrio molitor) meal on hepatic pathways of apoptosis and autophagy in three farmed fish species, gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss), fed diets at 25%, 50% and 60% insect meal inclusion levels respectively, was investigated. Hepatic proteome was examined by liver protein profiles from the three fish species, obtained by two-dimensional gel electrophoresis. Although cellular stress was evident in the three teleost species following insect meal, inclusion by T. molitor, D. labrax and O. mykiss suppressed apoptosis through induction of hepatic autophagy, while in S. aurata both cellular procedures were activated. Protein abundance showed that a total of 30, 81 and 74 spots were altered significantly in seabream, European seabass and rainbow trout, respectively. Insect meal inclusion resulted in individual protein abundance changes, with less number of proteins altered in gilthead seabream compared to European seabass and rainbow trout. This is the first study demonstrating that insect meal in fish diets is causing changes in liver protein abundances. However, a species-specific response both in the above mentioned bioindicators, indicates the need to strategically manage fish meal replacement in fish diets per species.
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Ração Animal , Apoptose , Autofagia , Insetos Comestíveis/embriologia , Proteínas de Peixes/metabolismo , Pesqueiros , Peixes/metabolismo , Fígado/metabolismo , Proteoma , Tenebrio/embriologia , Animais , Bass/metabolismo , Larva , Valor Nutritivo , Oncorhynchus mykiss/metabolismo , Proteômica , Dourada/metabolismo , Especificidade da EspécieRESUMO
In terms of sustainability and circular economy, agricultural by-products may be efficiently reused in insects' rearing for high-quality protein sources in human diet and animal feeds. The present study aimed to explore whether the utilization of carob pods as feeding substrate may beneficially affect Tenebrio molitor's growth, nutritional value, antioxidant status and cellular responses. Increasing levels of milled whole carob pods (0, 25, 50, 75, 100%) were used as alternative wheat bran (control) substrates for yellow mealworm rearing, while growth performance, proximate composition, total phenolic content, antioxidant enzyme activity and the expression of stress- and apoptotic-related proteins were evaluated in larvae. The results showed that carob pods' content up to 75% did not significantly differentiate larvae weight, development time and total dry matter. Larvae total phenolic content and antioxidant activity exhibited a significant increase at 75% content. Although the antioxidant enzymes' activity decreased at both 25 and 50% levels, higher carob content levels (75 and 100%) resulted in no significant changes compared to the control. Carob pods led to decreased apoptotic indicators and the low expression of most stress-related proteins compared to the control. The present findings demonstrate that carob pods and their antioxidant properties exert beneficial effects on T. molitor's rearing and nutritional status, although 100% carob content may impact adversely the larvae due to the high amounts of carob tannins.
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Insect meals are considered promising, eco-friendly, alternative ingredients for aquafeed. Considering the dietary influence on establishment of functioning gut microbiota, the effect of the insect meal diets on the microbial ecology should be addressed. The present study assessed diet- and species-specific shifts in gut resident bacterial communities of juvenile reared Dicentrarchus labrax and Sparus aurata in response to three experimental diets with insect meals from three insects (Hermetia illucens, Tenebrio molitor, Musca domestica), using high-throughput Illumina sequencing of the V3-V4 region of the 16S rRNA gene. The dominant phyla were Firmicutes, Proteobacteria and Actinobacteria in all dietary treatments. Anaerococcus sp., Cutibacterium sp. and Pseudomonas sp. in D. labrax, and Staphylococcus sp., Hafnia sp. and Aeromonas sp. in S. aurata were the most enriched shared species, following insect-meal inclusion. Network analysis of the dietary treatments highlighted diet-induced changes in the microbial community assemblies and revealed unique and shared microbe-to-microbe interactions. PICRUSt-predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly differentiated, including genes associated with metabolic pathways. The present findings strengthen the importance of diet in microbiota configuration and underline that different insects as fish feed ingredients elicit species-specific differential responses of structural and functional dynamics in gut microbial communities.
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Sustainability, circular economy and alternative production systems are urgent imperatives for humanity and animal husbandry. Unless wasted, agri-food by-products can offer a promising source of high value. We evaluated the effect of rice bran (RB), corncob (CC), potato peels (PP), solid biogas residues (BR), and olive-oil processing residuals (OR), as alternative substrates to wheat bran (WB as control), on the growth and nutritional value of Tenebrio molitor during its breeding for animal feeds and/or human consumption. Innovation-wise, we further investigated the substrate supplementation (0, 10, 20%) with post-distillation residues of Mediterranean aromatic-medicinal plants (MAPs: lavender, Greek oregano, rosemary, olive; 1:1:1:1 ratio). Tenebrio molitor larvae (TML) were reared in all the studied substrates, and TML and diets' proximate and fatty acid compositions as well as total phenol and flavonoid content and antioxidant potential were assessed using standard procedures. After statistical analysis of correlations, we observed that CC promoted oviposition and progeny survival; larval weight and dry matter were positively affected mainly by dietary energy and fat content; number of TML and/or larval weight increased using 10% MAPs inclusion in WB, RB and OR or RB, OR, BR and PP, respectively, which did not affect protein content; TML fatty acid composition decreased the content of saturated ones and increased that of mono-unsaturated ones; MAPs residues had an apparent favorable impact on total phenolic content and antioxidant activity of each substrate, with RB displaying the highest capacity and content. These findings indicate that alternative substrates can be exploited and their enrichment with natural phenolics is able to influence T. molitor growth, offering highly beneficial and nutritional value.
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Despite the technical progress in high-throughput sequencing technologies, defining the sample size which is capable of yielding representative inferences in metabarcoding analysis still remains debatable. The present study addresses the influence of individual variability in assessing dietary effects on fish gut microbiota parameters and estimates the biological sample size that is sufficient to imprint a statistically secure outcome. European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) were fed three alternative animal protein diets and a fishmeal control diet. Gut microbiota data from 12 individuals per diet, derived from Illumina sequencing of the V3-V4 region of the 16S rRNA gene, were randomized in all possible combinations of n-1 individuals. Results in this study showcased that increasing the sample size can limit the prevalence of individuals with high microbial load on the outcome and can ensure the statistical confidence required for an accurate validation of dietary-induced microbe shifts. Inter-individual variability was evident in the four dietary treatments where consequently misleading inferences arose from insufficient biological replication. These findings have critical implications for the design of future metabarcoding studies and highlight the urgency in selecting an adequate sample size able to safely elucidate the dietary effects on fish gut microbial communities.
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Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Dieta/veterinária , Proteínas Alimentares/administração & dosagem , Proteínas de Peixes/administração & dosagem , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Animais , Bass , DouradaRESUMO
Fish are constantly exposed to microorganisms in the aquatic environment, many of which are bacterial pathogens. Bacterial pathogens activate the innate immune response in fish involving the production of pro-inflammatory molecules that, in addition to their immune-related role, can affect non-immune tissues. In the present study, we aimed at investigating how inflammatory responses can affect metabolic homeostasis in the gilthead seabream (Sparus aurata), a teleost of considerable economic importance in Southern European countries. Specifically, we mimicked a bacterial infection by in vivo administration of lipopolysaccharide (LPS, 6 mg/kg body weight) and measured metabolic parameters in the blood and, importantly, the mRNA expression levels of the three isotypes of peroxisome proliferator activated receptors (PPARα, ß, and γ) in metabolically-relevant tissues in seabream. PPARs are nuclear receptors that are important for lipid and carbohydrate metabolism in mammals and that act as biological sensors of altered lipid metabolism. We show here that LPS-induced inflammatory responses result in the modulation of triglyceride plasma levels that are accompanied most notably by a decrease in the hepatic mRNA expression levels of PPARα, ß, and γ and by the up-regulation of PPARγ expression only in adipose tissue and the anterior intestine. In addition, LPS-induced inflammation results in an increase in the hepatic mRNA expression and protein activity levels of members of the mitogen-activated protein kinase (MAPK) family, known in mammals to regulate the transcription and activity of PPARs. Our results provide evidence for the involvement of PPARs in the metabolic response to inflammatory stimuli in seabream and offer insights into the molecular mechanisms underlying the redirection of metabolic activities under inflammatory conditions in vertebrates.