Dietary Tenebrio molitor larvae meal effects on cellular stress responses, antioxidant status and intermediate metabolism of Oncorhynchus mykiss.

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.

Diet replacement with whole insect larvae affects intestinal morphology and microbiota of broiler chickens.

Insect-based diets are gaining interest as potential ingredients in improving poultry gut health. This study assessed the dietary treatment with whole dried Tenebrio molitor larvae (TM) on broiler chickens' gut microbiota and morphology. 120 Ross-308 broilers received treated diets with 5% (TM5) and 10% (TM10) replacement ratio in a 35-day trial. Intestinal histomorphometry was assessed, as well as claudin-3 expression pattern and ileal and caecal digesta for microbial community diversity. Null hypothesis was tested with two-way ANOVA considering the intestinal segment and diet as main factors. The TM5 group presented higher villi in the duodenum and ileum compared to the other two (P < 0.001), while treated groups showed shallower crypts in the duodenum (P < 0.001) and deeper in the jejunum and ileum than the control (P < 0.001). Treatments increased the caecal Firmicutes/Bacteroidetes ratio and led to significant changes at the genus level. While Lactobacilli survived in the caecum, a significant reduction was evident in the ileum of both groups, mainly owed to L. aviarius. Staphylococci and Methanobrevibacter significantly increased in the ileum of the TM5 group. Results suggest that dietary supplementation with whole dried TM larvae has no adverse effect on the intestinal epithelium formation and positively affects bacterial population richness and diversity.

Combined Dietary Supplementation of Tenebrio molitor Larvae and Chitosan in Growing Pigs: A Pilot Study.

Nowadays, the global animal industry faces considerable challenges in securing sufficient feed resources. Responding to consumer demands for reduced use of antibiotics in animal nutrition, better animal welfare status, and reduced impact on the environment, there is an increased urgency to develop innovative functional feeds with a reduced environmental footprint and the ability to improve meat quality and safety. In an effort to explore innovative feed ingredients for growing pig diets, the combined dietary supplementation of Tenebrio molitor larvae and chitosan was investigated. An experimental trial was performed with 48 weaned pigs (34 days of life; mixed sex) that were randomly assigned to four treatment groups (with six males and six females each): Group A (control), Group B (supplemented with T. molitor larvae at 10%), Group C (supplemented with chitosan at 0.05%), and Group D (supplemented with both ingredients at 10% and 0.05%, respectively). On the 42nd day of the experimental trial, samples of blood, feces, and carcass parts were taken for analysis. The results indicated that the insect larvae meal significantly improved (p < 0.05) overall performance, increased (p < 0.05) blood red blood cell content, increased meat phenolic content (p < 0.05), improved meat oxidative stability (p < 0.05), and affected meat fatty acid profile (p < 0.05). On the other hand, chitosan had no significant effect on overall performance (p > 0.05), but it significantly increased blood lymphocyte content (p < 0.05), affected the fecal microbiota (p < 0.05), improved meat oxidative stability (p < 0.05), increased meat phenolic content (p < 0.05), and affected meat fatty acid composition (p < 0.05) and (p < 0.05) meat color. Finally, the combined use of both T. molitor and chitosan significantly affected some important zootechnical parameters (p < 0.05), fecal microbial populations (p < 0.05), meat color (p < 0.05), and fatty acid profile (p < 0.05). Further investigation into the potential interaction between insect larvae meals and chitosan in pig diets is advised.

Investigating the role of genetic variation in vgll3 and six6 in the domestication of gilthead seabream (Sparus aurata Linnaeus) and European seabass (Dicentrarchus labrax Linnaeus).

Gene function conservation is crucial in molecular ecology, especially for key traits like growth and maturation in teleost fish. The vgll3 and six6 genes are known to influence age-at-maturity in Atlantic salmon, but their impact on other fish species is poorly understood. Here, we investigated the association of vgll3 and six6 in the domestication of gilthead seabream and European seabass, both undergoing selective breeding for growth-related traits in the Mediterranean. We analysed two different sets of samples using two different genotyping approaches. The first dataset comprised farmed and wild populations from Greece, genotyped for SNPs within the two genes ('gene-level genotyping'). The second dataset examined 300-600 k SNPs located in the chromosomes of the two genes, derived from a meta-analysis of a Pool-Seq experiment involving farmed and wild populations distributed widely across the Mediterranean ('chromosome-level genotyping'). The gene-level analysis revealed a statistically significant allele frequency differences between farmed and wild populations on both genes in each species. This finding was partially supported by the chromosome-level analysis, identifying highly differentiated regions may be involved in the domestication process at varying distances from the candidate genes. Noteworthy genomic features were found, such as a CpG island in gilthead seabream and novel candidate genes in European seabass, warranting further investigation. These findings support a putative role of vgll3 and six6 in the maturation and growth of gilthead seabream and European seabass, emphasizing the need for further research on their conserved function.

Dietary Supplementation with Pomegranate and Onion Affects Lipid and Protein Oxidation in the Breast Meat, Thigh, and Liver, Cellular Stress Protein Responses, and Gene Expression of Liver Enzymes Involved in Protein Synthesis in Broilers.

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.

Breeding Substrate Containing Distillation Residues of Mediterranean Medicinal Aromatic Plants Modulates the Effects of Tenebrio molitor as Fishmeal Substitute on Blood Signal Transduction and WBC Activation of Gilthead Seabream (Sparus aurata).

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.

The Probiotic Phaeobacter inhibens Provokes Hypertrophic Growth via Activation of the IGF-1/Akt Pathway during the Process of Metamorphosis of Greater Amberjack (Seriola dumerili, Risso 1810).

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.

Genetic Identification and Traceability of Insect Meals.

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.

Utilization of Tenebrio molitor Larvae Reared with Different Substrates as Feed Ingredients in Growing Pigs.

The procurement of adequate feed resources is one of the most important challenges for the animal industry worldwide. While the need for feeds rich in protein is constantly increasing, their production cannot readily keep up. Consequently, to overcome this challenge in a sustainable way, it is necessary to identify and develop new feeding strategies and feed ingredients, such as insect meals. In the present study, Tenebrio molitor larvae that were reared on two different substrates (standard and enriched with medicinal aromatic plant material) were used as feed ingredients for growing pigs. A total of 36 weaned pigs (34 days old) were randomly allocated to three treatment groups and fed either the control diet (A) or diets supplemented at 10% with one of the two insect meals (B and C). At the end of the trial (42 days), blood, feces, and meat samples were collected for analysis. The insect meal supplementation did not affect (p > 0.05) overall performance but significantly modified (p < 0.001) the fecal microflora balance and the blood cholesterol (p < 0.001), while the rest of the blood parameters tested were not affected. Moreover, this dietary supplementation significantly affected some microbial populations (p < 0.001), improved the total phenolic content (p < 0.05), and the fatty acid profile (p < 0.001) of the meat cuts, but did not affect (p > 0.05) meat color or proximate composition. Further research is needed to evaluate the different types and levels of inclusion of insect meals in pig nutrition.

Advances in understanding the mitogenic, metabolic, and cell death signaling in teleost development: the case of greater amberjack (Seriola dumerili, Risso 1810).

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.

Carob (Ceratonia siliqua) as Functional Feed Is Beneficial in Yellow Mealworm (Tenebrio molitor) Rearing: Evidence from Growth, Antioxidant Status and Cellular Responses.

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.

Synergistic effect of long-term feed deprivation and temperature on the cellular physiology of meagre (Argyrosomus regius).

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.

Tenebrio molitor larvae meal inclusion affects hepatic proteome and apoptosis and/or autophagy of three farmed fish species.

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.

Dietary Tenebrio molitor Larvae Meal Inclusion Exerts Tissue-Specific Effects on Cellular, Metabolic, and Antioxidant Status in European Sea Bass (Dicentrarchus labrax) and Gilthead Seabream (Sparus aurata).

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.

Sex-biased dynamics of three-spined stickleback (Gasterosteus aculeatus) gene expression patterns.

The study of the differences between sexes presents an excellent model to unravel how phenotypic variation is achieved from a similar genetic background. Sticklebacks are of particular interest since evidence of a heteromorphic chromosome pair has not always been detected. The present study investigated sex-biased mRNA and small non-coding RNA (sncRNA) expression patterns in the brain, adipose tissues, and gonads of the three-spined stickleback. The sncRNA analysis indicated that regulatory functions occurred mainly in the gonads. Alleged miRNA-mRNA interactions were established and a mapping bias of differential expressed transcripts towards chromosome 19 was observed. Key players previously shown to control sex determination and differentiation in other fish species but also genes like gapdh were among the transcripts identified. This is the first report in the three-spined stickleback demonstrating tissue-specific expression comprising both mRNA and sncRNA between sexes, emphasizing the importance of mRNA-miRNA interactions as well as new presumed genes not yet identified to have gender-specific roles.

Antibacterial Effects of Essential Oils of Seven Medicinal-Aromatic Plants Against the Fish Pathogen Aeromonas veronii bv. sobria: To Blend or Not to Blend?

Despite progress achieved, there is limited available information about the antibacterial activity of constituents of essential oils (EOs) from different medicinal-aromatic plants (MAPs) against fish pathogens and the complex interactions of blended EOs thereof. The present study aimed to investigate possible synergistic antimicrobial effects of EOs from seven Greek MAPs with strong potential against Aeromonas veronii bv. sobria, a fish pathogen associated with aquaculture disease outbreaks. The main objective was to evaluate whether blending of these EOs can lead to increased antimicrobial activity against the specific microorganism. A total of 127 combinations of EOs were prepared and their effect on A. veronii bv. sobria growth was tested in vitro. We examined both the inhibitory and bactericidal activities of the individual EOs and compared them to those of the blended EOs. The vast majority of the investigated combinations exhibited significant synergistic and additive effects, while antagonistic effects were evident only in a few cases, such as the mixtures containing EOs from rosemary, lemon balm and pennyroyal. The combination of EOs from Greek oregano and wild carrot, as well as the combinations of those two with Spanish oregano or savoury were the most promising ones. Overall, Greek oregano, savoury and Spanish oregano EOs were the most effective ones when applied either in pure form or blended with other EOs.

Configuration of Gut Microbiota Structure and Potential Functionality in Two Teleosts under the Influence of Dietary Insect Meals.

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.

Towards Functional Insect Feeds: Agri-Food By-Products Enriched with Post-Distillation Residues of Medicinal Aromatic Plants in Tenebrio molitor (Coleoptera: Tenebrionidae) Breeding.

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.

Gut Microbiota of Five Sympatrically Farmed Marine Fish Species in the Aegean Sea.

In this study, we hypothesized that sympatrically grown farmed fish, i.e. fish which experience similar environmental conditions and nutritionally similar diets, would have more convergent gut microbiota. Using a "common garden" approach, we identified the core microbiota and bacterial community structure differences between five fish species farmed in the same aquaculture site on the west coast of the Aegean Sea, Greece. The investigated individuals were at similar developmental stages and reared in adjacent (< 50 m) aquaculture cages; each cage had 15 kg fish m-3. The diets were nutritionally similar to support optimal growth for each fish species. DNA from the midgut of 3-6 individuals per fish species was extracted and sequenced for the V3-V4 region of the bacterial 16S rRNA. Only 3.9% of the total 181 operational taxonomic units (OTUs) were shared among all fish. Between 5 and 74 OTUs were unique to each fish species. Each of the investigated fish species had a distinct profile of dominant OTUs, i.e. cumulative relative abundance of ≥ 80%. Co-occurrence network analysis for each fish species showed that all networks were strongly dominated by positive correlations between the abundances of their OTUs. However, each fish species had different network characteristics suggesting the differential significance of the OTUs in each of the five fish species midgut. The results of the present study may provide evidence that adult fish farmed in the Mediterranean Sea have a rather divergent and species-specific gut microbiota profile, which are shaped independently of the similar environmental conditions under which they grow.

Imprinting statistically sound conclusions for gut microbiota in comparative animal studies: A case study with diet and teleost fishes.

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.