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 () 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 () 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 () 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

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

Acknowledgements Financial support for the trial on European sea bass was provided by the AQUAEXEL Project PROINSECTLIFE (Ref. No. 0013/03/05/15B), the AQUAEXEL Project INDIFISH (Ref. No. 0125/08/05/15/TNA), and by the University of Turin (ex 60%) Grant (Es. fn. 2014). NP (Scholarship Code: 1752) has been fnancially supported by the General Secretariat for Research and Technology (GSRT) of Greece and the Hellenic Foundation for Research and Innovation (HFRI) and MM by the Operational Programme “Human Resources Development, Education and Lifelong Learning” in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432) as implemented by the State Scholarships Foundation (ΙΚΥ). Tanks to Evelyn Argo and Craig Pattinson (University of Aberdeen) for providing help with 2DE. EM was fnancially supported by Marine Alliance for Science and Technology Scotland (MASTS) visiting Fellowship.Peer reviewedPublisher PD

Nutritional challenges and gastrointestinal microbiota in reared aquatic organisms

During the last decade, the rapid advancement in next-generation sequencing technologies has facilitated the structural and functional characterization of microbial communities, revealing a multitude of microorganisms that colonize the intestinal lumen in an endosymbiotic regime. The multifaceted contribution of gut microbiota to the proper functioning of the host-organism has attracted interest in a variety of animal organisms, with the majority of research focusing on the responsive modulation under environmental and/or dietary influence. In the context of intensifying and establishing a sustainable aquaculture industry, driven by a seafood production with mitigated ecological footprint, alternative to fishmeal protein sources in aquafeed are urgently needed. Therefore, the present thesis aims to clarify the impact of partial fishmeal substitution with insect-based meals (Tenebrio molitor, Hermetia illucens and Musca domestica) on the gut microbial ecology of three aquatic animal species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax), as well as the Baltic shrimp (Palaemon adspersus), a potential decapod crustacean candidate for the Greek aquaculture. Bacterial assessment in mesenteric samples was implemented by recruiting Illumina MiSeq sequencing technology, targeting the V3-V4 region of the 16S rRNA gene. Prior to the gut microbiota investigation, the present thesis focused on the determination of a sample size sufficient to yield statistically reliable representative population-level inferences in metabarcoding analysis of microbial communities in reared fish species. In this regard, sequencing results from 12 individuals per diet were randomized in all possible combinations of n-1 individuals and the probability of recording the mean abundance and diversity parameters was subsequently calculated. The present findings indicated that increasing sample size limits the dominance of individuals with high microbial load in the results and additionally provides the required statistical confidence for reliable estimations of diet-induced shifts in gut microbial communities. Furthermore, the results suggest that the primary aim of the study should drive the sample size selection, since three samples were proven herein sufficient for partial mean diversity estimation, whereas nine samples were deemed necessary to determine true species richness and detect changes in response to nutritional challenges. Partial fishmeal substitution with insect meals maintained the predominance of Firmicutes, Proteobacteria and Actinobacteria as the main bacterial phyla in the gut microbiota of the two fish species without significant changes in diversity indices. However, species-specific shifts were observed in response to the diets, with the microbial communities of S. aurata showcasing more prominent differentiation and inter-individual variability between dietary groups. Insect meals favored niche shaping within the communities, which contributed to the emergence and/or dominance of several beneficial bacteria, including lactic acid bacteria widely used in aquaculture as probiotics. Moreover, co-occurrence network analysis revealed a plethora of unique insect meal-induced bacterial interactions, crucial in defining the functionality and ecological stability of microbial communities. The predicted functional profile of gut microbiota showed significant differences in bacterial genes associated with miscellaneous processes, including metabolic pathways. In addition, the dietary-induced modulations of microbial communities seem to exert tissue-specific effects on basal intermediary metabolism, as indicated by the estimated enzymatic activity. In regard to P. adspersus, the preliminary assessment showcased the prevalence of Proteobacteria in the microbial communities, while enrichment of Firmicutes was apparent following partial substitution with insect meals potentially due to the presence of chitin as well as changes in the carbohydrate source. Among the examined insect meals, H. illucens putatively promoted microbial balance and favored physiological status indicators such as the Firmicutes to Bacteroidetes ratio, indirectly contributing to the improvement of P. adspersus growth. On the contrary, the results regarding T. molitor as a fishmeal substitute indicated alterations in the diversity and the evenness of microbial ecosystems, which may provoked gut dysbiosis and favored the emergence of opportunistic pathogenic and non-pathogenic bacteria. Conclusively, the present doctoral thesis provides critical findings relevant to the experimental design of future metabarcoding research in farmed fish species and underlines the significance in selecting a sufficient sample size to reliably imprint and elucidate the dietary effects on gut microbial communities. Furthermore, the present findings emphasize the impact of diet on the configuration and equilibrium of microbial communities and demonstrate that insect meals as feed ingredients may trigger species-specific modifications of the structural and functional dynamics of gut microbial communities.Κατά τη διάρκεια της τελευταίας δεκαετίας, η ραγδαία βελτίωση των τεχνολογιών αλληλούχισης νέας γενιάς έχει διευκολύνει το δομικό και λειτουργικό χαρακτηρισμό των μικροβιοκοινότητων υπό το πρίσμα περιβαλλοντικών και/ή διατροφικών επιδράσεων, αποκαλύπτοντας ένα πλήθος μικροοργανισμών που αποικίζουν τον εντερικό αυλό σε ένα καθεστώς ενδοσυμβίωσης. Στα πλαίσια της ανάπτυξης και εδραίωσης μιας βιώσιμης βιομηχανίας υδατοκαλλιεργειών, όπου επικρατεί η παραγωγή θαλασσινών προϊόντων με μειωμένο οικολογικό αποτύπωμα, επιτάσσεται η εύρεση εναλλακτικών πηγών πρωτεϊνών για τις ιχθυοτροφές. Ως εκ τούτου, η παρούσα διατριβή αποσκοπεί στην αποσαφήνιση του αντίκτυπου της μερικής υποκατάστασης του ιχθυάλευρου με άλευρα από προνύμφες εντόμων (Tenebrio molitor, Hermetia illucens και Musca domestica) στην μικροβιακή οικολογία του εντέρου τριών ειδών υδρόβιων ζωικών οργανισμών, της τσιπούρας (Sparus aurata) και του λαβρακιού (Dicentrarchus labrax), καθώς και ενός δυνητικά εκτρεφόμενου δεκάποδου καρκινοειδούς, της γαρίδας της Βαλτικής (Palaemon adspersus). Η βακτηριακή εκτίμηση στο DNA των μεσεντερικών δειγμάτων υλοποιήθηκε επιστρατεύοντας την τεχνολογία Illumina MiSeq για την αλληλούχιση της περιοχής V3-V4 του γονιδίου 16S rRNA.Προτού πραγματοποιηθεί η μελέτη των μικροβιακών κοινοτήτων, η παρούσα διατριβή εστίασε στον καθορισμό του δειγματοληπτικού μεγέθους που είναι επαρκές να αποδώσει με στατιστική ασφάλεια αντιπροσωπευτικά συμπεράσματα για το σύνολο του πληθυσμού σε μεταγονιδιωματικές αναλύσεις μικροβιοκοινοτήτων εκτρεφόμενων ιχθύων. Για το σκοπό αυτό, τα αποτελέσματα της αλληλούχισης από 12 άτομα ανά διατροφική ομάδα τυχαιοποιήθηκαν σε όλους τους πιθανούς συνδυασμούς n-1 ατόμων και ακολούθως υπολογίστηκε η πιθανότητα καταγραφής των μέσων παραμέτρων αφθονίας και ποικιλότητας. Τα ευρήματα της παρούσας διατριβής ανέδειξαν ότι η αύξηση του μεγέθους δείγματος μπορεί να περιορίσει την επικράτηση ατόμων με υψηλό μικροβιακό φορτίο στα αποτελέσματα και να εξασφαλίσει τη στατιστική εμπιστοσύνη που απαιτείται για την αξιόπιστη εκτίμηση των διατροφικά επαγόμενων μετατοπίσεων στις μικροβιοκοινότητες του εντέρου. Περαιτέρω, τα αποτελέσματα υποδεικνύουν ότι η επιλογή του δειγματοληπτικού μεγέθους οφείλει να συνάδει με τον πρωταρχικό σκοπό της έρευνας, δεδομένου ότι τρία δείγματα καθίστανται επαρκή για τη μερική εκτίμηση της μέσης ποικιλότητας, ενώ αντίθετα για τον προσδιορισμό του πραγματικού πλούτου ειδών και την καταγραφή μεταβολών ως απόκριση σε διατροφικές προκλήσεις απαιτείται αριθμός δειγμάτων που ανέρχεται στα εννέα. Η μερική υποκατάσταση με εντομοάλευρα διατήρησε την επικράτηση των Firmicutes, Proteobacteria και Actinobacteria ως τα κυρίαρχα βακτηριακά φύλα στις μικροβιακές κοινότητες των δύο ειδών ιχθύων χωρίς σημαντικές μεταβολές στους δείκτες ποικιλότητας. Εντούτοις, τα δύο είδη υπέδειξαν ειδο-ειδική απόκριση στις ιχθυοτροφές, με τις μικροβιακές κοινότητες του S. aurata να παρουσιάζουν μεγαλύτερη διαφοροποίηση και δια-ατομική διακύμανση μεταξύ των διατροφικών ομάδων. Τα εντομοάλευρα ευνόησαν τη δημιουργία οικοθέσεων στις κοινότητες για εμφάνιση ή και κυριαρχία διάφορων ωφέλιμων βακτηρίων, συμπεριλαμβανομένων βακτηρίων του γαλακτικού οξέος που χρησιμοποιούνται ευρέως στις υδατοκαλλιέργειες ως προβιοτικά. Η ανάλυση δικτύων συνύπαρξης ανέδειξε πληθώρα μοναδικών βακτηριακών αλληλεπιδράσεων που ευνοούνται από τα εντομοάλευρα και συμβάλλουν στον καθορισμό της λειτουργικότητας και στην οικολογική σταθερότητα των μικροβιοκοινοτήτων. Επιπρόσθετα, το προβλεπόμενο λειτουργικό προφίλ των εντερικών κοινοτήτων υπέδειξε σημαντικές διαφοροποιήσεις σε γονίδια που σχετίζονται με ποικίλες λειτουργίες, συμπεριλαμβανομένων των μεταβολικών μονοπατιών. Οι επαγόμενες από τα εντομοάλευρα αλλαγές στις μικροβιακές κοινότητες ενδέχεται να επηρέασαν ιστο-ειδικά το βασικό ενδιάμεσο μεταβολισμό, όπως παρατηρήθηκε σύμφωνα με την ενζυμική δραστικότητα. Όσον αφορά το P. adspersus, η προκαταρκτική εκτίμηση υπέδειξε την επικράτηση του φύλου Proteobacteria στις μικροβιακές κοινότητες, ενώ η μερική υποκατάσταση με τα εντομοάλευρα συνέβαλε στον εμπλουτισμό των Firmicutes ενδεχομένως λόγω της παρουσίας της χιτίνης και επιπρόσθετων αλλαγών στην πηγή των υδατανθράκων. Ανάμεσα στα υπό μελέτη εντομοάλευρα, η μερική υποκατάσταση με H. illucens πιθανώς προώθησε τη μικροβιακή ισορροπία και ευνόησε δείκτες της φυσιολογικής κατάστασης όπως ο λόγος Firmicutes προς Bacteroidetes, συμβάλλοντας έμμεσα στη βελτίωση της αύξησης του P. adspersus. Εν αντιθέσει, τα αποτελέσματα σχετικά με το T. molitor ως υποκατάστατο του ιχθυαλεύρου υπέδειξαν διαταραχές στην ποικιλότητα και ομοιομορφία των μικροβιακών οικοσυστημάτων, ευνοώντας την ανάπτυξη εντερικής δυσβίωσης και την εμφάνιση ευκαιριακών παθογόνων και μη βακτηρίων.Εν κατακλείδι, η παρούσα διατριβή παραθέτει σημαντικά ευρήματα για τον πειραματικό σχεδιασμό μελλοντικών μεταγονιδιωματικών μελετών σε εκτρεφόμενους ιχθείς και επισημαίνει την επιτακτική ανάγκη επιλογής επαρκούς δειγματοληπτικού μεγέθους, ικανού να αποτυπώσει με αξιοπιστία τις διατροφικές επιδράσεις στις εντερικές μικροβιακές κοινότητες. Περαιτέρω, τα παρόντα ευρήματα υπογραμμίζουν τη συμβολή της διατροφής στη διαμόρφωση και ισορροπία των μικροβιοκοινοτήτων και υποδεικνύουν ότι η χρήση των αλεύρων από διάφορα έντομα ως συστατικά των ζωοτροφών δύναται να προκαλούν ειδικές για το εκάστοτε είδος διαφοροποιήσεις της δομικής και λειτουργικής δυναμικής των μικροβιακών κοινοτήτων του εντέρου

In Vivo Effects of Lipopolysaccharide on Peroxisome Proliferator-Activated Receptor Expression in Juvenile Gilthead Seabream (Sparus Aurata)

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, β, 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, β, 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

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

A phytobiotic extract, in an aqueous or in a cyclodextrin encapsulated form, added in diet affects meat oxidation, cellular responses and intestinal morphometry and microbiota of broilers

The present trial examined the effects of diet supplementation with an extract including Greek oregano, garlic, rock samphire, and camelina, administered either in aqueous form or encapsulated in cyclodextrin, on broiler chickens. The duration of the trial was 35 days. Mixed broiler chicks (Ross-308, 120 individuals, 1 day old) were randomly allocated to one of three groups, each with four replicates. Control group A (CONTROL) was fed a basal diet consisting of maize and soybean. The diet of the AQORGCC and CDORGCC groups was further supplemented with aqueous and cyclodextrin-encapsulated herbal extracts, respectively. Levels of lipid and protein oxidation were determined in breast and thigh meat samples. Furthermore, to address cellular stress and signaling responses, the expression patterns of heat shock proteins (Hsp60, Hsp70, and Hsp90), mitogen-activated protein kinases (P38 and P44/42 MAPKs), and apoptotic-related proteins (Bcl-2/Bad ratio) were investigated in breast and thigh tissues using Western blot analysis. The intestinal morphometry of the duodenum, jejunum, and ileum was also assessed. To investigate ileal and cecal bacterial community diversity, 16S rRNA gene high-throughput amplicon sequencing on the V3–V4 hypervariable region was performed. The results showed that the herbal extract in cyclodextrin delayed meat lipid oxidation. According to the protein expression patterns, the formulated diets elicited tissue-specific cellular responses. Compared with the CONTROL group, dietary supplementation with the encapsulated form resulted in significant Hsp induction and MAPK activation, whereas, in the group whose diet was supplemented with the aqueous form, the expression of most of the examined proteins decreased or was maintained at a constant level. Villus height and lamina propria width were mostly affected by the aqueous herbal extract, whereas the number of goblet cells remained unchanged among the groups. Firmicutes, Proteobacteria, and Bacteroidota were the major phyla in mean relative abundance in all diets in both cecal and ileal samples. Alpha-diversity indices highlighted higher species richness and diversity in the cecum than in the ileum, as well as in chicks treated with the aqueous extract of the herbal mixture, but only in the cecum. Cecal beta-diversity differed between the cyclodextrin and the CONTROL groups, while ileal beta-diversity varied only between the aqueous-treated group and the CONTROL group. In conclusion, the dietary mixtures of herbal extracts (particularly those encapsulated in cyclodextrin) improved protein and lipid oxidation and increased the number of beneficial lactic acid-producing bacteria in the cecum, whereas the aqueous herbal extract mostly affected bacterial activity in the proximal part of the chicken intestine. Similarly, intestinal morphometry in the duodenum, jejunum, and ileum was mostly affected by the aqueous herbal extract, which seems to inhibit proteins associated with stress signaling in meat

Carob (<em>Ceratonia siliqua</em>) as Functional Feed Is Beneficial in Yellow Mealworm (<em>Tenebrio molitor</em>) 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