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.

Lack of negative effects of fasting of gilthead seabream (Sparus aurata) breeders during the spawning period on maternal and egg nutrient composition, fertilization success, and early embryo/larval development.

The effect of fasting on spawning performance, maternal, and egg nutrient composition, and on embryo/larval development was monitored in gilthead seabream (Sparus aurata). Two broodstocks were fasted during two consecutive years, for a period of 43 and 54 days within the spawning season, in a preliminary (year 1, 5-year-old breeders) and the main study (year 2, 6-year-old breeders), respectively. Mean daily fecundity showed a declining trend during fasting in the main study only, while fertilization success was high in both years and it was not affected by fasting, as was hatching and 5-day larval survival. There was a loss of 23.5% of maternal body mass due to fasting, and a reduction in gonadosomatic and hepatosomatic indexes, as well as crude protein in maternal muscle and gonads, but not in the liver. After fasting, muscle Σω-6 PUFA and C18:3ω-3 were reduced while C20:4ω-6, 20:5ω-3/20:4ω-6, and C22:6ω-3/20:4ω-6 increased; in the liver, significant reductions were observed in C16:0, C18:3 ω-3, 20:5ω-3/C22:6ω-3 and increases in C18:0, C20:5ω-3, Σω-6 PUFA, and 20:5ω-3/20:4ω-6; in gonads, C15:0, ΣMUFA, 20:5ω-3/C22:6ω-3, 20:5ω-3/20:4ω-6 were increased, while C18:1ω-9 and C20:5ω-3 decreased. Contrary to maternal tissues, the energy density and proximate composition of the eggs did not change due to fasting. The study suggests that fasting of gilthead seabream breeders for 6-8 weeks during the spawning period does not affect spawning performance, egg proximate composition, or embryo and early larval development since maternal nutrient reserves are mobilized to maintain optimal egg nutrient composition.

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.

Understanding the Interaction Effects between Dietary Lipid Content and Rearing Temperature on Growth Performance, Feed Utilization, and Fat Deposition of Sea Bass (Dicentrarchus labrax).

This study was conducted to elucidate the interaction effects of temperature and dietary lipid levels (2 × 2 factorial experiment) on the growth performance, muscle, and liver composition in adult farmed European sea bass (Dicentrarchus labrax). Two groups of fish (190 g; 60 fish per group) were distributed in 12 tanks in triplicates and kept at two different temperature regimes; one starting at 23 °C and then changed to 17 °C for 61 days, and the other starting at 17 °C and then changed to 23 °C for 39 days. Two commercial diets containing both ~44% crude protein but incorporating different dietary lipid levels, 16.5% (D16) and 20.0% (D20) (dry matter (DM)), were fed to the fish to apparent satiation; the type of diet fed to each fish group remained constant throughout the experiment. Final body weight, weight gain, and specific growth rate were significantly higher for the fish group held at 23 °C compared to the fish group at 17 °C (before the temperature changes), while the dietary fat content did not have any profound effect in both groups. Furthermore, the different temperature regimes did not affect muscle or liver composition, but, on the contrary, dietary lipids affected hepatosomatic, perivisceral fat, and visceral indexes. Feed conversion ratio and specific growth rate were not affected by the dietary lipid level. An interaction of temperature and dietary lipid content was observed in daily feed consumption (DFC) and final body weight (FBW).

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.

Effect of water temperature on cellular stress responses in meagre (Argyrosomus regius).

Μeagre (Argyrosomus regius) is a newly introduced species in the aquaculture sector characterized by high growth rates and good nutritional value. However, the optimum conditions such as water temperature for the rearing of this species are still under research. The aim of the present study was to investigate several biochemical responses in the heart, muscle, liver, and intestine of meagre reared under different water temperatures (17, 23, and 26 °C) in comparison to the initial acclimation temperature at 20 °C. To assess its cellular stress response, the molecular markers of Hsp70 and Hsp90 induced levels, p38 MAPK and p44/42 MAPK phosphorylated levels, ubiquitin conjugates, and Bcl-2 levels were determined. Moreover, to investigate its antioxidant defense, the activities of superoxide dismutase, catalase, and glutathione reductase were measured. The obtained results in meagre showed a distinct tissue-specific response regarding the variations observed in the studied parameters under different acclimation temperatures. Specifically, most examined tissues presented a decrease in the levels of the examined markers compared to 20 °C, while in the intestine, an increase of Hsp70 and p38 MAPK levels was observed at 23 and 26 °C. However, data on the final weight and length of the fish lead to the conclusion that high water temperatures such as 26 °C might be the best for the rearing of this species. Therefore, it is assumed that the observed differential molecular responses may act in a cell-protective way at all temperatures, irrespectively of the effect of temperature on growth parameters.

Honey Bee Pollen in Meagre (Argyrosomus regius) Juvenile Diets: Effects on Growth, Diet Digestibility, Intestinal Traits, and Biochemical Markers Related to Health and Stress.

This research aimed to evaluate the effects of the inclusion of honey bee pollen (HBP) in meagre (Argyrosoumus regius) juveniles' diets on growth performance, diet digestibility, intestinal morphology, and immunohistochemistry. Furthermore, stress-related molecular markers and biochemical blood profile of fish were evaluated, together with mineral trace and toxic element concentration in pollen and diets. Specimens of meagre (360) of 3.34 ± 0.14 g initial body weight, were randomly allocated to twelve 500 L circular tanks (30 fish per tank). Four diets were formulated: a control diet and three experimental diets with 1%, 2.5%, and 4% of HBP inclusion. All the growth parameters and crude protein and ether extract digestibility coefficients were negatively linearly affected by increased HBP inclusion (p < 0.0001). Histology of medium intestine showed slight signs of alterations in group HPB1 and HPB2.5 compared to control. Fish from HBP4 group showed severe alterations at the intestinal mucosa level. Immunohistochemical detection of TNF-α in the medium intestine showed the presence of TNF-α+ cells in the lamina propria, which resulted in accordance with the increased level of the TNF-α protein detected by immunoblotting in the liver. This stress situation was confirmed by the increased hepatic level of HSP70 (p < 0.05) in fish fed the HBP4 diet and by the linear decrease of total serum protein levels in HBP-containing diets (p < 0.0001). These negative effects can be related to the ultrastructure of the bee pollen grain walls, which make the bioactive substances unavailable and can irritate the intestine of a carnivorous fish such as meagre.

Metals and elements in sardine and anchovy: Species specific differences and correlations with proximate composition and size.

Species - specific differences in the levels of 26 metals and elements in sardine and anchovy are investigated and the factors of proximate composition (proteins, lipids, ash) and body size (length, weight) that may affect the metal and elemental concentrations in fish are explored. Statistical analysis revealed that levels of metals and elements in fish seem to strongly depend on species. Significantly higher levels of most of the metals and elements studied have been observed in anchovy compared to sardine at each of six different sites. The observed species-specific differences in metal content could be attributed to different proximate composition and size among other factors. The significant (positive or negative) correlations found between lipids, proteins, ash, weight, length and metals or elements, suggest that higher levels of most of the metals and elements studied can be associated with lower lipid content, greater protein and ash content and smaller size.

Effects of long-term feed deprivation on body weight loss, muscle composition, plasma metabolites, and intermediate metabolism of meagre (Argyrosomus regius) under different water temperatures.

The effect of feed deprivation at four water temperatures (17, 20, 23, 26 °C) was investigated in meagre (Argyrosomus regius) of initial mean weight ± SD, 116.16 ± 4.74 g, in triplicate groups. Fish were deprived of feed for a period of 60 days and sampled on days 0, 14, 41, and 60, during which body weight, specific growth rate, somatic indices, muscle proximate composition, plasma metabolite levels (total lipids, proteins, cholesterol, triglycerides, glucose), and liver and muscle enzymatic activities [L-lactate dehydrogenase (L-LDH), citrate synthase (CS), malate dehydrogenase (MDH)] were evaluated. Long-term feed deprivation resulted in a significant decrease in body weight, condition factor (CF), hepatosomatic index (HSI), muscle lipids, and plasma metabolites (all except proteins) and increase in muscle moisture. Plasma glucose concentration decreased with time and became significantly lower at 41 and 60 days. Glucose concentration and weight loss expressed a different response in the short term (14 days) than in the long term (14 and 60 days) of feed deprivation, suggesting a change in glucose metabolic profile. After 60 days of feed deprivation, there was an increase in the L-LDH activity in the liver at all temperature levels, which reflects a rising glycolytic potential by activating the carbohydrate metabolism and an ATP-dependent demand. MDH activity increased in the liver and muscle, except at 17 °C in the muscle, which indicates aerobic glycolysis and lipolysis. CS activity in the liver increased after the 60 days, whereas that in the muscle decreased, indicating the muscle is less dependent on aerobic oxidation for energy reserves.

Effects of partial dietary supplementation of fish meal with soymeal on the stress and apoptosis response in the digestive system of common dentex (Dentex dentex).

BACKGROUND: Soybean is a common alternative protein source of plant origin in aquafeeds as it has a reasonably balanced amino acid profile and is widely available. This study aimed to investigate the influence of partial substitution of fish meal with soy meal on cytoprotective pathways and apoptosis in the digestive system of common dentex (Dentex dentex), using the activation of Hsp70, p38 MAPK, Bcl-2 and caspase-3. The experimental approach involved feeding of common dentex with three isoprotein and isoenergetic diets that contained fish meal as a protein source (FM), partial replacement of fish meal by soy meal 25% (SM25) and 40% (SM40) for 3 months. RESULTS: The SM40 diet induced Hsp70 activation only in the middle part of intestine. On the other hand, both SM25 and SM40 diets diminished the phosphorylation of p38 MAPK in the anterior and the middle part of intestine, whereas only SM25 induced p38 MAPK phosphorylation in the stomach. Moreover, a decrease in the levels of caspase-3 activity was observed in the middle and posterior intestine, as well as in the stomach after feeding with SM25 diets. Furthermore, Bcl-2 levels were increased by SM40 in the anterior and by SM25 in the middle part of intestine. CONCLUSIONS: SM25 and SM40 diets elicited a tissue and soy concentration specific cellular and cell protective response in the different parts of the digestive tract in common dentex.

Identification, Phylogeny, and Function of fabp2 Paralogs in Two Non-Model Teleost Fish Species.

Intestinal fatty-acid-binding protein (IFABP or FABP2) is a cytosolic transporter of long-chain fatty acids, which is mainly expressed in cells of intestinal tissue. Fatty acids in teleosts are an important source of energy for growth, reproduction, and swimming and a main ingredient in the yolk sac of embryos and larvae. The fabp2 paralogs, fabp2a and fabp2b, were identified for 26 teleost fish species including the paralogs for the two non-model teleost fish species, namely the gilthead sea bream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Despite the high similarity of fabp2 paralogs, as well as the identical organization in four exons, paralogs were mapped to different chromosomes/linkage groups supporting the hypothesis that the identified transcripts are true paralogs originating from a single ancestor gene after genome duplication. This was also confirmed by phylogenetic analysis using fabp2 sequences of 26 teleosts and by synteny analysis carried out with ten teleosts. Differential expression analysis of the gilthead sea bream and European sea bass fabp2 paralogs in the intestine after fasting and refeeding experiment further revealed their altered implication in metabolism. Additional expression studies in seven developmental stages of the two species detected fabp2 paralogs relatively early in the embryonic development as well as possible complementary or separated roles of the paralogs. The identification and characterization of the two fabp2 paralogs will contribute significantly to the understanding of the fabp2 evolution as well as of the divergences in fatty acid metabolism.

Starvation and re-feeding affect Hsp expression, MAPK activation and antioxidant enzymes activity of European sea bass (Dicentrarchus labrax).

In the context of food deprivation in fish (wild and farmed), understanding of cellular responses is necessary in order to develop strategies to minimize stress caused by starvation in the aquaculture section. The present study evaluates the effects of long term starvation (1F-3S: one-month feeding-three-month starvation) and starvation/re-feeding (2S-2F: two-month starvation-two-month re-feeding) compared to the control group (4F-0S: four-month feeding-zero month starvation) on cellular stress response and antioxidant defense in organs, like the intestine, the liver, the red and white muscle of European sea bass Dicentrarchus labrax. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK) and the extracellular signal-regulated kinases (ERK-1/2). For the determination of the effect of the oxidative stress caused by food deprivation and/or re-feeding, the (maximum) activities of antioxidant enzymes such as glutathione peroxidise (GPx), catalase (CAT) and superoxide dismutase (SOD) as well as the determination of thiobarbituric acid reactive substances (TBARS) were studied. The experimental feeding trials caused a tissue distinct and differential response on the cellular and antioxidant capacity of sea bass not only during the stressful process of starvation but also in re-feeding. Specifically, the intestine phosphorylation of ERKs and antioxidant enzymatic activities increased in the 2S-2F fish group, while in the 1F-3S group an increase was detected in the levels of the same proteins except for GPx. In the liver and the red muscle of 2S-2F fish, decreased Hsp70 and phosphorylated p38 MAPK levels and increased Hsp90 levels were observed. Additionally, SOD activity decreased in the red muscle of 2S-2F and 1F-3S groups. In the liver and red muscle of 1F-3S group Hsp70 levels increased, while the activation of p38 MAPK in the liver decreased. In the white muscle, Hsp90 levels decreased and the phosphorylation of p38 MAPK increased in both feeding regimes compared to control. In the same tissue, GPx and catalase levels were decreased in 2S-2F regime, while SOD levels were decreased in 1F-3S regime.