Prevalence of opportunistic pathogens and anti-microbial resistance in urban aquaculture ponds.

Bacterial antimicrobial resistance (AMR) has emerged as a significant concern worldwide. The microbial community profile and potential AMR level in aquaculture ponds are often undervalued and attract less attention than other aquatic environments. We used amplicon and metagenomic shotgun sequencing to study microbial communities and AMR in six freshwater polyculture ponds in rural and urban areas of Bangladesh. Amplicon sequencing revealed different community structures between rural and urban ponds, with urban ponds having a higher bacterial diversity and opportunistic pathogens including Streptococcus, Staphylococcus, and Corynebacterium. Despite proteobacterial dominance, Firmicutes was the most interactive in the community network, especially in the urban ponds. Metagenomes showed that drug resistance was the most common type of AMR found, while metal resistance was only observed in urban ponds. AMR and metal resistance genes were found mainly in beta and gamma-proteobacteria in urban ponds, while AMR was found primarily in alpha-proteobacteria in rural ponds. We identified potential pathogens with a high profile of AMR and metal resistance in urban aquaculture ponds. As these ponds provide a significant source of protein for humans, our results raise significant concerns for the environmental sustainability of this food source and the dissemination of AMR into the food chain.

Fish waste to sustainable additives: Fish protein hydrolysates alleviate intestinal dysbiosis and muscle atrophy induced by poultry by-product meal in Lates calcarifer juvenile.

Valorising waste from the processing of fishery and aquaculture products into functional additives, and subsequent use in aquafeed as supplements could be a novel approach to promoting sustainability in the aquaculture industry. The present study supplemented 10% of various fish protein hydrolysates (FPHs), obtained from the hydrolysis of kingfish (KH), carp (CH) and tuna (TH) waste, with 90% of poultry by-product meal (PBM) protein to replace fishmeal (FM) completely from the barramundi diet. At the end of the trial, intestinal mucosal barriers damage, quantified by villus area (VA), lamina propria area (LPA), LPA ratio, villus length (VL), villus width (VW), and neutral mucin (NM) in barramundi fed a PBM-based diet was repaired when PBM was supplemented with various FPHs (p < 0.05, 0.01, and 0.001). PBM-TH diet further improved these barrier functions in the intestine of fish (p < 0.05 and 0.001). Similarly, FPHs supplementation suppressed PBM-induced intestinal inflammation by controlling the expression of inflammatory cytokines (tnf-α and il-10; p < 0.05 and 0.001) and a mucin-relevant production gene (i-mucin c; p < 0.001). The 16S rRNA data showed that a PBM-based diet resulted in dysbiosis of intestinal bacteria, supported by a lower abundance of microbial diversity (p < 0.001) aligned with a prevalence of Photobacterium. PBM-FPHs restored intestine homeostasis by enhancing microbial diversity compared to those fed a PBM diet (p < 0.001). PBM-TH improved the diversity (p < 0.001) further by elevating the Firmicutes phylum and the Ruminococcus, Faecalibacterium, and Bacteroides genera. Muscle atrophy, evaluated by fiber density, hyperplasia and hypertrophy and associated genes (igf-1, myf5, and myog), occurred in barramundi fed PBM diet but was repaired after supplementation of FPHs with the PBM (p < 0.05, 0.01, and 0.001). Similarly, creatine kinase, calcium, phosphorous, and haptoglobin were impacted by PBM-based diet (p < 0.05) but were restored in barramundi fed FPHs supplemented diets (p < 0.05 and 0.01). Hence, using circular economy principles, functional FPHs could be recovered from the fish waste applied in aquafeed formulations and could prevent PBM-induced intestinal dysbiosis and muscular atrophy. GRAPHICAL ABSTRACT.

From waste to feed: Microbial fermented abalone waste improves the digestibility, gut health, and immunity in marron, Cherax cainii.

New aquafeed ingredients produced by a circular economy approach are the opportunity for sustainable and resilient aquaculture. In the light of this approach, the mixture of abalone waste and Sargassum spp (9:1) fermented by Saccharomyces cereviceae and Lactobacillus casei (Yakult®) (FMAS) were used to replace 0% (FMAS0), 25% (FMAS-25), 50% (FMAS-50), 75% (FMAS-75), and 100% (FMAS-100) of fishmeal (FM) protein in marron, Cherax cainii diet. The marron was fed these diets in triplicate for 90 days. Growth, feed utilization and protein efficiency ratio were unchanged in marron-fed all test diets. Improvement in apparent protein digestibility was aligned with an increase in the size and number of B-cells in the hepatopancreas. Most of the immune responses, except for haemocyte clotting time, hyaline cells and neutral red retention time (NRR time) were unchanged by 42- and 90-days feeding trials compared to those of the control group. 90 days post-feeding marron with FMAS25 showed a lower haemocyte clotting time than the post 42 days feeding marron with the same diet. Hyaline cells increased in marron fed FMAS75 for 90 days compared to marron fed the same diet for 42 days. The challenge test involved injecting marron with Vibrio mimicus resulted in a 100% survival rate after 96 h of exposure. During the challenge test, phagocytosis activity in 24 and 48-h post-challenged marron fed FMAS75 decreased which recovered after 96 h post-challenge. Marron fed FMAS50 also recorded a significantly higher proportion of granular cells after 24 h and NRR time at 96 h compared with that of other treatments. Given the above indicators of bio-growth, feed efficiency and immune responses, total replacement of FM protein of marron practical feed with FMAS are considered feasible and optimum to maintain health status and resistance to disease.

A Mixture of Full-Fat and Defatted Hermetia illucens Larvae and Poultry By-Products as Sustainable Protein Sources Improved Fillet Quality Traits in Farmed Barramundi, Lates calcarifer.

The physicochemical quality and shelf-life of fillets from barramundi, which were fed for 56 days on a mixture of poultry by-product meal (PBM), full-fat Hermetia illucens (FHI), and defatted HI (DHI), were investigated and compared to a fishmeal (FM) control diet. The proximate and total amino acids compositions of the fillets were unaffected by the test diets, while the mixture of PBM and HI larvae improved the sensory quality. An eight-day shelf-life study showed that PBM-HI-based diets improved the texture profile based upon the chewiness, cohesiveness, gumminess, and hardness, regardless of the storage time. The improved texture was aligned with comparatively less degradation of the microstructure of the muscle tissue in the same diets. An improvement in the quality index (QI) value, an increase in pH, and a decrease in lipid oxidation were also found in the fillets of barramundi fed test diets compared with the control diet during the storage time. The test diets positively influenced flesh lightness and redness, while the color profiles were negatively influenced by the storage time. Overall, the maintenance of compositional attributes; the enhancement of fillet sensory attributes, texture, and brightness; and the improved raw fillet shelf-life support the inclusion of PBM-HI-based diets in aquafeed.

Gas to protein: Microbial single cell protein is an alternative to fishmeal in aquaculture.

Methanotrophic bacteria represent an appealing opportunity to convert methane, a potent greenhouse gas, into a highly nutritious animal feed ingredient, single-cell protein (SCP). SCP has a comparable or superior nutritional profile that to most conventional protein sources and can be produced within a lower environmental footprint. The present study investigated the effect of replacing fishmeal (FM) with methanotrophic SCP in diets for barramundi (Lates calcarifer), a carnivorous fish with a high demand for dietary protein and energy. Dietary inclusion levels of 0 %, 10 %, 20 % and 30 % SCP (representing 0, 25, 50 and 75 % FM replacement) were tested, with and without additives. Triplicate groups of juvenile barramundi were fed the diets over 31 days. The inclusion of SCP significantly improved weight gain and feed conversion efficiency (FCE). Dietary SCP inclusion supported good gut health, with decreasing trends of hepatosomatic index, improved plasma biochemistry, and no adverse histopathological changes. Barramundi fed the SCP diets showed an intact intestinal barrier and a significant improvement in villi and lamina propria area when fed the additive supplemented SCP diets. This study demonstrates that this SCP is highly palatable to barramundi (even without dietary additives) and can replace up to 75 % FM with significant improvements in growth and FCE.

Zeolite mediated processing of nitrogenous waste in the rearing environment influences gut and sediment microbial community in freshwater crayfish (Cherax cainii) culture.

Zeolite is known to uptake toxic metals and filter nitrogenous waste from aquaculture effluents. The present study aimed to investigate the impacts of zeolite in three different applications namely, dietary zeolite (DZ), suspended zeolite (SZ) in the water column, and a combination of both (DZSZ) relative to unexposed freshwater crayfish, marron (control). At the end of the 56-days trial, the impact was assessed in terms of characterization of microbial communities in the culture environment and the intestine of marron. Alongside the microbial communities, the innate immune response of marron was also evaluated. The 16S rRNA data showed that marrons exposed to the suspended zeolite had a significant increase of bacterial diversity in the gut, including the restoration of marron core operational taxonomic units (OTUs), relative to other forms of exposures (DZ, DZSZ) and the control. Suspended zeolite alone also increased the number of unshared OTUs and genera, and improved predicted metabolic functions for the biosynthesis and digestion of proteins, amino acids, fatty acids, and hormones. In the tank sediment, the shift of microbial communities was connected more strongly with the time of experiment than the type of zeolite exposure. In the second case, only control marron had a different microbial ordination in terms of rare taxa present in the community. Nevertheless, the modulation in the gut environment was found more prominent in DZ, relative to modulation in the tank sediments. The taxa-environment correlation identified Rhodoferax as the most potential bacteria in removing nitrogenous waste from the rearing environment. Further analysis showed that SZ resulted in the upregulation of genes associated with the innate immune response of marron. Overall results suggest that SZ can be used to enrich microbial communities in the gut and tank sediments and better immune performance of marron.

Sources of protein diet differentially stimulate the gut and water microbiota under freshwater crayfish, marron (Cherax cainii, Austin 2002) culture.

To reduce the reliance on fishmeal (FM), other protein sources have been evaluated on cultured animals. In a 60-days feeding trial, marrons (Cherax cainii) were fed a FM diet and five test diets containing 100% of plant-based protein sources such as soybean, lupin and valorised animal-based proteins such as poultry-by-product, black soldier fly and tuna hydrolysate. At the end of the trial, DNA samples from marron gut and rearing water were investigated through DNA-based 16S rRNA gene sequencing. Plant-based diets increased abundance for Aeromonas, Flavobacterium and Vogesella, whereas animal and insect proteins influenced diverse bacterial groups in the gut linked to various metabolic activities. Insect meal in the water favoured the growth of Firmicutes and lactic acid bacteria, beneficial for the marron health. Aeromonas richness in the gut and reared water signified the ubiquitous nature of the genus in the environment. The higher bacterial diversity in the gut and water with PBP and BSF was further supported by qPCR quantification of the bacterial single-copy gene, rpoB. The overall results suggested that PBP and BSF can exhibit positive and influential effects on the gut and water microbial communities, hence can be used as sustainable ingredients for the crayfish aquaculture.

Transformation of fish waste protein to Hermetia illucens protein improves the efficacy of poultry by-products in the culture of juvenile barramundi, Lates calcarifer.

Promoting a circular economy via the transformation of food waste into alternative and high-value protein sources for aquaculture diets is a novel approach to developing alternative raw materials to fishmeal (FM). This approach can reduce the ecological impact on the aquatic environment and simultaneously can provide an option for sustainable food waste management. In this context, we report a 56-day trial of feeding barramundi, Lates calcarifer on four iso­nitrogenous and iso-lipidic diets where the control (0PBM-0HI) was a FM-based diet and the other test diets replaced FM protein with mixtures of a poultry by-product meal (PBM) and a full-fat Hermetia illucens (HI) larvae meal reared on fish waste: the test diets were 85% PBM + 15% HI (85PBM-15HI), 80% PBM + 20% HI (80PBM-20HI) and 75% PBM + 25% HI (75PBM-25HI). Fish fed PBM-HI-based diets showed an equal growth rate and amino acid profile when compared to the control group. Among all serum metabolites, alanine aminotransferase and glutamate dehydrogenase decreased in fish fed PBM-HI-based diets, whilst total protein levels improved in the same diets. Serum lysozyme and bactericidal activity were unchanged which supported the observation of similar infection rates against V. harveyi. Except for the kidney and intestine, catalase activity in the serum and liver increased in fish-fed PBM-HI-based diets. In assessing the gastrointestinal mucosal morphology, the goblet cells producing neutral mucins were higher in PBM-HI-fed fish than the control. PBM-HI diets also enhanced bacterial richness and diversity and increased abundance for Lactobacillus, Clostridium, and Ruminococcus. In summary, combining full-fat HI with PBM allowed complete replacement of FM with no negative effects on growth whilst improving gut health. Such diets would be beneficial for the aquaculture industry, both ecologically and economically, as well as providing value-adding to animal waste as alternative protein sources for aquafeed production.

Supplementation of tuna hydrolysate and insect larvae improves fishmeal replacement efficacy of poultry by-product in Lates calcarifer (Bloch, 1790) juveniles.

The effects of feeding different levels of poultry by-product meal (PBM) replacing fishmeal (FM) protein, supplemented with tuna hydrolysate (TH) and Hermetia illucens (HI) larvae, on the growth, fillet quality, histological traits, immune status, oxidative biomarker levels and gut microbiota of juvenile barramundi, Lates calcarifer were investigated for six weeks. Barramundi were fed four isonitrogenous and isolipidic diets in which a FM based diet was used as the Control diet (Diet1) and compared with other non-FM diets containing 80%, 85% and 90% PBM along with the concurrent supplementation of 5% and/or 10% TH and HI larvae meal. These treatment diets were designated as 80PBM10TH+10HI (Diet2), 85PBM5TH+10HI (Diet3) and 90PBM5TH+5HI (Diet4). The growth and condition factor of fish fed 80PBM10TH+10HI and 85PBM5TH+10HI were significantly higher than the Control. Total saturated, monounsaturated and polyunsaturated fatty acid retention in the fish muscle increased in fish fed PBM-based diets, supplemented with TH and HI larvae meal, with no adverse effect on post-harvest characteristics such as texture and colour of fish fillets. Improvement in serum total bilirubin and total protein content was found in all fish fed TH and HI larvae supplemented PBM. Similarly, immune response showed a significant increase in fish fed non-FM test diets than the Control. In the distal intestine, supplementation of any quantities of TH and HI larvae to PBM led to an increase in the microvilli density and neutral mucins while the number of goblet cells in the skin were unchanged. Liver, kidney, and spleen histology demonstrated a normal structure with no obvious changes in response to all test diets. Bacterial diversity increased in fish fed Diets 2 and 3 with a high abundance of Proteobacteria in Diets 1 and 4 and Firmicutes in Diets 2 and 3. The fish on test diets showed a lower abundance of genus Vibrio. Fish fed TH and HI larvae supplemented PBM diets showed lower infection rate to V. harveyi than the Control. Collectively, concurrent supplementation of TH and HI larvae could improve the quality of PBM diets with positive effects on growth, fillet quality, intestinal health, immunity, and disease resistance.

A Combination of Hermetia illucens Reared on Fish Waste and Poultry By-Product Meal Improves Sensory and Physicochemical Quality of Farmed Barramundi Filets.

The proximate composition, sensory attributes, and shelf life of filets from barramundi, Lates calcarifer, were fed a fishmeal (FM) based diet (0PBM-0HI) and three test diets replacing FM protein entirely with 85% poultry by-products meal (PBM) and 15% Hermetia illucens (HI) larvae meal protein (85PBM-15HI), 80% PBM and 20% HI (80PBM-20HI) and 75% PBM and 25% HI (75PBM-25HI) were investigated. After a 56-day feeding trial, the crude protein, moisture, and ash percentage were unchanged while the crude lipid increased in barramundi filet when fed with PBM-HI-based diets. The increase in C12:0 (lauric acid) and C14:0 (myristic acid) resulted in an increase in the total saturated fatty acid while the monounsaturated fatty acid elevated due to an increase in C16:1n7 and C18:1cis + trans in the filet of the barramundi fed with a PBM-HI based diet. While the decrease in the total polyunsaturated fatty acid (PUFA) content in PBM-HI based fed barramundi filet was mainly due to a decrease in essential fatty acids including C20:5n3 [eicosapentaenoic acid (EPA)] and C22:6n3 [docosahexaenoic acid (DHA)] when compared with the 0PBM-0HI fed barramundi filet. The sensory quality was improved by PBM-HI-based diets, manifested by the highest scores given by the panelists. Texture profiles were not affected by diet but cohesiveness, gumminess, and chewiness decreased with increasing storage time. On days 1 and 8, skin brightness decreased in the skin of the barramundi fed with 85PBM-15HI and 80PBM-20HI compared with the skin of the 0PBM-0HI fed barramundi. Skin redness improved in fish-fed PBM-HI-based diets. The flesh brightness and yellowness increased significantly in barramundi when fed with PBM-HI-based diets. On days 1 and 4, the flesh brightness of the barramundi fed with PBM-HI-based diets demonstrated an increase compared with 0PBM-0HI. PBM-HI diets suppress lipid oxidation while lipid oxidation increased over the storage time. In summary, the improvement in sensory quality and color coupled with the suppression of rancidity in barramundi filets underpinned the potentiality of using the mixture of PBM and HI transformed from food waste in the barramundi diet to improve the filet quality and thus support sustainability and circular economy in aquaculture.

Lactobacillus plantarum in black soldier fly (Hermetica illucens) meal modulates gut health and immunity of freshwater crayfish (Cherax cainii).

Probiotic supplements are being used to improve the growth and immune performance of aquaculture species over the last couple of decades. In recent times, black soldier fly (BSF) is considered as one of the promising sources of alternative protein to fishmeal protein in aqua-diets. Since the freshwater crayfish, marron (Cherax cainii), a Western Australian's native and iconic freshwater crayfish species, grows fairly slow under commercial farming environment, this study was aimed to investigate the supplemental effect of BSF and BSF with probiotic bacteria Lactobacillus plantarum (BSFLP) on overall health and immune performance of marron after 56 days of feeding under laboratory conditions. The post-trial data revealed insignificant influences of any diets on growth performance, however, both BSF and BSFLP based diets significantly improved some haemolymph parameters and gut health of marron. High throughput sequence data revealed that both BSF and BSFLP diets significantly improved the diversity of microbial communities including some beneficial bacteria for crustaceans in the hindgut of marron. Further analysis showed that both BSF and BSFLP diets upregulated the expression of some genes in the gut tissue and haemocytes associated with the innate immune response of marron at 48 h post injection. The up-regulation of some immune genes in BSFLP diet group was found significantly linked to OTU abundance for Lactobacillus. The findings of this study could be helpful for improving overall health status of marron.

Total replacement of fishmeal with poultry by-product meal affected the growth, muscle quality, histological structure, antioxidant capacity and immune response of juvenile barramundi, Lates calcarifer.

The present study investigates if the total replacement of dietary fishmeal (FM) with poultry by-product meal (PBM), supplemented with methionine influences the muscle fatty acids composition, normal gut morphology, histological traits of the liver, muscle, and gill, liver enzymes, immune and antioxidant response, and stress-related gene in juvenile barramundi, Lates calcarifer in relation to growth and feed utilization. Barramundi (3.58±0.01g) were randomly distributed into six 300 L seawater recirculating tanks (25 fish/tank) and fed two formulated isonitrogenous and isolipidic diets for 6 weeks. The control diet had FM as the sole animal protein source, whereas other test diet had only PBM as an animal protein source. Dietary PBM affected the fish performance and feed utilization. Regarding muscle fatty acid profile, total saturated fatty acids and monounsaturated fatty acids elevated while total PUFA particularly n-3 LC-PUFA and EPA decreased in PBM fed fish than control diet fed fish. Liver, muscle, gill, and intestinal histology showed no obvious alteration in control diet fed fish, however, more lipid droplets and hepatic vacuolization in the liver, necrotic myotome in muscle, hyperplasia in secondary lamellae in gill and short and broken folds in the intestine were observed in PBM fed fish. Similar to light microscopy observation of intestinal morphology, the transmission electron microscopy (TEM) analysis revealed shorter and smaller microvilli in fish fed PBM. Histopathological alterations in the liver of PBM fed fish were further associated with the elevated levels of aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) and the significant upregulation of stress-related genes, HSP70 and HSP90. Also, a negative influence on lysozyme activity, and antioxidant enzymatic activities were recorded in fish fed PBM. Overall, it can be concluded that a total substitution of FM protein by methionine supplemented PBM negatively influenced the growth performance, liver health, histological traits of different organs, immune and antioxidant response, and expression of stress-related genes in juvenile barramundi.

Impact of varied combinatorial mixture of non-fishmeal ingredients on growth, metabolism, immunity and gut microbiota of Lates calcarifer (Bloch, 1790) fry.

The search for suitable fish meal replacements in aqua-diets is a salient agenda in the constant effort of making aquaculture practices more sustainable. In this study, we tested four customised diets composed by systematic inclusion of pre-selected fish meal substitutes, lupin kernel meal, BSF meal, TH and PBM on growth, metabolism, cytokine profile, gut morphology and microbiota of juvenile Lates calcarifer. Five isoproteic and isoenergetic diets were prepared viz. FM100 as a control (without fish meal substitute), while FM75, FM50, FM25 and FM0 indicates replacement of fish meal (FM) at 25%, 50%, 75%, and 100%, respectively by a mixture of four different pre-selected non-fish meal (NFM) ingredients. Fish fed FM100, FM75, FM50, FM25 exhibited consistent growth and haematological response, while the fish fed no fishmeal (FM0) showed significant decline in final body weight (FBW) and specific growth rate (SGR). The poor growth performance was correlated with a decrease in villous width, microvilli height and goblet cells density. A significant shift in abundance profile of Psychrobacter in the gut microbial profile of fish fed FM50 was noticed compared to fish fed FM100. The results of qRT-PCR showed up-regulated expression of innate immune responsive genes in the FM50 group. The adverse impacts on growth performance and gut health of fish fed FM0 suggest that the complete substitution of fishmeal is not advisable and the inclusion range of these alternatives should be decided for a species only after examining their effect on maximal physiological performance.

The ameliorative effects of various fish protein hydrolysates in poultry by-product meal based diets on muscle quality, serum biochemistry and immunity in juvenile barramundi, Lates calcarifer.

In an effort to reduce the use of fishmeal (FM), the effect of using protein from poultry by product meal (PBM) along with the supplementation of three different fish protein hydrolysate (FPH) including yellowtail kingfish, carp and tuna hydrolysate (designated as KH, CH and TH, respectively) were evaluated in juvenile barramundi for growth performance, fillet quality, mucosal immunity, serum biochemistry, immune response and infection against Vibrio harveyi. Fish were fed a FM based control diet + three isonitrogenous and isolipidic diets containing 90% of PBM protein supplemented with different types of hydrolysates: 90% PBM +10% KH (90PBM + KH), 90% PBM + 10% CH (90PBM + CH) and 90% PBM + 10% TH (90PBM + TH). Growth performance and indices were unaffected by the hydrolysate supplemented diets when compared to the control. FPH supplemented PBM diets resulted in improved muscle quality by improving poly unsaturated fatty acids (PUFA), ∑n-3, ∑n-6 and ∑n-9, and health related lipid indexes were not affected. The internal architecture of spleen and kidney were not altered by test diets whilst FPH supplemented PBM modulated acidic mucins in intestine and skin of fish. Improved infection rate in response to two weeks post infection with V. harveyi in the FPH supplemented diets was further associated with an increased serum immune response and a concomitant regulation of proinflammatory and inflammatory cytokines in the head kidney. Serum biochemistry including alanine transaminase (ALT), glutamate dehydrogenase (GLDH) and total bilirubin (TB) showed a decreasing trend both in pre-challenge and post-challenge barramundi fed FPH supplemented diets whereas cholesterol level decreased significantly in post-challenge groups fed 90PBM + KH and 90PBM + TH than pre-challenge barramundi. This study signifies that supplementation of 10% with different three FPH, hydrolysed by an alcalase® enzyme in PBM-based diets for barramundi could be good strategies to overcome the negative consequences triggered by animal by-product ingredients.

Growth and health of juvenile barramundi (Lates calcarifer) challenged with DO hypoxia after feeding various inclusions of germinated, fermented and untreated peanut meals.

Peanut (Arachis hypogaea) is mainly grown for oil extraction and the remaining oil-free seed referred as peanut meal (PM) leaves with high protein content which can be a possible substitute for fishmeal in aqua-diets. This study evaluates the suitability of three types of processed peanut seeds, namely untreated PM (UPM), fermented PM (FPM), and germinated PM (GPM) from peanut seeds to replace fishmeal in barramundi (Lates calcarifer) diets cultured under a commercial production environment. Nine formulated diets having 3 inclusion levels from the 3 different peanuts (15%, 30% and 60% fishmeal replacement) were evaluated against a control without PM. The performance of various types and levels of PMs was assessed by examining the growth, gut and liver condition and survival of fish after eight weeks of feeding the test diets. The immunological responses of juvenile barramundi were assessed by exposing the fish to the hypoxic conditions for 4 hours. The results showed that fermentation and germination significantly (P<0.05) reduced the tannins and alkaloid contents in the PMs. The fish fed 15% GPM diet grew faster and had higher survival than fish fed control diet, while fish fed diet including 60% GPM showed a significant reduction in growth and survival, and an increase in food conversion rate (FCR). FPM and UPM at any inclusion levels did not alter the growth, survival and FCR. Histology analysis revealed that fish fed 60% GPM and UPM showed higher amount of lipid droplets in liver, myodigeneration in fish muscle and a decrease number of acidic mucins in distal gut compare to all other test diets. Stress caused by reduced dissolved oxygen did not change the sodium, potassium, chlorides and alanine aminotransferase concentrations of plasma of fish fed any diet. However, the stress did increase plasma cortisol significantly (P<0.05) in fish fed 60% GPM, 30% and 60% UPM diets. These results suggest that the PMs can partly replace the fishmeal in juvenile barramundi diet and the processing further improves the PMs quality by reducing its antinutritional factors which in turn can increase either its inclusion level in the barramundi diets or improved growth and health status of the species.

Supplementation of Hermetia illucens Larvae in Poultry By-Product Meal-Based Barramundi, Lates calcarifer Diets Improves Adipocyte Cell Size, Skin Barrier Functions, and Immune Responses.

A 6-week feeding trial was performed to examine the effects of supplementing Hermetia illucens (HI) larvae meal when fishmeal (FM) was replaced with poultry by-product meal (PBM) in juvenile barramundi, Lates calcarifer diet. The effect was evaluated in terms of barramundi growth, filet quality, internal tissue structure, serum biochemistry, skin neutral mucins, immune response, and resistance to Vibrio harveyi. Three isonitrogenous (48% crude protein) and isolipidic (18% crude lipid) diets: an FM-based diet (control) and two diets containing 60 and 75% of PBM supplemented with 10% HI larvae (60PBM + HI and 75PBM + HI) were formulated. A total of 225 barramundi, with an average weight of 15.87 ± 0.14 g, were randomly distributed into nine tanks, each holding 25 fish. There were no significant effects of test diets on growth, but feeding HI-supplemented PBM diets significantly increased the survival rate. A significantly reduced intraperitoneal fat index in HI-supplemented-PBM-fed fish was correlated to a decreased size of peritoneal adipocytes. The observation of no histopathological alteration of the liver in the HI-supplemented-PBM-fed fish was further supported by significant alterations in serum biochemistry, in particular, a decreasing tendency of alanine transaminase, glutamate dehydrogenase, and total bilirubin. A 14-day challenge with V. harveyi indicated that HI-supplemented PBM diets reduced the infection rate in barramundi. After 24 h of infection, increased serum (lysozyme) and skin barrier functions, down-regulation of interleukin-1beta, and upregulation of interleukin-10 were found in HI-supplemented-PBM-fed fish.

Influence of fish protein hydrolysate produced from industrial residues on antioxidant activity, cytokine expression and gut microbial communities in juvenile barramundi Lates calcarifer.

The present study investigated the supplemental effects of tuna hydrolysate (TH) in poultry by-product meal (PBM) and dietary fishmeal (FM) diets on antioxidant enzymatic activities, gut microbial communities and expression of cytokine genes in the distal intestine of juvenile barramundi, Lates calcarifer. Fish were fed with fermented (FPBM + TH) as well as non-fermented PBM (PBM + TH) and FM (FMBD + TH) diets with 10% TH supplementation for 10 weeks. A basal diet prepared without TH supplementation served as control. The results showed that the activity of glutathione peroxidase was significantly higher in FPBM + TH than the control, while the malondialdehyde and catalase activities were unchanged. FPBM + TH diet significantly (P < 0.05) upregulated the pro-inflammatory cytokines including IL-1ß and TNF-α while considerable downregulation (P < 0.05) was observed in the mRNA expression levels of anti-inflammatory cytokine, IL-10 in the distal intestine of fish. The 16SrRNA analysis using V3-V4 region evidenced the ability of FPBM + TH to modulate the distal intestinal gut microbiome, augmenting the richness of Firmicutes and Fusobacteriaat at phylum level and Bacillus, Lactococcus and Cetobacterium at genus level. All these results have shown that fermented PBM with TH supplementation could improve the antioxidant capacity and inflammatory responses of juvenile barramundi while influencing the microbial communities at both phylum and genera levels.

Insect larvae, Hermetia illucens in poultry by-product meal for barramundi, Lates calcarifer modulates histomorphology, immunity and resistance to Vibrio harveyi.

This study investigated the effects of replacement of fishmeal (FM) with poultry by-product (PBM) protein, supplemented with black soldier fly, Hermetia illucens (HI) larvae on growth, histomormhology, immunity and resistance to Vibrio harveyi in juvenile barramundi. Two hundred and twenty five barramundi averaging 3.51 ± 0.03 g were randomly allocated into three groups and fed isonitrogenous and isocalorific diets containing different levels of PBM supplemented with HI as follows: Control (FM based diet), 45PBM + HI (45% PBM supplemented with 10% HI), and 90PBM + HI (90% PBM supplemented with 10% HI) for 6 weeks. Results showed that dietary inclusion of 45PBM + HI significantly improved the growth performance than control whereas growth inhibition occurred in the 90PBM + HI. The 45PBM + HI groups demonstrated significant increases in histometric measurements (villus and enterocyte width, and microvilli height) and acidic mucins. The impaired growth in 90PBM + HI groups was further associated with multifocal necrosis in the liver, an upregulation of the stress related genes (HSP70 and HSP90) and increase in the levels of liver enzymes. When 45PBM + HI was fed, survival against V. harveyi increased significantly and also an increase in serum immunity and immune-related genes in the head kidney was observed after infection.

Marked variations in gut microbiota and some innate immune responses of fresh water crayfish, marron (Cherax cainii, Austin 2002) fed dietary supplementation of Clostridium butyricum.

This study aimed to investigate the effects of Clostridium butyricum as a dietary probiotic supplement in fishmeal based diet on growth, gut microbiota and immune performance of marron (Cherax cainii). Marron were randomly distributed into two different treatment groups, control and probiotic fed group. After 42 days of feeding trial, the results revealed a significant (P < 0.05) increase in growth due to increase in number of moults in marron fed probiotics. The probiotic diet also significantly enhanced the total haemocyte counts (THC), lysozyme activity in the haemolymph and protein content of the tail muscle in marron. Compared to control, the 16S rRNA sequences data demonstrated an enrichment of bacterial diversity in the probiotic fed marron where significant increase of Clostridium abundance was observed. The abundance for crayfish pathogen Vibrio and Aeromonas were found to be significantly reduced post feeding with probiotic diet. Predicted metabolic pathway revealed an increased activity for the metabolism and absorption of carbohydrate, degradation of amino acid, fatty acid and toxic compounds, and biosynthesis of secondary metabolites. C. butyricum supplementation also significantly modulated the expression level of immune-responsive genes of marron post challenged with Vibrio mimicus. The overall results suggest that C. butyricum could be used as dietary probiotic supplement in marron aquaculture.

Molecular characterization and interactome analysis of aerolysin (aer) gene from fish pathogen Aeromonas veronii: The pathogenicity inferred from sequence divergence and linked to histidine kinase (cheA).

Aerolysin (aer) is one of the most important and abundant virulence factors in the infection of fish by Aeromonas veronii. A comprehensive study on the molecular characterization and pathogenicity of the aer gene from 34 A. veronii isolates from diseased carp and catfish was carried out and its interactome was analysed to observe the functional correlations between aer and other proteins within the A. veronii network. The PCR-based amplification of aer from the 34 isolates of A. veronii showed more aer-positive isolates from catfish with a high pathogenic potential in the in vivo challenge test than the carp fish. The analysis of aer gene sequence from challenged fish revealed significant sequence divergence according to the types and geographical distribution of the fish. The networking analysis of aer from the model A. veronii B565 revealed histidine kinase (cheA) as the most functional interacting partner. The study of the interaction between aer from the experimental A. veronii and cheA demonstrated that the A chain of cheA plays a more important role than the corresponding B chain during contact, and a linker sequence of 15 residues controlled the entire interaction process. Therefore, cheA could be an excellent drug target for controlling A. veronii infection of fish.