Aquaculture sludge as feed for black soldier fly: Transfer of chemical and biological contaminants and nutrients.

Aquaculture sludge (uneaten feed and faeces) is nutrient rich and has potential as feed for insects. The aim of this study was to investigate the transfer of chemical and biological contaminants, as well as nutrients, from aquaculture sludge to black soldier fly larvae. The larvae were reared on a sludge mixture made of different sludges collected from Norwegian freshwater salmonid facilities. The sludge was spiked with four common salmon pathogens: Infectious Pancreatic Necrosis Virus, Infectious Salmon Anemia virus, Yersinia ruckeri or Mycobacterium salmoniphilum. During the 15 days of growth on sludge, the black soldier fly larvae accumulated valuable nutrients including protein, fat, eicosapentaenoic acid, iron, manganese, zinc and selenium. The larvae also accumulated undesirable substances including cadmium, mercury, dioxins and polychlorinated biphenyls. The concentrations of dioxins exceeded the EU maximum level set for animal feed. None of the salmon pathogens that were spiked to the sludge were detected in the black soldier fly larvae. This study reports low risk of transfer of salmon pathogens from sludge to insect larvae, and showed that the transfer of heavy metals, minerals and metalloids are in accordance with earlier studies. The large variations in levels of heavy metals between batches of sludge can cause levels in BSF exceeding the EU maximum levels, and thus indicate a need for monitoring of the proposed value chain. The transfer of dioxins from sludge to insects, reported for the first time in this paper, would be of special interest for future research, with special focus on risk mitigation.

Characterization of nutrients and contaminants in fish sludge from Atlantic salmon (Salmo salar L.) production sites - A future resource.

A total of 47 fish sludge samples from commercial land-based Atlantic salmon (Salmo salar) farms in Norway were assessed for their nutrient composition, presence of various legacy contaminants and a wide spectrum of contaminants of emerging concern, veterinary medicines as well as selected salmonid pathogenic bacteria and virus. The aim was to document the levels of desirable and undesirable components in fish sludge in relation to a potential future use of sludge as invertebrate feed. The samples had variable, but relatively high protein and fat contents, indicating a high load of undigested feed in some of the sludge samples. Fatty acid analysis showed the presence of essential omega-3 fatty acids. In terms of undesirable substances, 43% and 84% of the sludge samples contained levels of arsenic and cadmium, respectively, which exceeded the EU Maximum Levels established for complete animal feed. The concentrations of copper, zinc, iron and aluminum were highly variable in the sludge samples. The concentrations of dioxins, sum PCB6, and chlorinated pesticides were all below the Maximum Levels for animal feed. Of the 18 per- and polyfluoroalkyl substances (PFAS) only one compound (L-PFOS) was present at measurable levels. None of the samples had detectable levels of veterinary medicines, salmonid virus or bacteria. Performing a suspect and non-target screening of the sludge samples identified 18 compounds, including four pharmaceuticals, plastic-related products and the UV filter benzophenone, warranting further investigations. Overall, the results from this study show that fish sludge is a nutrient-rich resource; however, undesirable substances, originating from the feed or from treatment of sludge may be present.

Assessing amino acid solubility of black soldier fly larvae meal in Atlantic salmon (Salmo salar) in vivo and in vitro.

In vitro and in vivo methods were used to evaluate amino acids solubility of black soldier fly (BSF) larvae meal and two experimental diets (reference and test diets) for Atlantic salmon. The current study used in vitro method such as pH stat to compare and standardise the salmon extracted enzyme (SE), and commercial enzyme (CE) based on their hydrolytic capacity on a purified protein substrate. Further, an in vitro amino acid solubility of feed ingredients and diets were measured using the standardised enzyme volume from SE and CE. Results showed that SE and CE exhibit similar protein hydrolytic capacity upon standardisation on purified substrates. However, when using the two-stage hydrolysis (acidic and alkaline steps), significantly higher amino acid solubility was observed with CE except for glycine, and proline which were equally solubilised by both SE, and CE. No significant difference was observed between reference and test diet using the SE except for tyrosine, valine, leucine, and phenylalanine, which were significantly higher solubilised in reference diet than test diet. Whereas higher solubility of valine, isoleucine, aspartic acid, and glutamic acid was observed in test diet using CE than SE. Similarly, the solubility of valine, isoleucine, and glutamic acid were higher in BSF larvae meal when CE was used. The in vivo true protein digestibility of BSF larvae meal was 99%, and 81% for the test diet containing BSF larvae meal. The results demonstrated a positive correlation (r = 0.91; p < 0.01) between salmon and commercial enzymes but overall, no significant correlation was observed for amino acid solubility between in vivo and in vitro. However, there was a strong positive correlation for protein solubility using SE (r = 0.98) than CE (r = 0.74) with the in vivo true protein digestibility. The efficiency of SE, and CE can be compared, and standardised based on DH%, and hence correlates better with the in vivo protein digestibility but not with amino acid solubilities.

A rapid acid hydrolysis method for the determination of chitin in fish feed supplemented with black soldier fly (Hermetia illucens) larvae.

Insects are a natural source of feed for fish and have received more attention as a potential source of sustainable high-quality protein. However, contrasting results in different feeding trials have been ascribed to the chitin contained in the exoskeleton of insects and highlighted the importance of developing reliable methods for the quantification of chitin to draw meaningful conclusions about its effect on fish health. A rapid method based on the hydrolysis of chitin into glucosamine and further quantification by liquid chromatography tandem mass spectrometry is evaluated. The method offers good selectivity, linearity, limit of detection (1.08 × 10-5 % w/v or 5.38 × 10-4 % w/w), limit of quantification (3.26 × 10-5 % w/v or 1.63 × 10-3 % w/w), trueness (88.39-109.29 %) and precision (2.24-10.72 %). The quantitative method was successfully applied to real samples of fish feed supplemented with chitin from black soldier fly (Hermetia illucens) larvae.

The impact of seawater warming on fatty acid composition and nutritional quality indices of Trematomus bernacchii from the Antarctic region.

There is a growing interest in exploiting Antarctic fisheries for human consumption. However, information on how the nutritional qualities of these resources will respond to the predicted seawater warming in the region for the next century is poor. The present research investigates changes in various nutritional indices of dietary importance (e.g. the ratio polyunsaturated to saturated fatty acids, the atherogenicity index, the thrombogenicity index, the hypo-cholesterolemic to hyper-cholesterolemic index, the health-promoting index, the flesh lipid quality and the ratio omega-3 to omega-6 index) by determining the fatty acid composition in muscle of Trematomus bernacchii (an Antarctic fish species) in its natural habitat (-1.87 °C) and warmer temperatures (0.0, 1.0, 2.0 °C). Comparison of the estimated nutritional indices at -1.87 °C with those at warmer temperatures revealed that seawater warming caused changes in the nutritional indices in the range of -12%<Δ < 30%. The observed changes were not statistically significant and ascribed to biological variability. Therefore, the nutritional values of T. bernacchii muscle were preserved after increasing the temperature of its natural habitat by + 4 °C. The present research is the first report describing the nutritional quality indices for an Antarctic fish species and the consequences of seawater warming on the nutritional value of T. bernacchii.

Total replacement of dietary fish meal with black soldier fly (Hermetia illucens) larvae does not impair physical, chemical or volatile composition of farmed Atlantic salmon (Salmo salar L.).

BACKGROUND: The aquafeed sector has been replacing conventional dietary ingredients with more economic and eco-friendly ingredients. Insects embody a promising alternative as a result of being highly nutritious and showing traits leading to a circular bioeconomy. Atlantic salmon (Salmo salar L.) at the sea-water stage were fed diets with a partial or complete substitution of fishmeal with meal of Hermetia illucens larvae reared on a media containing Ascophyllum nodosum mixed with organic wastes (60:40). The present study aimed to assess the quality of fillets by characterizing its physico-chemical traits with conventional and innovative methods, such as the proton transfer reaction-time of flight-mass spectrometer technique, allowing the analysis of samples at room temperature. Finally, steamed fillets underwent a consumer test to investigate the liking of consumers and their intention of re-consumption. RESULTS: The main findings showed that a complete dietary substitution of fishmeal with H. illucens larvae meal did not impair the physico-chemical quality of A. salmon fillets. Notably, neutral n-3 polyunsaturated fatty acids (PUFA) slightly but significantly increased in the fillets of A. salmon fed H. illucens, also as a result of the additional fish oil present in the diets containing insect. The volatile organic profile was not altered by the different diets. The consumer-liking test revealed that Italian consumers appreciated the tested salmon irrespective of the administered feed. CONCLUSION: Tailoring the insect fatty acid profile by rearing the larvae on a PUFA-rich substrate, coupled with a dietary modulation of the oily source, can successfully maintain or even increase the cardioprotective characteristics of fillets. © 2019 Society of Chemical Industry.

Replacing fish meal with insect meal in the diet of Atlantic salmon (Salmo salar) does not impact the amount of contaminants in the feed and it lowers accumulation of arsenic in the fillet.

Insects are promising sources of protein and lipid in feeds for farmed animals. In the European Union, the use of insect meal (IM) and insect oil is permitted in fish feed. However, the European Food Safety Authority has highlighted the lack of data regarding the chemical safety of insects and products thereof. In this study, Atlantic salmon (Salmo salar) were fed diets in which fish meal (FM) was partially or fully substituted with IM, resulting in four diets with an FM replacement of 0%, 33%, 66% and 100% by IM. The IM was produced from Black soldier fly (Hermetia illucens) larvae fed media containing 60% seaweed (Ascophyllum nodosum). After 16 weeks of feeding, fish fillet samples were collected. The concentrations of undesirable substances, e.g., heavy metals, arsenic, dioxins, mycotoxins, pesticides, in the IM, the diets and fillets were determined. The concentrations of the analysed compounds in the IM were all below EU maximum levels for feed ingredients, except for arsenic. However, for complete feeds the concentrations of these compounds in the feeds, including arsenic, were all below EU MLs. Arsenic was transferred from seaweed to IM, resulting in arsenic levels in IM similar to what has been documented for FM. Transfer of arsenic from feed to fillet was observed; however, total arsenic concentrations in the fillet significantly decreased when fish were fed diets with more IM and less FM. Arsenic speciation analysis of the diets showed that although total arsenic levels were similar, the arsenic species were different. Arsenobetaine was the major organoarsenic species in the diets containing FM, while in diets containing IM several unidentified arsenic species were detected. The results suggest that the lower feed-to-fillet transfer of arsenic when FM is replaced by IM may be due to the presence of arsenic species with low bioavailability in the IM.

The Effect of Omega-3 and Omega-6 Polyunsaturated Fatty Acids on the Production of Cyclooxygenase and Lipoxygenase Metabolites by Human Umbilical Vein Endothelial Cells.

Although the correlation between polyunsaturated fatty acids (PUFA) and the production of pro- and anti-inflammatory metabolites is well documented, little is known about the simultaneous effect of different PUFA on the production of cyclooxygenase and lipoxygenase metabolites. The present research examines the association between different omega-3 (ω-3) and omega-6 (ω-6) PUFA and the release of four cyclooxygenase and six lipoxygenase metabolites in cell medium by human umbilical vein endothelial cells (HUVEC). The different combinations of ω-3 and ω-6 PUFA were prepared according to a full 24 factorial design that enables studying not only the main effects but also the different interactions between fatty acids. In addition, interactions diagrams and principal component analysis were useful tools for interpreting higher order interactions. To the best of our knowledge, this is the first report addressing the combined effect of ω-3 and ω-6 PUFA on the signaling of prostaglandins, prostacyclins, leukotrienes and resolvins by HUVEC.

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra.

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures.

Effect of dietary replacement of fish meal with insect meal on in vitro bacterial and viral induced gene response in Atlantic salmon (Salmo salar) head kidney leukocytes.

With the fast growth of today's aquaculture industry, the demand for aquafeeds is expanding dramatically. Insects, which are part of the natural diet of salmonids, could represent a sustainable ingredient for aquaculture feed. The aim of the current study was to test how a partial or total replacement of dietary fishmeal with insect meal affect gene responses involved in inflammation, the eicosanoid pathway and stress response in Atlantic salmon (Salmo salar L.) in isolated head kidney leukocytes after exposure to bacterial or viral mimic. Insect meal (IM) was produced from black soldier fly (BSF, Hermetia illucens) larvae. Seawater Atlantic salmon were fed three different diets for 8 weeks; a control diet (IM0, protein from fishmeal and plant based ingredients (25:75) and lipid from fish oil and vegetable oil (33:66); and two insect-meal containing diets, IM66 and IM100, where 66 and 100% of the fishmeal protein was replaced with IM, respectively. Leukocytes were isolated from the head kidney of fish (n = 6) from each of the three dietary groups. Isolated leukocytes were seeded into culture wells and added either a bacterial mimic (lipopolysaccharide, LPS) or a viral mimic (polyinosinic acid: polycytidylic acid, poly I: C) to induce an inflammatory response. Controls (Ctl) without LPS and poly I: C were included. The transcription of interleukins IL-1ß, IL-8, IL-10 and TNF-α were elevated in LPS treated leukocytes isolated from salmon fed the three dietary groups (IM0, IM66 and IM100). The inflammatory-related gene expression in head kidney cells were, however, not affected by the pre-fed substitution of fish meal with IM in the diet of salmon. Gene transcriptions of PTGDS and PTGES were neither affected by LPS, poly I: C or the experimental diets fed prior to cell isolation, while salmon fed with IM showed a lower expression of LOX5. The gene expression of TLR22 and C/EBP-ß were down-regulated by the LPS treatment in the cells isolated from salmon fed insect-based diets (IM66 and IM100) compared to fish fed the IM0. Similarly, the leukocytes challenged with LPS and isolated from fish fed with IM66 and IM100 down-regulated the expression of Mn-SOD, GPx1, HSP27 and HSP70 compared to salmon fed IM0. In general, these results suggested that replacement of fishmeal with IM in the diets of Atlantic salmon had no effect on the transcription of pro-inflammatory genes in the head kidney cells. There was, however, an effect of dietary IM on the transcription of antioxidant and stress related genes in the leukocytes.

Growth and Safety Assessment of Feed Streams for Black Soldier Fly Larvae: A Case Study with Aquaculture Sludge.

The production of food is an intensive source of environmental impact. In aquaculture, one source of impact is solid waste, which contains high concentrations of minerals, other nutrients, and metals. The larvae of Hermetia illucens are capable of consuming this material, but applying technology that is based on these larvae for managing waste streams, like those from aquaculture, requires careful examination of safety risks. A study is performed examining the growth performance of larvae that were fed on solid aquaculture waste. Subsequently, a thorough analysis of safety risks from inorganics, with detailed the results on microelements that have previously received little attention in the literature, is performed to serve as a guideline for how to assess the safety of waste streams such as these. Findings confirm existing results in the literature that Cd is bioaccumulative, but also that other elements, including Hg, Mn, and especially K, are bioaccumulative. To the authors' knowledge, this is the first research where the accumulation of Ag is also tested. The results of these tests are explained within the context of regulations in various countries where Hermetia illucens is cultivated, serving as a reference for practitioners to rigorously screen out high risk feed streams that they may consider using as feed sources. It is intended that these references and the demonstrated accumulation of a range of elements motivate comprehensive industry safety practices when evaluating new feed sources.

Chemical characterization of 21 species of marine macroalgae common in Norwegian waters: benefits of and limitations to their potential use in food and feed.

BACKGROUND: In the past few years, much effort has been invested into developing a new blue economy based on harvesting, cultivating and processing marine macroalgae in Norway. Macroalgae have high potential for a wide range of applications, e.g. as source of pharmaceuticals, production of biofuels or as food and feed. However, data on the chemical composition of macroalgae from Norwegian waters are scant. This study was designed to characterize the chemical composition of 21 algal species. Both macro- and micronutrients were analysed. Concentrations of heavy metals and the metalloid arsenic in the algae were also quantified. RESULTS: The results confirm that marine macroalgae contain nutrients which are relevant for both human and animal nutrition, the concentrations whereof are highly dependent on species. Although heavy metals and arsenic were detected in the algae studied, concentrations were mostly below maximum allowed levels set by food and feed legislation in the EU. CONCLUSION: This study provides chemical data on a wide range of algal species covering the three taxonomic groups (brown, red and green algae) and discusses both benefits of and potential limitations to their use for food and feed purposes. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay.

Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of ß-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo "autophagometer."

Dietary methionine availability affects the main factors involved in muscle protein turnover in rainbow trout (Oncorhynchus mykiss).

Methionine is a limiting essential amino acid in most plant-based ingredients of fish feed. In the present study, we aimed to determine the effect of dietary methionine concentrations on several main factors involved in the regulation of mRNA translation and the two major proteolytic pathways (ubiquitin-proteasome and autophagy-lysosomal) in the white muscle of rainbow trout (Oncorhynchus mykiss). The fish were fed for 6 weeks one of the three isonitrogenous diets providing three different methionine concentrations (deficient (DEF), adequate (ADQ) and excess (EXC)). At the end of the experiment, the fish fed the DEF diet had a significantly lower body weight and feed efficiency compared with those fed the EXC and ADQ diets. This reduction in the growth of fish fed the DEF diet was accompanied by a decrease in the activation of the translation initiation factors ribosomal protein S6 and eIF2α. The levels of the main autophagy-related markers (LC3-II and beclin 1) as well as the expression of several autophagy genes (atg4b, atg12 l, Uvrag, SQSTM1, Mul1 and Bnip3) were higher in the white muscle of fish fed the DEF diet. Similarly, the mRNA levels of several proteasome-related genes (Fbx32, MuRF2, MuRF3, ZNF216 and Trim32) were significantly up-regulated by methionine limitation. Together, these results extend our understanding of mechanisms regulating the reduction of muscle growth induced by dietary methionine deficiency, providing valuable information on the biomarkers of the effects of low-fishmeal diets.

Macronutrient composition of the diet affects the feeding-mediated down regulation of autophagy in muscle of rainbow trout (O. mykiss).

Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes through a lysosome dependent degradative pathway. Although the induction of autophagy by starvation has been extensively studied, we still know very little about how autophagy is regulated under normal nutritional conditions. The purpose of the present study was to characterize both in vivo and in vitro the response of the autophagy-lysosomal degradative pathway to nutrient (amino acids and carbohydrates) availability in the muscle of the carnivorous rainbow trout. We report that meal feeding is accompanied by a rapid activation of Akt, FoxO1 and the Target of Rapamycin (TOR) signaling pathways and a concomitant decrease of autophagosome formation. We also show that this effect occurs only when the proportion of dietary proteins increases at the expense of carbohydrates. Concurrently, our in vitro study on primary culture of trout muscle cells demonstrates an opposite effect of amino acids and glucose on the regulation of autophagy-lysosomal pathways. More specifically, the addition of amino acids in cell culture medium inhibited the formation of autophagosomes, whereas the addition of glucose had an opposite effect. The effect of amino acids was accompanied by an activation of TOR, considered as an important regulator of autophagosomal formation. However, the mechanisms involved in the effect of glucose were independent of Akt, TOR and AMPK and remain to be determined. Together, these results demonstrated the specific role of macronutrients as well as that of their interactions in the regulation of autophagy and highlight the interest to consider the macronutrient composition of the diets in the control of this degradative pathway.