Aquaculture has been a rapidly developing industry during the past four decades and is expected to continue in the foreseeable future to feed the growing population (Garlock et al., 2022). Atlantic salmon (Salmo salar) is one of the most farmed species (2.7 MT), accounting for 32.6 % of marine and coastal aquaculture of all finfish species in 2020 (FAO, 2022). Norway is the world’s leading producer of Atlantic salmon, producing around 1.4 MT in 2020. However, the downside of intensification of salmon industry has been the economic losses, due to mortality caused by salmon louse and treatments, infectious diseases, mucosal health, or multiple environmental stressors (Norwegian fish health report 2022). It has been shown that nutritional approach using functional feed ingredients (e.g., β-glucans or mannan-oligosaccharides, nucleotides, or plant extracts) can modulate the immune system of fish species (Ringø et al., 2012) and can protect fish from disease, parasites and various stressors (Hossain et al., 2023), thereby decreasing the mortality rate.

Recently, great attention has been given to the potential of using insect meal as sustainable feed ingredients due to its good nutritional profile (well-balanced content of amino acids, fatty acids, vitamins and minerals), presence of immunostimulant and bioactive compounds (Van Huis and Gasco, 2023). Among the approved insect species for its inclusion in aquafeed, black soldier fly larvae (BSFL) have gained widespread attention at research and commercial scale (Veldkamp et al., 2022b). This popularity of the black soldier fly might be due to its short life cycle, fast larval growth and ability to convert a variety of waste into valuable mass, thereby being an advantage for upscaling the insect production for use by the feed sector (Tran et al., 2022). Dietary BSFL meal can replace both marine and plant-based ingredients (such as fish meal (FM) and soy protein) and an inclusion up to 30% in aquafeed is possible without affecting the growth performance of fish species (Liland et al., 2021). However, dietary inclusion of BSFL meal should not only ensure growth but also modulate the immune responses and thus improve the health of farmed fish species (Mouithys-Mickalad et al., 2020).

The examination of the immunomodulatory effects of BSFL meal as a functional feed ingredient in fish serves to offer insights into the mechanisms and behavior of immune parameters in salmon fed BSFL-based diets. The BSFL meal contains diverse bioactive and immunostimulant compounds such as antimicrobial peptides (AMPs) (e.g., α-helical peptides, cysteine-rich peptides, proline-rich peptides, glycine-rich peptides), fatty acids (lauric acid) and polysaccharides (chitin and chitosan) (Veldkamp et al., 2022a). Insect AMPs play a key part in the innate immune response and have been shown to improve fish resistance to diseases (Hu et al., 2021; Rashidian et al., 2021). Lauric acid is known for its antiviral and antibacterial activities (Lieberman et al., 2006) and is also considered as a good source of energy for salmon (Belghit et al., 2019b). Furthermore, it has been shown that BSFL meal contains chitin, a polysaccharide that can function as a potential prebiotic in animal feed (Song et al., 2014). Studies in juvenile Mozambique tilapia (Oreochromis mossambicus) showed that incorporating small doses (0.5 g/kg feed) of dried prepupae from BSF in their diet had enhanced the hematological parameters (Ushakova et al., 2018). Moreover, dietary inclusion of housefly (Musca domestica) at low levels (between, 0.75% and 7.5%) increased the innate immunity and disease resistance of red sea bream (Pagrus major) (Ido et al., 2015) and black carp (Mylopharyngodon piceus) (Ming et al., 2013). An inclusion of dietary insect meals at low levels might act as a functional feed ingredient with potential health beneficial effects for fish species.

Therefore, based on this knowledge, the current study aimed to investigate the effects of dietary inclusion of BSFL meal as a functional feed ingredient in the Atlantic salmon diet (5% and 10%), reared in open sea-cages over the course of one year, aiming to simulate commercial farming conditions. The study aimed to assess the mucosal health and innate immune responses of salmon when exposed to chronic stress (pathogens and parasites, fluctuating temperature, salinity and water quality) and acute stress (delousing and handling).

The Atlantic salmon fed either 5% or 10% dietary BSFL meal showed a similar growth performance to the fish fed a diet with standard commercial protein sources. Earlier studies performed in laboratory scale in tanks on land, have also shown no effects on growth when using up to 12% full fat BSFL meal in pre smolts (Weththasinghe et al., 2021a) and up to 15% defatted BSFL meal in post smolt salmon (Belghit et al., 2019a) replacing the protein sources in the salmon feed (FM and plant-based protein). The current study thus confirms what has been seen earlier, but now verifying the lack of a negative impact on growth of Atlantic salmon fed BSFL meal in a situation much closer to a commercial aquaculture production.

The general welfare of salmon was monitored weekly (approximately 53 weeks) in terms of gill score and sea-lice count. These parameters were not affected by the experimental diet, except for December 2021, which is likely to be due to a random effect of sampling. Apart from that, the cumulative mortality rate was stable from the start of this trial (July-August 2021) until the mid-sampling (April 2022; 42 weeks post release into sea-cage) and was similar to the mortality pattern observed in Norwegian and Scottish salmon farms studied over the last 5 years (Soares et al., 2011; Bang Jensen et al., 2020), with an overall mortality rate of ~15% in Norwegian farms. However, after this time of the year till the end of the current trial, the mortality rate increased, which peaked in August 2022 (52 weeks post release into sea-cage), making the overall mortality rate of this trial ~34%. In fact, this rate was similar to the mortality rate (~23%) reported for salmon in a similar production zone in 2021 (Norwegian fish health report, 2022). This high mortality in the current study can be linked to various factors including multiple delousing, and diseases faced by salmon during that period. The salmon were exposed to thermal delousing to maintain the adult sea-lice population within the legal limits. Thermal delousing, a commonly used method in salmon farms to control and eliminate sea-lice, can contribute to increased mortality due to its exposure to higher water temperatures (up to 32 °C for ~ 30 sec), and repeated handling that create a highly stressful environment for the salmon (Moltumyr et al., 2021). During one of the delousing procedures, samples were taken before and after the process, indicating a notable increase in stress levels with higher plasma cortisol levels observed after one hour. However, the cortisol response was independent of the dietary BSFL meal in the salmon diet. Previous studies have also attempted different dietary modulation to reduce the delousing stress. For instance, Selvam et al. (2022) modulated the levels of dietary eicosapentaenoic acid and docosahexaenoic acid in Atlantic salmon but did not observe any significant dietary effect when exposed to mechanical delousing stress. Nonetheless, previous studies reported reducing the sea-lice infestations and mortalities in salmon through functional feed ingredients (Jensen et al., 2015) and dietary nucleotides (Burrells et al., 2001a). Apart from the delousing and handling stress, increased mortality might be also due to bacterial infection with Pasteurellosis, and Branchiomonas Cysticola, observed in salmon during the regular farm monitoring (diagnosed in May-June 2022, analyzed by Pharmaq Analytic, Bergen, Norway). These bacteria are known to affect the heart and gills in salmon, if occurred in heavy load, leading to high mortality (Gjessing et al., 2021; Sandlund et al., 2021). However, in the current study, very high gill infection was not observed in the three dietary groups. Hence, a direct correlation between gill score and bacterial occurrences was not observed. Nevertheless, it is plausible that diseases alone might not induce mortalities, but the combined stress from handling, delousing, and pathogenic pressure could contribute to increased mortality among salmon (Segner et al., 2012). There were, however, no effects of diets on growth or survival, demonstrating that diets containing up to 10% BSFL meal can give a long-term performance equal to fish fed a commercial diet in open sea-cages.

Studying hematological indices and mucosal health can be an easy and an important diagnostic tool for observing stress, physical anomalies, diseases symptoms and monitoring health (Assefa and Abunna, 2018; Seibel et al., 2021). The most used indicator for assessing stress levels in fish is plasma cortisol, which was measured at the mid and final sampling of this study to assess potential differences in basal stress level due to diet. At the final sampling, the plasma cortisol levels were higher in the salmon fed the control diet than the fish fed diets containing BSFL meal. The observed higher cortisol in the final sampling is hypothesized to be due to the exposure of salmon to multiple stressors mentioned in this study towards the end of the trial. The series of stressful events eventually resulted in elevated stress in salmon resulting in higher cortisol response in plasma. Furthermore, parallel to the above findings, the control group had a lower RBC and Hb count than the fish fed BSFL 5% at the same time point. This suggests that the elevated stress condition may have reduced the RBC’s oxygen-carrying capacity in control group compared to salmon fed BSFL meal, meaning that this group of salmon faced comparatively less stressful conditions in this experimental trial. A similar pattern of increased erythrocytes and decreased cortisol levels were observed in a study conducted in rainbow trout when fed with roselle (Hibiscus sabdariffa) meal, underlining the reason being the antioxidant capacity of the roselle meal (Hoseini et al., 2021). It has been shown that different insect species, including BSFL contain bioactive compounds with antioxidant properties (Priyadarshana et al., 2022; Veldkamp et al., 2022a) and is also known to modulate the oxidative stress in cellular and animal models (D’Antonio et al., 2021). Stress can affect the general health and welfare of fish. The results obtained in this study showed that salmon fed BSFL diets were able to better cope with the stressful events, probably due to the presence of bioactive compounds (chitin, AMPs) and antioxidant capacity, which might have reduced the oxidative stress in Atlantic salmon. The antioxidant capacity could also be confirmed by analyzing other potential stress biomarkers such as catalase, superoxide dismutase or respiratory burst activity. However, these analyses were not performed in the current study.

Lysozyme is one of the innate immune parameters studied in skin mucus (Pelusio et al., 2022). Differences in the mucus lysozyme activities mostly relate to the mucus composition (e.g., mucins, nucleotides, proteases, AMPs) (Fast et al., 2002) and an increase in lysozyme activity generally is triggered due to high microbial load (Ghafoori et al., 2014). Different studies have shown that plasma lysozyme activity increased in fish species fed diets containing insect meal, particularly after a challenge with pathogens (Su et al., 2017; Ido et al., 2019), triggering the innate immune responses. The evidence so far indicates a dose-dependent relationship between insect meal and plasma lysozyme activity in Atlantic salmon (6.25% vs 12.5% inclusion), as presented by Weththasinghe et al. (2021b). However, in the current study, the level of mucus lysozyme at the end of the feeding trial was lower in salmon fed BSF-based diets compared to the fish fed control diet. Apart from bacterial infection, it has been shown that higher lysozyme activity can also be due to high stress conditions (Dash et al., 2018). As discussed earlier, the control group exhibited high levels of cortisol, an indicator of stress condition, which might have increased the level of mucus lysozyme. A similar proportionality between plasma cortisol and skin mucus lysozymes was observed in Atlantic salmon when exposed to crowding stress (Demers and Bayne, 1997; Djordjevic et al., 2021). Fevolden et al. (1994), discussed that the elevated levels of plasma cortisol and rise in lysozyme could indicate higher susceptibility of organism to pathogens and this proportional relationship might be a compensatory adaptation to enhance innate immunity. Thus, lower levels of lysozyme in BSFL fed diets might indicate that salmon fed BSFL diet were comparatively less stressed due to immunostimulant property of BSFL.

The viscoelasticity of mucus is determined by the presence of various biopolymers such as DNA and mucins, responsible for trapping the pathogens and initiating the inflammatory responses (Kaitlyn et al., 2022). In humans, neutrophils are observed to actively release DNA, and this extracellular DNA plays a significant role in capturing harmful microorganisms (Brinkmann et al., 2004). This implies that DNA might have an immune function in mucus. In fish, the skin mucus contains DNA originating from host cells, as well as from beneficial or harmful bacteria (Brinchmann, 2016). Thus, higher secretions of DNA from neutrophils or other cell types into the skin’s surface can potentially increase the thickness of the mucus layer and thereby protecting the skin by increasing its viscoelasticity (Cameron and Endean, 1973; Al-Hassan et al., 1985; Caballero et al., 2020). In the present study, the DNA concentration in the skin mucus was comparatively lower in the control diet than BSFL fed at 5% (final sampling) but lacked any significant differences at the mid-sampling. Earlier work has shown that gill mucus viscosity was significantly lower in Atlantic salmon and brown trout (Salmo trutta L.) affected with amoebic gill disease compared to healthy fish (Roberts and Powell, 2005). Hender et al. (2021) reported an increase in the number of acidic mucins in Asian seabass (Lates calcarifer), that enhanced the gut and skin mucosal immunity when 30% FM and fish oil were replaced with partially defatted BSFL meal. Previous research findings suggest that supplementing of nucleotides and yeast cell extracts in the diet of Atlantic salmon enhances improved mucosal health and gut microbiota (Burrells et al., 2001b; Wang et al., 2022). Our results are therefore in line with earlier reports. Research has indicated that the low molecular weight antimicrobial peptides found in BSFL meal, specifically defensins, exhibit antibacterial properties. Defensins serve as active agents of the innate immune system, offering a first line of defense against infectious pathogens (Veldkamp et al., 2022a). Furthermore, proline-rich AMPs can interfere with DNA and RNA synthesis by binding to nucleic acids (Kragol et al., 2002) and exhibit antimicrobial properties against various bacteria (Mattiuzzo et al., 2007). Hence, there is a possibility that the presence of these antimicrobial peptides in the diet of salmon might have increased the mucus secretions in the form of DNA and mucins from neutrophils or by modulating the cellular process and producing more viscous mucus. However, further investigation is needed to comprehensively understand the link between diet and mucus defense systems, as well as more knowledge on how DNA concentrations can be used as a measure of mucus viscosity.

In the current study, higher mRNA expression of muc18 and mmp9 in gill (mid-sampling) were observed in salmon fed dietary BSFL at 5% compared to salmon fed control diet. These genes are mainly associated with mucus production and inflammatory processes such as remodeling and wound healing (Swain et al., 2006). An increased transcription of l1β in skin and gill (mid and final sampling) were also observed in salmon fed dietary BSFL meal. This observed transcription of il1β might be due to the bacterium Branchiomonas cisticola which is known to affect inflammatory genes (such as mmp25, mmp13, il1β, il8) (Gjessing et al., 2021). Thus, higher expression of il1β in salmon fed BSFL meal may indicate that they were able to comparatively resist better against pathogens. Furthermore, previous studies reported an upregulation of stress and immune related genes in gut of salmon fed with BSFL meal in vivo (Li et al., 2019; Li et al., 2020) and in vitro using isolated head kidney leucocytes from salmon challenged with bacterial and viral pathogens (Stenberg, 2018). Similar results were obtained in different tissues in Asian seabass, and European seabass when partially defatted BSFL meal and full fat BSFL meal was substituting 22% and 15% of the FM (Abdel-Latif et al., 2021; Hender et al., 2021). Furthermore, studies have also reported that supplementation of prebiotics such as Pediococcus acidilactici to the salmon diet could potentially stimulate the immune system and trigger the release of pro-inflammatory cytokines activation to fight possible pathogen attack (Jaramillo-Torres et al., 2019). In the current study, these observed effects might be linked to the immunostimulant properties of chitin and chitosan (Ghotloo et al., 2015). The chitin or chitosan from the BSFL meal is known to have the potential to stimulate the innate immune response (Veldkamp et al., 2022a). In the current study, however, the content of these components has not been measured. Therefore, the higher expression of these genes involved in the inflammatory process might be due to the content of chitin/chitosan as an immunomodulant in the BSFL meal or due to other compounds in the BSFL meal.

Inflammation triggers the innate immune system, which activates responses causing tissue damage and the release of AST and ALT into the body fluids (Samim and Vaseem, 2021). Compared to the other experimental diets, the diet with BSFL meal fed at 5% showed significantly lower levels of plasma ALT and AST at the mid-sampling. This suggests that incorporating BSFL meal into the diet may have a positive effect on the liver health of salmon. In the current study a reduction in plasma cholesterol and triglycerides was observed in the group fed BSFL which were similar to the studies reported in Japanese seabass (Lateolabrax japonicus) and African catfish (Clarias gariepinus) when FM was replaced by dietary BSFL meal (Wang et al., 2019; Fawole et al., 2020). This effect is likely due to the presence of chitin in BSFL, which has been documented to lower triglyceride and cholesterol levels (Wang et al., 2019). However, it is also possible that dietary cholesterol level or feeding status of salmon may have contributed to the observed effect, as the fish were sampled at a fed state.

Dietary inclusion of BSFL meal up to 10% in Atlantic salmon did not have any significant effects in general growth and survival. The salmon fed BSFL 5% showed an increase in the number of erythrocytes, and a reduced cortisol response when exposed to various stressors during the end of the trial and could indicate the antioxidant capacity of salmon fed BSFL diet. Moreover, this group of salmon exhibited a higher skin mucus DNA concentration which suggested higher mucus secretions enabling better protection against pathogens and stress conditions. Also, the inflammatory gene such as il1β, was upregulated in skin and gill tissue (mid and final samplings), while the plasma ALT and AST levels were lower in salmon fed dietary BSFL meal at 5% (mid sampling). It is worth mentioning that the health indicators observed in salmon behave differently across different fish sizes, time points and environmental conditions. In conclusion, the results of this study showed that dietary BSFL meal can modulate the immune responses in Atlantic salmon reared at large scale under farm-like conditions.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

This trial is exempt from application to the Norwegian Food Safety Authority, according to the regulation “FOR-2015-06-18-761 Regulation concerning the use of animals for scientific purposes, § 6. Godkjenning av forsøk". The approval requirement does not apply to experiments involving only the killing of animals to use organs or tissues from them. The study was conducted in accordance with the local legislation and institutional requirements.

GR: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources Data Curation, Writing - Original Draft Writing - Review & Editing, Visualization. AP: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources Data Curation, Review & Editing, Visualization, Supervision. MK: Conceptualization, Methodology, Investigation, Review & Editing, Project administration. NL: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Review & Editing, Visualization. E-JL: Conceptualization, Methodology, Validation, Investigation, Review & Editing. IB: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources Data Curation, Writing - Original Draft Writing - Review & Editing, Visualization, Supervision, Project administration. All authors contributed to the article and approved the submitted version.