Novel diets and fish health : evaluating the immuno-nutritional impact of filamentous fungus Paecilomyces variotii in aquafeeds for Atlantic salmon (Salmo salar)

Abstract

Filamentous fungi Paecilomyces variotii is an interesting microbial ingredient (MI) with high protein content and bioactive compounds such as β-glucans, mannans and nucleic acids. In the past, P. variotii has supported good growth of chicken, pigs and calves by replacing conventional ingredients. However, there is a paucity of research knowledge on the nutritional value and health benefits of P. variotii for fish. Thus, the objective of this thesis was to investigate the nutritional value and health impact of P. variotii in Atlantic salmon with focal interest on the growth performance, gut microbiota, local and systemic immune responses, as well as overall fish health and welfare in both freshwater and seawater under controlled conditions. The P. variotii used in this thesis was produced by Enifer (Espoo, Finland) on sulphite stillage through continuous fermentation and dried at 70 0C using fluidized bed. Regarding the articles related to this thesis, in paper I, we used a desk study method to review state-of-the-art literature in which in vitro salmonid models have been used to study microbial derived immunostimulants (β-glucans, LPS, dsRNA/poly[I:C]) and economically relevant salmonid pathogens (viruses, bacteria, parasites). We identified their strength and weaknesses and provided future perspectives on 3D models like bioreactors, organ-on-chip, organoids etc. Only partial information is obtained from current in vitro models (mostly 2D); hence they cannot be proxies or direct replacements but suitable complements of in vivo models. The study found several 3D models, where research into which are advanced in mammals but not fish. Future 3D models like gene editing, bioreactors, organ-on-chip models and organoids are interesting models that can be adapted for studies related to fish. Like the 2D models, 3D models in their current state cannot individually replace live animal testing. However, a combination of the 3D models and advanced bioengineering can enhance their usage as possible animal replacement models. In paper II we characterized the bioactive composition of P. variotii, its bioactivity on primary cultures of salmon head kidney leucocytes (HKLs) and spleen leucocytes (SLs) with or without heat inactivated M. viscosa (an important salmonid pathogen), as well as the impact of downstream processing (DSP) on the ultrastructure of P. variotii. We also investigated the impact of carbon source (sulphite stillage, glucose or glycerol) and choice of fermentation (batch or continuous) on the crude protein (CP) and glucan composition of P. variotii. Glucose or glycerol in combination with batch fermentation significantly increased the glucan but lowered CP contents of P. variotii and vice versa for sulphite stillage together with continuous fermentation. Drying also significantly reduced the thickness of the cell wall and mannan layer of P. variotii which could in turn improve digestibility and access to nutrients. Stimulating HKLs with different fractions of P. variotii induced an early pro-inflammatory (tnfα, il1β) and anti-microbial response (cath2) while in SLs, both pro- and anti-inflammatory response (tnfα, il6 and il10) was induced. Stimulating HKLs and SLs with M. viscosa alone upregulated pro-inflammatory cytokines (tnfα, il1β, il6), antimicrobial response (cath2), and receptors (sclra, sclrb) but suppressed regulatory response (il10). Prolonged co-stimulation (48h) with M. viscosa and P. variotii induced homeostasis (tgfb, il6) and antimicrobial (cath2) response in SLs. In paper III, to understand the impact of P. variotii on short-term growth performance and health responses, vaccinated Atlantic salmon pre-smolts were fed diets where P. variotii replaced CP from fishmeal (FM), wheat gluten meal (WGM), and soy protein concentrate (SPC) at 0%, 5%, 10% or 20% for 28 days in freshwater (FW). The mycoprotein supported the short-term growth performance of Atlantic salmon and induced a dose-dependent linear improvement of FCR with increasing inclusion of P. variotii. High CP replacement with P. variotii (20%) induced downregulation of receptors and cytokines (sclra, sclrc, cr3, il1b, tgfb) indicative of truncation of signal transduction in the distal intestine (DI). In the head kidney (HK), P. variotii activated and controlled immune response through c-type lectin receptors, signal molecules, cytokines, effector molecules, and coordinated communication between innate and adaptive immune response through antigen presenting cell markers (mhcii, cd83) and T cell transcription factors (gata3, rorc, foxp3). Interestingly, the HK proteomic profile of fish fed the 20% P. variotii diet showed reduced proteins related to inflammation (apoptosis, necroptosis etc), and signalling (TLRs, CLRs etc) contrary to the gene expression data. However, these proteins were increased in the skin mucus with other key proteins related to the complement activation (C1q, C4, C7), arginine and proline metabolism. Fish fed diets with CP replacement with 5% P. variotii showed high titres of specific IgM against V. anguillarum in the serum. Finally, 5% and 10% CP replacement with P. variotii increased both the α- and β-diversity and modulated lactic acid bacteria (LAB), including Ligilactobacillus. In paper IV, the fish from freshwater were transferred to saltwater (SW) and fed for additional 35 days with the same diets as in FW (paper III). The results indicate that P. variotii supports the short-term growth performance of Atlantic salmon post-smolts. Increasing inclusion of P. variotii in diets increased the DI simple fold height but reduced the area of epithelium occupied by goblet cells (GCs). Again, 5% CP replacement with P. variotii decreased the average size of supranuclear vacuoles in the DI of the fish. In addition, 20% CP replacement with P. variotii increased serum and skin mucus total IgM titres while 5% CP replacement with P. variotii increased epidermal muc-like proteins and cytokine il10 in the HK of the fish compared with the control diet group. We also observed a significantly lower serum creatine kinase (CK) in fish fed the 20% CP replacement diet compared with the control diet. Interestingly, we observed a high occurrence of welfare disorders (e.g., scale loss, fin rot/damage, skin haemorrhage) in fish fed the 20% CP replacement with P. variotii. Based on the results, it can be concluded that P. variotii is a promising novel MI whose nutritional and functional composition can support fish growth and meet the prophylactic needs of salmonids. Thus, beyond the nutritional value of P. variotii, its functional potential can be harnessed through immuno-nutritional strategies to improve the health and welfare of farmed salmonids in sustainable and eco-friendly manner.