Introduction
Microbial proteins including filamentous fungal biomass contain noticeable levels of protein, fatty acids and other nutritious components (Karimi et al., 2018). Also fungi cell wall contains chitin and chitosan and other polysaccharides as their bioactive components having immunomodulatory and antimicrobial properties (Mario et al., 2008, Esteban et al., 2001). Thus, fungi may play a role in improving the health of farmed fish. Several studies has been reported that environmental (abiotic) and host (biotic) factors play important role in shaping the gut communities (Ingerslev et al., 2014a; Ringø et al., 2016; Yan et al., 2016; Sun et al., 2020). Most of these studies investigated the short or long term dietary effect of diets on gut microbiota. However, a study targeting the gradual change in microbial communities over time with the type of diet that to our knowledge has not been described earlier. Diet can adversely modulate the gut microbial composition leading inflammation of distal intestine as in case of Atlantic salmon fed with higher levels of soy protein (Gajardo et al., 2017). It is necessary to understand the interaction between host, gut microbiota, diet and feeding strategy for the development of novel diets ensuring better fish health and welfare. Hence, this study was conducted to observe role of N. intermedia in modulating the intestinal microbiota of rainbow trout under provided feeding periods.
Materials and Methods
Three experimental diets were prepared, a reference diet (RD), a non-preconditioned diet (NPD) and a preconditioned diet (PD). Diet RD was prepared with fishmeal as a major protein source without fungal biomass. Formulation for diet NPD and PD were created by mixing 30 percent of Neurospora intermedia biomass and 70% of diet RD according to (Cho, 1979). Formulation for diets PD and NPD were same, however, diet PD was conditioned in a convection oven (Electrolux Professional, FCE061) at 105⁰C for 5 min in order to increase the degree of gelatinization of starch and emulate temperature treatment during extrusion conditions. Twenty fish were distributed randomly in each experimental tank (mean weight: 127± 4 g) and were acclimated for 9 days on commercial diet. Feeding was provided in excess at 1.5% of body weight in excess. Gut microbiota were analyzed on day 0, 10, 20 and 30. Similarity Percentage Analysis (SIMPER) and Analysis of Similarity (ANOSIM), Principal coordinate analysis (PCoA) and Principal component analysis (PCA) were performed using Paleontological Statistics Software version 4.03 (PAST).
Results
The bacterial OTUs abundance were dominated by two phyla Firmicutes (58%) and Proteobacteria (15%). From day 0 to day 30, Firmicutes ranged from 38 % to 79% followed by Proteobacteria which ranged from 8% to 24% for different diets. Peptostreptococcus (9%), Lactococcus (L. lactis, 7%), Brevinema (6%), Streptococcus (5%), Deefgea (5%) and Anaerotruncus (4%) were the most abundant over 30 days. Diet and day has significant effect on shaping the gut microbial composition. The pairwise comparison of the treatments groups were significantly dissimilar within and between day intervals for top 6 OTUs. PCA plot reveals the occurance of Peptostreptococcus and Streptococcus are correlated. Their abundance was correlated to initial days of feeding in all diets whereas abundance of Lactococcus was more correlated to final days of feeding. One way ANOSIM confirmed that the microbial composition was similar at day 0 and day 30 whereas dissimilar at day 10 and day 20. The overall microbial composition for diet RD is different from PD and NPD at day 10.
Discussion
The analysis reveals that the core gut microbiota was dominated by Firmicutes followed by Proteobacteria which is similar with other studies on salmonids (Nayak, 2010; Gajardo et al., 2017). Bacterial composition for plant protein based diets or fish meal free diets show increase in abundance of Lactobacillales in rainbow trout and salmon respectively (Schmidt et al., 2016; Michl et al., 2017). Earlier studies suggested that change in microbiota with change in diet was observed in salmon, rainbow trout and brown trout after first feeding (Ingerslev et al., 2014b; Michl et al., 2017; Michl et al., 2019). No change in the gut microbiota with the time was observed suggesting after certain days or longer feeding gut microbiota gains stability. Similarly, it was observed from the analysis that gut microbiota were significantly dissimilar at day 10 and day 20 and were similar at day 30 of feeding suggesting that the microbiota might be approaching stability.
Conclusion
References
Cho, C. (1979). Apparent digestibility measurements in feedstuffs for rainbow trout. In: Halver, J., Tiews, K. (ed. Proceedings of the world symposium on finfish nutrition and fishfeed technology Heenemann, pp. 239-247.
Gajardo, K., Jaramillo-Torres, A., Kortner, T.M., Merrifield, D.L., Tinsley, J., Bakke, A.M. & Krogdahl, Å. (2017). Alternative protein sources in the diet modulate microbiota and functionality in the distal intestine of Atlantic salmon (Salmo salar). Appl. Environ. Microbiol., 83(5), pp. e02615-16.
Ingerslev, H.-C., Strube, M.L., von Gersdorff Jørgensen, L., Dalsgaard, I., Boye, M. & Madsen, L. (2014a). Diet type dictates the gut microbiota and the immune response against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). Fish & shellfish immunology, 40(2), pp. 624-633.
Ingerslev, H.-C., von Gersdorff Jørgensen, L., Strube, M.L., Larsen, N., Dalsgaard, I., Boye, M. & Madsen, L. (2014b). The development of the gut microbiota in rainbow trout (Oncorhynchus mykiss) is affected by first feeding and diet type. Aquaculture, 424, pp. 24-34.
Karimi, S., Soofiani, N.M., Mahboubi, A. & Taherzadeh, M.J. (2018). Use of Organic Wastes and Industrial By-Products to Produce Filamentous Fungi with Potential as Aqua-Feed Ingredients. Sustainability, 10(9).
Michl, S.C., Beyer, M., Ratten, J.-M., Hasler, M., LaRoche, J. & Schulz, C. (2019). A diet-change modulates the previously established bacterial gut community in juvenile brown trout (Salmo trutta). Scientific reports, 9(1), pp. 1-12.
Michl, S.C., Ratten, J.-M., Beyer, M., Hasler, M., LaRoche, J. & Schulz, C. (2017). The malleable gut microbiome of juvenile rainbow trout (Oncorhynchus mykiss): Diet-dependent shifts of bacterial community structures. PLoS One, 12(5).
Nayak, S.K. (2010). Role of gastrointestinal microbiota in fish. Aquaculture Research, 41(11), pp. 1553-1573.
Ringø, E., Zhou, Z., Vecino, J.G., Wadsworth, S., Romero, J., Krogdahl, Å., Olsen, R., Dimitroglou, A., Foey, A. & Davies, S. (2016). Effect of dietary components on the gut microbiota of aquatic animals. A never‐ending story? Aquaculture nutrition, 22(2), pp. 219-282.
Schmidt, V., Amaral-Zettler, L., Davidson, J., Summerfelt, S. & Good, C. (2016). Influence of fishmeal-free diets on microbial communities in Atlantic salmon (Salmo salar) recirculation aquaculture systems. Appl. Environ. Microbiol., 82(15), pp. 4470-4481.
Sun, S., Su, Y., Yu, H., Ge, X. & Zhang, C. (2020). Starvation affects the intestinal microbiota structure and the expression of inflammatory-related genes of the juvenile blunt snout bream, Megalobrama amblycephala. Aquaculture, 517, p. 734764.
Yan, Q., Li, J., Yu, Y., Wang, J., He, Z., Van Nostrand, J.D., Kempher, M.L., Wu, L., Wang, Y. & Liao, L. (2016). Environmental filtering decreases with fish development for the assembly of gut microbiota. Environmental microbiology, 18(12), pp. 4739-4754.
Di Mario, F., Rapana, P., Tomati, U., & Galli, E. (2008). Chitin and chitosan from Basidiomycetes. International
journal of biological macromolecules, 43(1), 8-12.
Esteban, M.A., Cuesta, A., Ortuño, J. & Meseguer, J. (2001) Immunomodulatory effects of dietary intake of chitin
on gilthead seabream (Sparus aurata L.) innate immune system. Fish Shellfish Immunol. 11, 303– 315.