Introduction
Sustainable aquaculture is dependent on a variety of factors, including water quality and effective feed formulations. To increase sustainability in aquaculture maintaining fish health, alternative protein sources to replace fishmeal (FM) should be used. Insects represent a new world of sustainable and protein-rich sources for farmed fish diets. The yellow mealworm (Tenebrio molitor) is one of the most popular insect species used in aquafeeds. The larvae from this insect are an excellent substitute for FM (Chemello et al., 2020; Terova et al., 2021). While great research efforts have been made to evaluate feed formulations, focusing especially on the effects on fish gut microbiota, few studies have explored host-environment interactions. Here, we evaluated the effects of a T. molitor -based novel feed formulation on the microbiota at the water-fish interface, in an engineered ecosystem farming rainbow trout (Oncorhynchus mykiss).
Materials and methods
Rainbow trout of about 80 g mean initial weight were randomly distributed into 400 L tanks (3 tanks/diet, 21 fish/tank) and fed with either a control diet without insect meal (IM) (diet A), or diets B, C, and D formulated with T. molitor larvae meal at 5%, 10%, and 20% inclusion, respectively. Fish were fed twice a day (at 8 am and 3 pm), 6 days per week. Water samples and water tank biofilm samples were collected to quantify bacterial DNA load by using qPCR. For each water sample, total nitrogen, nitrites, nitrates, ammoniacal nitrogen, and phosphate were measured with a spectrophotometer. At the end of the trial, six fish/diet were sampled from the groups fed with diet A (without IM), and diet D (with 100% FM/IM replacement). The skin mucus microbiota was obtained by gentle scraping of the fish body with a cotton swab, whereas the gut autochthonous microbiota was obtained by scraping the mucosa of the entire intestine. Using 16S rRNA metabarcoding, we comprehensively analyzed the microbiota of water inlet, water, tank biofilm, fish mucus, fish cutis, and feed samples. Raw sequencing data was processed by QIIME2. The Kruskal-Wallis H test for all and pairwise tests were used to compare the groups. Statistical significance between groups was determined by the ADONIS (permutation-based ANOVA, PerMANOVA) test with 10000 permutation-based Bray-Curtis.
Results and Discussion
The microbiological and chemical analysis of water showed no significant differences due to different feeds consumed by fish, proving that FM/IM substitution does not affect water quality. Microbiota analysis revealed the presence of a highly reduced core microbiota, constituted by Aeromonas spp., for both the control group and the novel feed test group. Looking at tank biofilm, Acinetobacter, Pseudomonas, Rhodococcus, and Candidatus Amoebophilus were significantly more abundant in tanks in which diet A was administered. The skin microbial community composition of two dietary groups A and D displayed distinctive features. A decrease of Proteobacteria and specifically of the Deefgea genus in the skin mucus-associated microbiota was found in trout fed with IM. Similarly, at the gut mucosa level, the dietary T. molitor meal inclusion led to a significant reduction of gut Proteobacteria phylum, predominantly belonging to the Gammaproteobacteria class. Furthermore, with respect to the control fish group, feeding IM led to a lower abundance of Acinetobacter genus, another potential pathogen in aquaculture, commonly known as a microorganism transmitting antibiotic resistance genes. Therefore, altogether our findings on gut microbiota indicate that feeding trout with IM has a positive effect through inhibiting the growth of potential Gram- pathogen bacteria. By network analysis, we showed that the major driver of microbial community structure was the sample source, with the main differences found between environmental vs host-associated samples. Network analysis indicated that samples clustered based on sample source, with no significant differences related to the feed formulation. Thus, different feed formulations seemed to not affect the environment (water and tank biofilm) and the fish (skin and mucus) microbiota. Trying to disentangle the contribution of feed at a finer scale, we performed a differential abundance analysis, and we observed differential enrichment/impoverishment in specific taxa, comparing the samples belonging to the control diet group and the insect-based diet group (Figure. 1).
Conclusions
Our results highlight a link between the environment and the fish, and subtle but significant differences due to feed formulation.
Acknowledgements
This work was funded by the EU Horizon 2020 AquaIMPACT (Genomic and nutritional innovations for genetically superior farmed fish to improve efficiency in European aquaculture), number: 818367. The research was co-funded by the AGER project Fine Feed for Fish (4F), Rif. No. 2016-01-01.
References
Chemello G, Renna M, Caimi C, Guerreiro I, Oliva-Teles A, Enes P & Gasco L. Partially defatted Tenebrio molitor larva meal in diets for grow-out rainbow trout, Oncorhynchus mykiss (Walbaum): Effects on growth performance, diet digestibility and metabolic responses. Animals. 2020;10(2):229.
Terova G, Gini E, Gasco L, Moroni F, Antonini M, Rimoldi S. Effects of full replacement of dietary fishmeal with insect meal from Tenebrio molitor on rainbow trout gut and skin microbiota. J. Anim. Sci. Biotechnol. 2021;12(614):1-14.