In the last several decades, the importance of the gut microbiota has been documented in numerous studies showing that growth performance and fish health are closely related to the microbiota. As Butt and Volkoff (2019) commented, feeding habits influence the structure and composition of the gut microbiota . Additionally, plant-based proteins change the content and structure of the autochthonous microbiota of carnivorous species (Kononova et al. 2019) such as sea trout; in contrast, the use of a natural source of protein such as insect meals may play a role in maintaining the amount of these types of microorganisms that are part of the gut environment of the fish and enhance fish health . Furthermore, insect meals would be able to modulate the microbiota of these animals due to the chitin and antimicrobial peptide contents , .
In this trial, more than 40% of FM (fish meal) was replaced with insect meals in the two diets, although the insect meals produced similar growth and survival rates when observing the weight gain. When analyzing the bacteria present in the digesta, the dominant phylum in all the treatment groups was Firmicutes, in contrast to brown trout fed a commercial diet, in which the dominant phylum was Proteobacteria, ranging from 88.4 to 92.6% . However, Michl et al. (2019) reported a reduction in the amount of Proteobacteria and an increase in the amounts of Firmicutes and Bacteriodetes in the same species fed diets with more plant-based meals . In contrast, Rimoldi et al. (2019) detected a gradual increase in the abundance of Tenericutes and a reduction in the abundances of Proteobacteria and Firmicutes in n rainbow trout fed different amounts of black soldier fly meals . Moreover, Kononova et al. (2019) affirmed that Proteobacteria is more abundant in carnivorous species and Firmicutes is more abundant in herbivorous species . The results from the present trial showed that the abundance of Firmicutes would be conditioned by the amount of plant meal in the three diets, which was approximately 47% of the total, but not the inclusion of insect meals.
After performing a detailed analysis of the classes present in the digesta, Bacilli was the most abundant in all treatment groups, followed by Clostridia, both of which belong to the Firmicutes phylum, but the sum of Alphaproteobacteria and Gammaproteobacteria, which belong to the Proteobacteria phylum, presented similar amounts in all treatment groups, indicating that MW and SW meals exerted the same effect as FM on the digesta of sea trouts. In addition, that the phylum Firmicutes and class Clostridia have been repeatedly identified in the digestive tracts of herbivorous fish, and as described above, the higher abundance of this class would be related to the higher amount of plant meal present in the diets .
The order and family distribution followed a similar trend as the class distribution. Although the bacterial genera exhibited changes based on the type of protein meal source, the most representative genera in the CON group were Clostridium and Lactobacillus and those in the MW group were Enterococcus and Clostridium, but the most representative genera in the SW group were Pediococcus and Enterococcus. With the exception of Pediococcus, the other genera are used as probiotics in aquaculture, increasing bacterial diversity , which probably occurred in fish fed these insect meals. The type of meals exerted a direct effect on the abundance of different genera in the intestinal digesta.
An analysis of the species abundance showed that fish fed the diet with SM exhibited a decrease in the relative abundance of C. cadaveris compared to the CON and MW groups; this species is known as a component of the normal fecal flora of humans and animals, which affects people with a poor overall condition of immunosuppression . On the other hand, C. cadaveris is one of the most prominent bacterium present during the decay of dead bodies . Moreover, C. cadaveris might trigger bacteremia that is related to a high mortality rate in humans. In the present study, the relative abundance of the commensal species C. cadaveris was decreased in the SW group, but significant differences were not observed between the CON and MW groups. Therefore, the reduction in the relative abundance C. cadaveris in the gastrointestinal tract of fish induced by the diet containing SW should may considered as a positive effect on public health.
C. parietina belongs to phylum Cyanobacterium and was previously detected in alkaline and oxygenated freshwaters . The growth of Cyanobacteria is stimulated by the hypoxia of water reservoirs. Moreover, the contamination of dry food and feed with Cyanobacterium is considered a risk of toxin prevalence. Moreover, C. parietina is the bacteria with a higher potential for endotoxin production. The diet containing SW caused a decrease in the abundance of the bacterial species C. parietina in the fish GIT, which may reduce possible cyanobacterial toxin reservoirs in the fish GIT.
The diet containing SW improved the commensal probiotic microbiome in intestinal digesta of Salmo trutta vr. trutta. The SW diet increased the abundance of some bacterial genera, such as Pediococcus that is considered a positive fish GIT bacteria. Pediococcus is a genus of gram-positive lactic acid-producing bacteria belonging to the family Lactobacillaceae. In the SM group, an increase in the abundance of pediococci was observed, with the most abundant species identified as P. pentosaceus in the SW group. The bacterial species P. pentosaceus exerts bacteriocynogenic effects on Staphylococcus aureus and Escherichia coli . P. pentosaceus is mostly associated with food fermentation; it produces pendocins that are safe for food preservation and is used as a starter culture in the fermentation of meat products.
Enterococcus is a key component of the intestinal flora of humans and is widespread in the intestines of most animals, including fish. Some species belonging to the Enterococcus genus, such as Enterococcus faecalis from fish intestine, are use as aquatic probiotics . The SM diet increased the abundance of some Enterococcus species in the fecal digesta, among which E. durans may be considered a possible probiotic, because it potentially produces bacteriocins, namely, durancins . Another species with probiotic potential that have been isolated from fish is Enterococcus gallinarum that regulates the innate immune response . An increase in the abundance of Enterococcus gilvus was also observed in the fecal digesta of the analyzed SW group. The analysis of gene expression in Enterococcus gilvus has identified novel carotenoid biosynthesis genes that improve the multistress tolerance of Lactococcus lactis and promotes their activity toward methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE) . Additionally, W. cibaria, which was more abundant in the SW group, has shown to be an effective probiotic in hybrid surubim . Although significant differences among certain groups of bacteria were observed, the composition of the most representative species shows that they are part of the digestive tract flora, the environment, or part of the protein sources with probiotic properties that help the fish to thrive and achieve target growth and survival rates. In addition, Gajardo et al. (2016) commented that LAB are more abundant in salmon fed a plant-based diet than in fish fed a fishmeal-based diet , although, Ringø and Gatesoupe (1998) commented that LAB, such as the Lactobacillus, Carnobacterium, and Streptococcus genera, are also commonly detected in healthy fish microbiota of different fish species, including salmonids . However, insect meals also increase the amount of LAB, as observed in the present study.
Our research diet containing SW decreased the relative abundance of Streptococcus gallinaceus in the intestinal digesta. S. gallinaceus was first described in 2002 and was isolated from clinical samples of chickens. In 2003, S. gallinaceous was isolated from an outbreak of septicemia associated with a high prevalence of endocarditis in a flock of broiler parents. Chadfield et al. (2005) reported an association of this species with septicemia and endocarditis in chickens . The decrease in the relative abundance of S. gallinaceus detected in the intestinal digesta might be considered a positive dietary effect of SW.
Furthermore, when comparing alpha diversity parameters, the inclusion of insect meal in the diet did not modify the different parameters measured, such as richness, evenness, and dominance. The Shannon H values were similar to those obtained in rainbow trout fed only FM and greater than 60% of MW meal . Additionally, the Chao1 values obtained in the present study were similar to those observed in the digesta of the proximal intestine of salmon fed 45% FM and 38% plant meals . These authors obtained a higher Shannon H index than observed in our results. Moreover, in brown trout fries fed three experimental diets, 100% FM, 50% and 90% plant-based diets followed by a crossover feeding design, plant-based diets produced higher Chao1 and Shannon indexes than the FM diet, although the Chao1 values were lower than the values reported for sea trout in this experiment . In general, the diversity among treatments was similar. Additionally, the NMDS analysis and the similarities of the clusters showed that the microbiome of the MW group is more similar to the FM group than the SW group, which would be more useful for salmonid nutrition, as described by Antonopoulou et al. (2019) .
As mentioned above, the two insect meals exerted a similar effect to FM on maintaining the alpha diversity, and the values of dominance, equitability, and evenness were similar between all treatment groups, showing a balanced microbiome population that varied in abundance among bacterial classes, orders, genus and species as a natural consequence of the type of protein sources used. Nevertheless, the different meals that the fish consumed exerted positive effects on the microbiome, growth and survival performance of the sea trout, although the predominance of phylum Firmicutes in all treatment groups would be a consequence of the amount of plant meals, which were higher (47.17%) than animal meals (33.5%) in the diets, particularly for soybean meal, as highlighted by Kononova et al. (2019) and Michl et al. (2019) , . Nevertheless, we cannot forget that plant meals are part of all commercial diets because of their availability and lower prices than fishmeal, and they are used to study the effects of alternative meals, such as insect meals.