The diversity and composition of the bacterial microbiome in teleosts have been extensively studied. However, the understanding of intestinal fungi and the factors influencing the composition of different gut fungal groups in fish remains limited. Addressing this, our study aims to shed light on the previously unexplored realm of teleost associated fungi.
Here, we present, for the first time, a comprehensive analysis of the remarkable transitions occurring within fungal communities throughout the life cycle of Atlantic salmon. By examining different stages of ontogeny, we unravel the dynamic nature of fungal populations. Furthermore, we also assess the spatial variation of the mycobiome of Atlantic salmon across multiple farms.
4.1 Hatching do not cause a change in the beta diversity of the mycobiome in Atlantic salmon
Salmonids have a long embryonic developmental time and the eggs are susceptible to fungal infections during this phase (Liu et al., 2014). Therefore, there is immense interest in the commensal microbes present at these stages, as they could be used to combat pathogens. This potential of commensal organisms has already been demonstrated in Atlantic salmon, where a member of the Actinobacteria (genus: Frondihabitans) was able to limit the spread of Saprolegnia in eggs (Liu et al., 2014). In the present study, EBH had the highest diversity and the diversity scores were higher than those of the bacterial population. This could indicate involvement of the fungal population in host developmental processes or pathogen containment (Thambugala et al., 2020). Interestingly, in contrast to the drastic changes in the bacterial population from EBH to HL (Lokesh et al., 2018), there was no significant transition in the fungal composition from EBH to HL. This was surprising because hatching results in a complete change in the anatomical structures that normally supports independent populations (Bledsoe et al., 2022). Furthermore, after hatching, microenvironments such as the gills, mouth, and gut take shape and provide unique niches for specific phylotypes (Llewellyn et al., 2014). Although there were no changes in alpha- and beta diversity, the significant phylotypes belonging to HL were different, indicating a shift in the abundance of certain groups at the HL stage. This may indicate a specific functional significance of these groups in HL.
Hatching is also a critical stage in ontogeny because the animal becomes more exposed to pathogenic organisms without protection from the chorion. Although members of Malassezia restricta found in the HL stage are not well characterized in teleosts, there are reports of this species causing infective endocarditis in humans (Houhamdi Hammou et al., 2021). On the other hand, Trichoderma viride (abundant in HL) is known to be a potent antifungal species that efficiently kills pathogenic fungi in plants through its extracellular secretions consisting mainly of the enzymes chitinases and β-1,3-glucanases (Pradhan et al., 2022). It would be relevant to investigate whether the salmon phylotype has similar potential for pathogen control.
4.2 First feeding has a strong impact on the intestinal fungal diversity and composition
The transition from yolk to exogenous feeding is a critical window in the ontogeny of teleosts including salmon (Hamre et al., 2013). In contrast to the non-significant transition in bacterial alpha and beta diversity, there were significant changes in the diversity of fungal populations. The difference in diversity (both alpha and beta) between the 7 wph and 8 wph was minimal. This could be due to the fact that the sampling points were only one week apart, which is not sufficient to detect inter-individual differences in the onset of feeding. The other comparisons between the stages sampled at this phase were significantly different from each other, indicating a progressive transition in the fungal communities. This suggests that the fungal populations are more sensitive to the onset of exogenous feeding than the bacterial populations. The progressive decrease in alpha diversity could be an indication of host selection of the phylotypes or the predominance of the phylotypes that could utilize the dietary components. Such selection by the host and the effect of diet is extensively reported for the bacterial microbiome in teleosts (Bakke et al., 2015; Llewellyn et al., 2014; Zhang et al., 2019).
We sampled 3 late freshwater stages and observed a significant fluctuation in alpha diversities, with 44 wph showing a sharp decline in Shannon index. In addition, beta diversities also differed significantly between the three groups sampled in this phase, indicating a progressive transition in community composition with development. Although the factors that caused the sharp decline in alpha diversity are not known, it should be noted that there was one phylotype (Candida tropicalis) that was highly abundant at this stage. It needs to be further investigated whether such high abundance has negative effects on the occurrence of other groups. Although Candida tropicalis is known to interfere with biofilm formation and morphogenesis of Candida albicans in humans, its effect on other fungal groups is unknown (de Barros et al., 2018).
4.3 Transfer of Atlantic salmon to seawater leads to a transition in fungal diversity and composition
Transfer to seawater is another critical step in the life history of Atlantic salmon, as the fish undergoes a series of physiological changes before entering seawater (McCormick et al., 1998). In the present study, a significant decrease in alpha diversity was observed after the fish were transferred to seawater. Beta diversity was also significantly different between the freshwater (62 wph) and seawater (65 wph) groups. It is known that the change to seawater affects the composition of the bacterial community both on the skin and intestine (Dehler et al., 2017; Lokesh and Kiron, 2016). It is noteworthy that a clear shift in the phylogenetic content was observed in the case of the bacterial microbiome, whereas in the present study, the phylotypes observed as significant features of the seawater stages (80 wph) were consistently observed in other stages and their abundance increases in seawater, indicating the halophilic nature of the phylotypes.
Immediately after the seawater transfer, Candida tropicalis was identified as the most abundant phylotype in the seawater group (65 wph), although it was also present at a substantial level in the freshwater group (62 wph). This suggests that the Candida tropicalis strain found in Atlantic salmon thrives both in freshwater and seawater, with a preference for seawater as its abundance increased in seawater (65 wph). The three stages sampled during the seawater phase showed a gradual significant increase in alpha diversity indices. At the same time, beta diversities also differed significantly between the groups. The drastic reduction in alpha diversity immediately after transfer to seawater and the progressive increase in alpha diversity during the seawater phase could indicate a restructuring of the compositional profile in response to the changes in salinity (Kim et al., 2021; Lorgen-Ritchie et al., 2023). Several phylotypes, including Alternaria metachromatica, Davidiella tassiana and Debaryomyces prosopidis, were identified as significant features of the late seawater stages (80 wph). It is noteworthy that these phylotypes were also present in fish at freshwater stages. It will be interesting to study the symbiotic relationships between these phylotypes and the host in different salinity levels to understand their importance in host biology.
4.4 Spatial variations in the intestinal fungal communities of Atlantic salmon
The fungal communities from the three farms did not differ significantly from each other in terms of their alpha diversities, whereas their beta-diversities differed significantly from each other. The phylotypes observed in adult fish from the farms were broadly similar to those found in the early developmental stages and in the intestines of the juveniles, although the abundance of certain groups was significantly different between farms. Such differences in intestinal community composition between farms (spatial differences) could be due to several reasons, including water temperature, salinity, pH and other husbandry practices, as well as fish origin and host-derived factors (Giatsis et al., 2015; Sullam et al., 2012). Although there are not many studies showing the spatial variations in the intestinal fungal communities of teleosts, it has been shown that the fungal phylotypes in the wild and lab reared zebrafish are significantly different. The wild zebrafish being highly abundant with Dothideomycetes whereas the lab reared fish comprising mainly of Saccharomycetes (Siriyappagouder et al., 2018b).
4.5 Dominant fungal communities of Atlantic salmon during their early and adult life
A few phylotypes were present in the fish irrespective of the stages of development or the samples source (different farms), namely Humicola nigrescens, Saccharomyces cerevisiae, Candida tropicalis, Davidiella tassiana, and Alternaria metachromatica. Their ubiquitous presence in all life stages, including the eggs and hatchlings, and the intestine in both freshwater and marine phases, suggests that they are subject to direct host selection and that these core members may be vital to host welfare (Sharp and Foster, 2022). Different strains of Saccharomyces cerevisiae have already been isolated and characterised from the intestine of different fish species. Their role in the health and nutrition of farmed fish has also been studied in detail, showing a positive effect on the overall health of the host (Sharma et al., 2022). Members of Candida sp. have been reported from the intestinal population of marine fish and a member of the salmonid family rainbow trout (Oncorhynchus mykiss) (Andlid et al., 1995; Gatesoupe, 2007), although their specific role in host biology remains to be described.
Davidiella tassiana and Alternaria metachromatica are known plant pathogens (Bashir et al., 2014). These two phylotypes occur at different stages of ontogeny, including eyed eggs (EE). Although their functions in teleosts are generally uncharacterised, the susceptibility of fish eggs to fungal infections of multispecies origin may indicate that these groups are indeed opportunistic pathogens. The presence of Humicola nigrescens has been detected in environmental soil samples (Sharma-Poudyal et al., 2017), but its occurrence in the host-associated microbiome is not well established. These groups of organisms are known to produce extracellular phytase that cleaves phytic acid, resulting in higher bioavailability of organic phosphate, zinc and iron (Bala et al., 2014). It is likely that this phylotype provides these minerals, which are important for host development, in the early stages of development and in the intestine of young animals and adults.
The fungal microbiota in the early stages of ontogeny and in the intestine of juvenile and adult fish is a mixture of potentially opportunistic pathogenic phylotypes and some fungicidal and probiotic groups. Overall, these results warrant further investigation to understand whether it is indeed an arms race between pathogens and beneficial groups. Furthermore, understanding the role of abiotic factors such as temperature, pH and other water quality parameters in this interaction is crucial for the development of aquaculture health management strategies.
4.6 Fungal diversity is significantly lower than the bacterial diversity in most of the stages of ontogeny
In addition, we compared the alpha diversity of fungi with that of bacteria at the same developmental stages and found that fungal diversity was significantly lower at most stages, with the exception of a higher diversity value in the EBH group. Lower fungal diversity compared to bacterial diversity has been found in many species (Gatesoupe, 2007; Scanlan and Marchesi, 2008). Although fungal populations have lower diversity compared to bacteria, they are considered functionally important for the stability of the microbial ecosystem because there are specific interactions between bacterial and fungal groups that are important for the physiology of the host (Gatesoupe, 2007; Lapiere and Richard, 2022). Indeed, in zebrafish it has been shown that exposure of larvae to Debaryomyces sp. (isolated from the intestine of zebrafish) resulted in a significant increase in the abundance of potentially beneficial bacterial genera such as Pediococcus and Lactococcus, suggesting possible cross-kingdom interactions between these groups (Siriyappagouder et al., 2018a).