Discussion
Interactions between fish hosts and their microbiomes have been an under-studied area of research, perhaps due to the complexity of the host-microbiome relationship making the detection of specific microbial features that impact the host phenotype challenging. We approached this problem by manipulating gut microbiomes and measured the impact on key candidate gene regulation – such effects are likely mechanisms for microbes to affect host phenotype and health. We found that our treatments resulted in changes in host gene expression patterns, and those changes were mostly related to immune function and cell motility/integrity. By correcting for the direct effects of the treatment, as well as the quantitative genetic effects of family, we showed that changes in microbial communities do lead to changes in host physiology. Given the putative function of the responding genes, our work indicates a likely effect on host fitness as well. Indeed, many recent studies have shown that microbial symbionts are critical biological components for host traits closely associated with fitness, such as immune system development and function (Fuess et al., 2021; Langlois et al., 2021; Rosshart et al., 2017).
This is the first study to consider and compare the impact of probiotics and antibiotics administered to captive fish on the rearing water microbial communities and we found that the aquatic microbial communities in the rearing tanks were significantly influenced by the feed treatments. This was not expected as the fish food treatment itself represented a small proportion of the tank volume, especially given the low flow through water effect. One possible factor is that up 90% of administered antibiotics are excreted in the urine and faeces of the fish, still in the active form (Polianciuc et al., 2020). The common bacterial phyla we report in the tank water were also reported in other studies that showed Proteobacteria, Bacteroidota, Firmicutes are the dominant taxa in water where fish are held (Chiarello et al., 2015; He et al., 2018; Stevick et al., 2019; Uren Webster et al., 2018; Zhang et al., 2019). Nevertheless, we observed significant treatment effects on the rearing water BCs, one possible explanation would be antibiotic-associated diarrhea leading to more fish gut-associated microbial excretion. Another reason could be antibiotic-susceptible taxa being replaced by taxa resistant to antimicrobial agents (e.g.,Mycoplasmataceae (Firmicutes) (antibiotic (15%), control (1%), probiotic (3%)). Since the aquatic microbiome itself plays a role in maintaining fish health (Blancheton et al., 2013) quantifying the unexpected effects of feed-based treatment on the rearing water is unexpected and important as it may contribute to dysbiosis and poor health outcomes in the fish. Although the negative effects of antibiotics on healthy fish have been reported before, few studies have considered the effect of antibiotic treatment on the rearing water microbiome. Furthermore, our study showed that probiotic feed treatment also affected the water microbiome. Previous studies showed that that treating water with probiotics can improve water quality (Elsabagh et al., 2018; Tabassum et al., 2021).
The microbial communities present in fish rearing water are thought to affect the initial colonization of the fish microbiota during development (Llewellyn et al., 2014; Talwar et al., 2018). However, similar to other studies (Uren Webster et al., 2018; Wu et al., 2018), our fish gut microbiomes were distinct from the water sample microbiomes. This indicates that the fish host gut microbiome is likely largely independent of the water microbial community and that other factors such as diet and host genome may be contributing disproportionally (Talwar et al., 2018).
Our principal goal was to use probiotic and antibiotic treatments to alter the Chinook salmon gut microbiome to determine the potential role of gut microbiota composition variation in host-microbiome interactions. However, we also assessed how the gut microbial community reacted to the treatments. We found that, while fish gut BC alpha diversity was not affected by the treatments, beta diversity was significantly different among all three treatments. Similar results were reported in other studies, indicating community richness (alpha diversity) did not respond to treatment with probiotics and antibiotics, but beta diversity did (Hernandez-Perez et al., 2022; Kokou et al., 2020; Laursen et al., 2017). One possible reason for this is that using antibiotics does not necessarily mean a reduced diversity of bacterial taxa. Indeed, a review showed that individuals with dysbiosis (potentially caused by treatment) can have even more diverse microbial community than healthy individuals (Berg et al., 2020). For example, Rosado et al (2019) showed that treatment of farmed seabass (Dicentrarchus labrax ) with OTC caused a decrease in core BC diversity in the gill and an increase in the skin. One reason that our probiotic treatment did not change BC alpha diversity may be we treated healthy fish. Previous studies in human have shown that probiotics in healthy patients (healthy state) does not greatly impact the resident microbial populations (Eloe-Fadrosh et al., 2015; Lahti et al., 2013). In general, external stimuli that affect the intestinal environment can drive a hierarchical series of microbiome responses; resistance, resilience, redundancy or finally dysbiosis−depending on if the disturbance overcomes the intestinal microbial ecosystem (Lozupone et al., 2012; Moya and Ferrer, 2016; Sommer et al., 2017). It appears that the microbial responses to probiotics in our study is either resistance or resilience, as previous studies have shown that the BCs tended to be more resilient to external stimuli. On the other hand, treatment with antibiotics tends to result in either of resilience, redundancy or dysbiosis.
We predicted that the gut microbial community would respond to the treatments through an increase in beneficial gut bacteria (probiotic treatment) or through a decrease in the beneficial microbes with a related increase in the number of potential pathogens (antibiotic treatment). This was based on the expectation that antibiotics can cause dysbiosis in the gut, resulting in elevated levels of opportunistic pathogens (Dethlefsen and Relman, 2011; Francino, 2015), while prebiotics and probiotics are expected to increase the frequency of gut barrier-protecting bacteria such as Lactobacillaceae andBifidobacteriaceae (Xiao et al., 2014). In this study, bacteria with potential probiotic properties (Lactobacillaceae ,Bifidobacteriaceae , Streptococcaceae ) were higher in the probiotic group compared to other treatment groups, as expected. On the other hand, Pseudomonadaceae and Aeromonadaceae had higher relative abundances in the antibiotic treated fish. Similar patterns of response to probiotics and antibiotics in BC structure and composition have been reported by others (Falcinelli et al., 2016; Kokou et al., 2020; Navarrete et al., 2008; Rutten et al., 2015). For example, Kokouet al (2020) showed that after seven days of of antibiotic treatment, the European seabass (Dicentrarchus labrax ) microbiome increased in Staphylococcus , Pseudomonas genera (Proteobacteria). OTC treatment was reported to reduce gut microbial diversity in Atlantic salmon, while enhancing possible opportunistic pathogens belonging to Aeromonas spp. likely due to eliminating competing microorganisms (Navarrete et al., 2008). Moreover, Falcinelliet al (2016) showed that Firmicutes, specificallyLactobacillus genus, were significantly higher in probiotic treated Zebrafish (Danio rerio ) larvae relative to controls.
Studies in humans (Qin et al., 2010) and fishes (Boutin et al., 2014) have reported that the gut microbiome varies substantially at the individual and population level, and the transcriptome of the fish gut appears to correlate with this variation (Franzosa et al., 2014; Qin et al., 2010). Moreover, Thaiss et al (2016) showed that treatment with antibiotics will change the mouse gut microbiome, and that the microbiome in turn regulates fluctuations in the host transcriptome and epigenome. In our study, we showed that our treatment altered the gut microbiota, then we tested if these changes were associated with changes in host gene expression. Specifically, we showed that several genes related to cellular processes such as cell activation, cell communication, and cell death were upregulated after treatment with antibiotics in the feed. Although previous studies have shown a direct effect of antibiotic treatment on gene transcription in humans (Ryu et al., 2017), antibiotic treatment had a limited effect on gene expression in germ-free mice (Morgun et al., 2015, Ruiz et al., 2017), providing evidence that the microbiome mediates the effects of orally administered antibiotics on the host. In this study we found that our antibiotic treatment resulted in the upregulation of genes related to cell death. Moreover, bacteria from the Firmicutes and Bacteroidetes phyla were reduced while members of the Proteobacteria phylum increased. Zarrinpar et al (2018) showed a similar shift the BC in the mouse cecal; however, a cecal transcriptome analysis showed that the changes in the BC resulted in changes in the expression of genes related to cellular growth and proliferation, as well as cell death and survival pathways. This suggests that colonic remodeling after treatment with antibiotics is directly driving changes in the host transcriptome. Additionally, in our antibiotic treatment group, we showed increased transcription of the mrp7 (multidrug resistance-associated protein 7-like) gene. Moreover, our qPCR analyses showed upregulation of aifm3 gene in antibiotic group. A study by Stoddard et al (2019) in zebrafish showed that after introducing antibiotics to fish, inflammatory gene transcription was downregulated and apoptotic genes such as aifm3 were upregulated within 24 hours.
Antibiotics are designed to pass the gut barrier and become systemic; however, probiotics are live microorganisms that are not able to pass the lumen barrier. Probiotics can directly modulate host physiology by interacting with host cells (mostly immune cells), and through indirect changes in microbiome composition (Langlois et al., 2021). We showed that genes related to post-translation modifications were over-expressed in the probiotic treatment group, relative to the control and antibiotic treatment groups. Previous studies showed that probiotic diet supplements elicit a proinflammatory response in fish (Nayak, 2010) and honeybees (Daisley et al., 2020) which promotes more effective pathogen clearance and improved disease resistance. In this study we found that our treatment with probiotics indeed changes the BC composition with increased numbers of potential probiotics taxa (Lactobacillaceae and Bifidobacteriaceae ). Moreover, our treatment with probiotics showed fewer genes related to apoptosis process responding, relative to the antibiotics group. However, this was not the case for the control treatment, which was expected as the fish in control group were healthy. Finally, we noticed that our probiotic treatment did change the expression of several genes related to immune function as reported in other studies (Petrof et al., 2004; Tomosada et al., 2013). For example, Tomosada et al (2013), showed that Bifidobacteria strains can have immunoregulatory effect in the intestinal epithelial cells by modulation the ubiquitin-editing enzyme. Moreover, similar to this study, Willms et al. (2022) also showed that beneficial bacteria can promote intestinal angiogenesis in Zebrafish. The precise mechanism of action of probiotics remains to be elucidated, especially in heathy states.
One approach to characterize the bidirectional interactions between the host and the microbiome BC is to perturb the gut and measure the response of the host (such as in AIMD studies). In this study, we used antibiotics and probiotics to modify the microbial communities within the gut and measured host gene transcription responses to those modifications. We explored this effect using correlation between multiple common bacterial taxa and host gene transcription. The results of that analysis were consistent with a microbiome-mediated effect on the host. We found that specific microbial taxa are affecting the regulation of several host genes, for example, the abundance ofLactobacillaceae was positively and negatively associated with the transcription of the rabep2 and manf host genes, respectively. Previous work has shown that a single-nucleotide polymorphism (SNP) in therabep2 gene in humans is associated with ulcerative colitis, consistent with a strong association between rabep2 gene and gut bacterial taxa (Jostins et al., 2012). Furthermore, rabep2 gene is involved in ciliogenesis (Airik et al., 2016) and is important for the function of ciliated epithelial cells (Look et al., 2001) which is in close contact with bacteria. Moreover, upregulation of manf gene can active innate immune cells and repairing damaged tissue (Neves et al., 2016; Sereno et al., 2017). However, further studies will be required to determine the specific association of Lactobacillaceae with manf host gene.
The direction of interaction between fish gut and microbiome is not clear, yet it is the basis of the co-evolution of the host with it’s associated microbiomes. In this study we experimentally modified the fish gut microbiome and evaluated host gut tissue responses to those perturbation using transcriptome analysis and transcriptional profiling coupled with a controlled breeding design to control for host genome variation. Short term (10 days) perturbation of the juvenile Chinook salmon gut microbiome with antibiotics and probiotics affected the microbiome BC composition and host gene expression patterns. This study achieved a number of important goals: (1) characterized the effects of antibiotics and probiotics on the aquatic BC (2) characterized juvenile Chinook salmon gut microbiome BC response to antibiotic and probiotic treatment (3) characterized the host gut tissue transcriptional response to antibiotic and probiotic treatments. We showed that our treatments with antibiotics and probiotics not only changed the Chinook salmon microbiome BC (composition), but we also observed significant changes at the gene expression level in the gut tissue of the fish. This study provides insight into a long-standing co-evolved symbiotic relationship between fish gut tissue and its associated microbiome. Moreover, understanding factors influencing the fish gut microbiome and its influence on host health and fitness will help in better sustainable growth for the aquaculture.