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.