Intensive aquaculture has increased the severity and frequency of fish diseases. Given
the functional importance of gut microbiota in various facets of host physiology,
modulation of this microbiota is a feasible strategy to mitigate emerging diseases
in aquaculture. To achieve this, a fundamental understanding of the interplay among
fish health, microbiota, and invading pathogens is required. This commentary focuses
on current knowledge regarding the associations between fish diseases, dysbiosis of
gut microbiota, and immune responses. Furthermore, updated research on fish disease
from an ecological perspective is discussed, including colonization resistance imposed
by commensals and strategies used by pathogens to overcome resistance. We also propose
several directions for future research, such as exploration of the causal links between
fish diseases and specific taxa, and identification of universal gut microbial biomarkers
for rapid disease diagnosis.
Fish aquaculture is the fastest growing animal food sector to support the growing
human population, with a year-on-year growth rate of 10.4% (FAO, 2013). However, fish
production is threatened by numerous diseases (Lafferty et al., 2015). This is particularly
pertinent to aquacultural systems that impose various stressors on aquatic animals
(Lafferty et al., 2015; Li et al., 2017a). Traditionally, antibiotics have been widely
applied to prevent and treat diseases in aquacultures. However, antibiotic abuse has
been highlighted in the transfer of resistance genes among pathogens, and has raised
concerns regarding environmental pollution and consumer safety (Brandt et al., 2015).
In recent years, the introduction of probiotics has been considered a sustainable
strategy to improve fish health and protect them from emerging diseases (de Bruijn
et al., 2017). Despite the extensive list of candidate probiotics investigated in
previous studies (Dawood et al., 2016; Liu et al., 2018; Ramesh et al., 2017), successful
application has been limited, as reported in a survey of farmers (Xiong et al., 2016).
The lack of consistency in probiotic performance may be due to unsuccessful colonization
as a result of sudden changes in habitats, e.g., from aerobic culture conditions to
the anaerobic intestines (Giatsis et al., 2016). In addition, the fish gut is a main
pathogen transmission route and a portal of entry (de Bruijn et al., 2017; Li et al.,
2017a; Ringø et al., 2007; Zhang et al., 2015). Therefore, understanding the factors
that dictate the invasion of pathogens and establishment of probiotics in the intestine
will provide an initial step towards predicting and treating fish diseases.
Gut microbiota can affect fish physiology, development, life span, immunity, and barriers
against pathogens (Burns et al., 2016; Nie et al., 2017; Smith et al., 2017; Yan et
al., 2016). Therefore, the gut microbiota plays an indispensable role in fish fitness.
Several recent reviews have centered on the diversity and functions of bacterial communities
in healthy fish (de Bruijn et al., 2017), as well as on the external factors that
affect fish gut microbiota (Wang et al., 2017) and interactions between gut microbiota
and innate immunity in fish (Gómez & Balcázar, 2008; Nie et al., 2017). However, most
previous studies have focused on factors that govern healthy gut microbiota, such
as diet, rearing conditions, and fish genotype (Schmidt et al., 2015; Sullam et al.,
2012; Yan et al., 2016). In contrast, few studies have reported on the interplay among
gut microbiota, fish immunity, and disease (Nie et al., 2017). In this commentary,
we summarize current knowledge on the associations between fish immunity, gut microbiota,
and invading intestinal pathogens. We also highlight recent progress in uncovering
the ecological processes of fish diseases.
According to the diversity resistance hypothesis, a more diverse microbial community
harbors greater probability of having a species with an antagonistic trait toward
an invader or pathogen (Fargione & Tilman, 2005). Consistent with this assertion,
higher alpha diversity (mean species diversity at the habitat level) is frequently
detected in healthy fish compared with diseased fish, such as largemouth bronze gudgeon
(Coreius guichenoti) (Li et al., 2016), crucian carp (Carassius auratus) (Li et al.,
2017a), and ayu (Plecoglossus altivelis) (Nie et al., 2017). One possible explanation
for this pattern is that the invading pathogens out-compete the gut commensals, thereby
reducing diversity. Similarly, gnotobiotic zebrafish (Danio rerio Hamilton, 1822)
have been shown to be more sensitive to pathogenic infections (Oyarbide et al., 2015).
In addition, antibiotic administration generally reduces diversity of the gut microbiota,
which, in turn, facilitates colonization by external pathogens (He et al., 2017).
Indeed, gut microbial diversity has been used as a biomarker of fish health and metabolic
capacity (Clarke et al., 2014), with low diversity and stability of the microbiota
closely associated with fish disease (He et al., 2017; Li et al., 2017a; Nie et al.,
2017). A preponderance of evidence has demonstrated that more diverse gut communities
exert greater protective effects on the host (De Schryver & Vadstein, 2014; Johnson
et al., 2008; Zhu et al., 2016). In this regard, gut microbial diversity in fish should
be maximized to reduce pathogenic invasions in aquaculture systems.
Fish are in continual contact with a complex and dynamic planktonic microbiota. Therefore,
it is expected that gut microbiota in fish will be largely affected by microbes in
the environment. This has been demonstrated by the high similarity between water and
gut microbiotas of Atlantic cod larvae (Gadus morhua) (Bakke et al., 2013), rainbow
trout (Oncorhynchus mykiss) (Wong et al., 2013), and tilapia larvae (Giatsis et al.,
2015). Based on the co-evolution theory, however, to improve host fitness, mutualistic
relationships between fish and gut microbiota should be tightly regulated to ensure
suitable bacterial colonization (McFall-Ngai et al., 2013). As a result, gut bacterial
communities between recently caught and domesticated fish share similar community
structures (Roeselers et al., 2011). Intriguingly, reciprocal gut microbiota transplants
between zebrafish and mice have shown that the relative abundance of lineages changes
to resemble normal gut microbiota of the recipient host (Rawls et al., 2006). Similarly,
previous meta-analysis has revealed that host phylogeny determines the composition
of fish gut bacteria, even at the bacterial phylum level (Sullam et al., 2012). For
example, the gut microbiota of largemouth bronze gudgeon is dominated by phyla Proteobacteria,
Actinobacteria, and Tenericutes (Li et al., 2016), whereas Gammaproteobacteria, Alphaproteobacteria,
Firmicutes, and Bacteroides are predominant in the gut of ayu (Nie et al., 2017).
This pattern also holds true for different fish species (herbivorous Ctenopharyngodon
idellus, carnivorous Siniperca chuatsi, and Silurus meridionalis) reared in the same
pond (Yan et al., 2016). Indeed, it has been suggested that gut microbiotas of fish
are distinct from those in rearing water and/or sediment (Li et al., 2017a; Schmidt
et al., 2015; Zhang et al., 2018). However, this does not mean that the gut microbiota
is temporally stable during the entire lifetime of the fish; rather, gut bacterial
communities vary significantly during the developmental stages in healthy fish (Li
et al., 2017b; Stephens et al., 2016; Yan et al., 2016; Zhang et al., 2018). This
high temporal pattern is largely contributed to by maturation of the host (Burns et
al., 2016; Zhang et al., 2018) as selection of gut microbiota is reinforced with time.
Intriguingly, several species of fish exhibit core gut microbiota, including zebrafish
(Roeselers et al., 2011), rainbow trout (Wong et al., 2013), channel catfish (Ictalurus
punctatus), largemouth bass (Micropterus salmoides), and bluegill (Lepomis macrochirus)
(Larsen et al., 2014), though location-dependent variations in gut microbiota also
exist. These core lineages may be used as baselines for future probiotic trials.
It is worth emphasizing, however, that the tight link between fish and their gut microbiota
can be disrupted by diverse variables, with host disease being the primary factor
(Li et al., 2017a; Nie et al., 2017). Gut bacteria reside on mucosal surfaces, which
provide the first line of defense against pathogens. Specifically, commensal bacteria
compete for or modify the ecological niche and available nutrients to inhibit the
colonization and proliferation of incoming pathogens in the intestine (Kamada et al.,
2013). For example, well-known probiotic Bifidobacterium prevents pathogenic Escherichia
coli invasion via acidification of the intestinal environment (interspecies barrier
effect) (Fukuda et al., 2012). In addition, gut commensals can produce bacteriocins
and proteinaceous toxins that specifically inhibit members of the same or similar
bacterial species (intraspecies barrier effect). Therefore, susceptibility to pathogenic
infection seems to rely, at least in part, on the structure of the host’s gut microbial
community (Galindo-Villegas et al., 2012; He et al., 2017). Indeed, dysbiosis in the
gut microbiota is frequently associated with fish disease (He et al., 2017; Nie et
al., 2017). However, it is currently unclear whether changes in the microbial community
are a cause or consequence of these diseases.
Responses of a community to disturbance (e.g., disease) are not solely the sum of
the traits of individual species but are also dependent on interspecies interactions
(Faust & Raes, 2012; Zhu et al., 2016). Our recent work showed that pathogenic infections
have a significant impact on the gut microbiota, with diseased ayu exhibiting less
complex and diverse interspecies interactions (Nie et al., 2017). Indeed, interspecies
interaction analysis has been applied to identify candidate pathogens and/or probiotics
in gut diseases (Buffie et al., 2015; Dai et al., 2018). Furthermore, it is apparent
that populations, not clones, are the causal agents of some aquaculture diseases (Hou
et al., 2018; Lemire et al., 2015). This idea overturned the traditional view that
only a pathogen and/or virulence gene result in disease (Falkow, 1988), and led to
the ‘ecological Koch’s postulates’, which aims to untangle the interplay between host
health, microbiota, invading pathogens, and diseases (Vonaesch et al., 2018). However,
current understanding on the ecological processes that govern the gut microbiota in
fish is still in its infancy, and no consensus has yet emerged. For example, it has
been reported that the relative importance of determinism increases as zebrafish mature
(Burns et al., 2016), whereas other studies have shown the opposite trend (Li et al.,
2017b; Yan et al., 2016). Understanding the factors that govern the gut microbiota
provides an initial step to establishing and maintaining a healthy fish microbiome
(de Bruijn et al., 2017; De Schryver & Vadstein, 2014). In this regard, exploring
the underlying mechanisms of fish diseases will provide an integrated approach to
systems biology and ecology.
Going a further step, gut signatures can also be associated with fish diseases. For
example, taxa affiliated with genera Vibrio, Aeromonas, and Shewanella are overrepresented
in the gut microbiota of “red-operculum” disease in crucian carp, whereas Cetobacterium
species are indicators of healthy fish (Li et al., 2017a). Similarly, Aeromonas is
a biomarker for largemouth bronze gudgeon suffering from furunculosis (Li et al.,
2016). This phenomenon suggests that certain gut microbial signatures are indicative
of host health status irrespective of disease pathogeny, as has been demonstrated
in human gut diseases (Mancabelli et al., 2017). Recent mechanistic studies suggest
that the inflammatory host response produces reactive oxygen species, which facilitate
a competitive advantage to facultative anaerobic lineages, such as Aeromonas (Winter
& Bäumler, 2014). To date, however, surprisingly few studies have examined the association
between disease severity and degree of dysbiosis in the gut microbiota during disease
progression in fish. As a result, it is unclear whether the transition from healthy
to diseased gut microbiota is gradient-like or a discrete process (Knights et al.,
2014). If the transition is gradual, gut microbial signatures could serve as independent
variables for predicting the incidence of fish disease, similar to that observed in
shrimp diseases (Xiong et al., 2017; Xiong et al., 2018).
In addition to direct inhibition, the fish gut microbiota also plays critical roles
in epithelial renewal and maturation, which, in turn, regulate immune responses (Gómez
& Balcázar, 2008; Wang et al., 2017). Under normal conditions, goblet cells secrete
mucus, which functions as a barrier to inhibit migration of microorganisms out of
the intestinal lumen (Ringø et al., 2007). A mature gut mucosa is also essential for
distinguishing pathogens from commensals through pattern recognition receptors (PRRs,
such as toll-like receptors, RIG-I-like receptors, NOD-like receptors and AIM2-like
receptors), which detect bacterial antigens and activate signaling cascades to regulate
immune responses (cytokines) (Pérez et al., 2010). For example, the toll-like receptor
family, a representative member of PRRs, recognizes conserved structures in pathogens,
which can recruit and regulate the immune and inflammatory cells that initiate and
mediate systemic immune responses (Fasano & Sheadonohue, 2005). Additionally, commensals
can protect the host by depriving invading pathogens of nutrients, secreting a range
of antimicrobial substances and occupying the niche (de Bruijn et al., 2017; Gómez
& Balcázar, 2008; Pérez et al., 2010). However, if this balance is disrupted, such
as during pathogenic infections, the innate and adaptive immune systems are activated
to prevent disease exacerbation. Conversely, there is a correlation between colonization
of probiotics and innate immune responses, such as phagocytic and alternative complement
pathway activities, which protect fish against pathogens (Balcázar et al., 2007; Kim
& Austin, 2006).
Studies on gnotobiotic zebrafish demonstrate that the gut microbiota enhances the
stability of β-catenin via activation of Wnt signaling, thereby promoting intestinal
cell proliferation over normal ontogenesis (Cheesman et al., 2011; Rawls et al., 2006).
Compared with germ-free zebrafish, conventionally raised zebrafish exhibit a greater
abundance of genes associated with epithelial proliferation and innate immune response
(Rawls et al., 2004). However, germ-free zebrafish with a commensal microbiota can
robustly activate NF-κB and its target genes in intestinal and extra-intestinal tissues
(Kanther et al., 2011). Similarly, colonization of commensals in larvae stimulates
neutrophils and activates pro-inflammatory genes through the TLR/MyD88 signaling pathway
and phagocytes, which can enhance disease resistance in zebrafish (Galindo-Villegas
et al., 2012). Specifically, the gut microbiota induces intestinal macrophages by
upregulating pro-IL-1β. The mature form of IL-1β (activated by pathogen infection)
recruits neutrophils, thereby priming macrophages to eradicate pathogens (Kamada et
al., 2013). Significant association between the gut microbiota and transcription level
of secreted immunoglobulin M (sIgM, a proxy for adaptive immune development) has been
reported during healthy zebrafish development (Stephens et al., 2016). Compared with
functional B- and T-cell receptor immune-deficient zebrafish, wild-type zebrafish
exhibit an individualized gut microbiota and increased determinism of gut microbiota
assembly (Stagaman et al., 2017). Our recent work also showed pro-inflammatory cytokines
IL-1β and TNF-α to be activated in response to pathogenic infections in ayu (Nie et
al., 2017). On the other hand, administration of probiotics to sea bass (Dicentrarchus
labrax L.) results in the downregulation of IL-1β and transforming growth factor-β
(Picchietti et al., 2008). Collectively, these results indicate a normal gut microbiota
contributes indispensable roles in regulating the fish immune system, and vice versa.
As described above, the host and gut microbiota have co-evolved multiple strategies
to not only prevent colonization by external pathogens, but also suppress resident
pathogens. However, pathogens have developed various strategies to overcome these
barriers, including entry into the host, occupation of a unique niche, circumvention
of commensals and host defense barriers, and acquisition of nutrients from fish hosts
(Ringø et al., 2007). Specifically, pathogens express sortases and adhesins for anchoring
to host intestinal cells. After attachment to the intestinal tract, pathogens produce
toxins and hemolysins to aggressively damage the intestinal lining and induce inflammatory
responses (Mazmanian et al., 2001; Ringø et al., 2007). There is evidence that the
inflamed environment induces production of reactive oxygen and/or nitrogen species
by the host, resulting in a bloom of facultative anaerobic bacteria (e.g., Proteobacteria)
and reduction in obligate anaerobic bacteria (Winter & Bäumler, 2014). This shift
in community composition compromises colonization resistance imposed by gut microbiota,
thereby facilitating the overgrowth of potentially harmful indigenous bacterial species
(Galindo-Villegas et al., 2012; He et al., 2017). To escape from host immune clearance,
some enteric pathogens harbor a modified form of siderophore (chelating iron under
iron-limiting conditions) that is not inhibited by host cell-secreted lipocalin 2,
which can further promote the growth of pathogens (Fischbach et al., 2006). Additionally,
pathogenic capsules promote virulence by reducing host immune responses (Singh et
al., 2011). Gram-negative pathogens commonly encode the type 6 secretion system (T6SS),
which enables pathogens to attack the resident microbiota and to confer them with
a competitive advantage (Russell et al., 2014; Vonaesch et al., 2018). In addition,
to counteract nutritional competition by commensals, some pathogens can use alternative
or pathogen-specific nutrients, which expand the nutrient niche for their colonization
(Fabich et al., 2008). Alternatively, invaders can also occupy a distinct niche during
replication to reduce competition with commensals. For example, pathogenic Citrobacter
rodentium expresses intimin, which enables its localization to the intestinal epithelial
surface, where commensals do not normally occur (Kamada et al., 2012). Intriguingly,
pathogens can sense cues (e.g., bile acids, temperature, and nutrient availability)
from their host to regulate virulence genes at the appropriate location (Fraser &
Brown, 2017; Vonaesch et al., 2018). This regulatory mechanism can therefore maximize
the chance of successful invasion.
Once a pathogen escapes colonization resistance imposed by gut commensals and host
immunity, it can replicate and further express diverse virulence factors to attack
fish and cause disease. There is increasing evidence that pathogenic infections cause
profound disturbances to the fish gut microbiota and immune responses (He et al.,
2017; Nie et al., 2017; Ringø et al., 2007). Notably, variations in the gut microbiota
of ayu are significantly associated with TNF-α and IL-1β expression levels (Nie et
al., 2017). Similarly, antibiotic administration can also cause imbalance in the gut
microbiota of zebrafish, resulting in a compromised immune response, which further
increases susceptibility to infections (He et al., 2017). Molecular experiments further
suggest that decreased water quality can promote pathogen virulence (Penttinen et
al., 2016). Therefore, disease onset in fish can be attributed to a variety of disturbances,
such as environmental stress and antibiotic administration, which disrupt the gut
microbiota in stressed fish and enhance the virulence of pathogens.
In summary, the introduction of pathogens into hosts is antagonized by environmental
pressure, fish filtering, and colonization resistance of gut commensals (Mallon et
al., 2015). In healthy fish, the gut microbiota directly antagonizes the colonization
or overgrowth of pathogens (Nie et al., 2017). These effects include competition for
resources, niche exclusion, and suppression of virulence factors. In addition, pathogens
are suppressed by immune clearance. In diseased fish, balances in the protective commensal
microbial community and host immunity are disturbed by external factors. For example,
antibiotic usage can decrease species diversity and alter gut microbial community
structure in fish (He et al., 2017). Pathogenic infections have been shown to significantly
disrupt interspecies interactions in the fish gut microbiota (Nie et al., 2017). These
alterations may open up ecological niches for pathogenic invasions. Furthermore, environmental
stresses may impose additional pressure on fish, leading to compromised immunity.
Lastly, the expression of virulence genes in pathogens can also be induced by poor
water quality (Penttinen et al., 2016; Zhou et al., 2012). These detrimental effects
cumulatively attenuate resistance to colonization by pathogens and allow overgrowth
of harmful colonies that may lead to disease.
Given the functional importance of the gut microbiota in improving host fitness, introduction
or augmentation of beneficial microbes may be a promising approach for protecting
fish from emerging diseases (de Bruijn et al., 2017). However, various studies have
identified long lists of implicated microbes that may contribute to the gut microbiota
dysbiosis-disease relationship, and these associations may reflect biomarkers of disease.
Therefore, future work is required to explore the causal links between fish disease
and specific taxa, which may enable us to optimize gut microbiota composition to mitigate
fish disease. Pathogenic infections involve several phases: introduction, establishment,
spread, and impact, which are governed by the environment, host, and gut microbiota
(Mallon et al., 2015). To understand the mechanisms underlying fish disease, one should
focus on the infection process from an ecological prospective (De Schryver & Vadstein,
2014; Xiong et al., 2016) instead of isolating potential pathogens from diseased fish.
Next generation sequencing has allowed the identification of universal gut microbial
biomarkers (common features of affected individuals) in various fish diseases from
different regions. Therefore, we recommend that relevant information should be deposited
into a public database, which could enable convenient cross-disease comparisons. This
would facilitate rapid diagnosis as well as promote prediction of the course and prognosis
of disease.