Discussion
Environmental gradients can influence ecological interactions and
phenotypic characteristics (e.g., physiology, microbiome) of natural
populations. In this work, we investigated the extent to which an
environmental gradient of pollution in one of the most urbanized coastal
areas in the world, modulates the interactions of animal hosts and their
associated microbial communities. By assessing intra-specific variation
in the diversity, structure, and function of environmental and
animal-associated microbiota in the tropical sea cucumberHolothuria leucospilota , we tested the interplay between
deterministic (e.g., environmental/host filtering) and stochastic (e.g.,
random microbial dispersal) processes underpinning host-microbiome
interactions and microbial assemblages. Overall, our results indicate
that microbial communities are complex and vary in structure and
function between the environment and the animal hosts. However, these
differences are modulated by the level of pollution across the gradient
with marked clines in alpha and beta diversity. Yet, such clines and
overall differences showed opposite directions when comparing
environmental- and animal-associated microbial communities. These
findings suggest that the interplay between both, environmental and host
filtering underpins microbial community assembly in H.
leucospilota along the pollution gradient in Hong Kong.
Excessive nutrient and metal loading, driven by rapid urban development,
is a major threat to coastal and marine ecosystems worldwide, leading to
profound changes in biodiversity, biochemical processes, and ecosystem
functioning (Duprey et al., 2016; Heery et al., 2018; Hong et al., 2021;
Johnston et al., 2015; Mayer-Pinto et al., 2015, 2018; Woodward et al.,
2012). In Hong Kong waters, at a larger scale, this chemical pollution
is influenced by the Pearl River (Geeraert et al., 2021), generating a
strong west-east gradient in nitrogen (west: nitrated dominated; east:
dissolved organic nitrogen dominated) and heavy metals, with levels
exceeding thresholds for sediment toxicity (Geeraert et al., 2021; Hong
et al., 2021). At smaller scales, other independent west-east pollution
gradients can be found in some areas of Hong Kong (e.g., Tolo Harbour)
influenced by the high sewage loading, the tidal hydrodynamics, and the
seascape structure (Figure 1). Both, large- and small-scale gradients of
pollution in Hong Kong have been linked with recent faunal changes in
benthic species (Hong et al., 2021; Nicholson et al., 2011), and the
alteration of the spatial distribution and loss of foundational species
such as hard corals (Duprey et al., 2016). Our study revealed that
environmental microbial communities are also influenced by the
geographic trend in pollution that exists along the Tolo Harbour
(west-east), a potential consequence of clinal differences in nutrient
availability, especially for nitrogen and phosphorus. Similar
correlations between microbial composition in sediments and seawater
have been documented along pollution gradients in other marine regions
as a function of geographic clines in phosphorus (Stevick et al., 2021),
heavy metals, and nitrogen availability (J. Chen et al., 2019; L. Chen
et al., 2019; Di Cesare et al., 2020). In our study, the west-to-east
gradient of pollution showed negative clines in the contribution of some
dominant alphaproteobacterial groups such as the PS1 Clade,
Balneola , and Acholeplasma, while positive trends inAEGEAN-169, Candidatus actinomarina, and Ilumatobacter .
Such geographic differences could be attributed to shifts in the
microbial ecological niche and some degree of local adaptation. In fact,
it has been shown that microbial communities in more polluted sites can
exhibit higher capacity to reduce intracellular levels of heavy metals,
hydrocarbons, and other environmental contaminants compared to less
polluted areas (J. Chen et al., 2019; Dell’Anno et al., 2020). For
instance, Balneola sp. is a competitive organic-degrading
bacteria that is known to proliferate in coastal areas with high levels
of N enrichment (Y.-F. Xu et al., 2022). In our study, this group
exhibited an increased abundance in the highly polluted site (SFB) and
decreased in the medium-low pollution areas (LCC–TPC) of the Tolo
Harbour. This trend was also observed in Acholeplasma , a group
that typically dominates coastal areas characterized by high chemical
oxygen demand driven by wastewater discharges (G. Zhang et al., 2021). A
contrasting pattern was found for Candidatus actinomarina , a
bacteria that has the capacity to proliferate in oligotrophic marine
waters (López-Pérez et al., 2020), thanks to physiological adaptations
that facilitate efficient nutrient acquisition and processing
(Giovannoni et al., 2014; Lauro et al., 2009).
Despite the general geographic trend in microbial communities, we found
micro-habitat differences in the functional profiles within the more
polluted site. In the inner part of the Harbour (west), significant
group dispersion was observed in environmental microbiota compared to
the outer parts (east: less polluted sites). The high to low variation
in bacterial community composition along the cline was particularly
evident between microbial communities in seawater and sediments, a
pattern that may be explained by differences in their enzymatic
capabilities (broader in sediments) and strategies to access organic
matter that has already been degraded during passage through the water
column (Teske et al., 2011). In seawater, the lower microbial diversity
was mainly dominated by Alphaproteobacteria followed byActinobacteria and Bacteroidota . However, such dominance
declined with the level of pollution along the Tolo harbour. These
findings are partially aligned with previous studies along a
eutrophication gradient in the South China Sea (Li et al., 2020; J.
Zhang et al., 2019). Actinobacteria in particular, has been well
documented as a dominant group in eutrophic environments (Gong et al.,
2019; Yun et al., 2017), in which these organisms are suggested to play
a wide range of ecological functions such as the decomposition of
organic matter (Puttaswamygowda et al., 2019). In sediments, on the
other hand, the higher diversity was dominated byActinobacteriota , Alphaproteobacteria, andBacteroidota. Of these, only Actinobacteriota showed major
changes along the cline, together with other groups such asGammaproteobacteria (copiotrophs and involved in nitrate
metabolism, Herlambang et al., 2021; Newton et al., 2011) andChloroflexi . Such dominance and clinal trend in the sediments
contrast with the profiles observed in the seawater, highlighting the
differential ecological influence of physicochemical conditions in these
environments (i.e., water column and benthos). This is particularly true
for nutrient load and chemical pollution, as environmental differences
in these factors are known to influence variation in microbial abundance
across marine gradients (Conte et al., 2018; Jiang et al., 2013).
The sea cucumber- associated microbiome does not fully reflect the
microbial composition of the environment along the pollution gradient.
Similar to the sediments, the diversity of microbial communities in the
guts and skin of the sea cucumbers was higher than in the surrounding
seawater. However, contrary to the sediments, sea cucumber microbiome
diversity was higher in moderated (for skin) and less polluted (for
guts) sites. These clinal differences were also reflected in the overall
community composition where sea cucumber microbiota was dominated byAlphaproteobacteria, Bacteroidota, Actinobacteria, Firmicutes,
Gammaproteobacteria, and Cyanobacteria. While the initial three
phyla also exhibited dominance in environmental samples, there were
evident differences in their composition between the host and
environmental samples. These findings are in line with previous studies,
highlighting the role of host filtering as a “modulatory tool” shaping
the microbial composition from the external microbial source pool (Gao
et al., 2022; Weigel, 2020). In fact, these phyla have been documented
as dominant groups in the guts of diverse species of sea cucumbers (Feng
et al., 2021; Pagán-Jiménez et al., 2019; H. Zhang et al., 2019),
suggesting a conserved filtering mechanism, likely mediated by secondary
metabolites (Darya et al., 2020) or immune factors (Dolmatova et al.,
2004; Gowda et al., 2008; X. Wang et al., 2017). Such mechanism
promotes/favours specific sets of microbes with potential beneficial
effects for the health and survival of the sea cucumber host (Zhao et
al., 2024), or that display neutral and/or transient characteristics (Q.
Wang et al., 2018). However, other mechanisms, uncouple from the sea
cucumber host, seem to have an additional influence on the microbiome ofH. leucospilota as can be seen by the clinal differences in some
microbial groups across the pollution gradient. These mechanisms are
most likely associated with the regulatory influence of environmental
factors and their role in shaping microbial assemblies (Deng et al.,
2022; H. Xu et al., 2019). In our study, sea cucumbers in the high
polluted site harbour microbiomes dominated by families such asRhodobacteraceae , Flavobacteriaceae , Rhizobiaceae ,Sphingomonadaceae and Cyclobacteriaceae . The dominance of
these microbial groups in coastal polluted areas has been associated
with their physiological tolerances (e.g., to heavy metals) and
capacities to degrade organic pollutants (e.g., members of Rhizobiaceae;
Teng et al., 2015). The abundance of these microbes exhibited a
decreasing trend along the west-east pollution gradient, further
cementing the notion of its adaptation to more heavily polluted regions
compared to less contaminated areas. Overall, the distinctions observed
between the environmental and host samples suggest that, although the
environment has an influence on the sea cucumber microbiota, the host
filtering capacity plays a major role in regulating the composition and
abundance of their associated microbial communities. This filtering
capacity, however, may vary along geographic ranges depending on the
magnitude of the environmental gradient, in this case, the level of
pollution.
Intra-individual differences in sea cucumber microbiome reflect
tissue-specific control of microbial communities. Group dispersion was
lower in the gut microbiome across the cline while the dispersion of the
skin microbiome was higher, particularly in the more polluted site. This
observation can be attributed to the extent of exposure to the external
environment, where the skin directly interacts with both seawater and
sediments. Due to the high variability in the environment and the direct
exposure of the skin, there is a higher variation in microbial
composition for the skin samples. Contrary, the gut is an enclosed
system with strong control by the host on their microbial assemblages
(Lachnit et al., 2019; Weigel, 2020), resulting in lower variation.
Apart from the differential dispersion between the skin and gut samples,
the predominant microbial groups evidenced distinct structural and
functional profiles (e.g., degradation pathways associated with the
skin, while both biosynthesis and degradation pathways elevated in the
gut). Such intra-individual differences in the microbiome between body
parts are potentially associated with their unique characteristics
(e.g., biochemistry, nutrient and oxygen content), and regulatory
mechanisms (e.g., secondary metabolites, Pagán-Jiménez et al., 2019; B.
Wang et al., 2015; Y. Wang et al., 2023), as well as the influence of
the external environmental (Sylvain et al., 2020). For example,
biochemical characteristics in gastrointestinal systems in diverse
metazoans, including echinoderms, are suggested to favour members ofBacteroidota (the second most dominant phyla in the sea cucumber
gut in our study), supporting a symbiotic interaction with their host
(Balakirev et al., 2008; Thomas et al., 2011). This phylum is composed
of diverse physiological types that exist from strictly anaerobic
bacteria like Bacterodetes sp. (present in the gut samples of
this study), to facultative anaerobes such as Lutibacter sp. (a
dominant genus found in H. leucospilota ,), and strictly aerobic
bacteria —Flavobacteria (Choi & Cho, 2006; Lee et al., 2012;
Thomas et al., 2011). It has been increasingly recognised that members
of Bacteroidota, are an integral part of their host metabolism
due to their specialised capacity for degradation of high molecular
weight organic matter such as protein and carbohydrates, as well as
organic pollutants (Mayer et al., 2016; Thomas et al., 2011; H. Zhang et
al., 2019). In our study, such function (e.g., carbon degradation) was
found significantly expressed in the gut of the sea cucumber, suggesting
the beneficial and important existence of Bacteroidota members
(e.g., Flabacteriaceae which was in high abundance compared to
skin and environmental samples), for the breakdown complex molecules, as
well as for biosynthesis activity (less expressed in the skin microbiome
functional profile, Kirchman, 2002). Our results here support the
hypothesis that the interplay between host-selective mechanisms and
inherent host conditions modulate contrasting intra-host microbiome
composition (skin and gut) in the sea cucumber H. leucospilota .
The Tolo Harbour, like many urbanised and industrialised estuaries
around the globe, has been radically altered by historical and ongoing
anthropogenic activities. Such alterations have impacted local
biodiversity and the overall ecosystem functioning (L. Chen et al.,
2019; Fleddum et al., 2011; Lei et al., 2018). At the organismal level,
urbanization and pollution are known to influence the physiology,
behaviour and life history of diverse marine animals (Elizabeth Alter et
al., 2021; Morroni et al., 2023; Weis, 2014). These effects can also be
observed in the complex assembly of animal-associated microbial
communities and the functions they provide to their hosts (Fu et al.,
2023; Pei et al., 2022; Q. Wang et al., 2018; Wei et al., 2022).
Alterations in host-associated microbiomes driven by urbanization and
pollution are typically characterized by two types of outcomes: 1) host
showing resilience due to the presence and enhancement of beneficial
microbial members (Fragoso ados Santos et al., 2015; Palladino et al.,
2023) or 2) dysbiosis as a result of host dysregulation (Huang et al.,
2020; Lachnit et al., 2019). Our study is likely to indicate the former,
as we detected multiple dominant microbes with a beneficial role, such
as Rhodobacteriaceae (keystone species in sea cucumber intestinal
system, J. Yu et al., 2023; H. Zhang et al., 2019) andRhizobiaceae (potentially aiding in pollutant breakdown, Teng et
al., 2015) in sea cucumber within the highly polluted region. However,
further studies are needed to test this hypothesis, disentangling the
microbial contribution to the host’s survival and tolerance to marine
pollution.