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.