Holosystemics Part VII: Chlorophyll, Eutrophication, and Cyanotoxin-Derived Microbial Perturbations of Freshwater Fish

by | Jun 25, 2024

Whereas it may seem investigations of freshwater fish are of little use to reef enthusiasts due to oceanic and riverine disparities, the way salt- and fresh-water microbial consortia and their hosts respond to abiotic and biotic variations are remarkably similar. Furthermore laboratory investigations housing fish in aquaria reliably yield analogous results which repudiates the myth and age-old paradigm that “all systems run differently”.

Blooms of Cyanobacteria including Microcystis aeruginosa cause eutrophication-derived ecological hypoxia in freshwater; and manufacture antimicrobials, enzyme inhibitors, and cytotoxins, while fish are routinely exposed throughout their lifetime. The hepatotoxin microcystin-LR (MC-LR) amasses in liver where it instigates inflammation and necrosis through oxidative stress. Fish are affected after ingestion of contaminated prey or water (Gallet et al. 2023), albeit freshwater fish do not have to drink like saltwater teleosts. Finfish gut microbiota appear less diverse compared to higher vertebrates where the phyla Bacteroidetes, Firmicutes and Proteobacteria predominate (Egerton et al. 2018; Llewellyn et al. 2018). Extracts of various Microcystis strains caused perturbations of fish gut microflora, whereas MC-LR did not (Duperron et al. 2019). “Algae” manufacturer allelopathic compounds that affect the life cycle and the reproductive competence (fecundity) of other species (Birrell et al. 2008; Gallet et al. 2023) whereas microcystin has been isolated from the cyanobacterial mat of Floridian and Bahamian black band disease of corals (Richardson et al. 2007). Cyano-metabolites may exert a more profound effect on microbiomes and the development of a pathobiome (Gallet et al. 2023).

Fig 1. Experiments evaluating the effects of a cyanobacterial bloom of Microcystis aeruginosa on the gut microbial populations of freshwater medaka fish (Oryzias latipes). The first phase lasted 28 days where fish were kept in the growth medium (d28_Z8; yellow), water (d28_0; blue) or the Microcystis aeruginosa chlorophyll a (Chla) densities of 1, 10, and 100 micrograms per litre (µg l-1; ppb) designated d28_1 (light green), d28_10 (medium green), and d28_100 (dark green) which equate to negligeable, ~0.4, and ~10.4 µg l-1 microcystin-LR (MC-LR). All treatments were purged on day 28 in freshwater until day 33, whereafter all fish were exposed to 100 micrograms per litre (µg l-1) of Chla until day 39. Image courtesy of Gallet et al. 2023 and the Creative Commons Licence 4.0. http://creativecommons.org/licenses/by/4.0/

The first phase of freshwater experiments lasted 28 days, where controls included medaka fish (Oryzias latipes) kept in freshwater (d28_0) or the nutrient-enriching (eutrophication-analogous) cyanobacterial growth medium (d28_Z8), whilst the treatments exposed fish to Microcystis aeruginosa chlorophyll a (Chla) densities of 1, 10, and 100 micrograms per litre (µg l-1; d28_1; d28_10; d28_100) which equated to ~0.4 and ~10.4 µg l-1 (ppb) MC-LR in the latter two, whereas the toxin was below detection levels in the former (Fig 1.; Gallet et al. 2023).

The gut mucosal communities of all fish comprised 42 to 219 amplicon sequence variants (ASVs; @microbial lineages), where fish exposed to the growth medium had higher than average richness of ~136 ± 56 which was curtailed in fish kept in just water at ~82 ± 25 ASVs. Fish gut microbes increased marginally in a stepwise fashion in proportion to increased Chla, MC-LR, and Microcystis aeruginosa.

The treatments (d28_1, d28_10, d28_100) and growth medium (d28_Z8) affected microbial community composition and density in comparison to freshwater controls (d28_0), where d28_Z8 had an analogous effect to d28_1 and d28_10 (Gallet et al. 2023). The gut populations in the d28_0 control fish were dominated by 50, 28, 10, and 5 percent of reads belonging to the bacterial phyla Fusobacteriota, Firmicutes, Proteobacteria, and Bacteroidota respectively. The relative abundances of these phyla changed according to treatments however the greatest difference was observed in d28_100 where Firmicutes were found in merely 3 percent of reads. Aeromonas and Flavobacterium species were more abundant in the fish guts of the d28_Z8 and d28_100 groups, while Reyranella species were merely more plentiful in the latter. The Firmicutes genus ZOR0006 was significantly reduced in the fish gut mucosae exposed to 100 µg l-1 Chla, yet this genus was the most plentiful of this phylum in other fish intestinal communities. Cetobacterium species remained core affiliates of all fish ranging from 27 to 48 percent of reads (Gallet et al. 2023).

Negligeable microeukaryotes and Archaea were detected in the gut populations, while the genera Flavobacterium, Aeromonas, Gemmobacter, and Rhizobiales had proliferated in the d28_100 group. The profusions of three Firmicutes assigned genera were decreased in d28_100 compared with d28_0 where ZOR0006 had accounted for nearly 60 precent (Fig 2.; Gallet et al. 2023).

Fig 2. The relative abundances of the bacterial phyla and families of the fish gut communities after the initial 28-day phase of experiments, where d28_0, d28_Z8, d28_1, d28_10, and d28_100 refer to fish kept in freshwater, the growth medium, or the Microcystis aeruginosa Chla densities of 1, 10, and 100 µg l-1 respectively. Image courtesy of Gallet et al. 2023 and the Creative Commons Licence 4.0. http://creativecommons.org/licenses/by/4.0/

The metabolism of a microbiome is referred to as the metabolome, where 1,674 metabolites were identified on day 28. 53 percent of the extracellular products found in medaka fish gut were implicated in the biosynthesis of cobalamin (vitamin B12) like those from Cetobacterium species, whereas ZOR0006 (Erysipelotrichaceae) exhibited carbohydrate-degradative competencies, while both genera could interconvert lactate and pyruvate. Lactate has biocidal properties and is thought vital for gut repair and function despite pyruvate being implicated in the proliferation of pathogens. Gene homologues associated with amino-acid, carbohydrate, and lipid metabolism are common to most prokaryotes, and their products were enriched in the d28_0 and d28_100 groups, whereas enzymes for cofactor and secondary metabolite biosynthesis, and porphyrin and chlorophyll metabolism were amplified in merely the latter. Significant differences were observed between the metabolomes of d28_0 and d28_100 and all other classes, yet those of d28_Z8, d28_1 and d28_10 were comparable (Gallet et al. 2023). Liver metabolites were unaffected yet accumulations in muscle were evident where those from d28_0 and both d28_Z8 and d28_1 were markedly different, whilst those of d28_100 were dissimilar to all other groups except for d28_0 (Gallet et al. 2023).

The least abundant microbes in the d28_0 control group were negatively correlated to several metabolites, where Barnesiellaceae and Epulopiscium were the most plentiful genera, while comprehensive metabolome databases were lacking (Gallet et al. 2023).

Fish were transferred to freshwater on the 28th day as a purgative until day 33 (d33_0_0; d33_Z8_0; d33_1_0; d33_10_0; d33_100_0) whereafter all groups were exposed to 100 µg l-1 Chla Microcystis aeruginosa until day 39 (d39_0_0_100; d39_Z8_0_100; d39_1_0_100; d39_10_0_100; d39_100_0_100). The richness of gut microbiota in these fish decreased from day 28 (82 ± 25) to 33 (50 ± 25), whereafter richness increased until day 39 (72 ± 22). By day 33 all fish gut communities had similar profiles to d28_0, whereas the prokaryotic gut affiliates in d28_100 were mostly identical to d39_100_0_100 (Fig 3.; Gallet et al. 2023).

Microcystis holobiont-associated microbes from water and intestines ranged from 9.8 to 18.1 percent of reads and approached 4.6 percent of reads on day 39 respectively. The communities were somewhat similar between day 33 and day 39 in comparison to the disparity between day 33 and all other treatments, and the difference between day 39 and d28_Z8.

Gut-bound microbes of the phyla Bacteroidota, Fusobacteriota, and Proteobacteria were comparable between day 33 and day 39, whereas Firmicutes were represented by an unclassified ASV at day 33 which was mostly absent by day 39. Remarkably Cetobacterium species were conserved core affiliates throughout all fish from all treatments (Gallet et al. 2023).

Fig 3. The relative abundances of the bacterial phyla of the fish gut microbiomes after a depuration phase from 28 to day 33, and a further six days of 100 µg l-1 Chla from day 33 to day 39. Image courtesy of Gallet et al. 2023 and the Creative Commons Licence 4.0. http://creativecommons.org/licenses/by/4.0/

There was little difference between the gut metabolomes of the fish from d28_Z8 and d28_1 and those at day 33, or from d28_Z8 and d28_10 and those of day 39. These triads differed from each other and the metabolomes of fish gut communities from other classes. Two Vibrio and two Reyranella species were negatively and positively associated with several metabolites. Liver metabolites were unaffected whilst muscle metabolites were similar at day 33, day 39, and in fish from d28_100, which were distinct from all other groups (Gallet et al. 2023).

Cetobacterium species were most abundant in gut, while ZOR0006 and Flavobacterium species were the most enriched in faeces, while environmental biofilms were monopolised by the genus Reyranella. All fish appeared healthy and survived, while blooms of Microcystis aeruginosa impact gut bacteria such as Firmicutes which are likely implicated in host metabolic processes. ZOR0006 were environmentally negligible and thus may be obligate intestinal microbiota of medaka fish. Cetobacterium species (Fusobacteria) are likely symbionts that manufacture 54 gut metabolic modules including one for vitamin B12 biosynthesis, while ZOR0006 utilise 15 conventional intestinal metabolites. Carbohydrate exploitation appears central to the metabolism of fish intestinal communities, while exceeding a bloom threshold seems to cause gut populations to shift. Reyranella species may be transitory gut residents whereas the genera Aeromonas, Flavobacterium, and Shewanella may be opportunistic pathogens or common core affiliates (Gallet et al. 2023).

Cyanopeptides like aerucyclamides and bacteriocins are potent antimicrobials and cytotoxins (Riley & Wertz 2002; Martins & Vasconcelos 2015). A change in microbial community structure does not necessarily spell disfunction especially with raised operational redundancy, however the metabolome shifted which may reflect host adaptive or immunological responses. Nevertheless there was a strong correlation between the microbial community compositions and the emergence of metabolites, where muscle variations were noted yet they were trivial compared to those of the gut (Gallet et al. 2023).

Remarkably, the growth medium modified prokaryotic gut communities and thus analogous effects are likely to arise within eutrophic fish-only recirculating systems or sea cage mariculture. Next we expand and consolidate our comprehension in part VIII as we delve deeper into pathobiome development.


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