Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Summary: The filamentous chlorophyte Ostreobium sp. dominates shallow marine carbonate microboring communities, and is one of the major agents of reef bioerosion. While its large genetic diversity has emerged, its physiology remains little known, with unexplored relationship between genotypes and phenotypes (endolithic versus free‐living growth forms). Here, we isolated nine strains affiliated to two lineages of Ostreobium (>8% sequence divergence of the plastid gene rbc L), one of which was assigned to the family Odoaceae, from the fast‐growing coral host Pocillopora acuta Lamarck 1816. Free‐living isolates maintained their bioerosive potential, colonizing pre‐bleached coral carbonate skeletons. We compared phenotypes, highlighting shifts in pigment and fatty acid compositions, carbon to nitrogen ratios and stable isotope compositions (δ ¹³ C and δ ¹⁵ N). Our data show a pattern of higher chlorophyll b and lower arachidonic acid (20:4ω6) content in endolithic versus free‐living Ostreobium . Photosynthetic carbon fixation and nitrate uptake, quantified via 8 h pulse‐labeling with ¹³ C‐bicarbonate and ¹⁵ N‐nitrate, showed lower isotopic enrichment in endolithic compared to free‐living filaments. Our results highlight the functional plasticity of Ostreobium phenotypes. The isotope tracer approach opens the way to further study the biogeochemical cycling and trophic ecology of these cryptic algae at coral holobiont and reef scales.

Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral’s capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature. Three coral fragments were inoculated with 15N-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. 15N-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative 15N-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of 15N-enriched hotspots, suggesting these tissues act as a “collection and digestion” site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these 15N-hotspots revealed these to be host phagosomal and secretory cells/vesicles. This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

Microscopic filaments of the siphonous green algae Ostreobium (Ulvophyceae, Bryopsidales) colonize and dissolve the calcium carbonate skeletons of coral colonies in reefs of contrasted salinities. Here, we analyzed their bacterial community’s composition and plasticity in response to salinity. Multiple cultures of Pocillopora coral-isolated Ostreobium strains from two distinct rbcL lineages representative of IndoPacific environmental phylotypes were pre-acclimatized (>9 months) to three ecologically relevant reef salinities: 32.9, 35.1, and 40.2 psu. Bacterial phylotypes were visualized for the first time at filament scale by CARD-FISH in algal tissue sections, within siphons, at their surface or in their mucilage. Ostreobium-associated microbiota, characterized by bacterial 16S rDNA metabarcoding of cultured thalli and their corresponding supernatants, were structured by host genotype (Ostreobium strain lineage), with dominant Kiloniellaceae or Rhodospirillaceae (Alphaproteobacteria, Rhodospirillales) depending on Ostreobium lineage, and shifted Rhizobiales’ abundances in response to the salinity increase. A small core microbiota composed of seven ASVs (~1.5% of thalli ASVs, 19–36% cumulated proportions) was persistent across three salinities in both genotypes, with putative intracellular Amoebophilaceae and Rickettsiales_AB1, as well as Hyphomonadaceae and Rhodospirillaceae also detected within environmental (Ostreobium-colonized) Pocillopora coral skeletons. This novel knowledge on the taxonomic diversity of Ostreobium bacteria paves the way to functional interaction studies within the coral holobiont.

The filamentous chlorophyte Ostreobium sp. dominates shallow marine carbonate microboring communities, and is one of the major agents of reef bioerosion. While its large genetic diversity has emerged, its physiology remains little known, with unexplored relationship between genotypes and phenotypes (endolithic versus free-living growth forms). Here, we isolated 9 strains affiliated to 2 lineages of Ostreobium (>8% sequence divergence of the plastid gene rbc L), one of which was assigned to the family Odoaceae, from the fast-growing coral host Pocillopora acuta Lamarck 1816. Free-living isolates maintained their bioerosive potential, colonizing pre-bleached coral carbonate skeletons. We compared phenotypes, highlighting shifts in pigment and fatty acid compositions, carbon to nitrogen ratios and stable isotope compositions (δ ¹³ C and δ ¹⁵ N). Our data show a pattern of higher chlorophyll b and lower arachidonic acid (20:4ω6) content in endolithic versus free-living Ostreobium . Photosynthetic carbon fixation and nitrate uptake, quantified via 8h pulse-labeling with ¹³ C-bicarbonate and ¹⁵ N-nitrate, showed lower isotopic enrichment in endolithic compared to free-living filaments. Our results highlight the functional plasticity of Ostreobium phenotypes. The isotope tracer approach opens the way to further study the biogeochemical cycling and trophic ecology of these cryptic algae at coral holobiont and reef scales.