The microbiome has recently emerged as a central theme in environmental toxicology because microbes respond to contaminants, facilitate biotransformation and chemical detoxification, and interact with the host (6). Recent surveys of the toxicological mice and rat models have shown that the gut microbiota of contemporary colonies in these rodents can differ markedly depending on the animal origin and other factors associated with their husbandry (23, 24). The same concerns are fully applicable to ecotoxicological models. In this study, we present evidence that despite genetic similarities and controlled environments in D. magna, a well-established animal model, their microbiomes are clone-dependent even within the same laboratory (Fig. 2a and Figure S4). This clone-dependent variation results in divergent responses to chemical exposure (Fig. 2b).
Consistent with our hypothesis, the transplantation of the microbiome yielded statistically indistinguishable dose-response curves in the T-lines, whereas in the animals with their original microbiomes (CR-lines), significant differences in LC50 values were observed between the F clone and the two other clones. The observed inter-clonal variability in the LC50 values was substantial (0.5–1.1 mg/L) but within the reported range for this assay (0.29–2.1 mg/L; for 24–48 h test) (24, 43) in D. magna and other cladocerans (44). Furthermore, the transplant treatment resulted in an increased similarity of microbial diversity, the community structure and the reconstructed metabolic capacities among different clones. Therefore, the daphnid tolerance to CrIV exposure was affected by the gut microbial assembly and the genetic background of the clone (Table S2).
These findings support the potential application of microbiome transplantation to reduce inter-clonal variations in gut microbiota and improve the uniformity of host reactions to exposure. Developing a microbiome transplant methodology necessitates careful consideration of several factors, such as donor selection, the bacterial diversity of the transplant inoculum, and the target bacteria taxa. These considerations are essential to effectively manipulate the microbiome and standardize its impact on the host giving the substantial roles of individual bacterial species, inter-species interactions (45, 46), and bacterial metabolites (47) in influencing the host's physiological state.
Additionally, selecting germ-free animals as test organisms can be a viable option to decrease the variability in test outcomes due to microbiome effects. Another possibility is to utilize germ-free animals as recipients of the microbiome transplant, as done here, which alleviates competition between introduced and resident microbes. Indeed, starting with a germ-free host gives the introduced microbiome a better opportunity to establish and colonize without competition from preexisting resident microbes. This approach can enhance the success of the microbiome transplant and allow for a more controlled manipulation of the microbiome composition (48).
Microbiome transplants can introduce bacteria that play a crucial role in maintaining a healthy status, such as the influential genus Limnohabitans which exhibited a strong correlation with daphnid survivorship across the clones and lines (Fig. 4). Thus, this genus should be explored as a predictor of clone sensitivity, which may help interpret differences in LC50 values observed in different clones and/or laboratories. Limnohabitans is a common inhabitant of the Daphnia gut (49, 50), with often reported positive correlations to fecundity and population stability (51). Furthermore, previous studies have linked Limnohabitans to tolerance towards heavy metals, such as lead and cadmium (52), and have proposed it as a potential candidate for bioremediation (53). These findings help explain the observed decrease in Limnohabitans relative abundance in F-T-lines, which aligns with the lower LC50 compared to F-CR. Conversely, the Limnohabitans increase in Nies-T-lines was associated with increased LC50 (Figs. 2a and 4, Table S5). Other taxa associated with high LC50, such as Hydrogenophaga, Fluviicola and Williamsia (Table S7), were previously linked to contaminated environments and bioremediation (54, 54, 55), although not specifically in the context of chromium pollution. These results provide novel insights into the involvement of Limnohabitans and other influencial taxa within Daphnia microbiome, shedding light on the microbiome and host responses to different conditions and their potential impact on toxicity levels.
Metabolic reconstructions based on the phylogenies for poorly described microbiomes should be approached cautiously, as accuracy depends on the proportion of sequenced genomes in the studied taxa. Well-described systems like the human gut allow for high accuracy, while less researched environments with undescribed taxa have higher uncertainty. Daphnia's microbiome is relatively understood (50), so the presented metabolic reconstruction should reasonably approximate bacterial metabolic capabilities. Whereas no specific pathways contributing to chromium metabolism (e.g., synthesis chromate reductase, resistance proteins, etc.) were identified by the Picrust2 analysis, the metabolic reconstruction indicates that a general upregulation of the reductive, degradative capacities of the microbiome was associated with high tolerance to CrVI across the clones and lines (Table S8). One can speculate that under reductive conditions, CrVI can be reduced to CRIII, which is both less toxic and not efficient in crossing cell membranes resulting in reduced chromium uptake by the gut epithelium (56). The mechanisms through which bacterial strains reduce CrVI to CRIII are variable and species-dependent, and enzymatic chromate reduction in aerobic bacteria can be conveyed by reductases evolved on other substrates (56).
In summary, we demonstrated that microbiome transplants could be applied to standardize daphnids (and, perhaps, other invertebrate models) to increase taxonomical and functional microbiome similarity. This similarity would improve the reproducibility of the effect studies within and among laboratories. In line with previous work, we showed that the host genotype also shapes microbiome composition in Daphnia (20, 57, 58). Therefore, regular microbiome monitoring in the laboratory-reared daphnids is needed to enable comparisons between laboratories and test occasions and to understand the variability sources. Addressing the role of the microbiome in mediating ecotoxicological responses becomes crucial for achieving an appropriate level of environmental protection and enhancing the repeatability, reliability and predictive power of hazard assessment.