Elasmobranchs are exposed to coastal pollution, yet molecular biomarkers are scarce, and their bile proteins have so far been described only for a single shark individual. Bile from two endangered batoids caught by artisanal fisheries off Rio de Janeiro, Brazil, namely the spiny butterfly ray Gymnura altavela (n = 20) and the groovebelly stingray Dasyatis hypostigma (n = 21), was characterized. Total and heat-stable (70 °C) bile protein fractions were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and quantified by densitometry, gelatinolytic activity was assessed by zymography, and immunoreactive matrix metalloproteinase-2 (MMP-2) was measured by enzyme-linked immunosorbent assay (ELISA). Bile resolved into five recurrent molecular-weight-defined band groups in G. altavela and six in D. hypostigma, between ~15 and ~ 75 kDa. A ~ 15 kDa group was present in 80 of 81 lanes and accounted for a median 47–67% of lane intensity. A ~ 75 kDa group persisted in the heat-stable fraction of both species. Across 104 band contrasts, no significant sex or site differences were detected after correction for multiple testing. Juveniles showed stronger bands than adults on three gels, but a replicate gel of the same animals did not confirm this. Gelatinolytic activity appeared as broad lysis zones, and immunoreactive MMP-2 was detected in all 44 samples tested (group means about 115–139 ng mL−1), with no significant sex difference. These data provide the first multi-individual descriptive baseline for batoid bile, a matrix obtainable from by-catch without additional mortality. The bands remain unidentified and no protein has yet been validated as a contamination biomarker; mass spectrometry and paired contaminant data are needed.
Freshwater food webs are increasingly exposed to eutrophication and warming, which can shift phytoplankton community composition and reduce the availability of long-chain polyunsaturated fatty acids to higher trophic levels. We investigated whether the model freshwater crustacean Daphnia magna can compensate for a dietary lack of eicosapentaenoic acid (EPA) through endogenous EPA synthesis from its precursors, and whether this response depends on food quality, temperature, and genetic background. Two D. magna strains, isolated from contrasting temperate regions, were exposed to four diets ranging from edible cyanobacteria-dominated to cryptophyte-enriched communities at 15, 20, and 25 °C. Using deuterium labelling, compound-specific hydrogen isotope analysis, fatty acid quantification and fitness measurements, we assessed EPA bioconversion and its consequences for consumer performance. Cyanobacteria-rich food reduced biomass accrual, delayed reproduction, decreased body size, and lowered offspring production. Despite these pronounced fitness effects, endogenous EPA production remained stable across all dietary treatments. Instead, bioconversion was primarily governed by water temperature, reaching a maximum at approximately 20 °C, and was slightly higher in the cold-adapted strain, consistent with underlying biochemical constraints on metabolic processes. Slight differences between the two strains suggest an important contribution of genetic background in determining EPA biosynthesis. These results indicate that endogenous EPA synthesis in D. magna was not actively regulated but was constantly active and potentially buffered against low dietary EPA levels, but did not fully compensate for poor food quality.
Blue mussels (Mytilus edulis) from the southern North Sea are exposed to multiple natural and anthropogenic stressors, including parasite infestation and microplastic pollution. For example, the intestinal copepods Mytilicola intestinalis and Mytilicola orientalis are common endoparasites of blue mussels in the coastal areas. We investigated the prevalence and composition of Mytilicola species in mussels from the Wadden Sea. The infestation rate of M. edulis by Mytilicola spp. was 78%. One third of infested mussels harboured both parasite species. These parasites may feed on intestinal tissue, detached cells, or the gut content of the host which also may contain natural and anthropogenic microparticles that were ingested by the mussel. In a 5-day laboratory experiment, mussels either received no food, were fed with microalgae, or were exposed to microplastics. Activities of selected digestive enzymes were measured in the midgut gland of the mussels and in whole parasites. Digestive enzyme profiles revealed significant functional differences between the host and either parasite. Mussels showed high esterase and (3-glucosidase activities, reflecting the utilization of algal food. In contrast, both Mytilicola species showed low (3-glucosidase activities. Elevated alpha-glucosidase and N-acetyl-(3-D-glucosa-minidase activities in M. orientalis suggest the utilization of glycogen and mucus from the host. Treatments involving starvation, feeding, and microplastics had no significant effects on the enzyme profiles of the host, and there was no correlation between the enzyme activities of the host and its parasites. Enzyme profiles proved useful for interpreting trophodynamic interactions between mussels and their Mytilicola endoparasites, but there were limited responses to exposure conditions for all species.
In freshwater biomonitoring, the zebra mussel Dreissena polymorpha is commonly used as a sentinel organism, with individuals usually standardised by shell length as a proxy for age to limit inter-individual variability. However, the relationship between shell length and estimated biological age can vary both within and across populations. This raises questions about the biological relevance of size-based standardisation and the comparability of data across studies using different size ranges. The present study examined how a battery of biochemical biomarkers, covering energy metabolism, antioxidant and detoxification defences, and cellular damage, varied across five defined size classes distributed along juvenile and adult life stages, sampled during a pre-spawning and a post-spawning period. Biochemical biomarker profiles were differentiated across size classes during the pre-spawning period, consistent with distinct physiological strategies between juveniles and adults, whilst discrimination was reduced during the post-spawning. These results were consistent with body mass patterns, which diverged among classes during pre-spawning but were less differentiated following gamete release. Biomarker profiles thus differed across size classes and sampling period, with a significant interaction between size class and sampling period detected for six of the eleven biomarkers analyzed, highlighting the importance of accounting for the size-age relationship in biomonitoring studies to achieve a biologically realistic interpretation of results.
The tetrazolium-based MTT assay is widely used to assess cellular metabolic activity, yet the subcellular origin and mechanistic basis of MTT reduction remain incompletely defined. Here, we investigated the localization and biochemical determinants of MTT reductive activity using subcellular fractionation of lobster cockroach Nauphoeta cinerea head tissue and zebrafish Danio rerio brain, combined with substrate specificity, protein-dependence analysis, and mitochondrial electron transport chain (ETC) inhibition. Succinate-driven MTT reduction was predominantly localized to mitochondrial fractions (P2 and P3), accounting for similar to 60-65% of total activity in both species, while post-mitochondrial supernatants exhibited minimal activity. MTT reduction increased linearly with protein concentration across all fractions (R-2 = 0.95-0.99), with mitochondrial fractions displaying the highest protein-normalized reductive capacity. MTT reduction was highly sensitive to malonate and significantly inhibited by antimycin A and sodium azide, confirming dependence on Complex II-mediated electron entry and downstream ETC activity, whereas rotenone produced only partial inhibition, consistent with bypass of Complex I under succinate-driven conditions. Collectively, these findings demonstrate that MTT reduction is strongly dependent on substrate selection and experimental conditions. Under succinate-driven conditions, MTT reduction reflects mitochondrial ETC activity, with minimal non-mitochondrial contribution. This work provides a mechanistic framework that clarifies the intracellular origin of MTT reduction and supports its use as a substrate-dependent indicator of mitochondrial function in bioenergetic and toxicological studies. The consistent findings obtained in insect and fish tissues further support the conservation of the underlying mechanisms across phylogenetically distinct species and reinforce the value of alternative experimental models for investigating mitochondrial bioenergetics.
