Cadmium Highlights Common and Specific Responses of Two Freshwater Sentinel Species, Dreissena polymorpha and Dreissena rostriformis bugensis.

Zebra mussel (ZM), Dreissena polymorpha, commonly used as a sentinel species in freshwater biomonitoring, is now in competition for habitat with quagga mussel (QM), Dreissena rostriformis bugensis. This raises the question of the quagga mussel's use in environmental survey. To better characterise QM response to stress compared with ZM, both species were exposed to cadmium (100 µg·L-1), a classic pollutant, for 7 days under controlled conditions. The gill proteomes were analysed using two-dimensional electrophoresis coupled with mass spectrometry. For ZM, 81 out of 88 proteoforms of variable abundance were identified using mass spectrometry, and for QM, 105 out of 134. Interestingly, the proteomic response amplitude varied drastically, with 5.6% of proteoforms of variable abundance (DAPs) in ZM versus 9.4% in QM. QM also exhibited greater cadmium accumulation. Only 12 common DAPs were observed. Several short proteoforms were detected, suggesting proteolysis. Functional analysis is consistent with the pleiotropic effects of the toxic metal ion cadmium, with alterations in sulphur and glutathione metabolisms, cellular calcium signalling, cytoskeletal dynamics, energy production, chaperone activation, and membrane events with numerous proteins involved in trafficking and endocytosis/exocytosis processes. Beyond common responses, the sister species display distinct reactions, with cellular response to stress being the main category involved in ZM as opposed to calcium and cytoskeleton alterations in QM. Moreover, QM exhibited greater evidence of proteolysis and cell death. Overall, these results suggest that QM has a weaker stress response capacity than ZM.

Spatial and seasonal use of biomarkers in dreissenids: implications for biomonitoring.

In addition to pollution, organisms are exposed to natural variations of the biotic and abiotic factors of their environment. A battery of sub-cellular biomarkers has been measured seasonally in several populations of both Dreissena polymorpha and Dreissena rostriformis bugensis. To observe and understand the variability associated with biomarker responses, water physicochemistry, sediment contamination, and internal concentrations of contaminants in soft tissues were also considered. Results evidenced seasonal, inter-specific, and inter-populational variability of the measured responses, highlighting the needs (1) to acquire long-term data on the studied populations and (2) to incorporate environmental parameters and contamination in the interpretation of biological responses. From a biomonitoring perspective, significant relationships were identified between biomarkers, internal concentrations of contaminants in soft tissues, and sediment contamination in D. r. bugensis and, to a lesser extent, in D. polymorpha. The detailed interpretation of each biomarker of the battery measured is complex, but a global analysis of all biomarkers at once allows to obtain this signature of the contamination of the studied sites.

Dreissena polymorpha responses under thermal and hypoxic stress: New insights in the tolerance of this freshwater sentinel species.

Dreissena polymorpha is a sentinel freshwater mussel providing key functional ecosystemic services like nutrient recycling and suspended matter filtration. Global warming and especially extreme events imply rapid fluctuations of environmental parameters that sessile organisms could not escape. The increase occurrence of heat waves and the subsequent expansion of hypoxic areas could challenge the survival of mussels. This study provided a deeper knowledge of energy management and cellular function during thermal (+15 °C) or hypoxic (30% of dissolved oxygen saturation) stress for 7 days. A potential metabolic rate depression was highlighted in D. polymorpha under hypoxia through a decline in the mitochondrial activity and a constant AMP content over time. A contrasted pattern of response was observed in thermal-stressed mussels between 24 h and 7 days of exposure. A global increase of metabolic activity was noticed in mussels after 24 h while a return to control level was noticed at the end of the experiment. Although D. polymorpha is considered as a temperature tolerant species, a significant increase of ADP:ATP ratio, related to a decrease of mitochondrial activity and density, suggested an overwhelming of organisms. This study pointed to the importance of considering time of exposure to natural factor variations in tolerance window of organisms in a long-term changing environment. The apparent short-term tolerance of D. polymorpha could hide much more deleterious consequences, i.e. mortality, if abiotic stresses persist, as suggested by climate change models.

Effect of reproduction cycle stage on energy metabolism responses in a sentinel species (Dreissena polymorpha) exposed to cadmium: What consequences for biomonitoring?

Metal trace elements such as cadmium (Cd) are commonly present in ecosystems and could lead to impairment of mitochondrial functions and energy imbalance in aquatic organisms including molluscs. Combined exposure to increasing temperatures and Cd could enhance such an impact on animals. Seasonal fluctuations, such as temperature, and the corresponding reproduction cycle can affect biomarker responses. However, the reproduction cycle stage is rarely taken into account in ecotoxicological studies. Thus, this work aimed at understanding energy metabolism responses in a sentinel species, Dreissena polymorpha. Mussels were collected during the rest and the reproduction periods and were exposed to 10 µg.L-1 of cadmium (Cd) at two temperatures (in situ temperature and in situ temperature + 5°C) during 7 days. Energy metabolism was monitored by measuring reserves and energy nucleotides charge and by assessing aerobic and anaerobic metabolism markers, and upstream regulation pathways. Markers related to OXPHOS activity revealed seasonal variations under laboratory conditions. Conversely, adenylate nucleotides, glycogen, lipid and transcript levels of AMP-activated protein kinase, citrate synthase, ATP synthase and cytochrome b encoding genes remained steady after the acclimation period. No evident effect of Cd on energy metabolism markers was noticed for both exposures although the transcript level of succinate dehydrogenase and citrate synthase encoding genes decreased with Cd during the rest period. Cellular stress, revealed by lipid peroxidation and catalase mRNA levels, only occurred in Cd and warming co-exposed mussels during the reproduction period. These results suggest that contaminant impact might differ according to the reproduction cycle stage. The effect of confounding factors on biomarker variations should be further investigated to have a deeper knowledge of metabolism responses under laboratory conditions.

Seasonal monitoring of cellular energy metabolism in a sentinel species, Dreissena polymorpha (bivalve): Effect of global change?

Aquatic organisms such as bivalves are particularly sensitive to seasonal fluctuations associated with climate changes. Energy metabolism management is also closely related to environmental fluctuations. Changes in both biotic and abiotic conditions, such as the reproduction status and temperature respectively, may affect the organism energy status. A bivalve sentinel species, Dreissena polymorpha was sampled along its one-year reproduction cycle in situ (2018-2019) to study natural modulations on several markers of energy metabolism regarding seasonal variations in situ. A panel of different processes involved in energy metabolism was monitored through different functions such as energy balance regulation, mitochondrial density, and aerobic/anaerobic metabolism. The typical schema expected was observed in a major part of measured responses. However, the monitored population of D. polymorpha showed signs of metabolism disturbances caused by an external stressor from April 2019. Targeting a major part of energy metabolism functions, a global analysis of responses suggested a putative impact on the mitochondrial respiratory chain due to potential pollution. This study highlighted also the particular relevance of in situ monitoring to investigate the impacts of environmental change on sentinel species.

Environmental transcriptomes of invasive dreissena, a model species in ecotoxicology and invasion biology.

Dreissenids are established model species for ecological and ecotoxicological studies, since they are sessile and filter feeder organisms and reflect in situ freshwater quality. Despite this strong interest for hydrosystem biomonitoring, omics data are still scarce. In the present study, we achieved full de novo assembly transcriptomes of digestive glands to gain insight into Dreissena polymorpha and D. rostriformis bugensis molecular knowledge. Transcriptomes were obtained by Illumina RNA sequencing of seventy-nine organisms issued from fifteen populations inhabiting sites that exhibits multiple freshwater contamination levels and different hydrosystem topographies (open or closed systems). Based on a recent de novo assembly algorithm, we carried out a complete, quality-checked and annotated transcriptomes. The power of the present study lies in the completeness of transcriptomes gathering multipopulational organisms sequencing and its full availability through an open access interface that gives a friendly and ready-to-use access to data. The use of such data for proteogenomic and targeted biological pathway investigations purpose is promising as they are first full transcriptomes for this two Dreissena species.

Energy allocation in two dreissenid species under metal stress.

Measurements of biological responses on living organisms are essential in aquatic biomonitoring. In freshwaters, Dreissena polymorpha is an invasive bivalve commonly used in ecotoxicological studies and considered as a model organism. However, D. polymorpha abundances are declining while another species colonizes most of the freshwaters: Dreissena rostriformis bugensis. This species has already been studied in ecophysiology but there is still a lack of data concerning its responses to stressors before its use as a bioindicator of environmental pollution. This study aims to compare the responses of the two species exposed to metal stress. Responses at different levels of biological organization were targeted with measurement of sub-cellular and individual biomarkers following an exposure of up to 7 days to cadmium at 10 µg.L-1. At the individual level, the scope for growth (SFG) was measured. It corresponds to the energy allocated to growth and reproduction. D. polymorpha exhibits variations in biomarker measurements as well as in the SFG in presence of Cd. D. r. bugensis shows no variation in its responses at the different targeted levels. According to the present results, energy metabolism seems to have an essential role for these species when facing a metal stress. Different energy allocation strategies were evidenced between the two species, although the link with biochemical biomarkers is more evident for D. polymorpha than for D. r. bugensis.

Differential tolerance to nickel between Dreissena polymorpha and Dreissena rostriformis bugensis populations.

Differential tolerance to stress is partly responsible for the heterogeneity of biomarker responses between populations of a sentinel species. Although currently used for freshwater biomonitoring, studies concerning inter-populational variability in tolerance to contaminants for the zebra mussel (Dreissena polymorpha) are scarce. Moreover, this well-known invader is currently replaced by another, the quagga mussel (Dreissena rostriformis bugensis). To evaluate the differential tolerance between dreissenids, several populations of both species were exposed to a high concentration of nickel. A LT50 (time when 50% of individuals were dead) was established for each population. Biomarker responses and internal nickel concentration were also measured, to link tolerance with physiological status. Results evidenced that D. polymorpha populations are more heterogeneous and more tolerant than D. r. bugensis ones. For D. polymorpha populations only, LT50 values were positively correlated with the nickel contamination in situ, with higher anti-oxidative defences and a higher Integrated Biomarker Response value in the field. Such findings may be explained by local adaptation and invasion dynamic within each species. The significance of this differential tolerance when using biomarker responses for biomonitoring purposes is thus discussed.