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

Abstract

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.

Keywords: biomonitoring; proteoforms; quagga mussel; stress; zebra mussel.

Figure 1

F1F2 Factorial planes of Principal Component Analysis (PCA) computed on normalised spot volume. (a): PCA on the 88 spots × 17 gels obtained for ZM. F1 = 34.5%, F2 = 10%. (b): PCA on the 134 spots × 13 gels obtained for QM. F1 = 49.2%, F2 = 9%.

Figure 2

Heat maps of the differentially abundant gill proteoforms after cadmium exposure for (a) zebra and (b) quagga mussel. After a 7-day exposure to cadmium (100 µg·L⁻¹), gill proteins from exposed and control mussels were extracted and separated by 2DE. Images were analysed using Delta 2D (Decodon, Gmbh). The heat maps show the 60 up-regulated and 28 down-regulated spots in zebra mussels, and the 71 up- and 63 down-regulated spots in quagga mussels.

Figure 3

STRING analysis of cadmium-induced DAPs for zebra (ZM) and quagga (QM) mussels. Protein–protein interaction networks were constructed using the STRING database (https://string-db.org, accessed on 6 September 2023) [17]). Nodes in the network represent DAPs. Protein abbreviations are reported in Table 1. Confidence in protein interactions is shown by the thickness of the lines connecting each node. G refers to Gene Ontology terms: G1. GO:0033554_Cellular response to stress; G2. GO:0006790_Sulfur compound metabolic process; G3. GO:0019752_Carboxylic acid metabolic process; G4. GO:0005856_Cytoskeleton and G5. GO:0006417_Regulation of translation; G6. GO:0042981_Regulation of apoptotic process; G7. GO:0045055_Regulated exocytosis.

Figure 4

Shorter phosphoenolpyruvate kinase enzyme (PCK) proteoforms are more abundant after cadmium exposure. Structure of full-length PCK and shorter proteoforms of quagga (QM) and zebra (ZM) mussels. Top: PCK functional domains (NCBI CD batch search). Bottom: DAP localisation on 2DE gels; sequence coverage is highlighted in yellow.

Figure 5

Several proteoforms for one protein: the case of a 60 kDa neurofilament protein-like. In quagga mussel (QM), several spots were identified using homology with the zebra mussel (ZM) XP_052223977.1 sequence: Spots 250, 435 and 431. Spot 250 is a 71 kDa protein and probably the precursor form of the 60 kDa neurofilament protein (blue circle on the 2DE gel). It presented no variation after cadmium exposure. Spots 435 and 431 are shorter sequences of 55 kDa (red and orange circles on the 2DE gel, respectively), corresponding to the first part of the XP_052223977.1 sequence (see MS peptide coverage highlighted in yellow). The XP_052223977.1 sequence of the 60 kDa neurofilament protein-like of ZM is presented in the black box, with the NCBI Smartblast result showing its alignment with lamins.

Figure 6

A quagga mussel (QM) shorter enolase proteoform is a putative c-myc promoter-binding protein-1 isoform. (a) 37 kDa DAP QM 828 was identified as enolase (ENO1) and lacks the first part of the enzyme; (b) the shorter enolase is highly similar to the c-myc promoter-binding protein-1 isoform (MBP-1); (c) 48 kDa DAP QM 517 is also identified as enolase; coverage (highlighted in yellow) on the full-length sequence confirms this DAP correspond to the whole enzyme.

Figure 7

Pie Chart Diagram of GO biological process enrichment after Dreissena exposure to cadmium. Genome Ontology (GO) enrichment analysis of Cd induced DAPs-related biological processes. Allocation of biological processes is detailed in Table S4. Left, zebra mussel (ZM) and right, quagga mussel (QM).

Figure 8

Modulations of Dreissena gill proteomes are consistent with Cd exposure but partly differ reflecting species-specific cellular strategies in fighting homeostasis rupture.