Recombinant Mytilus edulis Cytochrome c oxidase subunit 2 (COII)

How can Mytilus edulis be distinguished from other Mytilus species at the molecular level?

Mytilus edulis can be distinguished from other blue mussel congeners using molecular markers. The ITS-1 locus amplifies fragments of similar size (~950 bp) in all three major Mytilus species, but restriction enzyme analysis can differentiate between them. After HhaI restriction digest of PCR products, M. trossulus produces a unique 280 bp fragment, while M. galloprovincialis and M. edulis produce a 450 bp fragment .

For more specific identification, researchers can use the following approach:

• PCR amplification of both ITS-1 and Glu-5' loci

• Restriction digest of ITS-1 PCR products with HhaI enzyme

• Gel electrophoresis on 2% agarose stained with ethidium bromide

• Analysis of fragment patterns to determine species identity or hybrid status

What are the optimal storage conditions for recombinant COII protein?

For optimal storage of recombinant Mytilus edulis COII protein, researchers should follow these guidelines:

• Store the lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

• For long-term storage, keep at -20°C/-80°C

• The protein is typically reconstituted in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

Repeated freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of activity.

How can recombinant COII be used in host-pathogen interaction studies in Mytilus edulis?

Recombinant COII can serve as a valuable tool in host-pathogen interaction studies in Mytilus edulis, particularly when investigating mitochondrial function during pathogen challenges. Researchers can use the following methodological approach:

• Develop a standardized challenge test protocol for M. edulis larvae or adult mussels

• Identify potential pathogens through literature research and laboratory experiments

• Challenge adult mussels through direct injection in the adductor muscle with candidate pathogens

• Manipulate environmental conditions (temperature, salinity, physical stress) to assess their impact on pathogen virulence

• Use ERIC-PCR for DNA fingerprinting of bacterial strains

• Correlate changes in COII expression or function with immune response genes such as lysozyme, mytilin B, and defensin

These studies can provide insights into how mitochondrial proteins like COII respond to bacterial challenges and their potential role in the immune response, particularly when correlated with known immune system markers.

What are the physiological implications of species-specific variations in COII between different Mytilus species?

Species-specific variations in COII between different Mytilus species may contribute to their differential responses to environmental stressors. Research comparing M. trossulus, M. edulis, and M. galloprovincialis has shown significant differences in physiological responses to temperature and salinity stress:

• M. trossulus appears to be the most sensitive to heat stress, followed by M. edulis, with M. galloprovincialis being the most heat-tolerant

• These differences correlate with heart rate responses (Hcrit) under stress conditions

• The acclimatory plasticity varies between species:

  • M. trossulus increases Hcrit by 1.8–3.4°C during acclimation to 21°C

  • M. galloprovincialis shows greater plasticity, increasing Hcrit by 2.4–5.2°C

  • M. edulis increases Hcrit by 2.0–4.7°C

These physiological differences may partially explain the distinct distribution patterns and invasive success of certain species. Researchers can use recombinant COII to investigate whether functional differences in this mitochondrial protein contribute to these physiological adaptations.

How can recombinant COII be utilized in in vitro cell culture systems derived from M. edulis?

Recombinant COII can be incorporated into in vitro cell culture systems derived from M. edulis to study mitochondrial function in different tissue types. Based on established protocols for M. edulis cell culture:

• Isolate cells from mantle tissue, which has shown superior attachment and proliferation characteristics compared to gill tissue

• Prepare substrate using Poly-D-Lysine (50 μg/ml) rather than Poly-L-Lysine for better cell attachment

• Dissociate tissues enzymatically (trypsin method preferred over mechanical dissociation to reduce contamination)

• Supplement culture media with recombinant COII to study:

  • Mitochondrial function in different cell types

  • Cellular responses to oxidative stress

  • Energy metabolism in germ cells versus other cell types

• Use antibodies against DEAD-box helicase 4 (DDX4/VASA) and cytokeratin to distinguish between germ cells and epithelial cells in culture

This approach is particularly valuable for studying cell-type specific responses in male germ cells, which have been shown to proliferate well in culture conditions for up to 25 days.

What is the recommended protocol for reconstituting recombinant COII protein?

The following protocol is recommended for reconstitution of recombinant Mytilus edulis COII protein:

• Centrifuge the vial briefly prior to opening to bring contents to the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is typically recommended)

• Mix gently by inversion or brief vortexing

• Aliquot into smaller volumes for storage to prevent repeated freeze-thaw cycles

• For long-term storage, keep at -20°C/-80°C

• For short-term use, working aliquots can be stored at 4°C for up to one week

This reconstitution protocol ensures optimal protein stability and activity for downstream applications.

How can researchers develop a challenge test for M. edulis using recombinant COII as a marker?

To develop a challenge test for M. edulis using recombinant COII as a marker, researchers should follow these steps:

• Preliminary Protocol Development:

  • Establish standardized rearing conditions for M. edulis larvae or adults

  • Identify potential pathogens through literature research and preliminary experiments

• Pathogen Preparation:

  • Culture selected bacterial strains in appropriate growth media

  • Harvest and adjust bacterial concentration to standardized levels

• Challenge Method:

  • For adults: Direct injection into the adductor muscle

  • For larvae: Addition of pathogens to rearing water

  • Include environmental stressors like temperature, salinity changes, or physical stress

• Monitoring and Analysis:

  • Track mortality rates and physiological responses

  • Monitor COII expression or activity as a marker for mitochondrial stress

  • Analyze immune response genes (lysozyme, mytilin B, defensin) in parallel

  • Use ERIC-PCR for DNA fingerprinting of bacterial strains

This protocol can provide insights into host-pathogen interactions and the role of mitochondrial proteins in disease resistance.

What methods can be used to study species-specific variations in COII function between Mytilus species?

To study species-specific variations in COII function between Mytilus species, researchers can employ the following methods:

• Genetic Characterization:

  • PCR amplification of ITS-1 locus with primers F-GTTTCCGTAGGTGAACCTG and R-CTCGTCTGATCTGAGGTCG

  • Restriction digest with HhaI enzyme

  • Analysis of fragment patterns on 2% agarose gel

• Physiological Response Measurements:

  • Heart rate monitoring as a proxy for whole-animal stress

  • Determination of critical heart rate (Hcrit) under temperature and salinity stress

  • Acclimation experiments at different temperatures (e.g., 14°C vs. 21°C)

• COII Functional Analysis:

  • Recombinant expression of species-specific COII variants

  • Enzymatic activity assays under varying temperature and salinity conditions

  • Protein stability assessments

  • Structural analysis of species-specific amino acid substitutions

These methods can help elucidate how variations in COII contribute to the differential stress responses observed between M. trossulus, M. edulis, and M. galloprovincialis.

Comparative Properties of Mytilus edulis COII and Related Proteins

Optimal Culture Conditions for M. edulis Cells for COII Expression Studies

Storage and Handling Parameters for Recombinant COII Protein