Recombinant Choristoneura rosaceana Cytochrome c oxidase subunit 2 (COII)

What is Choristoneura rosaceana and why is its COII protein significant for research?

Choristoneura rosaceana, commonly known as the obliquebanded leafroller, is a pest species that affects various plants including raspberry (Rubus spp.), apple (Malus domestica), pear (Pyrus communis), cherry (Prunus spp.), and filbert (Corylus avellana) among other deciduous trees and bushes in North America . The cytochrome c oxidase subunit 2 (COII) protein is a mitochondrial-encoded protein that serves as an important genetic marker for evolutionary studies, population genetics, and species delimitation in Lepidoptera. Research on this protein contributes to understanding phylogenetic relationships within the Tortricidae family and developing effective pest management strategies .

How is recombinant COII protein typically stored and reconstituted for experimental use?

For optimal stability, recombinant COII protein should be stored at -20°C/-80°C upon receipt, with aliquoting recommended to prevent repeated freeze-thaw cycles, which can significantly degrade protein quality. The lyophilized protein is typically reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (commonly 50%) before aliquoting and storing at -20°C/-80°C . Working aliquots may be stored at 4°C for up to one week to minimize degradation from repeated freezing and thawing .

What are the recommended protocols for expressing and purifying Recombinant Choristoneura rosaceana COII protein?

For efficient expression and purification of Recombinant Choristoneura rosaceana COII protein, researchers typically employ the following methodology:

• Expression System Selection: E. coli is the preferred expression system due to its rapid growth rate, high protein yield, and cost-effectiveness . BL21(DE3) or Rosetta strains are commonly used for membrane proteins like COII.

• Vector Design: The gene encoding COII (full length 1-227aa) is cloned into an expression vector with a His-tag at the N-terminus to facilitate purification .

• Expression Conditions:

  • Culture in LB medium supplemented with appropriate antibiotics

  • Induce expression with IPTG (0.1-1.0 mM) when OD600 reaches 0.6-0.8

  • Incubate at lower temperatures (16-25°C) for 16-20 hours to enhance proper folding

• Purification Process:

  • Lyse cells using sonication or pressure-based methods in Tris/PBS-based buffer

  • Purify using Ni-NTA affinity chromatography

  • Elute with imidazole gradient (20-250 mM)

  • Perform buffer exchange to remove imidazole

  • Verify purity by SDS-PAGE (>90% purity is typically achieved)

• Quality Control: Confirm protein identity by Western blot and/or mass spectrometry.

How can researchers effectively apply PCR-based methods to study COII gene variations in Choristoneura rosaceana populations?

To study COII gene variations in Choristoneura rosaceana populations, researchers should implement the following PCR-based methodological approach:

• Sample Collection: Collect specimens from diverse geographical regions, ensuring adequate representation of different host plant associations and ecological conditions .

• DNA Extraction:

  • Extract total DNA from individual specimens using standard protocols

  • For higher throughput studies, consider non-destructive DNA extraction methods to preserve morphological characters

• PCR Amplification:

  • Design primers specific to the 470-bp region of the COII gene

  • Standard PCR conditions: initial denaturation at 94°C for 2 min; 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 45s; final extension at 72°C for 10 min

• Sequencing and Analysis:

  • Sequence amplicons in both directions for accuracy

  • Check sequences for ambiguities using software like Sequencher 4.0

  • Align sequences by eye in programs such as PAUP 4.0b10

• Haplotype Identification:

  • Compare sequences to previously recorded haplotypes

  • Conduct phylogenetic analyses to determine relationships among haplotypes

  • Integrate with other genetic markers for comprehensive analysis

This approach has successfully identified distinct lineages within Choristoneura species complexes in previous studies .

What are the standard applications of Recombinant Choristoneura rosaceana COII protein in biochemical assays?

Recombinant Choristoneura rosaceana COII protein finds application in various biochemical assays, including:

• Enzyme Activity Assays:

  • Measure cytochrome c oxidase activity by monitoring the oxidation of reduced cytochrome c

  • Quantify enzyme kinetics (Km, Vmax) under various conditions

• Protein-Protein Interaction Studies:

  • Pull-down assays to identify interaction partners

  • Co-immunoprecipitation to verify protein complexes in vivo

  • Surface Plasmon Resonance (SPR) to determine binding affinities

• Structural Characterization:

  • Circular Dichroism (CD) spectroscopy to assess secondary structure

  • Limited proteolysis to identify stable domains

  • X-ray crystallography or NMR for high-resolution structural analysis

• Immunological Applications:

  • Development of antibodies against specific epitopes

  • Immunohistochemistry to localize the protein in tissue samples

  • Western blotting for protein expression analyses

• Functional Reconstitution:

  • Incorporation into liposomes to study membrane transport properties

  • In vitro reconstitution of electron transport chain components

The protein's purity (>90% as determined by SDS-PAGE) makes it suitable for these applications .

How can integrative taxonomy approaches be applied to resolve species boundaries in Choristoneura using COII as a molecular marker?

Integrative taxonomy offers a robust framework for resolving species boundaries in the Choristoneura complex by combining multiple lines of evidence. When using COII as a molecular marker, researchers should implement the following comprehensive approach:

• Multi-locus Molecular Analysis:

  • Sequence mitochondrial COII (470-bp region) from extensive sampling across geographic ranges

  • Complement with nuclear markers (e.g., microsatellite/SSR markers) to detect hybridization and introgression

  • Perform population genetic analyses using software like STRUCTURE to identify genetic clusters

• Behavioral Data Integration:

  • Document adult flight phenology, which is a key life-history trait maintaining genomic integrity

  • Record pheromone attraction patterns using field traps with species-specific lures

  • Analyze mating behavior under laboratory conditions

• Ecological Characterization:

  • Document host plant associations and feeding preferences

  • Record larval development rates under standardized conditions

  • Map ecological niches using GIS-based approaches

• Morphological Analysis:

  • Perform detailed morphometric analyses of taxonomically informative characters

  • Use both traditional and geometric morphometrics

  • Compare results with molecular phylogenies to identify diagnostic traits

When applied to Choristoneura populations in isolated habitats like Cypress Hills, this approach successfully identified multiple species (C. fumiferana, C. occidentalis, C. lambertiana) and hybrid forms, demonstrating the importance of extensive sampling and character analysis for understanding species boundaries .

What techniques are recommended for investigating potential post-translational modifications of Recombinant Choristoneura rosaceana COII protein?

To investigate post-translational modifications (PTMs) of Recombinant Choristoneura rosaceana COII protein, researchers should consider the following advanced methodological approaches:

• Mass Spectrometry-Based Techniques:

  • Use MALDI-TOF MS to analyze peptide fragments and identify PTMs

  • Apply LC-MS/MS for high-resolution mapping of modification sites

  • Implement top-down proteomics to analyze intact protein and preserve PTM patterns

• Site-Directed Mutagenesis Approach:

  • Generate serine-to-alanine mutations at potential phosphorylation sites

  • Create recombinant viruses containing these mutations (e.g., S92A, S93A, S9293A)

  • Analyze structural and functional differences using confocal microscopy and biochemical assays

• Phosphorylation-Specific Analysis:

  • Use phospho-specific antibodies in Western blotting

  • Apply Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Implement in vitro kinase assays to identify responsible kinases

• Temporal Analysis of Modifications:

  • Examine changes in modification patterns at different time points (e.g., 72 hpi vs. 96 hpi)

  • Correlate modifications with functional changes or protein-protein interactions

• Functional Impact Assessment:

  • Compare wild-type and mutant proteins in enzyme activity assays

  • Analyze structural changes using CD spectroscopy or limited proteolysis

  • Examine effects on protein-protein interactions using pull-down assays

Previous studies with similar proteins have shown that phosphorylation can induce significant structural changes that affect protein function .

How does the evolutionary history of COII in Choristoneura rosaceana compare with other Tortricidae species, and what are the implications for speciation research?

The evolutionary history of COII in Choristoneura rosaceana, when compared with other Tortricidae species, reveals important patterns with significant implications for speciation research:

• Phylogenetic Relationships:

  • COII sequences place C. rosaceana within a complex of closely related Choristoneura species

  • Molecular evidence suggests that C. rosaceana is more closely related to western North American species than to the eastern spruce budworm (C. fumiferana)

  • Analysis of 470-bp region of mtDNA from the COII gene has been instrumental in resolving these relationships

• Hybridization Patterns:

  • COII sequences, combined with microsatellite data, have identified hybrid zones between C. rosaceana and other Choristoneura species

  • In isolated forest patches like Cypress Hills, hybridization between distinct lineages has been documented

  • These natural hybridization events provide valuable models for studying speciation mechanisms

• Life-History Traits and Genetic Integrity:

  • Despite gene flow potential, species maintain genomic integrity through:

    • Differences in adult flight phenology

    • Species-specific pheromone attraction

    • Host plant specialization

  • These reproductive isolating mechanisms prevent complete genetic homogenization even in sympatry

• Biogeographical Implications:

  • The earliest-branching tortricid lineages show southern/tropical distributions

  • This pattern supports a hypothesized Gondwanan origin for the family Tortricidae

  • Speciation in northern temperate regions appears to be more recent

• Methodological Considerations:

  • Integration of COII data with nuclear markers (SSRs) provides more comprehensive evolutionary insights

  • Population assignment analyses at different k-values (k=2 and k=6) reveal hierarchical population structure

  • This integrated approach has identified six distinct population clusters within North American Choristoneura species

These findings underscore the value of COII as a molecular marker in speciation research and highlight the importance of integrative approaches that combine molecular, ecological, and behavioral data.

What are the optimal expression conditions for maximizing yield and solubility of Recombinant Choristoneura rosaceana COII protein?

To maximize yield and solubility of Recombinant Choristoneura rosaceana COII protein, researchers should optimize the following expression parameters:

• Expression System Selection:

  • E. coli BL21(DE3) or Rosetta strains are recommended for membrane proteins

  • Consider Shuffle T7 Express for proteins requiring disulfide bond formation

  • For complex post-translational modifications, insect cell systems may be considered

• Vector Design Optimization:

  • N-terminal His-tag placement has shown good results for COII protein

  • Consider fusion partners (MBP, SUMO, TrxA) to enhance solubility

  • Include a TEV protease cleavage site for tag removal if needed for functional studies

• Culture Conditions:

  Parameter	Recommended Range	Notes
  Temperature	16-25°C	Lower temperatures reduce inclusion body formation
  IPTG concentration	0.1-0.5 mM	Lower concentrations favor soluble expression
  OD600 at induction	0.6-0.8	Optimal cell density for induction
  Post-induction time	16-20 hours	Extended time at lower temperatures
  Media composition	LB with glucose	0.5-1% glucose suppresses basal expression

• Solubilization Strategies:

  • Use mild detergents (DDM, LDAO) for membrane protein extraction

  • Include glycerol (10%) and reducing agents in lysis buffers

  • Consider osmotic shock procedures for periplasmic expression

• Additives to Enhance Solubility:

  Additive	Concentration	Function
  Glycerol	5-10%	Stabilizes hydrophobic regions
  Trehalose	6%	Preserves protein structure during lyophilization
  NaCl	100-300 mM	Reduces non-specific interactions
  Imidazole	5-10 mM	Reduces non-specific binding during purification
  β-mercaptoethanol	5 mM	Maintains reducing environment

• Refolding Protocols (if needed):

  • Gradual dialysis from denaturing conditions

  • On-column refolding during purification

  • Pulsed dilution into refolding buffer

By systematically optimizing these parameters, researchers can achieve protein purity greater than 90% as determined by SDS-PAGE, suitable for various downstream applications .

How can researchers effectively design primers for amplifying and sequencing the complete COII gene from Choristoneura rosaceana and related species?

Designing effective primers for amplifying and sequencing the complete COII gene from Choristoneura rosaceana and related species requires a systematic approach:

• Sequence Alignment and Analysis:

  • Collect and align available COII sequences from Choristoneura species and related Tortricidae

  • Identify conserved regions flanking the COII gene

  • Note regions of interspecific variation for species-specific primers

• Primer Design Parameters:

  Parameter	Recommendation	Rationale
  Primer length	18-25 nucleotides	Balance between specificity and annealing efficiency
  GC content	40-60%	Ensures stable annealing
  3' end stability	No more than 2 G/C in last 5 bases	Prevents mispriming
  Tm value	55-65°C	Optimal for PCR amplification
  Complementarity	Avoid self-complementarity	Prevents primer-dimer formation

• Degenerate Primer Strategy:

  • For amplifying COII across multiple Choristoneura species, incorporate degenerate bases at variable positions

  • Limit degeneracy to 2-3 positions per primer to maintain specificity

• Recommended Primer Sets:

  • For the 470-bp region used in phylogenetic studies:

    • Forward: Based on conserved regions in tRNA-Leu

    • Reverse: Based on conserved regions in COII

  • For complete COII gene:

    • Design primers in flanking tRNA genes or CO1/CO3 genes

    • Consider using nested PCR approach for difficult templates

• Validation Protocol:

  • Test primers on known templates (positive controls)

  • Perform gradient PCR to determine optimal annealing temperature

  • Sequence PCR products to confirm target amplification

  • Test specificity across multiple Choristoneura species

• Troubleshooting Strategies:

  • For difficult templates, add PCR additives (DMSO, betaine)

  • For length polymorphisms, use long-range PCR polymerases

  • For AT-rich regions, adjust annealing temperature and buffer composition

This approach has been successfully applied in studies examining the 470-bp region of mtDNA from the COII gene in Choristoneura species, enabling reliable amplification and sequencing for phylogenetic analysis .

What are the most sensitive methods for detecting low-level expression of COII in Choristoneura rosaceana tissue samples?

For detecting low-level expression of COII in Choristoneura rosaceana tissue samples, researchers should employ these highly sensitive methodological approaches:

• Quantitative Real-Time PCR (qPCR):

  • Design COII-specific primers and probes

  • Use SYBR Green or TaqMan chemistry for detection

  • Include appropriate reference genes (e.g., actin, GAPDH) for normalization

  • Implement standard curve method for absolute quantification

  • This method can detect as few as 10 copies of target sequence

• Digital PCR (dPCR):

  • Partition sample into thousands of individual reactions

  • Provides absolute quantification without standard curves

  • Higher precision and reproducibility than qPCR

  • Less susceptible to inhibitors in tissue samples

• Droplet Digital PCR (ddPCR):

  • Combines microfluidics with digital PCR

  • Provides absolute quantification with improved sensitivity

  • Can detect single-molecule differences between samples

• RNA-Seq with Targeted Enrichment:

  • Pre-amplify COII transcripts using specific primers

  • Perform deep sequencing of enriched libraries

  • Analyze data using specialized low-abundance transcript detection algorithms

• In Situ Hybridization Techniques:

  Technique	Sensitivity	Application
  RNAscope	Single-molecule	Cellular localization with high specificity
  FISH	~10-20 copies/cell	Spatial distribution in tissues
  smFISH	Single-molecule	Quantitative analysis at single-cell level

• Immunological Methods with Signal Amplification:

  • Develop high-affinity antibodies against COII

  • Use tyramide signal amplification for immunohistochemistry

  • Employ proximity ligation assay for protein-protein interactions

  • Implement Western blotting with chemiluminescent substrates

• Mass Spectrometry-Based Proteomics:

  • Selected Reaction Monitoring (SRM) for targeted detection

  • Implement SWATH-MS for data-independent acquisition

  • Use isotope-labeled standards for accurate quantification

These methods have been successfully applied in studies of microsporidia detection in insect hosts, where qPCR was able to detect low-intensity infections easily overlooked through light microscopy . The choice of method depends on specific research questions, available tissue quantities, and required detection limits.

How can Recombinant Choristoneura rosaceana COII protein be used in studies of insecticide resistance mechanisms?

Recombinant Choristoneura rosaceana COII protein offers valuable applications in studying insecticide resistance mechanisms through these methodological approaches:

• Target-Site Resistance Studies:

  • Compare wild-type and mutant COII proteins for structural differences

  • Assess binding affinities of insecticides to recombinant proteins

  • Perform site-directed mutagenesis to introduce known or suspected resistance mutations

  • Evaluate enzymatic activity of mutant proteins compared to wild-type

• Biomarker Development:

  • Generate antibodies against recombinant COII protein

  • Develop immunoassays to detect expression changes in field populations

  • Compare COII expression levels between susceptible and resistant strains

  • Correlate expression changes with resistance phenotypes

• Functional Genomics Approaches:

  • Use recombinant COII to validate gene editing experiments (CRISPR/Cas9)

  • Perform protein-protein interaction studies to identify resistance-associated pathways

  • Develop activity-based assays to screen for novel insecticides

• Resistance Monitoring Tools:

  • Design molecular diagnostics based on COII sequence variations

  • Establish baseline susceptibility using biochemical assays

  • Track resistance allele frequencies in field populations

  • Develop rapid detection methods for known resistance mutations

This research is especially relevant as C. rosaceana has developed resistance to multiple insecticides, including spinetoram, chlorantraniliprole, and emamectin benzoate, making it challenging to control in agricultural settings .

What role does COII play in understanding the phylogeography and speciation patterns of Choristoneura species across North America?

COII has played a crucial role in elucidating the phylogeography and speciation patterns of Choristoneura species across North America:

• Identification of Species Complexes:

  • COII sequence analysis helped identify distinct species within what was previously considered a single widespread species

  • The 470-bp region of mtDNA from COII gene has been particularly informative for species delimitation

  • Integration with SSR markers allowed assignment of individuals to species with high confidence

• Biogeographic Patterns:

  • COII data revealed two major population clusters in North America:

    • Population 1: Species in boreal regions and eastern United States (C. fumiferana, C. pinus)

    • Population 2: Western species (C. occidentalis, C. biennis, C. orae, C. lambertiana, C. retiniana, C. carnana)

  • This east-west division reflects historical biogeographic events and ecological adaptations

• Hybridization Zone Identification:

  • In isolated forest patches like Cypress Hills, COII sequencing identified multiple species (C. fumiferana, C. occidentalis, C. lambertiana) and hybrid forms

  • These contact zones provide natural laboratories for studying speciation mechanisms

• Ecological Speciation Evidence:

  • COII data, combined with ecological information, showed that:

    • Life-history traits (adult flight phenology, pheromone attraction) maintain genomic integrity

    • Feeding habits (internal versus external) are strongly conserved phylogenetically

    • A clade characterized by eggs deposited in large clusters shows elevated incidence of polyphagy

• Temporal Patterns of Diversification:

  • The five earliest-branching tortricid lineages (identified using COII and other markers) are species-poor tribes with mainly southern/tropical distributions

  • This pattern supports a hypothesized Gondwanan origin for the family

  • More recent diversification events correlate with host plant radiation

These findings demonstrate the value of COII as a molecular marker in understanding the complex evolutionary history of Choristoneura species and highlight the importance of integrating molecular data with ecological and behavioral information for comprehensive phylogeographic studies .

How can researchers effectively use recombinant COII protein in developing molecular diagnostics for pest management of Choristoneura rosaceana?

Researchers can leverage recombinant COII protein to develop effective molecular diagnostics for Choristoneura rosaceana pest management through the following methodological approaches:

• Antibody-Based Detection Systems:

  • Generate highly specific polyclonal or monoclonal antibodies against recombinant COII

  • Develop ELISA-based detection kits for field use

  • Create lateral flow immunoassays for rapid identification

  • Implement immunofluorescence techniques for tissue localization

• DNA-Based Detection Methods:

  • Design species-specific primers targeting COII gene variations

  • Develop multiplex PCR assays to differentiate C. rosaceana from related species

  • Implement loop-mediated isothermal amplification (LAMP) for field-deployable detection

  • Create qPCR assays for quantitative assessment of infestation levels

• Population Monitoring Applications:

  • Develop molecular markers based on COII sequence polymorphisms

  • Track population movements and dispersal patterns

  • Monitor changes in population genetic structure in response to control measures

  • Identify source populations of new infestations

• Resistance Management Tools:

  • Screen for known resistance mutations in field populations

  • Develop functional assays using recombinant protein to detect phenotypic resistance

  • Create biosensors for rapid resistance screening

  • Implement multiplexed assays to detect multiple resistance mechanisms simultaneously

• Integration with Biological Control Strategies:

  • Use molecular diagnostics to monitor effectiveness of biological control agents

  • Develop detection systems for parasitoids, predators, and entomopathogens

  • Track infection rates of biocontrol agents like Bacillus thuringiensis and Nosema fumiferanae

  • Quantify the impact of Trichogramma releases on pest populations

These molecular diagnostic tools would significantly enhance integrated pest management programs for C. rosaceana, which is an important pest of various fruit crops and deciduous trees in North America . Previous research has established optimum sample sizes and multistage sampling plans for related Tortricidae species, which could be adapted for C. rosaceana using these molecular tools .