Recombinant Choristoneura biennis Cytochrome c oxidase subunit 2 (COII)

What is Choristoneura biennis and what is its ecological significance?

Choristoneura biennis, commonly known as the two-year-cycle budworm moth, is a species of moth belonging to the family Tortricidae. It is native to Canada, specifically found in Alberta and British Columbia . The species has a wingspan of approximately 26 mm, with adults typically emerging in July . C. biennis is a specialist herbivore feeding primarily on conifers, with larvae consuming Abies lasiocarpa (subalpine fir), Picea engelmanni (Engelmann spruce), and Picea glauca (white spruce) .

The ecological significance of C. biennis stems from its position in the Choristoneura genus, which includes several notable forest pests. While less infamous than its relative C. fumiferana (spruce budworm), understanding C. biennis biology contributes to our comprehension of conifer forest ecology and pest dynamics in North American ecosystems.

How does COII differ from other mitochondrial markers used in Lepidoptera research?

Cytochrome c oxidase subunit 2 (COII) is one of several mitochondrial genes commonly used in molecular phylogenetic studies of Lepidoptera. Unlike some other mitochondrial markers, COII provides several advantages:

• Evolutionary rate: COII evolves at an intermediate rate, making it particularly useful for resolving relationships at both species and genus levels.

• Conserved regions: COII contains sections that are highly conserved across Lepidoptera, facilitating primer design for PCR amplification.

• Variable regions: Despite conserved sections, COII also possesses regions with sufficient variability to distinguish closely related species within genera like Choristoneura.

• Size and manageability: The COII gene is of moderate length, making it relatively easy to sequence and analyze compared to longer mitochondrial regions.

COII has been effectively used in phylogenetic studies of Tortricidae, complementing other mitochondrial markers such as COI (the barcode region) and 16S rRNA .

What is the taxonomic classification of Choristoneura biennis?

Choristoneura biennis has the following taxonomic classification:

This species is formally recognized as Choristoneura occidentalis biennis, a subspecies within the broader Choristoneura spruce budworm complex .

What is the phylogenetic relationship between C. biennis and other Choristoneura species?

Choristoneura biennis belongs to the conifer-feeding group of Nearctic Choristoneura species. Phylogenetic analyses based on mitochondrial DNA suggest that:

• C. biennis is closely related to other North American conifer feeders including C. fumiferana, C. pinus, and C. occidentalis occidentalis .

• It is more distantly related to broad-leaf feeding Choristoneura species like C. rosaceana and C. conflictana .

• The genus Choristoneura appears to have originated in the Holarctic region approximately 23 million years ago, with major diversification occurring around 16 million years ago .

• The evolutionary history of Choristoneura shows two main clades: a primarily Nearctic clade (including C. biennis) and a predominantly Palearctic clade .

• Cladogenesis in the genus appears to have been synchronized with herbivorous specialization, with each clade divided into coniferophagous or polyphagous lineages .

Why is recombinant expression of COII from C. biennis valuable for research?

Recombinant expression of C. biennis COII provides several research advantages:

• Protein structure analysis: Enables structural studies without extracting limited quantities from natural sources.

• Functional characterization: Facilitates investigation of enzymatic properties and substrate specificity.

• Antibody production: Recombinant proteins can be used to generate specific antibodies for immunodetection studies.

• Comparative biochemistry: Allows comparison of COII properties between C. biennis and other Choristoneura species to understand adaptations.

• Evolutionary studies: Recombinant proteins can be modified to test hypotheses about the functional significance of amino acid substitutions observed in evolutionary comparisons.

What are the best expression systems for producing functional recombinant C. biennis COII?

The optimal expression system for recombinant C. biennis COII depends on research objectives:

Bacterial Expression Systems (E. coli):

• Advantages: Fast growth, high yield, cost-effective, well-established protocols

• Limitations: Lack of post-translational modifications, potential for inclusion body formation

• Recommended strains: BL21(DE3) for basic expression; Rosetta or CodonPlus strains for addressing codon bias issues

Insect Cell Expression Systems:

• Advantages: Proper folding, post-translational modifications similar to native protein

• Recommended: Sf9 or High Five™ cells with baculovirus expression vectors

• Considerations: COII is a membrane protein, so expression with appropriate membrane-targeting signals is essential

Yeast Expression Systems:

• Advantages: Eukaryotic processing, good for membrane proteins

• Recommended: Pichia pastoris for high-density culture and inducible expression

Cell-Free Expression Systems:

• Advantages: Bypasses toxicity issues, direct incorporation of labeled amino acids

• Particularly useful for structural studies requiring isotopic labeling

For studies focused on structure-function relationships, insect cell systems are preferable despite higher costs and technical complexity.

How can researchers differentiate between C. biennis and other closely related Choristoneura species using COII?

Differentiating C. biennis from other Choristoneura species requires an integrated approach:

Sequence-Based Differentiation:

• PCR amplification and sequencing of the COII gene

• Alignment with reference sequences

• Identification of species-specific nucleotide polymorphisms

Key Nucleotide Positions for Differentiation:
While specific COII polymorphisms for C. biennis are not detailed in the search results, mitochondrial DNA analysis has been successfully used for species delimitation within the Choristoneura genus .

Integrated Approach:
Combining COII sequence data with morphological characteristics provides the most accurate identification. This integrated approach has been effective for resolving the Choristoneura fumiferana species complex .

Phylogenetic Analysis:
Construction of phylogenetic trees using Maximum Likelihood or Bayesian methods can place unknown samples within the established evolutionary framework of Choristoneura species .

What challenges exist in expressing mitochondrial proteins like COII in heterologous systems?

Expressing mitochondrial proteins like COII in heterologous systems presents several challenges:

Codon Usage Bias:

• Mitochondrial genomes use a different genetic code from nuclear genomes

• Solution: Codon optimization of the COII gene for the chosen expression system

Membrane Protein Folding:

• COII is normally embedded in the inner mitochondrial membrane

• Solutions:

  • Use of specialized expression strains (C41/C43)

  • Expression as fusion proteins with solubility-enhancing tags

  • Addition of detergents or lipids during purification

Post-Translational Modifications:

• Native modifications may be absent in heterologous systems

• Solution: Choose eukaryotic expression systems when modifications are critical

Protein Toxicity:

• Overexpression may be toxic to host cells

• Solutions:

  • Tight regulation of expression using inducible promoters

  • Lower induction temperatures (16-20°C)

  • Cell-free expression systems

Protein Verification:

• Confirming proper folding and function is challenging

• Solutions:

  • Spectroscopic analysis

  • Activity assays comparing recombinant and native proteins

How does COII sequence variation correlate with ecological adaptations in Choristoneura species?

COII sequence variation in Choristoneura correlates with ecological adaptations in several ways:

Host Plant Specialization:
The diversification of Choristoneura species shows a pattern related to herbivorous specialization, with distinct clades specialized for feeding on conifers (like C. biennis) versus polyphagous species . COII sequences often reflect these ecological divergences.

Geographic Distribution:
Sequence variation in mitochondrial genes correlates with the biogeographic distribution of Choristoneura species across Nearctic and Palearctic regions .

Thermal Adaptation:
Sequence variations in mitochondrial genes like COII may reflect adaptations to different thermal environments, particularly relevant for species like C. biennis and C. fumiferana that must survive harsh winter conditions .

Divergence Timing:
Molecular clock analyses using mitochondrial genes suggest that major diversification events in Choristoneura occurred around 16 million years ago , potentially coinciding with climatic changes affecting host plant distributions.

What is the role of COII in understanding the evolutionary history of the Choristoneura genus?

COII plays a crucial role in elucidating the evolutionary history of Choristoneura:

Phylogenetic Reconstruction:
COII sequences provide data for reconstructing evolutionary relationships within Choristoneura, helping resolve the position of C. biennis relative to other species .

Divergence Time Estimation:
As a mitochondrial gene with a relatively stable evolutionary rate, COII can be used in molecular clock analyses to estimate divergence times between species lineages .

Historical Biogeography:
COII data contribute to biogeographical analyses, supporting hypotheses about the Holarctic origin of Choristoneura approximately 23 million years ago and subsequent colonization events .

Species Delimitation:
COII sequence data, integrated with morphological characteristics, helps identify cryptic species within species complexes, providing insights into speciation processes within the genus .

Evolutionary Rate Heterogeneity:
Analyzing variation in evolutionary rates of COII across different Choristoneura lineages can identify branches under different selective pressures, potentially correlating with ecological shifts.

What are the recommended protocols for extracting mitochondrial DNA from C. biennis specimens?

Recommended Protocol for mtDNA Extraction from C. biennis:

• Specimen Preparation:

  • Use fresh or properly preserved specimens (≤95% ethanol, -80°C storage)

  • For adult moths, remove wings and use thoracic muscle tissue

  • For larvae, use whole specimens or body sections avoiding gut contents

• Tissue Homogenization:

  • Homogenize in buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA, and 1% SDS

  • Add proteinase K (final concentration 100 μg/ml)

  • Incubate at 55°C for 3-4 hours with gentle agitation

• DNA Extraction Methods:

  • Commercial kits: Qiagen DNeasy Blood & Tissue Kit with modifications for small specimens

  • CTAB method: Particularly effective for museum specimens or degraded samples

  • For difficult samples, phenol-chloroform extraction may yield better results

• Quality Control:

  • Assess DNA quality using spectrophotometry (A260/A280 ratio)

  • Verify mtDNA enrichment by PCR amplification of mitochondrial and nuclear markers

  • Evaluate DNA integrity by gel electrophoresis

• Storage:

  • Store extracted DNA at -20°C for short-term or -80°C for long-term preservation

  • Avoid repeated freeze-thaw cycles by creating working aliquots

What primers are most effective for amplifying the complete COII gene from C. biennis?

While specific primers for C. biennis COII are not detailed in the search results, the following primer sets have been effective for COII amplification in Lepidoptera and specifically in Tortricidae:

Universal Lepidoptera COII Primers:

PCR Conditions:

• Initial denaturation: 95°C for 5 minutes

• 35 cycles of:

  • Denaturation: 94°C for 30 seconds

  • Annealing: 50-52°C for 45 seconds

  • Extension: 72°C for 1 minute

• Final extension: 72°C for 10 minutes

For Difficult Samples:

• Use nested PCR approach with external primers followed by internal primers

• Add 3% DMSO to reduce secondary structure formation

• For museum specimens, design overlapping shorter amplicons (<300 bp)

For expression studies, design primers with appropriate restriction sites for cloning into expression vectors, accounting for the desired reading frame and fusion tags.

What vector systems are optimal for expressing recombinant C. biennis COII?

The choice of vector system depends on the expression host and research objectives:

For E. coli Expression:

For Insect Cell Expression:

• Baculovirus vectors (e.g., pFastBac™)

• Features: Polyhedrin promoter, high-level expression, post-translational modifications

• Consider adding C-terminal His-tag for purification

For Yeast Expression:

• pPICZ series for Pichia pastoris

• Features: Methanol-inducible promoter, secretion signals, multiple copy integration

Key Considerations:

• Include affinity tags (His, FLAG, GST) for purification

• Add protease cleavage sites for tag removal

• Optimize codon usage for the expression host

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

• Include appropriate signal sequences for membrane protein targeting

What purification strategies yield the highest quality recombinant COII protein?

Purifying recombinant COII requires strategies appropriate for membrane proteins:

Extraction and Solubilization:

• Lyse cells using mechanical disruption (sonication or homogenization)

• Isolate membrane fraction by differential centrifugation

• Solubilize membranes using detergents:

  • Mild detergents: n-Dodecyl β-D-maltoside (DDM), digitonin

  • Intermediate detergents: LDAO, OG

  • Test detergent screening panel to optimize

Affinity Chromatography:

• Immobilized metal affinity chromatography (IMAC) for His-tagged COII

• Anti-FLAG affinity for FLAG-tagged constructs

• GST-affinity chromatography for GST fusion proteins

Additional Purification Steps:

• Size exclusion chromatography to remove aggregates

• Ion exchange chromatography for further purification

• Affinity tag removal using specific proteases (TEV, thrombin)

Quality Assessment:

• SDS-PAGE and Western blotting to verify purity and identity

• Mass spectrometry for accurate molecular weight determination

• Circular dichroism to assess secondary structure

• Functional assays to confirm activity

Storage Conditions:

• Store in buffers containing appropriate detergent at concentrations above CMC

• Add glycerol (10-20%) for stability

• Store at -80°C in small aliquots to avoid freeze-thaw cycles

How can researchers verify the structural integrity of recombinant COII?

Verifying structural integrity of recombinant COII requires multiple approaches:

Spectroscopic Methods:

• Circular Dichroism (CD)

  • Far-UV CD (190-250 nm): Secondary structure content

  • Near-UV CD (250-350 nm): Tertiary structure fingerprint

• Fluorescence Spectroscopy

  • Intrinsic tryptophan fluorescence for tertiary structure assessment

  • Changes in emission maximum indicate folding status

• Fourier Transform Infrared Spectroscopy (FTIR)

  • Particularly useful for membrane proteins like COII

  • Provides information on secondary structure elements

Hydrodynamic Properties:

• Size Exclusion Chromatography (SEC)

  • Monodisperse peak indicates properly folded protein

  • Multiple peaks suggest aggregation or degradation

• Dynamic Light Scattering (DLS)

  • Measures particle size distribution

  • Monodisperse sample indicates uniform protein state

Functional Assays:

• Enzymatic Activity

  • Cytochrome c oxidase activity using reduced cytochrome c as substrate

  • Polarographic measurement of oxygen consumption

• Ligand Binding

  • Spectroscopic detection of heme and copper cofactor binding

  • Differential scanning fluorimetry for thermal stability assessment

Structural Analysis:

What bioinformatic pipelines are recommended for analyzing COII sequence data in Choristoneura species?

A comprehensive bioinformatic pipeline for COII analysis includes:

Quality Control and Sequence Processing:

• Trim adapter sequences and low-quality bases (Trimmomatic or CutAdapt)

• Perform quality assessment (FastQC)

• Assemble paired-end reads if applicable (SPAdes or Velvet)

Sequence Alignment:

• Multiple sequence alignment using MAFFT or MUSCLE with G-INS-i strategy

• Manual inspection and refinement in AliView or Geneious

• Removal of poorly aligned regions using Gblocks or TrimAl

Phylogenetic Analysis:

• Model selection using ModelTest-NG or jModelTest

• Tree reconstruction methods:

  • Maximum Likelihood: IQ-TREE or RAxML

  • Bayesian Inference: MrBayes or BEAST

• Branch support assessment:

  • Bootstrap replicates (1000+) for ML trees

  • Posterior probabilities for Bayesian trees

Species Delimitation Analysis:

• Distance-based methods: ABGD or PTP

• Coalescent-based methods: GMYC or BPP

• Integrative approaches combining multiple lines of evidence

Divergence Time Estimation:

• Calibrate molecular clock using fossil evidence

• BEAST2 or MCMCTree for time-calibrated phylogenies

• FigTree or TimeTree for visualization

Sequence Polymorphism Analysis:

• DnaSP for nucleotide diversity, haplotype diversity, and neutrality tests

• MEGA or PAML for selection analysis (dN/dS ratios)

This pipeline has been successfully applied to analyze mitochondrial genes in Tortricidae and specifically in Choristoneura species .

How should researchers interpret conflicting signals between mitochondrial (COII) and nuclear gene phylogenies in Choristoneura?

When COII-based phylogenies conflict with nuclear gene trees in Choristoneura, researchers should:

Potential Causes of Conflict:

• Incomplete Lineage Sorting (ILS):

  • Recent rapid speciation events may result in random sorting of ancestral polymorphisms

  • Test with coalescent-based methods (ASTRAL, *BEAST)

• Introgression/Hybridization:

  • Mitochondrial capture through hybridization

  • Test using ABBA-BABA tests or similar methods

• Selection Pressures:

  • Adaptive evolution in mitochondrial genes

  • Analyze using selection tests (PAML, HyPhy suite)

• Sex-Biased Dispersal:

  • Maternally inherited mtDNA reflects female dispersal patterns

  • Compare geographic structure in mitochondrial vs. nuclear markers

Resolution Approaches:

• Multi-locus Approaches:

  • Analyze multiple nuclear loci alongside mitochondrial data

  • Use species tree methods that account for gene tree discordance

• Integrated Analysis:

  • Combine genetic data with morphological, ecological, and geographical information

  • Use integrative taxonomy approaches as demonstrated for the Choristoneura fumiferana complex

• Network-Based Methods:

  • Phylogenetic networks can visualize conflicting signals better than bifurcating trees

  • Methods: SplitsTree, PhyloNetworks

• Simulation Studies:

  • Simulate data under different evolutionary scenarios

  • Compare simulated patterns with observed discordance

Discordance between mitochondrial and nuclear markers is common in Lepidoptera and has been documented in Choristoneura species, often reflecting complex evolutionary histories rather than methodological artifacts .

What statistical approaches are most effective for determining species boundaries using COII in closely related Choristoneura species?

Determining species boundaries in closely related Choristoneura species requires multiple complementary approaches:

Distance-Based Methods:

• Automatic Barcode Gap Discovery (ABGD)

  • Identifies barcode gaps in pairwise genetic distances

  • Parameters: Pmin=0.001, Pmax=0.1, Steps=10, X=1.5

• Barcode Index Number (BIN)

  • Clusters sequences into operational taxonomic units

  • Particularly useful for initial sorting of specimens

Tree-Based Methods:

• Poisson Tree Processes (PTP)

  • Models speciation in terms of substitution events

  • Infers species boundaries directly from phylogenetic trees

• General Mixed Yule Coalescent (GMYC)

  • Identifies transition points between speciation and coalescent processes

  • Requires ultrametric trees (time-calibrated)

Coalescent-Based Methods:

• Bayesian Phylogenetics & Phylogeography (BPP)

  • Multispecies coalescent model

  • Incorporates uncertainty in gene tree estimation

  • Requires prior assignment to populations

Integrated Approaches:

• Iterative Taxonomic Validation

  • Cross-validate results from multiple methods

  • Identify consensus species hypotheses

• Integrative Taxonomy

  • Combine COII data with:

    • Morphological characters

    • Ecological data (host plant associations)

    • Geographical distribution

    • Behavioral traits (pheromone composition)

This integrative approach has proven effective for species delimitation within the Choristoneura fumiferana cryptic species complex, where mitochondrial DNA and morphological data were combined to resolve taxonomic uncertainties .

How can COII sequence data contribute to molecular clock analyses and divergence time estimation in Choristoneura?

COII sequence data provides valuable information for molecular dating in Choristoneura:

Calibration Points:

• Fossil Calibrations

  • Tortricid fossils can provide minimum age constraints

  • Calibration should account for preservation biases

• Secondary Calibrations

  • Derived from broader Lepidoptera dating studies

  • Example: The crown age of Choristoneura has been estimated at approximately 16 million years ago

Molecular Clock Models:

• Strict Clock

  • Assumes constant rate across all lineages

  • Rarely appropriate for Lepidoptera due to rate heterogeneity

• Relaxed Clocks

  • Uncorrelated lognormal relaxed clock (UCLN) in BEAST2

  • Allows rates to vary among lineages

  • Better accommodates rate variation in Choristoneura

Dating Methods:

• Bayesian Methods

  • BEAST2 with appropriate tree priors (Yule or Birth-Death)

  • MCMCTree in PAML package

• Penalized Likelihood

  • r8s software for single gene trees

  • Useful for comparison with Bayesian estimates

Analytical Considerations:

• Partition Schemes

  • Treat codon positions separately

  • Test different substitution models for each partition

• Prior Settings

  • Use informative but not overly restrictive priors

  • Perform sensitivity analyses varying key parameters

COII-based divergence time estimation has contributed to understanding the biogeographic history of Choristoneura, supporting hypotheses about the timing of major diversification events and their correlation with host plant evolution .

What methods should be used to analyze selection pressures acting on COII in different Choristoneura lineages?

Analyzing selection pressures on COII in Choristoneura lineages requires:

Site-Specific Selection Analyses:

• Maximum Likelihood Methods

  • PAML (CodeML) site models:

    • M0 (one-ratio) vs. M3 (discrete)

    • M1a (nearly neutral) vs. M2a (positive selection)

    • M7 (beta) vs. M8 (beta+ω>1)

  • HyPhy suite methods:

    • SLAC (Single Likelihood Ancestor Counting)

    • FEL (Fixed Effects Likelihood)

    • MEME (Mixed Effects Model of Evolution) for episodic selection

Branch-Specific Selection Analyses:

• Branch Models (PAML)

  • Two-ratio model: conifer-feeding vs. broad-leaf feeding lineages

  • Free-ratio model: separate ω for each branch

• Branch-Site Methods

  • PAML branch-site test for positive selection

  • aBSREL (adaptive Branch-Site Random Effects Likelihood) in HyPhy

Codon Substitution Models:

• Mechanistic Codon Models

  • MutSel models accounting for mutation bias and selection

  • Implemented in PhyloBayes-MPI

Structural Considerations:

• Structure-Based Analyses

  • Map selected sites to structural models

  • COII structure prediction using AlphaFold2

  • Analysis of functional domains and sites

Population Genetics Approaches:

• McDonald-Kreitman Test

  • Compares polymorphism within species to divergence between species

  • Implementation in DnaSP or PopGenome

• Tajima's D and related statistics

  • Detect deviations from neutrality

  • Particularly informative when comparing different Choristoneura lineages

These methods can reveal how selection pressures on COII differ between conifer-feeding Choristoneura species (like C. biennis) and those that feed on other host plants, potentially correlating with adaptations to different ecological niches.

What are the future research directions for C. biennis COII studies?

Future research on C. biennis COII should focus on:

• Complete characterization of recombinant COII properties

  • Biochemical and biophysical studies of protein function

  • Comparison with COII from other Choristoneura species

• Comparative genomics and transcriptomics

  • Integration of COII data with whole genome sequence data

  • Analysis of nuclear-mitochondrial gene co-evolution

• Ecological adaptation studies

  • Correlation of COII sequence variation with adaptation to different host plants

  • Investigation of cold tolerance mechanisms in relation to mitochondrial function

• Applied research applications

  • Development of molecular markers for pest monitoring

  • Exploration of species-specific targets for pest management

• Integrative phylogenomics

  • Combination of COII with genome-wide data for comprehensive phylogeny

  • Resolution of species boundaries in taxonomically challenging Choristoneura groups

The integration of molecular, morphological, and ecological data will continue to enhance our understanding of Choristoneura biennis and its evolutionary relationships, contributing to both basic science and applied forest management.

How can COII studies contribute to conservation and forest management strategies?

COII studies can make significant contributions to conservation and forest management:

• Species identification and monitoring

  • Molecular diagnostics for early detection of pest species

  • Tracking population dynamics and range expansions

• Evolutionary potential assessment

  • Genetic diversity analysis to predict adaptation capacity

  • Identification of locally adapted populations

• Climate change response prediction

  • Correlation of COII variants with thermal tolerance

  • Projection of range shifts under climate change scenarios

• Integrated pest management

  • Species-specific control strategies based on molecular differences

  • Resistance monitoring in managed populations

• Ecosystem impact assessment

  • Understanding the role of Choristoneura species in forest succession

  • Modeling interactions between pest outbreaks and forest dynamics