Recombinant Rhinolophus hipposideros Cytochrome b (MT-CYB)

What is MT-CYB and why is it important for bat species identification?

Mitochondrially encoded cytochrome b (MT-CYB) is a housekeeping gene involved in oxidative phosphorylation in cellular respiration. It has emerged as a preferred molecular marker for species identification due to its specific characteristics. MT-CYB shows low intraspecific variation and high interspecific variation, making it particularly suitable for accurate species identification . As a housekeeping gene, it exhibits relatively low evolutionary rates, which provides stability in phylogenetic analyses .

In bat research specifically, MT-CYB gene sequences (typically 1,140 bp in length) serve as reliable molecular markers for species identification. Studies have demonstrated that MT-CYB can distinguish between morphologically similar bat species with high accuracy, which is particularly valuable for cryptic species within the Rhinolophus genus . In comparative studies with other molecular markers, cytochrome b has shown superior performance in reconstructing mammalian phylogeny at various taxonomic levels, correctly assigning 95.85% of mammal species to Super Order, 94.31% to Order, and 98.16% to Family .

How does the MT-CYB gene structure facilitate phylogenetic analysis?

The MT-CYB gene in Rhinolophus hipposideros and other bat species consists of a full coding sequence of approximately 1,140 base pairs. This gene encodes a protein that contains two heme components: cytochrome bL and cytochrome bH . The structure includes functional sites such as the quinol oxidizing site (Qo or Qz) and the quinone reducing site (Qi or Qc), which are critical for electron transfer in the respiratory chain .

For phylogenetic analysis, the full MT-CYB gene sequence provides sufficient genetic information to resolve relationships at various taxonomic levels. Researchers typically use software like MEGA X for bioinformatic analysis, applying tools such as MUSCLE for sequence alignment and appropriate substitution models (e.g., GTR) for phylogenetic reconstruction . The gene's conserved regions allow for reliable alignment across diverse bat species, while variable regions provide the discriminatory power needed for species identification.

What methodological approaches are recommended for MT-CYB sequencing from bat samples?

For optimal MT-CYB sequencing from Rhinolophus hipposideros samples, researchers should consider the following methodological approach:

• Sample collection: Non-lethal sampling methods are preferred, typically using 3mm wing membrane biopsies or small amounts of blood.

• DNA extraction: Standard DNA extraction protocols using commercial kits designed for animal tissues are effective for bat tissue samples.

• PCR amplification: The full MT-CYB gene (1,140 bp) can be amplified using universal primers designed for mammalian MT-CYB. Optimized PCR conditions typically include an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation (94°C, 1 min), annealing (50-55°C, 1 min), and extension (72°C, 1.5 min), with a final extension at 72°C for 10 minutes .

• Sequencing: Both forward and reverse sequencing should be performed to ensure accuracy. Modern approaches use automated sequencing methods with dye-terminator chemistry.

• Sequence analysis: Raw sequences should be analyzed using software such as Geneious Prime, where sequences can be trimmed and assembled to generate consensus sequences . These consensus sequences should then be compared with reference sequences in databases like NCBI using BLAST for species identification .

This approach has successfully yielded full MT-CYB sequences from bat species including Rhinolophus rouxii, which is closely related to R. hipposideros .

How can site-directed mutagenesis be applied to study functional domains of MT-CYB?

Site-directed mutagenesis represents a powerful approach for investigating functional domains within the MT-CYB protein. When studying recombinant Rhinolophus hipposideros MT-CYB, researchers should target highly conserved residues that are likely involved in crucial functions of the protein.

Based on previous research, several key amino acid residues in cytochrome b have been identified as critical for function. For example, studies have targeted conserved residues such as A52, H217, K251, and D252 in bacterial systems, which are positioned at or near the quinone reductase site . A similar approach can be applied to bat MT-CYB.

Methodology for site-directed mutagenesis of MT-CYB:

• Identify conserved residues through multiple sequence alignment of MT-CYB across bat species and other mammals.

• Design primers with specific nucleotide substitutions to create the desired mutations.

• Perform PCR-based mutagenesis on a recombinant expression vector containing the R. hipposideros MT-CYB gene.

• Confirm mutations by sequencing before proceeding to expression and functional studies.

• Express both wild-type and mutant proteins in appropriate expression systems.

• Conduct functional assays to assess the impact of mutations, particularly focusing on electron transfer rates and interaction with other components of the respiratory chain.

Previous research has demonstrated that mutations in conserved residues can selectively impair electron transfer rates. For instance, mutations in H217 and D252 have been shown to significantly affect the reoxidation of cytochrome bH by ubiquinone without disrupting the reduction of cytochrome bH, indicating a specific role in electron transfer to the Qc-site .

What experimental challenges exist in expressing functional recombinant MT-CYB and how can they be overcome?

Expressing functional recombinant MT-CYB from Rhinolophus hipposideros presents several challenges due to the protein's hydrophobic nature and need for proper incorporation into membranes. Key challenges and their solutions include:

• Membrane protein expression: As MT-CYB is a membrane protein with multiple transmembrane domains, conventional expression systems often result in protein aggregation or improper folding.

  • Solution: Utilize specialized expression systems such as cell-free systems supplemented with liposomes or nanodiscs, which provide a membrane-like environment. Alternatively, consider Pichia pastoris or insect cell systems, which are often more successful for membrane protein expression.

• Heme incorporation: Functional MT-CYB requires proper incorporation of heme groups.

  • Solution: Supplement expression media with δ-aminolevulinic acid (ALA), a heme precursor, and ensure adequate iron availability. Co-express heme lyase or other chaperones that facilitate heme incorporation when necessary.

• Protein toxicity: Expression of MT-CYB may be toxic to host cells, particularly if it disrupts electron transport chain function.

  • Solution: Use tightly regulated inducible promoters, lower induction temperatures (16-20°C), and shorter induction times to minimize toxicity while maximizing yield.

• Functional assessment: Verifying that recombinant MT-CYB is functionally active can be challenging.

  • Solution: Develop specialized assays that measure electron transfer activity, such as reduction/oxidation of cytochrome bH or interaction with ubiquinone at the Qi site. Spectroscopic methods (particularly absorption spectroscopy at wavelengths characteristic of reduced and oxidized cytochromes) can provide evidence of proper folding and heme incorporation.

These approaches have been successfully applied to cytochrome b from other species and can be adapted for R. hipposideros MT-CYB, taking into account species-specific characteristics of the protein.

How can recombinant MT-CYB be used to study potential drug targeting of the Qi site?

Recombinant Rhinolophus hipposideros MT-CYB provides a valuable tool for investigating drug targeting of the Qi site, particularly in comparative studies with pathogen cytochrome b proteins. Recent research has identified the Qi site of cytochrome b as a promiscuous drug target in parasites such as Leishmania donovani and Trypanosoma cruzi , suggesting the potential for similar approaches with other organisms.

Methodological approach for drug targeting studies:

• Comparative structural analysis: Generate structural models of R. hipposideros MT-CYB and pathogen cytochrome b proteins, focusing on the Qi site architecture. Identify key differences that could be exploited for selective drug targeting.

• Binding site characterization: Use recombinant proteins to characterize the Qi binding site through site-directed mutagenesis of conserved residues. Mutations that confer resistance to known Qi site inhibitors can help map the binding pocket .

• Screening platform development: Establish a screening platform using recombinant MT-CYB to test the binding affinity and specificity of potential inhibitors. This can include:

  • Biochemical assays measuring disruption of electron transport

  • Binding assays using labeled inhibitors

  • Thermal shift assays to detect ligand binding

• Resistance mutation analysis: Generate a panel of Qi site mutations to identify potential resistance mechanisms. This approach has been successfully used with the Qi site of cytochrome b in pathogens, where a panel of resistant clones bearing cytochrome b mutations was created to screen compounds for their mode of action .

• Structural diversity assessment: Analyze structurally diverse compounds that target the Qi site to understand the promiscuity of this binding pocket. Research has shown that despite structural diversity, compounds can specifically target the Qi site of cytochrome b .

This research approach not only contributes to understanding the structure-function relationship of MT-CYB but also provides insights into comparative biochemistry and potential selective targeting of pathogen cytochrome b proteins.

How does MT-CYB compare to COI for bat species identification and phylogenetic reconstruction?

MT-CYB has distinct advantages over Cytochrome Oxidase I (COI) for bat species identification and phylogenetic analysis, particularly at higher taxonomic levels. A comparative analysis of these two genetic markers reveals important differences in their performance:

Based on comparison of DNA sequences from 217 mammalian species, MT-CYB more accurately reconstructs phylogeny and known relationships between species at Super Order, Order, Family, and generic levels . The superior performance of MT-CYB is particularly evident at the Super Order level, where it correctly assigns nearly 96% of species compared to only 78% for COI.

For species delimitation, using a Kimura 2-parameter p-distance threshold of 1.5-2.5, MT-CYB provides better resolution with a lower false positive rate and higher positive predictive value than COI . This makes MT-CYB particularly valuable for resolving relationships among closely related bat species, including within the genus Rhinolophus, where morphological identification can be challenging.

The improved performance of MT-CYB for mammalian phylogenetics, including bats, is likely due to its evolutionary rate and pattern of sequence conservation, which is well-suited for resolving relationships at multiple taxonomic levels.

How can researchers detect and analyze recombination events in MT-CYB evolution?

Detecting and analyzing recombination events in MT-CYB evolution requires specialized methodological approaches, as recombination can significantly impact phylogenetic inference and evolutionary interpretations. Evidence of recombination has been documented in the evolutionary history of coronaviruses in bats, involving structural protein genes , and similar methodologies can be applied to study MT-CYB recombination.

Methodological approach for recombination analysis:

• Sequence dataset preparation: Compile a comprehensive dataset of MT-CYB sequences from Rhinolophus hipposideros and closely related species. Include outgroups for comparative analysis.

• Alignment quality control: Generate a high-quality multiple sequence alignment using tools such as MUSCLE implemented in MEGA X software . Manually inspect and refine the alignment to ensure accuracy.

• Recombination detection methods: Apply multiple methods to detect potential recombination events:

  • Similarity plots: Identify unusual patterns of sequence similarity that may indicate recombination events.

  • Statistical tests: Use programs such as RDP4, which implements multiple algorithms (RDP, GENECONV, Chimaera, MaxChi, BootScan, SiScan, and 3Seq) to detect recombination.

  • Phylogenetic incongruence: Conduct phylogenetic analyses of different gene regions and identify topological conflicts that may indicate recombination.

• Breakpoint determination: For detected recombination events, determine the precise location of breakpoints using maximum likelihood approaches.

• Parental sequence identification: Identify potential parental sequences involved in the recombination event.

• Biological significance assessment: Evaluate the functional implications of recombination, particularly if it affects coding regions or functional domains of the MT-CYB protein.

• Visualization: Create graphical representations of recombination events, such as similarity plots or bootscan analyses, to effectively communicate findings.

When recombination is detected, researchers should consider its impact on phylogenetic analyses and possibly exclude recombinant regions or utilize methods that can accommodate recombination when reconstructing evolutionary relationships.

What evolutionary patterns have been observed in bat MT-CYB genes in relation to ecological adaptations?

The evolution of MT-CYB genes in bats, including Rhinolophus hipposideros, shows distinctive patterns that may reflect adaptations to their unique ecological niches and metabolic requirements. As a component of the electron transport chain, MT-CYB plays a crucial role in energy metabolism, making it a potential target of selection related to the high energy demands of flight and echolocation in bats.

Key evolutionary patterns observed in bat MT-CYB genes include:

Understanding these evolutionary patterns provides insights into the molecular basis of bat adaptations and can inform broader questions about metabolic evolution in mammals with high energy demands.

What bioinformatic tools and approaches are most effective for analyzing MT-CYB sequence data?

For comprehensive analysis of Rhinolophus hipposideros MT-CYB sequence data, researchers should employ a systematic bioinformatic workflow that integrates multiple tools and approaches. Based on current research practices, the following methodology is recommended:

• Sequence Assembly and Quality Control:

  • Use Geneious Prime software for initial sequence processing, trimming, and de novo assembly to obtain high-quality consensus sequences .

  • Apply quality filters to remove low-quality base calls and contamination.

• Sequence Identification and Comparison:

  • Compare consensus sequences with reference databases using BLAST to confirm species identification and calculate statistical significance of matches .

  • Calculate percentage identity with reference sequences to quantify genetic similarity.

• Multiple Sequence Alignment:

  • Use MUSCLE alignment tool implemented in MEGA X for accurate alignment of MT-CYB sequences .

  • Manually inspect and refine alignments to ensure accuracy, particularly around indel regions.

• Substitution Model Selection:

  • Determine the best substitution model for your dataset using jModelTest or the model test function in MEGA X .

  • For bat MT-CYB sequences, the GTR (General Time Reversible) model has been effectively applied in previous studies .

• Phylogenetic Analysis:

  • Implement Bayesian inference using MrBayes with MCMC (Markov chain Monte Carlo) methods for robust phylogenetic reconstruction .

  • Recommended parameters include: chain length of 10 million, burn-in of 30%, and sampling frequency of 200 .

  • Use FigTree for visualization of phylogenetic trees and calculation of posterior probabilities for each node .

• Sequence Divergence Analysis:

  • Calculate genetic distances using appropriate models (e.g., Kimura 2-parameter) in MEGA X.

  • For species delimitation, apply a p-distance threshold of 1.5-2.5, which has been shown to provide good resolution for mammalian species using MT-CYB .

• Visualization of Sequence Similarity:

  • Generate heatmaps based on nucleotide alignment and percentage of identity to visualize relationships between sequences .

  • Use color gradients (e.g., 70% identity in red to 100% identity in green) to represent sequence similarity .

This comprehensive approach ensures robust analysis of MT-CYB sequence data, facilitating accurate species identification and reliable phylogenetic inference.

How should researchers address contradictory phylogenetic signals in MT-CYB data?

Contradictory phylogenetic signals in MT-CYB data from Rhinolophus hipposideros and related bat species can arise from various biological phenomena and methodological artifacts. Addressing these contradictions requires a systematic approach:

• Identify the source of contradiction:

  • Sequence quality issues: Check for sequencing errors, chimeric sequences, or contamination that might introduce noise.

  • Incomplete lineage sorting: Recent speciation events may result in gene trees that differ from species trees.

  • Hybridization or introgression: Past gene flow between species can create conflicting signals.

  • Recombination: Evidence of recombination events has been observed in the evolutionary history of some bat viruses , and similar processes could affect MT-CYB interpretation.

  • Heterogeneous evolutionary rates: Variable rates across lineages or sites can distort phylogenetic inference.

• Methodological approaches to resolve contradictions:

  • Partition analysis: Analyze different regions of MT-CYB separately to identify localized contradictory signals.

  • Multiple phylogenetic methods: Apply both distance-based (Neighbor-Joining), maximum likelihood, and Bayesian approaches to assess consistency.

  • Test for recombination: Use specialized software like RDP4 to detect potential recombination events.

  • Coalescent-based methods: Implement methods that account for incomplete lineage sorting, such as *BEAST or ASTRAL.

  • Integrate multiple loci: Combine MT-CYB with other genetic markers (e.g., nuclear genes) to obtain a more comprehensive phylogenetic signal.

• Statistical assessment of competing hypotheses:

  • Topology tests: Apply AU (Approximately Unbiased), SH (Shimodaira-Hasegawa), or KH (Kishino-Hasegawa) tests to evaluate alternative tree topologies.

  • Bayesian model comparison: Use Bayes factors to compare different evolutionary models.

  • Bootstrap and posterior probability: Assess the statistical support for conflicting nodes.

• Biological interpretation:

  • When contradictions persist despite methodological approaches, consider biological explanations such as rapid radiation events or adaptive introgression.

  • Correlate conflicting patterns with ecological, behavioral, or geographical data that might explain evolutionary patterns.

By systematically addressing contradictory signals, researchers can develop more robust phylogenetic hypotheses and gain deeper insights into the evolutionary history of Rhinolophus hipposideros and related bat species.

What standards should be applied when depositing MT-CYB sequences in public databases?

To ensure high-quality data accessibility and reproducibility in research involving Rhinolophus hipposideros MT-CYB sequences, researchers should adhere to the following standards when depositing sequences in public databases:

• Sequence Quality and Completeness:

  • Deposit complete MT-CYB gene sequences (1,140 bp) whenever possible, rather than partial sequences .

  • Ensure bidirectional sequencing coverage with high quality scores (Phred > 30) throughout the sequence.

  • Clearly indicate if the sequence represents a consensus from multiple reads and document the assembly methodology.

• Metadata Requirements:

  • Provide comprehensive specimen information including:

    • Precise collection location with GPS coordinates

    • Collection date

    • Sex and age of the specimen

    • Voucher specimen information and repository

    • Morphological measurements (e.g., forearm length)

    • Ecological data (habitat, roosting site)

  • Document the methodological approach including:

    • DNA extraction method

    • PCR primers and conditions

    • Sequencing technology and coverage

• Taxonomic Verification:

  • Include both morphological and molecular identification methods .

  • Report percentage identity with reference sequences to validate taxonomic assignment .

  • Document any discrepancies between morphological and molecular identification.

• Database-Specific Requirements:

  • GenBank/NCBI: Use appropriate feature annotation for the MT-CYB gene, indicating coding regions and translation.

  • BOLD (Barcode of Life Database): Include additional standardized metadata fields specific to the BOLD system.

  • ENA (European Nucleotide Archive): Adhere to ENA-specific metadata requirements.

• Data Integration Standards:

  • Use established ontologies and controlled vocabularies for metadata when available.

  • Include cross-references to related datasets or specimens in other databases.

  • Provide ORCID identifiers for all contributing authors.

• Ethical and Legal Compliance:

  • Document permits for specimen collection and export.

  • Adhere to the Nagoya Protocol on Access and Benefit Sharing when applicable.

  • Document compliance with CITES regulations for protected species.

Adherence to these standards enhances the value of deposited MT-CYB sequences, facilitates meta-analyses and comparative studies, and supports reproducibility in bat molecular research.