Recombinant Sturnira tildae Cytochrome b (MT-CYB)

What is Sturnira tildae and where is it found in bat taxonomy?

Sturnira tildae (Tilda's yellow-shouldered bat) belongs to the genus Sturnira, a diverse and abundant group of bats distributed throughout the Neotropical region. The yellow-shouldered bats represent one of the most widespread and diverse groups of bats in Central and South America . Phylogenetic analyses have revealed that Sturnira species originated in the Andean region and later diversified, with S. tildae being part of the broader radiation of this genus that colonized various habitats across the Neotropics .

What is the biological function of cytochrome b (MT-CYB)?

Cytochrome b functions as a critical component of the electron transport chain in cellular respiration. This mitochondrial protein is formally known as "Complex III subunit 3" and is also referred to as "Cytochrome b-c1 complex subunit 3" or "Ubiquinol-cytochrome-c reductase complex cytochrome b subunit" . The protein plays an essential role in energy production by facilitating electron transfer within the mitochondria. The Sturnira tildae cytochrome b consists of a 134 amino acid sequence with specific structural domains that enable its function within the inner mitochondrial membrane .

Why has the cytochrome b gene become a standard marker in bat phylogenetic studies?

The cytochrome b gene has become a cornerstone in bat phylogenetic research for several key reasons. First, it evolves at a rate that makes it particularly suitable for examining relationships between closely related species and populations, providing sufficient variation for species-level discrimination . Second, its structured rate of evolution allows researchers to resolve both recent and moderately ancient divergence events. In comprehensive studies of Sturnira phylogeny, researchers have utilized cytochrome b alongside other markers (ND2, D-loop, RAG1, RAG2) to establish evolutionary relationships with high confidence . Finally, the extensive historical use of cytochrome b in bat systematics has created a rich comparative database that enhances the interpretative value of new sequences .

How is recombinant Sturnira tildae cytochrome b produced and purified for research use?

Recombinant Sturnira tildae cytochrome b production involves a multi-step process beginning with gene cloning and expression. The MT-CYB coding sequence is typically inserted into a suitable expression vector and transformed into a host system (commonly E. coli, though yeast, insect, or mammalian cells may be used for specific applications). The recombinant protein is often engineered with an affinity tag to facilitate purification, though "the tag type will be determined during production process" based on optimization requirements . Following expression, the protein undergoes purification through affinity chromatography, potentially followed by size exclusion and ion-exchange chromatography to achieve high purity. The final product is formulated in a Tris-based buffer containing 50% glycerol to maintain stability .

What are the optimal storage and handling conditions for maintaining recombinant cytochrome b activity?

For optimal preservation of recombinant Sturnira tildae cytochrome b activity, researchers should:

• Store the protein at -20°C for regular storage periods

• Use -80°C for extended long-term storage

• Store working aliquots at 4°C for no more than one week

• Avoid repeated freeze-thaw cycles that can cause protein degradation

• Maintain the protein in its supplied Tris-based buffer with 50% glycerol

These conditions ensure protein stability while preserving the native conformation and functional integrity of the recombinant protein for experimental applications.

What methodological approaches can be used to verify the identity and purity of recombinant cytochrome b preparations?

Researchers can employ multiple complementary techniques to verify the identity and purity of recombinant Sturnira tildae cytochrome b:

• SDS-PAGE analysis to assess molecular weight and initial purity assessment

• Western blotting using anti-cytochrome b antibodies for identity confirmation

• Spectrophotometric analysis to assess heme incorporation and functional properties

• Size exclusion chromatography to evaluate oligomeric state and homogeneity

These methods collectively provide comprehensive validation of the recombinant protein preparation before experimental use.

How has cytochrome b sequence analysis contributed to understanding Sturnira phylogeny?

• Strong support for the monophyly of genus Sturnira but rejection of the subgenus Corvira

• Identification of 21 monophyletic, putatively species-level groups, including three previously unnamed groups

• Resolution of the "S. lilium complex" into seven distinct species, with true S. lilium restricted to the Brazilian Shield

• Clarification of evolutionary relationships through maximum parsimony, maximum likelihood, and Bayesian inference analyses

The average pairwise genetic distance in cytochrome b between Sturnira species pairs was calculated at 7.09% (SD=1.61), providing a quantitative measure for species discrimination .

What insights has cytochrome b analysis provided about the biogeography and diversification of Sturnira?

Cytochrome b data, integrated with other genetic markers, has enabled researchers to reconstruct the biogeographic history of Sturnira with remarkable detail. Using time-calibrated phylogenies and ancestral range reconstruction methods (both parsimony-based DIVA and likelihood-based Lagrange), researchers determined that:

• The Sturnira lineage diverged from other stenodermatines during the mid-Miocene

• By the end of the Miocene (5.3 Ma), three basal lineages had emerged (bidens, nana, and aratathomasi)

• Two major clades (A and B) appeared and diversified during the Pliocene

• The radiation of Sturnira originated in the Andes, with all three basal lineages having strictly or mainly Andean distributions

• Subsequent colonization occurred in this sequence:

  • Pacific lowlands (Chocó)

  • Central America (after the Panamanian landbridge formed ~3 Ma)

  • Lesser Antilles (during the Pleistocene)

This robust biogeographic reconstruction demonstrates how cytochrome b analysis can illuminate complex patterns of diversification across evolutionary time and geographic space.

What limitations should researchers consider when using cytochrome b for phylogenetic inference?

Despite its utility, researchers should be aware of several important limitations when using cytochrome b for phylogenetic inference:

• Maternal inheritance and lack of recombination: As a mitochondrial gene, cytochrome b provides only a maternal perspective on evolutionary history, which may not align with the species tree derived from nuclear markers.

• Saturation at deeper taxonomic levels: The study results indicate that deeper nodes in bat phylogenies based on cytochrome b (relationships between bat families) often show poor statistical support (posterior probabilities <0.85) compared to more recent divergences .

• Introgression risks: Hybridization events can lead to mitochondrial introgression between species, potentially creating misleading phylogenetic signals.

• Variable evolutionary rates: Different regions of the cytochrome b gene evolve at different rates, and these rates may vary across lineages, complicating phylogenetic analyses.

• Incomplete resolution: For comprehensive phylogenetic reconstruction, cytochrome b data should be integrated with multiple markers, as demonstrated in the Sturnira study that combined cytochrome b with four other genetic markers .

These limitations emphasize the importance of using cytochrome b as part of a multi-marker approach rather than as a standalone solution for phylogenetic inference.

How does Sturnira tildae cytochrome b compare to its homologs in other bat species?

Comparative analysis of cytochrome b sequences across bat taxa reveals important evolutionary patterns:

Phylogenetic analyses suggest high conservation of cytochrome b function across bat lineages despite sequence divergence. This is evident in the clustering patterns observed in phylogenetic trees, where bats of the genus Myotis form distinct clades despite being sampled from different continents . The sequence differences reflect both neutral evolution and potential adaptive changes related to metabolic requirements in different bat lineages.

What role can cytochrome b analysis play in conservation genetics of threatened bat populations?

Cytochrome b analysis serves multiple critical functions in bat conservation genetics:

• Species identification and delimitation: Accurate identification of cryptic Sturnira species that are morphologically similar but genetically distinct, as demonstrated by the discovery of previously unrecognized species in the genus .

• Population structure assessment: Analysis of cytochrome b variation within species can reveal population genetic structure, helping identify isolated populations that may require separate conservation management.

• Genetic diversity monitoring: Measuring genetic diversity through cytochrome b polymorphisms provides an indicator of population health and resilience.

• Evolutionary significant unit (ESU) identification: Cytochrome b data helps define evolutionarily significant units within species, allowing conservation efforts to preserve genetic diversity across a species' range.

• Hybridization detection: Cytochrome b analysis can help identify instances of hybridization between closely related species, which may have conservation implications.

These applications make cytochrome b an invaluable tool in conservation genetics, particularly for Neotropical bats facing habitat loss and other anthropogenic threats.

How might Sturnira tildae cytochrome b research intersect with studies on bat-borne zoonotic diseases?

While cytochrome b itself is not directly involved in disease transmission, its study intersects with zoonotic disease research in several important ways:

• Host phylogeny clarification: Cytochrome b-based phylogenies help establish the evolutionary relationships among bat species, providing essential context for understanding host-pathogen dynamics and pathogen spillover risk .

• Co-evolutionary analysis: Comparing bat phylogenies (based on markers like cytochrome b) with pathogen phylogenies enables the identification of co-evolutionary patterns and potential cross-species transmission events.

• Species identification in surveillance: Cytochrome b can serve as a reliable marker for species identification in disease surveillance programs targeting bat reservoirs.

• Vaccine development strategies: Research on bat betaherpesviruses (such as DrBHV found in vampire bats) has identified their potential as vectors for transmissible vaccines against bat-borne diseases like rabies . Understanding bat phylogeny through cytochrome b analysis can inform the development of species-specific vaccine delivery strategies that account for evolutionary relationships.

The integration of cytochrome b research with disease ecology studies represents an important frontier in bat conservation and public health research.

What emerging technologies might enhance future research using recombinant Sturnira tildae cytochrome b?

Several cutting-edge technologies show promise for advancing research with recombinant Sturnira tildae cytochrome b:

• CRISPR-engineered expression systems: Development of optimized expression systems using CRISPR technology to enhance yield and purity of recombinant cytochrome b.

• Nanobody development: Engineering single-domain antibodies (nanobodies) against specific epitopes of Sturnira tildae cytochrome b for improved detection and functional studies.

• Cryo-EM structural analysis: Application of cryo-electron microscopy to determine high-resolution structures of cytochrome b in membrane environments, providing insights into function and evolution.

• Single-molecule biophysics: Investigating the functional properties of individual cytochrome b molecules to understand electron transfer dynamics and species-specific adaptations.

• Third-generation sequencing: Long-read sequencing technologies that can capture complete mitochondrial genomes in single reads, providing cytochrome b sequences in their full genomic context.

These technologies promise to deepen our understanding of cytochrome b structure, function, and evolution across bat lineages while enhancing its utility as a research tool.

What are the most significant contributions of Sturnira tildae cytochrome b research to bat evolutionary biology?

Cytochrome b research has fundamentally transformed our understanding of Sturnira evolution through several key contributions:

• Resolution of species complexes: Uncovering cryptic diversity, particularly in the S. lilium complex, with seven distinct species identified where previously only one was recognized .

• Biogeographic origin clarification: Establishing the Andean origin of Sturnira and documenting subsequent dispersal patterns across the Neotropics .

• Temporal framework: Dating key divergence events in Sturnira evolution from the mid-Miocene through the Pleistocene.

• Taxonomic revision support: Providing molecular evidence that refuted the validity of the subgenus Corvira while confirming the monophyly of Sturnira .

• Standardized genetic distance metrics: Establishing a 7.09% average cytochrome b sequence divergence threshold for species discrimination in this genus .

These contributions have not only advanced our understanding of Sturnira evolution but have also provided a model for integrating molecular and biogeographic approaches in bat systematics more broadly.

What promising research directions might emerge from continued study of Sturnira cytochrome b?

The future of Sturnira cytochrome b research holds several promising directions:

• Comprehensive phylogenomics: Integration of cytochrome b data with whole-genome sequencing to develop a more complete evolutionary picture of the genus.

• Fine-scale phylogeography: Expanded sampling across the geographical range of Sturnira species to reveal detailed patterns of population structure and historical biogeography.

• Functional genomics: Investigation of potential adaptive evolution in cytochrome b related to the ecological diversity of Sturnira species.

• Molecular clock refinement: Improvement of divergence date estimates through integration of fossil calibrations and advanced molecular clock models.

• Conservation applications: Development of rapid, non-invasive genetic tools based on cytochrome b for field identification and monitoring of threatened Sturnira populations.