Recombinant Lucilia cuprina FMRFamide-17

What is Recombinant Lucilia cuprina FMRFamide-17 and what is its significance in research?

Recombinant Lucilia cuprina FMRFamide-17 (also known as LucFMRFamide-17) is a neuropeptide originally derived from the Australian sheep blowfly (Lucilia cuprina). It belongs to the FMRFamide-related peptide (FaRP) family, which represents an important group of messenger molecules that regulate numerous physiological processes in insects, including behavior . The recombinant version is produced using baculovirus expression systems to provide researchers with a consistent, high-purity reagent for investigating neuropeptide signaling mechanisms . This particular neuropeptide is significant because it offers insights into neuromodulatory processes across dipteran species, potentially informing our understanding of neural circuit function and behavior regulation.

What is the molecular structure and composition of Lucilia cuprina FMRFamide-17?

Lucilia cuprina FMRFamide-17 has a defined amino acid sequence of PDNFMRF, containing 7 amino acid residues . It features the characteristic C-terminal RFamide motif that defines this neuropeptide family. The peptide is part of a larger group of 45 neuropeptides identified in Lucilia cuprina through mass spectrometry analysis . When produced recombinantly, the protein demonstrates >85% purity as measured by SDS-PAGE . The molecular structure includes the amidated C-terminus that is typical of FMRFamide-family peptides, which is critical for receptor recognition and biological activity.

What expression systems are most effective for producing Recombinant Lucilia cuprina FMRFamide-17?

The baculovirus expression system has proven most effective for producing Recombinant Lucilia cuprina FMRFamide-17 . This eukaryotic expression platform utilizes insect cells infected with recombinant baculovirus containing the gene of interest. The system offers several advantages for neuropeptide production:

• Native-like post-translational modifications

• Proper protein folding machinery

• High expression levels

• Reduced endotoxin contamination compared to bacterial systems

• Scalability for different research needs

When implementing this system, researchers should optimize infection parameters including multiplicity of infection, harvest timing, and culture conditions to maximize yield while maintaining protein quality. The baculovirus system's ability to process the peptide correctly makes it particularly suitable for producing biologically active FMRFamide-17.

What purification strategies ensure optimal purity and biological activity?

While specific purification protocols for Lucilia cuprina FMRFamide-17 are not detailed in the search results, a methodological approach based on the protein's characteristics would include:

• Initial clarification by centrifugation to remove cellular debris

• Primary capture using affinity chromatography if the recombinant protein contains a purification tag

• Intermediate purification by ion exchange chromatography, leveraging the peptide's charge properties

• Polishing step using size exclusion chromatography to remove aggregates and achieve final purity

• Quality control testing including SDS-PAGE to confirm purity >85%

Each purification step should be optimized with appropriate buffer conditions to maintain peptide stability and activity. Monitoring biological activity throughout the purification process using functional assays would ensure that the final product retains its neuromodulatory properties.

How can researchers validate the identity and purity of purified FMRFamide-17?

Validation of Recombinant Lucilia cuprina FMRFamide-17 should employ multiple complementary techniques:

For comprehensive validation, researchers should combine these methods to confirm both identity and purity before proceeding with experimental applications. This multi-modal approach ensures data reliability and experimental reproducibility.

What are the optimal storage conditions for maintaining FMRFamide-17 stability?

Optimal storage of Recombinant Lucilia cuprina FMRFamide-17 requires careful attention to temperature and formulation. According to product specifications, the protein should be stored at -20°C for routine storage, while extended storage should be at either -20°C or -80°C . The stability data indicates that:

• Repeated freezing and thawing cycles should be avoided as they can compromise peptide integrity

• Working aliquots can be maintained at 4°C for up to one week

• The shelf life in liquid form is approximately 6 months at -20°C/-80°C

• Lyophilized preparations demonstrate extended stability up to 12 months at -20°C/-80°C

These conditions are designed to minimize degradation processes including oxidation, deamidation, and aggregation that could affect the peptide's biological activity and experimental performance.

What reconstitution protocol maximizes FMRFamide-17 recovery and stability?

The recommended reconstitution protocol for maximizing recovery and maintaining stability of Recombinant Lucilia cuprina FMRFamide-17 involves a specific procedure:

• Briefly centrifuge the vial before opening to collect the material at the bottom

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

• Add glycerol to a final concentration between 5-50% (with 50% being the default recommendation)

• Prepare small aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term preservation

This protocol ensures optimal solubilization while the addition of glycerol serves as a cryoprotectant to prevent damage during freezing. Researchers should validate the concentration of reconstituted peptide using spectrophotometric methods before experimental applications.

How can researchers monitor potential degradation of FMRFamide-17 during storage?

Monitoring degradation of Recombinant Lucilia cuprina FMRFamide-17 during storage is essential for maintaining experimental consistency. While not explicitly detailed in the search results, a systematic approach would include:

• Regular analytical testing at defined intervals (e.g., monthly)

• Comparative RP-HPLC analysis to detect appearance of degradation products

• Mass spectrometry to identify specific degradation mechanisms (oxidation, deamidation, etc.)

• Functional assays to assess retention of biological activity

• SDS-PAGE to monitor potential aggregation or fragmentation

Establishing a stability-indicating analytical method at the beginning of research would allow for quantitative tracking of any degradation over time. This proactive approach enables researchers to establish appropriate expiration dates and ensure experimental reproducibility throughout a research project.

What immunohistochemical techniques are most effective for FMRFamide-17 detection in tissue samples?

Effective immunohistochemical detection of FMRFamide-17 in tissue samples requires optimized protocols as evidenced by published methodologies:

• Fixation: Use 4% paraformaldehyde with 0.15% glutaraldehyde in 0.1 M PBS (pH 7.4) to preserve peptide antigens while maintaining tissue architecture

• Permeabilization: Apply a permeabilization and blocking medium containing 0.05% Triton-X 100, 10% normal goat serum, and 0.25% bovine serum albumin to facilitate antibody penetration while reducing background

• Primary antibody: Utilize validated rabbit anti-FMRFamide antisera (e.g., Sigma Millipore AB15348) at dilutions of 1:800 to 1:1000, with extended incubation (24 hours at 6°C) and periodic agitation

• Secondary detection: Apply Cy3-conjugated goat-anti-rabbit antibodies (1:500 dilution) with nuclear counterstaining using Hoechst Blue (1:3000)

• Imaging: Employ confocal microscopy with appropriate filter sets to visualize the distribution and co-localization patterns

This comprehensive approach enables quantitative assessment of FMRFamide-expressing neurons, with studies in arthropods indicating that approximately 3.4-12.6% of neurons may show immunoreactivity with varying labeling intensities .

What mass spectrometry approaches provide the highest sensitivity for FMRFamide-17 analysis?

Mass spectrometry offers powerful tools for analyzing FMRFamide-17 with high sensitivity and specificity. Based on published research, the most effective approaches include:

• Matrix-assisted laser desorption ionization time of flight (MALDI-TOF): Effective for initial peptide identification and distribution studies in tissue samples

• Electrospray ionization quadrupole time of flight (ESI-Q-TOF): Provides higher sensitivity for complex samples and can be coupled with liquid chromatography for enhanced separation

• Multiple reaction monitoring (MRM): Enables targeted quantification with increased sensitivity for specific peptide sequences

• Peptide fragmentation analysis: Allows confirmation of the PDNFMRF sequence through characteristic fragment ions

These techniques have successfully identified and characterized 45 neuropeptides in Lucilia cuprina, including FMRFamide-17 . When implementing these methods, researchers should optimize sample preparation protocols to minimize peptide loss and maximize extraction efficiency from complex biological matrices.

How can researchers effectively distinguish between different FMRFamide-related peptides in analytical samples?

Distinguishing between closely related FMRFamide peptides requires a multi-faceted analytical approach:

• Chromatographic separation: Utilize high-resolution techniques such as nano-LC with appropriate column chemistry to separate peptides based on subtle differences in hydrophobicity

• High-resolution mass spectrometry: Employ instruments with sufficient mass accuracy to differentiate between peptides with small mass differences

• MS/MS fragmentation patterns: Analyze characteristic fragment ions that can distinguish between similar sequences

• Immunological specificity: Use antibodies with validated epitope specificity, recognizing that cross-reactivity between related FMRFamide peptides may occur

• Targeted MRM approaches: Develop specific transitions for each FMRFamide variant based on unique fragment ions

This integrated approach allows researchers to reliably identify and quantify Lucilia cuprina FMRFamide-17 (PDNFMRF) even in the presence of related neuropeptides. Careful method validation using synthetic standards is essential to confirm specificity and sensitivity.

What are the primary physiological roles of FMRFamide-17 in Lucilia cuprina?

While the specific physiological roles of FMRFamide-17 in Lucilia cuprina require further characterization, research on FMRFamide-related peptides in insects provides insight into potential functions:

• Neuromodulation: FMRFamide-related peptides act as important modulators of neural transmission, influencing the activity of specific neural circuits

• Behavioral regulation: As neuropeptides that regulate physiological processes including behavior, FMRFamides likely influence complex behavioral patterns in Lucilia cuprina

• Motor control: Studies in arthropods indicate that FMRFamide-immunoreactive neurons include characteristic motor neurons, suggesting roles in movement coordination and muscle control

• Sensory processing: Evidence suggests involvement in modulating sensory signal transduction, potentially including mechanosensory pathways

The diversity of these potential functions highlights the importance of FMRFamide-17 as a multifunctional signaling molecule within the insect nervous system, warranting detailed functional studies to elucidate its specific roles in Lucilia cuprina.

How does FMRFamide-17 interact with neuronal receptors to exert its biological effects?

The molecular mechanisms by which FMRFamide-17 interacts with its receptors have not been fully characterized in Lucilia cuprina, but research on related FMRFamide peptides suggests:

• G-protein coupled receptor activation: FMRFamides typically bind to GPCRs, initiating intracellular signaling cascades through G-protein activation

• Co-transmission with classical neurotransmitters: Evidence indicates that FMRFamides may be co-released with glutamate, suggesting complex interactions at the synaptic level

• Presynaptic modulation: Large electron-dense vesicles characteristic of neuropeptides have been observed at presynaptic terminals, indicating potential roles in modulating neurotransmitter release

• Receptor heterogeneity: Different neurons may express different receptor subtypes, explaining the observation of both weakly and strongly labeled neurons in immunohistochemical studies

Further research using heterologous expression systems, receptor binding assays, and electrophysiological recordings would help elucidate the specific receptor interactions and downstream signaling pathways activated by FMRFamide-17.

What evidence exists for co-localization of FMRFamide-17 with other neurotransmitters?

Research on FMRFamide-related peptides in arthropods provides compelling evidence for co-localization with classical neurotransmitters:

• Double-labeling experiments have demonstrated co-expression of FMRFamide-related peptides and glutamate within the same neurons, suggesting potential co-transmission

• Ultrastructural investigations revealed mixed presynaptic vesicle populations, including both small clear vesicles (typical for classical neurotransmitters) and large electron-dense vesicles characteristic for neuropeptides

• Among FMRFamide-immunoreactive neurons, characteristic motor neurons previously shown to express γ-aminobutyric acid or glutamate have been identified

• The differential labeling intensity (weak vs. strong) observed in immunohistochemical studies may reflect varying degrees of co-expression with other neurotransmitters

This co-localization pattern suggests complex neuromodulatory roles where FMRFamide-17 may fine-tune neuronal communication by modifying the effects of classical neurotransmitters at specific synapses.

How can FMRFamide-17 be utilized in neurophysiological research models?

Recombinant Lucilia cuprina FMRFamide-17 offers several valuable applications in neurophysiological research:

• Receptor characterization studies: The purified peptide can be used to identify and characterize FMRFamide receptors in heterologous expression systems

• Electrophysiological investigations: Application of defined concentrations to neuronal preparations allows examination of acute modulatory effects on membrane properties and synaptic transmission

• Calcium imaging experiments: FMRFamide-17 administration during calcium imaging can reveal patterns of neuronal activation and network effects

• Comparative physiology: The well-defined sequence allows comparison of physiological responses across different insect species to evaluate evolutionary conservation of function

• Structure-activity relationship studies: Synthetic variants can be compared with the recombinant peptide to identify critical residues for biological activity

These applications provide important insights into neuropeptide signaling mechanisms and can inform broader questions about neural circuit modulation and behavior regulation.

What control experiments should be included when working with FMRFamide-17?

Robust experimental design with appropriate controls is essential when working with Recombinant Lucilia cuprina FMRFamide-17:

How can researchers design experiments to investigate potential synergistic effects between FMRFamide-17 and other neurotransmitters?

Given the evidence for co-localization of FMRFamide-related peptides with classical neurotransmitters , investigating potential synergistic effects requires carefully designed experiments:

• Sequential application protocols:

  • Apply neurotransmitter (e.g., glutamate) alone and measure response

  • Apply FMRFamide-17 alone and measure response

  • Pre-apply FMRFamide-17 followed by neurotransmitter

  • Co-apply FMRFamide-17 and neurotransmitter simultaneously

  • Compare responses to identify potentiation, inhibition, or independent effects

• Concentration matrix experiments:

  • Test multiple concentrations of both FMRFamide-17 and the co-transmitter

  • Generate 3D response surfaces to identify non-linear interactions

  • Calculate synergy scores using appropriate mathematical models

• Temporal dynamics analysis:

  • Examine how FMRFamide-17 affects the kinetics of neurotransmitter responses

  • Measure onset, peak, decay, and duration parameters

  • Assess whether modulation is transient or persistent

• Receptor pharmacology:

  • Use selective receptor antagonists to block specific signaling pathways

  • Determine whether effects involve direct receptor interactions or downstream convergence

These experimental approaches can reveal the complex modulatory mechanisms through which FMRFamide-17 interacts with classical neurotransmission systems.

What evolutionary insights can be gained from comparative studies of FMRFamide-17 across different insect species?

Comparative analysis of FMRFamide-17 across insect species offers valuable evolutionary insights:

• Sequence conservation: Research has revealed remarkable similarity in the peptidome of cyclorraphan flies, suggesting strong evolutionary pressure to maintain FMRFamide structure and function

• Receptor evolution: While peptide sequences may be conserved, receptor divergence could drive species-specific responses to the same peptide

• Expression pattern variations: Different species may express FMRFamide-17 in distinct neuronal populations, reflecting adaptive specialization

• Functional divergence: Despite structural conservation, the physiological roles may vary between species based on ecological niches and behavioral specializations

This evolutionary perspective provides context for understanding how neuropeptide signaling systems adapt to different environmental pressures while maintaining core functional properties. The high degree of conservation observed in cyclorraphan flies suggests fundamentally important roles for these signaling molecules .

What are the challenges and opportunities in developing FMRFamide-based approaches for insect pest control?

The development of FMRFamide-based pest control strategies presents both challenges and opportunities:

Challenges:

• The "remarkable similarity of the peptidome of cyclorraphan flies" makes it difficult to develop species-specific approaches targeting the peptides themselves

• Mass spectrometric approaches do not cover the entire peptidome, leaving potential species-specific variants undetected

• Delivery methods for peptide-based control agents into target insects remain technically challenging

• Potential for rapid evolution of resistance through receptor modifications

Opportunities:

• Differences at the receptor level could potentially be exploited even if peptides are conserved

• Group-specific peptide ligands might exist that escaped detection in current studies

• Advanced delivery technologies such as RNA interference could target peptide production pathways

• Combination approaches targeting multiple neuropeptide systems simultaneously could enhance efficacy

These considerations suggest that while peptidome similarity presents obstacles, sophisticated approaches focusing on receptor differences or regulatory mechanisms might still yield effective control strategies.

How might genomic and transcriptomic approaches enhance our understanding of FMRFamide-17 biology?

Genomic and transcriptomic approaches offer powerful tools for advancing FMRFamide-17 research:

• Genome mining: Analysis of the Lucilia cuprina genome can identify all potential FMRFamide precursor genes and their regulatory elements, providing a comprehensive view of the FMRFamide gene family

• Transcriptome profiling:

  • Single-cell RNA sequencing can identify which neuronal populations express FMRFamide-17

  • Differential expression analysis across developmental stages can reveal temporal regulation

  • Comparative transcriptomics across species can identify conserved and divergent expression patterns

• Regulatory network analysis:

  • Identification of transcription factors controlling FMRFamide expression

  • Epigenetic profiling to understand chromatin states associated with neuropeptide gene expression

  • Analysis of non-coding RNAs that may regulate FMRFamide production or signaling

• Receptor identification:

  • Transcriptome analysis can identify candidate FMRFamide receptors

  • Co-expression network analysis can reveal potential downstream signaling partners

  • Comparative analysis across tissues can map receptor distribution patterns

These approaches provide a systems-level understanding of FMRFamide-17 biology beyond what is possible with traditional biochemical or pharmacological methods alone.

What are the most promising research directions for advancing our understanding of FMRFamide-17 biology?

The study of Recombinant Lucilia cuprina FMRFamide-17 offers several promising research directions:

• Receptor identification and characterization: Identifying the specific receptors for FMRFamide-17 in Lucilia cuprina would significantly advance our understanding of its signaling mechanisms and potential for targeted interventions.

• Circuit-level functional studies: Examining how FMRFamide-17 modulates specific neural circuits could reveal its roles in regulating complex behaviors relevant to insect ecology and pest management.

• Interactome mapping: Comprehensive investigation of proteins interacting with FMRFamide-17 and its receptors would illuminate the broader signaling networks and potential regulatory mechanisms.

• Comparative neuromodulation: Systematic comparison of FMRFamide-17 effects across dipteran species could reveal subtle functional differences despite sequence conservation.

• Technological developments: Creating biosensors for real-time monitoring of FMRFamide-17 release and activity in vivo would transform our understanding of its dynamic signaling properties.

These research directions would collectively advance both fundamental understanding of neuropeptide biology and potential applications in entomology and pest management.

How might emerging technologies advance FMRFamide-17 research in the next decade?

Emerging technologies are poised to revolutionize FMRFamide-17 research:

• CRISPR/Cas9 genome editing: Precise modification of FMRFamide genes or receptors in Lucilia cuprina will enable detailed functional studies through directed mutagenesis and reporter insertions.

• Advanced imaging techniques:

  • Expansion microscopy for super-resolution imaging of neuropeptide distribution

  • Light-sheet microscopy for whole-brain imaging of FMRFamide networks

  • Genetically encoded sensors for real-time visualization of peptide release

• Proteomics advances:

  • Enhanced sensitivity in mass spectrometry for detection of low-abundance peptide variants

  • Improved crosslinking mass spectrometry for identifying receptor-peptide interactions

  • Single-cell proteomics for cell-specific neuropeptide profiling

• Computational approaches:

  • Artificial intelligence for predicting peptide-receptor interactions

  • Molecular dynamics simulations of neuropeptide binding mechanisms

  • Systems biology modeling of neuropeptide signaling networks

These technological advances will provide unprecedented insights into the molecular, cellular, and systems-level functions of FMRFamide-17, potentially revealing novel applications in neuroscience and entomology.