Recombinant Sarcophaga bullata FMRFamide-2

What is the relationship between FMRFamide-like peptides and the sulfakinins found in Sarcophaga bullata?

The sulfakinins isolated from Sarcophaga bullata (also known as Neobellieria bullata) share significant structural similarities with FMRFamide-related peptides, particularly in their C-terminal regions. Specifically, the last three amino acids of these sulfakinins are identical to those found in FMRF-amide related peptides . This structural similarity suggests an evolutionary relationship between these peptide families and potentially overlapping functions. The neosulfakinins (Neb-SK-I and Neb-SK-II) isolated from S. bullata exhibit myotropic activity similar to some FMRFamide-like peptides, though with distinct tissue specificity that differentiates them functionally .

What methods are used to isolate and purify FMRFamide-related peptides from Sarcophaga bullata?

The isolation of FMRFamide-related peptides from S. bullata requires a multi-step purification process. Researchers typically begin with extract preparation from a large number of specimens (approximately 42,000 fleshfly heads for neosulfakinins) . The purification protocol involves a series of high-performance liquid chromatographic (HPLC) fractionations performed on columns with different characteristic features:

• Initial separation on p-Bondapak phenyl columns

• Secondary fractionation on LC-1 columns

• Tertiary purification on LC-8 columns

• Final purification on Protein Pak columns

Throughout this process, fractions are monitored for biological activity using appropriate bioassays, such as the Leucophaea hindgut motility assay, to track the presence of active peptides . This methodical approach allows for the isolation of biologically active fractions suitable for subsequent amino acid sequence analysis.

How is the primary structure of Sarcophaga bullata FMRFamide-related peptides determined?

Primary structure determination of S. bullata FMRFamide-related peptides involves several analytical steps:

• Enzymatic analysis tests are first conducted to determine if N-terminals are blocked

• For unblocked peptides, direct amino acid sequencing can be performed

• Sequencing is typically carried out on purified fractions from HPLC, with requirements for sample amounts typically in the picomole range (e.g., 30 pmol for Neb-SK-I)

For Neb-SK-I, this approach revealed a primary structure of Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe (MW: 1047) . For Neb-SK-II, the sequence was determined to be X-X-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe (Putative MW: 1700), where the first two N-terminal amino acids could not be identified due to limited sample availability .

What are the key post-translational modifications of FMRFamide-related peptides in Sarcophaga bullata and how do they affect functionality?

FMRFamide-related peptides in S. bullata undergo important post-translational modifications that significantly impact their biological activity. Research indicates that these peptides are likely:

• C-terminally amidated, which is critical for receptor binding and biological activity

• Sulfated at tyrosine residues, particularly at the Tyr⁴ position in Neb-SK-I

How do recombinant expression systems compare to natural extraction for studying Sarcophaga bullata neuropeptides?

While natural extraction has historically been the primary method for isolating S. bullata neuropeptides, recombinant expression systems offer several advantages:

• Yield: Natural extraction requires enormous numbers of specimens (42,000 fleshfly heads yielded only about 100 ng of Neb-SK-I and 26 ng of Neb-SK-II) . Recombinant systems can potentially produce significantly larger quantities.

• Structural precision: Recombinant systems allow for controlled production of peptides with specific post-translational modifications, enabling structure-function studies.

• Mutation analysis: Synthetic or recombinant approaches facilitate the creation of modified peptides to study the importance of specific amino acids or modifications.

• Consistency: Recombinant production provides more consistent yields and purity compared to natural extracts, which can vary between preparations.

What methodological approaches are used to characterize the biological activity of Sarcophaga bullata FMRFamide-related peptides?

Researchers employ several bioassay systems to characterize the biological activity of S. bullata FMRFamide-related peptides:

• Heterologous bioassays using tissues from other insect species:

  • Leucophaea hindgut motility assay: Measures the stimulatory effect on hindgut contractions

  • Locusta oviduct bioassay: Tests for effects on oviduct muscle contractions

• Activity quantification: The activity of purified natural peptides is measured in molar concentrations, with Neb-SK-I showing activity in the 1.5 × 10⁻⁷ M range and Neb-SK-II in the 5 × 10⁻⁸ M range on the Leucophaea hindgut assay .

• Comparative analysis: Comparing activity across different tissues helps determine tissue specificity (e.g., neosulfakinins were active on Leucophaea hindgut but inactive on Locusta oviduct) .

These approaches provide crucial information about the functional roles and specificity of these peptides in insect physiology.

What are the essential controls needed when studying myotropic activity of FMRFamide-related peptides?

When designing experiments to study the myotropic activity of FMRFamide-related peptides from S. bullata, researchers should include the following controls:

• Tissue-specific negative controls: Testing the peptide on tissues known not to respond (e.g., Locusta oviduct for neosulfakinins) helps confirm specificity .

• Concentration gradients: Testing multiple concentrations establishes dose-response relationships and determines threshold concentrations for biological activity.

• Synthetic replicas: Using both synthetic amidated and acid forms of the peptides helps determine the importance of C-terminal amidation and tyrosine sulfation for activity .

• Cross-species comparison: Testing the peptides on tissues from different insect species provides insights into evolutionary conservation of response mechanisms.

• Antagonist controls: Where available, specific receptor antagonists should be used to confirm receptor-mediated effects.

These controls enhance the reliability and interpretability of bioassay results when characterizing novel peptides.

How can immunolocalization techniques be applied to study the distribution of FMRFamide-related peptide receptors?

Immunolocalization provides valuable insights into the distribution of receptors for FMRFamide-related peptides in the insect nervous system. The methodology involves:

• Antibody production: Developing antisera against specific peptide regions of receptors, as demonstrated with antisera raised against two distinct peptide regions of the Drosophila neurokinin-like receptor NKD used to immunolocalize tachykinin-receptor-like proteins in S. bullata .

• Tissue preparation: Careful dissection and fixation of central nervous system tissues to preserve receptor structures and accessibility.

• Immunostaining protocols: Applying primary antibodies against the receptor of interest, followed by labeled secondary antibodies for visualization.

• Comparative analysis: Examining staining patterns across different insect species to identify evolutionarily conserved receptor distributions .

In S. bullata, such techniques have revealed immunoreactivity in nerve terminals of the retrocerebral complex, suggesting presynaptic localization of receptors, as well as in specific cell bodies in the subesophageal ganglion . These findings help map the neural circuits involved in peptide signaling.

What evolutionary relationships exist between insect FMRFamide-related peptides and vertebrate regulatory peptides?

FMRFamide-related peptides from S. bullata exhibit remarkable evolutionary relationships with vertebrate regulatory peptides:

• Structural homology: Neosulfakinins display structural similarities with vertebrate gastrin- and cholecystokinin-related peptides, particularly in key amino acid positions .

• Conserved functional domains: Neb-SK-II shares six amino acids at identical positions with gastrin II (specifically Glu³, Glu⁴, Tyr⁹, Gly¹⁰, Met¹¹, and other positions not specified in the search results) .

• Functional conservation: Both insect sulfakinins and vertebrate gastrin/cholecystokinin peptides have gastrointestinal myotropic functions, suggesting conservation of physiological roles across distant phyla .

These similarities provide compelling evidence for a long evolutionary history of this peptide family, with core structural and functional elements preserved over hundreds of millions of years of divergent evolution between vertebrates and invertebrates .

How do the sulfakinins from Sarcophaga bullata compare to similar peptides isolated from other insect species?

Comparative analysis of sulfakinins from S. bullata with those from other insect species reveals important patterns:

• Drosophila sulfakinins: Neosulfakinins exhibit high homology to putative drosulfakinin sequences, though the latter have not been isolated but were deduced from cloned Drosophila genomic DNA .

• Leucophaea maderae sulfakinins: Lem-SK-I and Lem-SK-II were among the first insect sulfakinins characterized and serve as reference points for comparing newly discovered sulfakinins .

• Periplaneta americana: Both a sulfated form (Pea-SK-I) and a non-sulfated form (identical to Lem-SK-II) have been isolated from this species, providing insights into the variability of post-translational modifications .

• Locusta migratoria: Additional sulfakinins have been purified and identified from this species (Lom-SK), showing structural similarities to the others .

This cross-species comparison highlights both conservation of core structural elements and species-specific variations that may relate to functional adaptations in different insect lineages.

How can research on Sarcophaga bullata neuropeptides contribute to understanding insect diapause regulation?

Research on S. bullata neuropeptides can significantly enhance our understanding of insect diapause regulation through several avenues:

• MicroRNA-mediated regulation: Studies have identified ten evolutionarily conserved miRNAs differentially expressed in diapausing pupae compared to nondiapausing counterparts in S. bullata . Neuropeptide signaling may interact with these miRNA pathways to coordinate developmental timing during diapause.

• Stress response mechanisms: S. bullata responds to environmental stressors by accumulating glycerol, which functions as both an anti-desiccant and cryoprotectant . The brain plays a critical role in initiating this response, suggesting potential involvement of neuropeptides in stress signal integration.

• Maternal effect regulation: Some miRNAs (miR-263a-5p, miR-100-5p, miR-125-5p, and let-7-5p) are significantly overexpressed in flies prevented from entering diapause by maternal effects . Investigating how neuropeptide signaling interacts with these maternal effect pathways could reveal important regulatory mechanisms.

Understanding these interconnected regulatory networks can provide insights into how insects adapt to seasonal changes and environmental challenges, with potential applications in pest management and conservation biology.

What emerging techniques might advance our understanding of FMRFamide-related peptide function in insects?

Several emerging techniques show promise for advancing research on FMRFamide-related peptides in insects:

• CRISPR-Cas9 gene editing: Enabling precise modification of neuropeptide genes and their receptors in model insects to study loss-of-function and gain-of-function phenotypes.

• Advanced mass spectrometry: Improved techniques for detecting and quantifying post-translational modifications like amidation and sulfation with greater sensitivity from smaller tissue samples.

• Single-cell transcriptomics: Allowing identification of neuropeptide expression patterns in specific neuronal populations to map peptidergic circuits with unprecedented resolution.

• Optogenetic and chemogenetic approaches: Providing tools to selectively activate or inhibit peptidergic neurons to determine their precise roles in physiological and behavioral processes.

• Computational modeling: Developing predictive models of neuropeptide-receptor interactions based on structural data to guide experimental design and drug development.

These techniques, applied to S. bullata and other model insects, will help unravel the complex functions of FMRFamide-related peptides in insect physiology and behavior.