Recombinant Lucihormetica subcincta Pyrokinin-5

What is Lucihormetica subcincta Pyrokinin-5 and what is its structure?

Lucihormetica subcincta Pyrokinin-5 is a neuropeptide belonging to the pyrokinin family found in the "Glow spot roach" (Lucihormetica subcincta). Structurally, it consists of the amino acid sequence GGES-SNEAKGMWFGPRLa, where 'a' indicates C-terminal amidation . This peptide is characterized by the conserved C-terminal FXPRLamide motif common to pyrokinins, though with slight variation. The full-length peptide contains approximately 17 amino acids, with the N-terminal region showing greater sequence variation compared to the highly conserved C-terminus .

Methodology for structure determination typically involves:

• Mass spectrometry of isolated peptides from perisympathetic organs

• Tandem mass spectrometry for sequence confirmation

• Comparison with homologous peptides from related species

How should Recombinant Lucihormetica subcincta Pyrokinin-5 be stored and handled in laboratory settings?

For optimal stability and activity of Recombinant Lucihormetica subcincta Pyrokinin-5:

• Reconstitution protocol:

  • Dissolve in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% to prevent freeze-thaw damage

• Storage conditions:

  • Short-term storage: -20°C

  • Extended storage: -20°C to -80°C

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Handling precautions:

  • Use sterile techniques to prevent contamination

  • Keep on ice during experimental setup

  • Centrifuge briefly before opening to collect material at the bottom of the tube

What are the primary biological functions of pyrokinins in insects?

Pyrokinins exhibit multiple physiological roles in insects, including:

• Myotropic activity: Regulation of muscle contraction, notably identified as having a myoinhibitory role in specific tissues like the ileum where PK2-R transcript is enriched

• Pheromonotropic effects: Modulation of pheromone biosynthesis and release in some insect species

• Melanotropic activity: Involvement in pigmentation and melanization processes

• Hindgut regulation: Expression of pyrokinin receptors is particularly high in the posterior hindgut (rectum) for PK1-R and anterior hindgut (ileum) for PK2-R, suggesting tissue-specific roles in digestive tract function

• Reproductive function: Significant pyrokinin receptor expression in reproductive organs indicates potential roles in reproduction

The function determination methodologies typically involve:

• Receptor characterization in heterologous cell systems

• Transcript-level expression analysis

• Immunohistochemical localization using specific antibodies

• Physiological assays measuring tissue response to applied peptides

How can phylogenetic relationships be analyzed using pyrokinin sequences?

Pyrokinins, as part of CAPA peptides, have proven valuable for phylogenetic analysis due to their combination of conserved and variable regions:

• Methodological approach:

  • Sequence multiple CAPA peptides including pyrokinins from target species

  • Align sequences with known reference sequences

  • Conduct Maximum Parsimony and Bayesian Inference analyses

• Phylogenetic information content:

  • C-terminal regions tend to be highly conserved and less informative

  • N-terminal sequences contain more variable, phylogenetically informative characters

  • From the example of cockroach studies, approximately 33 of 58 characters in CAPA peptide alignments are parsimony-informative

• Resolution capacity:

  • Produces phylogenetic trees largely consistent with molecular and morphological analyses

  • Effectively resolves relationships between major insect taxa

  • Successfully confirms monophyly of taxonomic groups like Blaberoidea (Blattellidae + Blaberidae)

• Complementary approach:

  • Combining pyrokinin sequence data with other neuropeptide families (e.g., adipokinetic hormones, sulfakinins) significantly increases bootstrap values in phylogenetic analyses

What experimental systems are most appropriate for studying pyrokinin function?

Several experimental systems offer advantages for investigating pyrokinin function:

• Heterologous cell expression systems:

  • Enables characterization of receptor activation and signaling pathways

  • Allows determination of dose-response relationships and receptor selectivity

  • Commonly used lines include HEK293 or CHO cells transfected with receptor constructs

• Ex vivo tissue preparations:

  • Isolated hindgut preparations (particularly ileum and rectum regions)

  • Allows direct measurement of myotropic effects

  • Enables study of tissue-specific responses where receptor expression is enriched

• Immunohistochemical approaches:

  • Utilizes PRXa-like antibodies (where X = V or L)

  • Allows visualization of peptidergic innervation along target tissues like the hindgut

  • Requires controls with pre-incubated antibody solution to confirm specificity

• Transcript expression analysis:

  • RT-PCR or qPCR for receptor expression profiling across tissues

  • Identifies potential target sites for peptidergic action

  • Correlates with functional significance of specific tissues

How do sequence variations in Lucihormetica subcincta Pyrokinin-5 compare with related species?

The sequence analysis of pyrokinins across cockroach species reveals significant patterns:

• Conservation patterns:

  • The Lucihormetica subcincta Pyrokinin (GGES-SNEAKGMWFGPRLa) shows identical sequence to Lucihormetica verrucosa and Lucihormetica grossei

  • This conservation within the genus suggests strong evolutionary constraints

  • The C-terminal FGPRLa motif is highly conserved across most cockroach species

• Comparative analysis across Blaberidae family:

  Species	Pyrokinin Sequence
  L. subcincta	GGES-SNEAKGMWFGPRLa
  L. verrucosa	GGES-SNEAKGMWFGPRLa
  L. grossei	GGES-SNEAKGMWFGPRLa
  Archimandrita tesselata	EGAN-SNEAKGMWFGPRLa
  Panchlora spec.	GGET-GNDAKAMWFGPRLa
  Panchlora viridis	GGET-GSDAKAMWFGPRLa

• Evolutionary implications:

  • The N-terminal region shows greater variability and contains more phylogenetically informative sites

  • Within subfamilies, sequence conservation is typically high

  • Sequence variations align with established taxonomic groupings

• Intraspecific consistency:

  • No sequence variations observed within populations of the same species

  • Consistent across different sexes and developmental stages

  • Stable even across geographically separated populations

What methodologies are recommended for functional characterization of recombinant pyrokinins?

For comprehensive functional characterization of recombinant pyrokinins:

• Receptor activation assays:

  • Heterologous expression of receptor in cell lines

  • Fluorescence-based calcium mobilization assays

  • FLIPR (Fluorescent Imaging Plate Reader) for high-throughput screening

  • Dose-response curve generation with EC50 determination

• Tissue-specific physiological assays:

  • Force transduction measurements on isolated hindgut preparations

  • Video microscopy to quantify changes in motility patterns

  • Comparison of effects on different gut regions (ileum vs. rectum)

• Structure-activity relationship studies:

  • Alanine scanning mutagenesis to identify critical residues

  • N-terminal or C-terminal truncations to determine minimal active core

  • Comparison of natural variants across species

• Competitive binding assays:

  • Radiolabeled or fluorescent-labeled peptide analogs

  • Displacement curves to determine binding affinities

  • Receptor subtype selectivity determination

• Molecular dynamics simulations:

  • In silico prediction of peptide-receptor interactions

  • Conformational analysis of peptide structure

  • Binding pocket identification and characterization

What is the significance of hybrid studies in understanding pyrokinin evolution and function?

Hybrid studies, such as those between Lucihormetica verrucosa and Lucihormetica subcincta, provide valuable insights:

• Evolutionary compatibility:

  • Successful hybridization between L. verrucosa females and L. subcincta males demonstrates reproductive compatibility

  • Direction-specific success (hybrid offspring only from L. verrucosa females × L. subcincta males, not the reverse)

  • Suggests recent evolutionary divergence and potential for introgression

• Implications for neuropeptide research:

  • Hybridization success indicates conservation of developmental pathways regulated by neuropeptides

  • Presents opportunity to study receptor-ligand coevolution

  • May indicate functional conservation of pyrokinins despite species divergence

• Methodological considerations for hybrid studies:

  • Control for prior mating by using female nymphs raised to adulthood in isolation

  • Standardized housing conditions (temperature 20-26°C, appropriate substrate depth)

  • Careful documentation of morphological traits in hybrids

• Hybrid fertility assessment:

  • Although hybrids can mate, fertility appears compromised

  • Testing with pure-bred individuals can assess hybrid fertility status

  • Implications for understanding reproductive isolation mechanisms

What challenges exist in expressing and purifying functional recombinant insect neuropeptides?

Several technical challenges complicate the production of functional recombinant insect neuropeptides:

• Post-translational modifications:

  • C-terminal amidation critical for biological activity

  • Requires specialized expression systems with appropriate amidating enzymes

  • E. coli systems typically lack necessary post-translational machinery

• Size constraints:

  • Small peptide size (17aa for pyrokinins) presents purification challenges

  • Often requires fusion protein strategies for initial expression

  • Precise cleavage methods necessary to obtain native sequence

• Folding and structure:

  • Secondary structure may be critical for receptor recognition

  • Disulfide bond formation must be properly controlled

  • Buffer conditions during purification can affect final conformation

• Solubility issues:

  • Hydrophobic residues in pyrokinins can cause aggregation

  • Addition of glycerol (5-50%) improves stability

  • May require specialized solubilization strategies

• Activity verification:

  • Functional equivalence to native peptide must be confirmed

  • Receptor activation assays necessary to verify biological activity

  • Comparative analysis with synthetic peptide standards recommended

How can pyrokinin research contribute to understanding evolutionary relationships in insects?

Pyrokinin sequences provide a valuable tool for evolutionary studies:

• Phylogenetic signal:

  • CAPA peptides including pyrokinins contain sufficient sequence variation for phylogenetic analysis

  • Generated phylogenies show remarkable agreement with trees based on molecular and morphological data

  • Particularly useful for resolving relationships between higher insect taxa

• Conservation across evolutionary time:

  • High sequence conservation within taxonomic groups reflects evolutionary constraints

  • Specific variations align with established taxonomic boundaries

  • Supports the reclassification of termites within cockroaches

• Multi-peptide approach:

  • Combining pyrokinin data with other neuropeptide families significantly increases phylogenetic resolution

  • Adipokinetic hormones and sulfakinins provide complementary information

  • Increases bootstrap values when incorporated into analyses

• Methodological advantages:

  • Can be obtained from single specimens

  • Direct sequencing via tandem mass spectrometry

  • Not dependent on genomic data availability

  • Applicable to ancient or poorly characterized taxa