Recombinant Blaberus craniifer Periviscerokinin-1

What is Periviscerokinin-1 from Blaberus craniifer, and what is its amino acid sequence?

Periviscerokinin-1 (Lem-PVK-1) is a neuropeptide first identified in blaberoid cockroaches, including Blaberus craniifer (death's head cockroach). It belongs to the CAPA peptide family and has the primary structure GSSGLIPFGRT-NH₂ with amidation at the C-terminus. The molecular formula is C₄₈H₇₉N₁₅O₁₄ with a molecular weight of 1090.2 Da . This peptide contains a highly conserved N-terminus, while only the penultimate amino acid residue (Arg) is consistently found across all members of this peptide family .

How are PVK-1 peptides stored and released in insect systems?

Periviscerokinin peptides are primarily stored and released from perisympathetic organs (PSOs), which are neurohaemal release sites in insects. These organs are the major storage and release centers for neurohormones produced in the ventral nerve cord . In cockroaches, the abdominal PSOs (approximately 70-90 μm in diameter) contain these neuropeptides . The peptides are produced in neurosecretory cells of the brain and transported to the PSOs for storage and release. Interestingly, these neuropeptides appear to be absent in the retrocerebral complex but are abundant in abdominal PSOs of blaberoid cockroaches .

What are the most effective techniques for identifying and characterizing PVK neuropeptides from insect tissue samples?

The most effective techniques for identifying and characterizing PVK neuropeptides include:

• Mass Spectrometry: Electrospray ionization-quadrupole time of flight (ESI-QTOF) MS has been successfully used to identify PVKs directly from extracts of single abdominal perisympathetic organs . This technique allows for the analysis of extremely small samples (single PSOs).

• Edman Degradation: This technique is used to confirm sequences identified by mass spectrometry .

• Liquid Chromatography: Reversed-phase high-performance liquid chromatography (RP-HPLC) with trifluoroacetic acid as an ion-pairing reagent is commonly used during isolation of these peptides .

• Bioassays: Myotropic bioassays using the isolated hindgut from cockroaches (particularly Leucophaea maderae) have been effective for monitoring the activity of these peptides .

For optimal results, researchers should combine these techniques, starting with the isolation of single PSOs, followed by extraction, separation by RP-HPLC, identification by ESI-QTOF MS, sequence confirmation by Edman degradation, and functional characterization using bioassays.

How can recombinant PVK-1 be most effectively expressed and purified for experimental studies?

Based on research methodologies, recombinant PVK-1 can be effectively expressed and purified using the following approaches:

• Expression Systems:

  • E. coli expression systems are commonly used for cost-effective production

  • Yeast and baculovirus expression systems may provide better post-translational modifications for functional studies

  • For the BNGR receptor studies, expression of receptor constructs was performed in HEK293 cells

• Cloning Strategy:

  • PCR amplification of the target sequence using primers with appropriate restriction sites

  • Cloning into suitable expression vectors (examples from receptor studies include pCMV-FLAG, pEGFP-N1)

• Purification Process:

  • HPLC purification with C-18 columns provides high purity (>95%)

  • TFA removal service is recommended for peptides used in cellular assays

For recombinant expression, researchers should consider using the DNA sequence coding for the mature peptide with a suitable fusion tag to facilitate purification, followed by tag removal and verification of the final product by mass spectrometry.

What are the primary physiological functions of PVK-1 in insects, and how are they experimentally demonstrated?

The primary physiological functions of PVK-1 include:

• Myotropic Activity: PVK-1 mediates visceral muscle contractile activity. This is demonstrated using myotropic bioassays where the peptide increases the tonus, frequency, and amplitude of contractions in isolated hindgut preparations . The experimental setup involves suspending the hindgut in an aerated saline solution and connecting it to a muscle transducer to monitor contractile activity.

• Neuromodulation: As a neuropeptide, PVK-1 is involved in fine-tuning the activity of other glands, particularly those controlled by the brain .

• Possible Diuretic Activity: Based on studies of related PVKs, these peptides may play a role in water and ion balance, though this would need specific demonstration for Blaberus craniifer PVK-1 .

Experimental approaches to demonstrate these functions include:

• Isolated organ bioassays (particularly hindgut contractility)

• Receptor binding and activation studies using cell lines expressing the receptor

• RNA interference (RNAi) to silence the receptor and observe physiological effects, as demonstrated in studies with Rhipicephalus microplus

How do the receptors for PVK-1 function, and what signaling pathways do they activate?

PVK-1 peptides interact with G protein-coupled receptors (GPCRs). Based on studies of similar systems:

• Receptor Activation: PVK-1 binds to its cognate GPCR, which primarily couples to Gq proteins rather than Gs or Gi proteins .

• Signaling Cascade:

  • Upon activation, the receptor triggers a Gq-dependent PKC/CREB cascade

  • This leads to increased intracellular calcium mobilization in a Gq inhibitor–sensitive manner

  • The activated receptor does not significantly increase cAMP production, confirming Gq rather than Gs involvement

• Receptor Dynamics:

  • After activation, the receptor undergoes internalization from the cell surface to the cytoplasm via a β-arrestin–dependent pathway

  • This internalization regulates the strength and duration of receptor-mediated cell signaling

• Downstream Effects:

  • Activation leads to ERK1/2 phosphorylation

  • The CRE-driven luciferase activity increases significantly upon receptor stimulation by the peptide

Experimental approaches to study these pathways include using specific inhibitors, measuring second messenger production, monitoring receptor internalization with fluorescently tagged receptors, and using reporter gene assays.

How do PVK-1 peptides vary across different insect species, and what does this reveal about their evolutionary conservation?

PVK-1 peptides show both conservation and variation across insect species, revealing important evolutionary insights:

• Sequence Conservation:

  • The N-terminus is highly conserved across species

  • Only the penultimate amino acid residue (Arg) is consistently found in all members of this peptide family

  • The sequence GSSGLIPFGRT-NH₂ is found in Leucophaea maderae (Lem-PVK-1)

  • In Rhipicephalus microplus, the sequence is pQGLIPFPRVa, which differs from the previously identified sequence PALIPFPRVa in their two N-terminal residues

• Phylogenetic Implications:

  • Analysis of CAPA peptides from 61 species has generated cladograms that align with recent molecular and morphological phylogenetic analyses

  • These analyses support the recent phylogenetic arrangement placing termites within cockroaches

  • The topology remains consistent when data from other neuropeptides (adipokinetic hormones and sulfakinins) are included

• Intraspecies Variation:

  • No sequence variation has been observed between males, females, and larvae within cockroach populations

  • Even when comparing laboratory-raised populations with field-collected specimens of species like Blaberus craniifer, no sequence variations were found

This high degree of conservation suggests strong evolutionary pressure to maintain these sequences, indicating their critical physiological importance across diverse insect taxa.

What structural and functional differences exist between recombinant and naturally occurring PVK-1?

Comparing recombinant and naturally occurring PVK-1:

• Structural Differences:

  • Recombinant PVK-1 may lack certain post-translational modifications present in the natural peptide, particularly when expressed in bacterial systems

  • The C-terminal amidation, critical for bioactivity, must be properly incorporated in recombinant systems

  • Different expression systems (E. coli, yeast, baculovirus) may yield varying degrees of authentic modification

• Functional Considerations:

  • Properly produced recombinant peptides should exhibit similar receptor binding and activation properties

  • Studies have shown that synthetic PVK peptides can activate receptors at nanomolar concentrations (EC₅₀ = 64 nM for Rhimi-CAPA-PVK1)

  • The presence of trifluoroacetic acid (TFA) salts in synthetic or recombinant peptides may affect cellular assays, causing interference with cellular proliferation

• Experimental Implications:

  • For cellular assays, TFA removal service is recommended as TFA can act as an unintended allosteric modulator of certain receptors

  • When using recombinant peptides, researchers should verify bioactivity through functional assays to ensure equivalence to natural peptides

How can RNAi approaches be used to study PVK-1 receptor function in vivo?

RNA interference (RNAi) provides powerful tools for studying PVK-1 receptor function in vivo:

• dsRNA Design Strategy:

  • Multiple dsRNA targets should be designed and tested (e.g., ds680-805, ds956-1109, ds1102-1200 for the Rhipicephalus microplus CAP2b receptor)

  • BLASTn searches should be conducted to assess the risk of non-target effects

  • Sequences with minimal identity to other genes should be selected to ensure specificity

• Delivery Methods:

  • Direct injection of dsRNA into the hemocoel is effective for larger insects

  • Controls should include non-injected specimens and dsRNA targeting irrelevant genes (e.g., beta-lactamase)

  • Positive controls using essential genes (e.g., beta-actin) help validate the RNAi technique

• Phenotypic Assessment:

  • Multiple physiological parameters should be monitored, including survival, weight, and reproductive output

  • Tissue-specific effects can be evaluated by dissecting relevant organs

  • Silencing efficiency should be verified by quantitative reverse-transcriptase PCR in whole specimens and dissected tissues

• Experimental Design Considerations:

  • Statistical validity requires proper blocking of treatments

  • Multiple biological replicates are essential

  • Time-course studies may reveal developmental or stage-specific effects

RNAi provides unique insights into receptor function that complement in vitro studies, revealing the integrated physiological roles of these signaling systems in the whole organism.

What are the most promising approaches for using PVK-1 knowledge in the development of selective insect control strategies?

Leveraging PVK-1 research for insect control strategies:

• Receptor-Targeted Approaches:

  • Since PVK-1 receptors are G-protein coupled receptors, they represent targetable sites for novel insecticides

  • The silencing of CAP2b/PVK receptor in Rhipicephalus microplus caused reduced survival, weight, and reproductive output, indicating potential for pest control applications

  • Selective antagonists of these receptors could disrupt critical physiological processes in pest species

• Peptide-Based Strategies:

  • Modified PVK-1 analogs could function as competitive antagonists

  • Understanding species-specific differences in PVK sequences allows for the design of compounds that selectively target pest species while sparing beneficial insects

  • Research on structure-activity relationships can guide the design of metabolically stable analogs with enhanced potency

• Genetic Approaches:

  • CRISPR-Cas9 technology could potentially be used to modify PVK receptor genes in pest populations

  • Gene drive systems incorporating PVK receptor targets might spread deleterious traits through pest populations

• Methodological Considerations:

  • Efficacy testing should include both in vitro receptor binding/activation assays and in vivo testing on target pests

  • Environmental safety assessments must evaluate effects on non-target organisms, particularly beneficial insects

  • Resistance management strategies should be incorporated into development plans

This approach represents an example of rational insecticide design based on understanding of invertebrate-specific neuroendocrine systems.

What are the optimal storage and handling conditions for recombinant PVK-1 to maintain its stability and bioactivity?

For optimal handling of recombinant PVK-1:

For long-term storage, the lyophilized peptide is most stable. When working with solutions, consider that these peptides can adhere to glass and certain plastics, so low-binding materials are recommended. The use of protease inhibitors may be beneficial when working with cell or tissue preparations.

What are the key considerations when designing a functional assay to measure PVK-1 activity?

When designing functional assays for PVK-1 activity:

• Receptor Activation Assays:

  • Cell lines expressing the PVK receptor (e.g., HEK293 cells) can be used

  • CRE-driven luciferase assay is effective for measuring receptor activation

  • Calcium mobilization assays detect Gq-mediated signaling

  • Include controls with specific G-protein inhibitors to confirm signaling pathway

• Myotropic Bioassays:

  • Isolated hindgut preparations from cockroaches provide a classical bioassay system

  • The preparation should be suspended in aerated saline solution and connected to a muscle transducer

  • Allow approximately one hour for equilibration before testing

  • With proper setup, up to 80 samples can be tested per day

  • Monitor alterations in the pattern of spontaneous contractile activity (stimulatory or inhibitory)

• Binding Assays:

  • Use fluorescently labeled PVK-1 (e.g., 5-FAM–tagged peptide) for direct binding studies

  • This approach can confirm whether the peptide directly binds to its putative receptor

• Receptor Internalization Assays:

  • Use receptors tagged with fluorescent proteins (e.g., EGFP) to monitor internalization

  • This provides a visual confirmation of receptor activation and downstream processing

These complementary approaches provide a comprehensive assessment of PVK-1 activity, from receptor binding to physiological effects, and should be selected based on the specific research questions being addressed.

What are the most significant gaps in our understanding of PVK-1 biology that warrant further investigation?

Despite considerable advances, several significant knowledge gaps remain:

• Receptor Structure-Function Relationships:

  • The precise binding pocket and activation mechanism of PVK-1 receptors remain undefined

  • Structural studies using techniques like cryo-EM would illuminate receptor-ligand interactions

  • Understanding these details could guide the design of selective agonists and antagonists

• Integrated Physiological Roles:

  • While myotropic effects are established, the broader physiological significance of PVK-1 in development, reproduction, and stress responses requires investigation

  • The interplay between PVK-1 and other neuropeptide systems remains largely unexplored

  • The potential role in insect immunity or response to pathogens warrants examination

• Evolutionary Aspects:

  • The evolutionary origin of the PVK family and its relationship to similar peptides in other invertebrates need clarification

  • Functional evolution of these peptides across diverse insect orders could reveal adaptation mechanisms

  • The genomic organization of PVK genes and their regulatory elements remains incompletely characterized

• Applied Research Needs:

  • Development of high-throughput screening systems for PVK receptor modulators

  • Investigation of species-specific receptor pharmacology to enable selective targeting

  • Understanding of resistance mechanisms that might develop against PVK-targeted control strategies

Addressing these gaps will require interdisciplinary approaches combining molecular biology, structural biology, physiology, and evolutionary studies.

How might advanced techniques like CRISPR-Cas9 genome editing enhance our ability to study PVK-1 function in model insects?

CRISPR-Cas9 technology offers transformative possibilities for PVK-1 research:

• Receptor Modification Strategies:

  • Precise knockout of PVK-1 receptor genes to create loss-of-function models

  • Introduction of point mutations to study structure-function relationships

  • Creation of fluorescently tagged receptors at endogenous loci for live imaging of receptor dynamics

  • Generation of conditional knockout systems to study developmental stage-specific effects

• Peptide Precursor Editing:

  • Modification of the PVK-1 precursor gene to alter peptide sequence or processing

  • Creation of null mutants to study complete loss-of-function phenotypes

  • Introduction of reporter genes under the control of native PVK promoters to track expression patterns

• Methodological Considerations:

  • Delivery of CRISPR components via microinjection into embryos

  • Screening strategies using phenotypic markers or molecular techniques

  • Validation of edits using sequencing and functional assays

  • Use of appropriate controls, including off-target analysis

• Research Applications:

  • Comparative studies across multiple insect models using identical modifications

  • Investigation of tissue-specific functions through conditional expression systems

  • Creation of humanized insect models expressing mammalian receptors to study evolutionary conservation

  • Development of gene drive systems for potential pest control applications

CRISPR technology allows for previously impossible precision in manipulating the PVK system, potentially revealing functions that have been difficult to discern using traditional pharmacological or RNAi approaches.