Recombinant Panchlora sp. Sulfakinin-1

What is Panchlora sp. Sulfakinin-1 and how does it compare to other insect sulfakinins?

Panchlora sp. Sulfakinin-1 is a neuropeptide belonging to the sulfakinin (SK) family found in the cockroach genus Panchlora. Sulfakinins function as important signal molecules in invertebrates, mediating various behavioral processes and physiological functions through interaction with G-protein-coupled receptors (GPCRs) .

Like other insect sulfakinins, Panchlora sp. Sulfakinin-1 likely contains characteristic tyrosine (Tyr) residues that serve as potential sulfation sites and glycine (Gly) residues that function as amidation sites . The amino acid sequence would be expected to show high homology with sulfakinins from other cockroach species, such as those identified in Periplaneta americana and other Blattodea.

Structurally, insect sulfakinins share similarities with mammalian cholecystokinin (CCK) and gastrin peptides, suggesting evolutionary conservation of these signaling molecules across diverse animal phyla .

What are the primary physiological roles of sulfakinin in insects?

Sulfakinins serve as multifunctional neuropeptides in insects with several well-documented physiological roles:

• Feeding regulation: Sulfakinin acts as a satiety signal that reduces food intake in multiple insect species. Injection of sulfated and nonsulfated analogs of SK has been shown to inhibit food consumption in beetles like Tribolium castaneum . In Drosophila melanogaster, it suppresses the digestive system .

• Behavioral modulation: Sulfakinin mediates the transition between foraging and mating behaviors through peripheral and central nervous system actions . In the fruit fly Bactrocera dorsalis, the Sk-SkR1 signaling is required to promote foraging by enhancing antennal sensitivity to food odorants during starvation .

• Metabolic regulation: Injection of sulfakinin in Dendroctonus armandi caused significant changes in metabolic parameters, including an increase in trehalose levels and decreases in glycogen and free fatty acid concentrations .

• Sexual behavior modulation: Sulfakinin can inhibit both male and female sexual behavior in D. melanogaster, although there are some contradictory reports suggesting it may promote receptivity in virgin females .

How are sulfakinin receptor systems organized in insects?

The sulfakinin signaling system in insects consists of SK peptides and their receptors (SKRs), which belong to the G-protein-coupled receptor (GPCR) family. These receptors show significant homology to mammalian cholecystokinin receptors (CCKRs) .

Typically, insects possess two distinct sulfakinin receptors:

• SkR1: In B. dorsalis, SkR1 is significantly upregulated in the antennae of starved flies and plays a crucial role in the behavioral switch from mating to foraging . SkR1 is expressed in odorant receptor (OR) neurons, indicating that sulfakinin acts directly on these neurons to alter olfactory perception .

• SkR2: While less is known about SkR2 than SkR1, it represents the second type of sulfakinin receptor found in insects. In B. dorsalis, SkR2 was not upregulated during starvation, suggesting differential roles for the two receptor types .

The sulfakinin receptors trigger downstream signaling cascades that result in altered gene expression patterns, particularly affecting odorant receptor genes during changes in physiological states such as starvation .

What are the most effective methods for producing recombinant Panchlora sp. Sulfakinin-1?

While the search results don't specifically address recombinant production of Panchlora sp. Sulfakinin-1, established methodologies for recombinant neuropeptide production can be applied:

• Gene synthesis and cloning: The first step involves identifying and amplifying the sulfakinin gene from Panchlora sp. Using degenerate primers designed from conserved regions of known sulfakinin genes, PCR amplification can be performed to obtain the full-length sequence . This approach was successfully used for cloning tropomyosin from Periplaneta americana .

• Expression system selection: For small peptides like sulfakinins, bacterial expression systems using E. coli are common, though eukaryotic systems like yeast or insect cells may provide better post-translational modifications.

• Fusion protein strategy: To improve expression and facilitate purification, the target peptide is often expressed as a fusion protein with tags like His6, GST, or MBP.

• Purification protocol: A typical workflow would include:

  • Metal affinity chromatography for His-tagged proteins

  • Enzymatic cleavage to remove the fusion tag

  • Reverse-phase HPLC for final purification

  • Mass spectrometry to confirm identity and purity

• Peptide sulfation: Since the biological activity of sulfakinins often depends on tyrosine sulfation, enzymatic sulfation using tyrosylprotein sulfotransferase or chemical sulfation methods may be necessary for producing biologically active recombinant peptide.

How can researchers verify the biological activity of recombinant Panchlora sp. Sulfakinin-1?

Verification of biological activity for recombinant Panchlora sp. Sulfakinin-1 would involve:

• Receptor binding assays: Using cells expressing sulfakinin receptors (SkR1 or SkR2) to measure binding affinity and activation potential of the recombinant peptide.

• Calcium mobilization assays: Since sulfakinin receptors are GPCRs, their activation can be monitored by measuring intracellular calcium flux in cells expressing these receptors .

• Feeding behavior assays: Microinjection of the recombinant peptide into test insects followed by quantification of food intake. Based on studies with other sulfakinins, the peptide should reduce feeding when injected into insects .

• Weight change monitoring: In D. armandi, sulfakinin injection caused significant reduction in body weight and increased mortality . Similar parameters could be measured after administering recombinant Panchlora sp. Sulfakinin-1.

• Metabolic parameter measurements: Analyzing trehalose, glycogen, and free fatty acid levels before and after peptide administration .

• Electrophysiological studies: Electroantennogram (EAG) recordings to assess changes in olfactory sensitivity in response to food or pheromone odors after peptide treatment .

What RNA interference techniques are most effective for studying sulfakinin function?

Based on the search results, effective RNA interference (RNAi) techniques for studying sulfakinin function include:

• dsRNA design and synthesis:

  • Design gene-specific primers for the SK and SKR genes based on obtained sequences

  • Amplify target regions (typically 300-500 bp)

  • Synthesize dsRNA using in vitro transcription

• Delivery methods:

  • Microinjection using Hamilton Microliter syringes with fine needles (32G) has proven effective for delivering dsRNA into adult and larval insects

  • The typical dosage is approximately 0.15 μL dsRNA solution at a concentration of 1,000 ng/μL

• Controls:

  • Include a control group injected with dsRNA targeting a non-insect gene like GFP (green fluorescent protein)

• Time course:

  • Collect samples at multiple time points (24, 48, and 72 hours post-injection) to monitor the progression of gene silencing

• Validation of knockdown:

  • Use quantitative RT-PCR to verify the reduction in transcript levels of target genes

  • Immunohistochemistry with specific antibodies can confirm protein-level reduction

• Phenotypic analysis:

  • Monitor food intake, body weight, metabolic parameters, and behavioral changes

  • In D. armandi, RNAi knockdown of SK and SKR resulted in increased body weight

  • In B. dorsalis, disruption of the sulfakinin gene using CRISPR/Cas9 confirmed the absence of sulfakinin immunoreactive signal in the central nervous system

How does sulfakinin signaling modulate olfactory perception in insect antennae?

Sulfakinin signaling plays a crucial role in modulating olfactory perception in insect antennae, particularly during physiological state changes like starvation. The process involves:

• Receptor expression in antennae: SkR1 is significantly upregulated in the antennae of starved insects, suggesting a direct role in olfactory modulation .

• Localization in OR neurons: Immunohistochemistry studies have shown that a subset of clustered odorant receptor (OR) neurons in the antennae express SkR1, indicating that sulfakinin acts directly on these neurons .

• Differential regulation of OR genes: Sulfakinin signaling regulates the expression of different sets of OR genes:

  • Starvation-induced OR genes (OR7a.4, OR7a.8, OR10a) that detect food volatiles are upregulated

  • Starvation-suppressed OR genes (OR49a, OR63a) that detect pheromones are downregulated

• Functional consequences: This reprogramming of the OR repertoire leads to:

  • Enhanced sensitivity to food odors (ethyl benzoate, ethyl butyrate, diethyl maleate, methyl eugenol)

  • Reduced sensitivity to sex pheromone components

• Integration with central signaling: The peripheral modulation by sulfakinin coordinates with central neural circuits also controlled by sulfakinin to trigger successful behavioral switches between foraging and mating .

This mechanism represents a sophisticated way for insects to adapt their sensory systems based on their physiological needs, prioritizing either food-seeking or reproductive behaviors.

What is the relationship between sulfakinin and other neuropeptides in regulating insect behavior?

Sulfakinin functions as part of a complex network of neuropeptides that collectively regulate insect behavior. Key relationships include:

• Complementary actions with short neuropeptide F (sNPF):

  • Both sNPF and sulfakinin receptors (sNPFR and SkR1) are upregulated in the antennae of starved flies

  • sNPF enhances sensitivity to food odors, similar to sulfakinin's role

  • These peptides likely work in parallel to control foraging behavior

• Interaction with SIFamide signaling:

  • SIFamideR1 is also upregulated during starvation

  • SIFamide regulates mating behavior, though its precise action on olfactory circuits remains to be clarified

• Functional overlap with tachykinin:

  • While sulfakinin enhances sensitivity to food odors, tachykinin inhibits sensitivity to aversive odors

  • Tachykinin also inhibits courtship behavior by mediating the perception of anti-aphrodisiac pheromones

• Relationship with neuropeptide F (NPF):

  • NPF regulates foraging behavior at the level of the olfactory circuit

  • NPF neurons are required to detect female sex pheromones

• Coordination with ion transport peptide (ITP):

  • ITP-like peptide is induced by starvation, suggesting a potential role in the behavioral switch

This network of neuropeptides provides insects with a sophisticated regulatory system for adapting behavior according to their physiological state. While sulfakinin plays a major role, the incomplete suppression of foraging behavior in sulfakinin mutants suggests that these other neuropeptide systems provide redundancy or complementary control mechanisms .

How do mutations in sulfakinin receptor genes affect insect physiology and behavior?

Research using CRISPR/Cas9-generated mutants has revealed several important effects of sulfakinin receptor gene mutations on insect physiology and behavior:

These findings demonstrate that sulfakinin receptor mutations have pleiotropic effects on insect physiology and behavior, affecting feeding, foraging, sensory perception, and reproductive activities. The effects are consistent with sulfakinin's role in coordinating behavioral responses to changing physiological states.

How conserved is sulfakinin structure and function across different insect orders?

Sulfakinin structure and function show remarkable conservation across different insect orders, though with some lineage-specific adaptations:

This high degree of conservation makes sulfakinin research in one insect species often translatable to others, while still requiring attention to species-specific differences.

What are the molecular mechanisms of sulfakinin-mediated regulation of feeding behavior?

Sulfakinin mediates feeding regulation through multiple molecular mechanisms operating at different levels of the nervous system:

• Central nervous system effects:

  • Sulfakinin acts as a satiety signal in the central nervous system

  • In Drosophila, knocking down SK in the brain leads to increased food intake

  • It affects the ability to distinguish different quality foods

• Peripheral sensory modulation:

  • In B. dorsalis, sulfakinin signaling via SkR1 in the antennae alters the expression of odorant receptor genes during starvation

  • This reprogramming enhances sensitivity to food odors, thereby increasing foraging success

  • The process involves upregulation of specific OR genes (OR7a.4, OR7a.8, OR10a) that detect food volatiles

• Gustatory system regulation:

  • In D. melanogaster, sulfakinin inhibits the sugar receptor GR64 in the proboscis and proleg tarsi

  • This inhibition reduces food ingestion by dampening the detection of sweet compounds

• Metabolic effects:

  • Injection of sulfakinin in D. armandi leads to:

    • Increased trehalose levels

    • Decreased glycogen concentrations

    • Reduced free fatty acid levels

  • These metabolic changes likely contribute to the satiety effect

• Receptor-mediated signaling pathways:

  • Sulfakinin receptors are G-protein-coupled receptors that trigger intracellular signaling cascades

  • Calcium mobilization assays have demonstrated that OR neurons respond to sulfakinin through SkR1

  • These signaling events ultimately lead to changes in gene expression and neuronal activity

• Integration with other neuropeptide systems:

  • Sulfakinin works in concert with other neuropeptides such as sNPF, NPF, tachykinin, and SIFamide

  • This integration allows for fine-tuned regulation of feeding behavior based on physiological state

What are the methodological challenges in studying recombinant sulfakinin in different insect species?

Researchers face several methodological challenges when studying recombinant sulfakinin across insect species:

• Sequence identification and cloning:

  • Species-specific variations in sulfakinin sequences necessitate custom primer design

  • Degenerate primers based on conserved regions can help identify novel sulfakinin genes

  • Complete gene characterization often requires both 5' and 3' RACE techniques to capture the full sequence

• Post-translational modifications:

  • Sulfakinins require specific post-translational modifications, particularly tyrosine sulfation, for full biological activity

  • Producing properly sulfated recombinant peptides is technically challenging

  • Bacterial expression systems lack the enzymes for sulfation, necessitating either:

    • Chemical sulfation post-purification

    • Use of eukaryotic expression systems with co-expression of sulfotransferases

• Functional testing across species:

  • Cross-species variation in sulfakinin receptors may affect binding affinity and response

  • Receptor activation assays need to be optimized for each species

  • Standardized functional assays for comparing activities across species are lacking

• Delivery methods:

  • Microinjection techniques must be adapted to the size and anatomy of different insect species

  • Dosage optimization is necessary for each species to avoid non-specific effects

  • The injection site can significantly affect peptide distribution and biological response

• Genetic manipulation challenges:

  • CRISPR/Cas9 protocols need to be optimized for each insect species

  • Efficiency of RNAi varies dramatically across insect orders, with some showing robust responses and others being relatively refractory

  • Generation of stable transgenic lines is still difficult for many non-model insect species

• Physiological assay standardization:

  • Feeding assays, metabolic measurements, and behavioral tests need to be tailored to each species' biology

  • Controlling for developmental stage, sex, and physiological state is essential for meaningful comparisons

How can recombinant sulfakinin be used for pest control applications?

Recombinant sulfakinin offers several potential applications for pest control strategies:

• Appetite suppression:

  • As sulfakinin acts as a satiety signal, recombinant peptides could be developed to reduce feeding in pest insects

  • In D. armandi, sulfakinin injection caused significant reduction in body weight and increased mortality

  • Delivery systems could include transgenic plants expressing the peptide or peptide-based sprays

• Disruption of foraging behavior:

  • By targeting the sulfakinin signaling pathway, it may be possible to disrupt the switch to foraging behavior during starvation

  • This could reduce crop damage by preventing efficient food location by pest insects

• Interference with mating behavior:

  • Since sulfakinin also regulates mating behavior, manipulating this pathway could potentially disrupt reproductive success

  • This approach could complement existing mating disruption strategies

• Target validation for novel insecticides:

  • Sulfakinin receptors (SkR1 and SkR2) represent potential targets for the development of small-molecule insecticides

  • Recombinant sulfakinin can be used in high-throughput screening assays to identify compounds that mimic or block its activity

• Species-specific control strategies:

  • While sulfakinin is conserved across insects, species-specific variations exist

  • These differences could potentially be exploited to develop more selective control methods

  • Recombinant Panchlora sp. Sulfakinin-1 could be used as a tool to identify such species-specific differences

The search results specifically note that understanding sulfakinin signaling "provides a potential molecular target for the control of this pest" and that the authors' work with B. dorsalis "provided new insight into the control of behavior by sulfakinin in B. dorsalis, which is one of the most widely distributed, fecund, and invasive insect pests to threaten global agriculture" .

What are the most promising directions for future research on insect sulfakinins?

Several promising research directions for insect sulfakinins emerge from the current literature:

• Comprehensive receptor characterization:

  • Further characterization of SkR1 and SkR2 in diverse insect species

  • Investigation of receptor-specific signaling pathways and downstream effects

  • Development of receptor-specific agonists and antagonists for experimental manipulation

• Neuromodulatory network mapping:

  • Detailed mapping of interactions between sulfakinin and other neuropeptide systems

  • Understanding how these different signals are integrated to produce coordinated behavioral outputs

  • Investigation of potential feedback loops between different neuropeptide systems

• Peripheral sensory modulation mechanisms:

  • Further exploration of how sulfakinin modulates peripheral sensory systems

  • Investigation of the transcriptional and post-transcriptional mechanisms by which sulfakinin regulates OR gene expression

  • Identification of the complete set of sensory neurons expressing sulfakinin receptors across different sensory modalities

• Evolutionary analysis:

  • Comparative genomic analysis of sulfakinin systems across arthropod lineages

  • Investigation of how sulfakinin signaling has evolved and diversified

  • Understanding the relationship between sulfakinin and related peptides in other invertebrates and vertebrates

• Applied research:

  • Development of stable, bioavailable sulfakinin analogs for potential pest control applications

  • Investigation of the potential for sulfakinin-based approaches to complement existing integrated pest management strategies

  • Exploration of sulfakinin receptor antagonists as novel insecticide leads

• Integration with other physiological systems:

  • Investigation of how sulfakinin signaling interacts with hormonal systems, immune function, and developmental processes

  • Understanding the role of sulfakinin in stress responses and adaptation to changing environments

The continued characterization of the Sk-SkR system at molecular, cellular, and behavioral levels promises to yield important insights into insect physiology and potentially valuable applications in agricultural pest management.

How do contradictory findings about sulfakinin function inform our understanding of its biological complexity?

The literature contains several seemingly contradictory findings about sulfakinin function that actually reveal the complexity of this signaling system:

• Dual roles in foraging versus satiety:

  • Contradiction: Sulfakinin is generally known as a satiety signal that reduces food intake , yet the Sk-SkR1 signal is required to promote foraging during starvation .

  • Resolution: This apparent contradiction highlights the context-dependent nature of sulfakinin signaling. It appears to have distinct roles in different tissues and physiological states:

    • In the central nervous system, it acts as a satiety signal

    • In the peripheral olfactory system, it enhances food detection during starvation

  • This dual action allows for coordinated regulation of both food-seeking and food consumption behaviors.

• Effects on sexual behavior:

  • Contradiction: Some studies show that sulfakinin inhibits both male and female sexual behavior in D. melanogaster , while others report that it promotes receptivity in mating in virgin females .

  • Resolution: These differences likely reflect:

    • Sex-specific effects of sulfakinin

    • Differential roles of Sk1 versus Sk2 peptides

    • Context-dependent actions based on reproductive status

    • Involvement of different receptor subtypes (SkR1 vs. SkR2)

• Species-specific differences:

  • Different insect species show variations in sulfakinin responses despite sequence conservation

  • This suggests that downstream signaling pathways or receptor distribution patterns may have evolved differently

  • These differences underscore the importance of studying sulfakinin across diverse insect taxa rather than generalizing from model species

• Systemic versus local effects:

  • Contradiction: Sk gene expression doesn't change in antennae during starvation, yet SkR1 is upregulated .

  • Resolution: This suggests that circulating Sk released from the central nervous system, rather than locally produced Sk, activates antennal SkR1 receptors .

  • This illustrates how sulfakinin functions both as a local neuromodulator and as a systemic hormone.

• Integration with multiple neuropeptide systems:

  • The incomplete suppression of foraging in Sk and SkR1 mutants indicates redundancy with other systems

  • This suggests that sulfakinin is one component of a complex, integrated network of neuropeptide signals

  • Different experimental conditions may reveal different aspects of this network, leading to apparently contradictory results

These contradictions and their resolutions demonstrate that sulfakinin signaling is highly complex, with context-dependent actions modulated by physiological state, sex, developmental stage, and species-specific adaptations. This complexity makes it a fascinating but challenging subject for scientific investigation.

What are the key structural characteristics of known insect sulfakinins?

Table 1: Structural Characteristics of Representative Insect Sulfakinins

Note: All sulfakinins share the characteristic C-terminal RF-amide motif and contain tyrosine residues that can be sulfated post-translationally. The sulfation status significantly affects biological activity in most cases. The exact sequence of Panchlora sp. Sulfakinin-1 would need to be determined experimentally but would likely follow similar structural patterns to other cockroach sulfakinins.

How does sulfakinin affect insect metabolic parameters across different species?

Table 2: Effects of Sulfakinin on Insect Metabolic Parameters

This table demonstrates that across multiple insect species, sulfakinin generally acts to reduce food intake and body weight, while also modulating carbohydrate and lipid metabolism. The specific effects on trehalose, glycogen, and free fatty acids have been most thoroughly documented in D. armandi, where SK injection increased trehalose while decreasing glycogen and free fatty acids .

What gene expression changes are induced by sulfakinin receptor activation in insect antennae?

Table 3: Odorant Receptor Gene Expression Changes in Response to Sulfakinin Signaling in Bactrocera dorsalis Antennae

This table summarizes how sulfakinin signaling via SkR1 regulates the expression of odorant receptor genes in B. dorsalis antennae during starvation. The starvation-induced changes in gene expression (upregulation of food odor receptors and downregulation of pheromone receptors) are diminished in both sk−/− and skr1−/− mutants . This reprogramming of the OR repertoire represents a molecular mechanism by which sulfakinin mediates the behavioral switch from mating to foraging during starvation.