Divergent venoms among two closely related co-distributed centipede species, Scolopendra morsitans and S. hardwickei in tropical Asia

This study utilizes an integrated proteo-transcriptomic approach to demonstrate that two closely related, co-distributed centipede species in peninsular India, *Scolopendra morsitans* and *S. hardwickei*, exhibit significant divergence in their venom protein repertoires and abundances, suggesting that distinct venom compositions may facilitate their ecological coexistence.

The Gist

Imagine two neighbors living in the same crowded apartment building in a tropical forest. They are very similar: they are the same size, they eat similar food, and they often bump into each other in the hallway. In the animal kingdom, these neighbors are two types of giant centipedes: Scolopendra morsitans and Scolopendra hardwickei.

For a long time, scientists wondered: If these two neighbors are so similar and live in the same place, do they use the same "weapons" to hunt?

This paper is like a forensic investigation into the venom (the poison) these centipedes carry. Here is the story of what they found, explained simply.

1. The "Chemical Cocktail" Analogy

Think of centipede venom not as a single poison, but as a giant, complex cocktail served at a bar.

• Some ingredients are neurotoxins (like a heavy sedative) that shut down the prey's nervous system so it can't move.
• Some are enzymes (like digestive juices) that start breaking down the prey's body before it's even eaten.
• Some are pore-forming toxins (like tiny drills) that punch holes in the prey's cells.

Usually, when two species live together, you might expect them to have the exact same cocktail because they are hunting the same bugs. This is called "convergence." But this study asked: Do they actually have different recipes?

2. The Investigation: A "Proteomic" Taste Test

The researchers didn't just guess; they did a deep dive. They collected venom from 39 different centipedes (28 of one species, 11 of the other) across India.

They used two high-tech tools:

• The Transcriptome (The Recipe Book): They looked at the centipede's DNA instructions to see what could be made.
• The Proteome (The Actual Drink): They analyzed the actual liquid venom to see what was actually poured into the glass.

By combining these, they got a perfect picture of the "menu" each species was serving.

3. The Big Discovery: Different Recipes for Different Neighbors

The result was surprising. Even though these centipedes are close relatives and live in the same neighborhoods, their venoms are completely different.

• The "Morsitans" Bar: This species had a very complex menu with 110 different proteins. It was like a fancy cocktail lounge with 110 unique ingredients. They had 24 special ingredients that the other species didn't have at all.
• The "Hardwickei" Bar: This species had a simpler menu with 84 proteins. They only had 4 unique ingredients that the other species didn't have.

The Key Difference: It wasn't just that they had different ingredients; they used them in different amounts.

• Analogy: Imagine both neighbors have a bottle of "Hot Sauce" (a specific toxin). The Morsitans neighbor puts a whole cup of it in their drink, while the Hardwickei neighbor only adds a single drop. This changes the entire flavor and effect of the drink.

4. Why Do They Have Different Venoms? (The "Niche" Theory)

So, why would two neighbors living in the same building have different weapons?

The paper suggests it's a case of avoiding competition.

• If both neighbors used the exact same poison, they would be fighting over the exact same prey.
• By evolving slightly different venoms, they might be targeting slightly different types of bugs or hunting in slightly different ways.
• The Metaphor: Imagine two pizza shops on the same street. If they both sell the exact same "Pepperoni Supreme," they will fight for every customer. But if one specializes in "Spicy Veggie" and the other in "Cheesy Meat Lovers," they can both survive side-by-side. The venom is their way of saying, "I hunt these bugs; you hunt those bugs."

5. Why This Matters

This study is a big deal for a few reasons:

1. It solves a mystery: It proves that even closely related predators can evolve to be very different to avoid fighting each other.
2. It's a new method: The researchers combined DNA reading with actual protein testing. This is like checking a chef's recipe book and tasting the soup to make sure they match. This is crucial for animals like centipedes, where we don't have much data yet.
3. Future Medicine: Centipede venom is full of powerful chemicals. By understanding exactly what's in these "cocktails," scientists might find new medicines to treat pain, heart disease, or even cancer in the future.

The Bottom Line

These two centipede species are like twin siblings who grew up in the same house but decided to wear different clothes and eat different foods to avoid stepping on each other's toes. Their venom is their unique style, and by being different, they can both thrive in the same tropical forest without killing each other off.

Technical Summary

1. Problem Statement

Functional traits, such as venom composition, are critical for understanding species coexistence and ecological niche differentiation. While venom is a key adaptive trait for predation and defense, its role in mediating competition between closely related, co-occurring arthropod predators remains poorly understood.

• The Gap: There is a lack of empirical evidence regarding whether co-existing venomous species evolve similar venoms due to shared ecological pressures (trait convergence) or divergent venoms to minimize competition (trait divergence/character displacement).
• The Specific Challenge: Studying venom in non-model arthropods is difficult due to the scarcity of genomic resources, the difficulty of extracting sufficient venom for individual-level quantification, and the limitations of using single-method approaches (proteomics or transcriptomics alone) which can lead to false positives or incomplete diversity estimates.
• The Study System: The authors focused on two large, closely related, and co-distributed centipede species in peninsular India: Scolopendra morsitans (widely distributed) and S. hardwickei (endemic to the Indian subcontinent). Both species share similar body sizes and habitats, making them an ideal model to test hypotheses regarding ecological competition and venom evolution.

2. Methodology

The study employed an integrated proteo-transcriptomic approach combined with ecological niche modeling to characterize venom phenotypes across multiple individuals.

• Taxon Sampling & Phylogeny:
  • Collected 59 individuals (28 S. morsitans, 11 S. hardwickei) from 35 unique localities across the Western and Eastern Ghats of India.
  • Reconstructed a molecular phylogeny using mitochondrial (COI, 16S) and nuclear (28S) markers to confirm evolutionary relationships and species boundaries.
• Species Distribution Modeling (SDM):
  • Utilized MaxEnt with bioclimatic variables (WorldClim) and occurrence records (including iNaturalist data) to model geographic ranges.
  • Quantified spatial overlap to confirm co-occurrence and validate the potential for ecological interaction.
• Venom Extraction & Processing:
  • Extracted venom from live individuals via electrostimulation (TENS unit) at the individual level.
  • Dissected venom glands for RNA extraction 4 days post-extraction.
• Transcriptomics:
  • Generated de novo venom gland transcriptomes for one adult of each species using Illumina NovaSeq 6000.
  • Assembled transcripts using Trinity, reduced redundancy with Cd-hit, and quantified expression (TPM) using Kallisto.
  • Used these custom transcriptomes as reference databases for proteomic identification.
• Proteomics:
  • Analyzed venom from all 39 individuals using bottom-up LC-MS/MS (Thermo Easy-nLC 1200 coupled to Q-Exactive).
  • Searched spectra against UniProt and the custom de novo transcriptome-derived protein databases.
  • Applied strict False Discovery Rate (FDR) thresholds (0.01/0.05) to validate protein identifications.
• Statistical Analysis:
  • Compared venom profiles using Jaccard (presence/absence) and Bray-Curtis (abundance) dissimilarity indices.
  • Employed ordination methods (PCA, nMDS) and statistical tests (Kruskal-Wallis, Dunn's post-hoc, PERMANOVA, ANOSIM) to assess intra- vs. interspecific variation.

3. Key Results

• Ecological Overlap: SDMs confirmed significant geographic overlap between S. morsitans and S. hardwickei in peninsular India, particularly in the central and northern Western Ghats, validating their status as co-distributed species.
• Venom Complexity:
  • Protein Count: S. morsitans venom contained 110 proteins, while S. hardwickei contained 84 proteins.
  • Toxin Diversity: S. morsitans exhibited higher toxin richness (mean 49 toxins) and abundance compared to S. hardwickei (mean 41 toxins).
  • Uniqueness: 24 toxin proteins were unique to S. morsitans, whereas only 4 were unique to S. hardwickei.
• Divergence in Phenotypes:
  • Statistical Significance: Interspecific dissimilarity was significantly higher than intraspecific dissimilarity for both presence/absence (Jaccard) and abundance (Bray-Curtis) metrics ($p < 0.001$).
  • Composition: While both species shared a core set of toxins (~65.5% similarity in protein presence), they differed significantly in the relative abundance of specific toxin classes.
  • Key Drivers of Divergence: Principal Component Analysis (PCA) revealed that the primary axes of variation were driven by:
    • PC1: $\beta$-pore-forming toxins ($\beta$-PFTx), γ-glutamyl transpeptidases (GGT), and CUB domain-containing metalloproteases.
    • PC2: Specific scoloptoxins (SLPTX15, SLPTX11).
• Transcriptomic Insights: High-expression transcripts were dominated by structural/metabolic genes (e.g., actin, troponin) rather than toxins, though specific toxins (SLPTX, $\beta$-PFTx, GGT) were highly expressed. The integrated approach identified 26 SLPTX proteins, whereas proteomics alone would have detected only 9, highlighting the necessity of transcriptomic references.

4. Key Contributions

1. First Comprehensive Venom Profile for S. hardwickei: This study provides the first detailed proteomic and transcriptomic characterization of the venom of S. hardwickei, a large endemic Indian centipede.
2. Validation of the Proteo-Transcriptomic Workflow: The study demonstrates that integrating de novo transcriptomes with proteomics is essential for non-model organisms. It significantly improves toxin detection rates and reduces false negatives caused by database limitations (only ~1-2% of proteins matched UniProt entries without custom references).
3. Evidence of Trait Divergence: The research provides empirical evidence that closely related, co-existing arthropod predators exhibit divergent venom phenotypes rather than convergence. This supports the hypothesis that venom variation is a mechanism for niche differentiation and reduced interspecific competition.
4. Functional Annotation: The study expands the functional understanding of centipede venom components, confirming the dominance of $\beta$-PFTx (cytolytic), scoloptoxins (neurotoxic), and enzymes (GGT, metalloproteases) in prey capture and tissue digestion.

5. Significance

• Ecological Theory: The findings support the concept of character displacement, suggesting that ecological competition drives the divergence of functional traits (venom) in sympatric species. This allows S. morsitans and S. hardwickei to potentially exploit distinct dietary niches or microhabitats despite overlapping geographic ranges.
• Evolutionary Biology: The study highlights that venom evolution in centipedes is not static; even closely related species with similar body sizes and habitats can evolve distinct chemical arsenals.
• Methodological Advancement: By overcoming the "database gap" in non-model taxa, this work sets a precedent for future venomics studies in understudied invertebrates, ensuring more accurate assessments of biodiversity and functional traits.
• Biomedical Potential: The identification of novel toxin isoforms and the characterization of high-abundance proteins (like $\beta$-PFTx and GGT) open new avenues for drug discovery, particularly given the known pharmacological potential of centipede venoms.

In conclusion, the paper establishes that venom composition is a dynamic trait shaped by ecological interactions, serving as a critical mechanism for the coexistence of predatory arthropods in tropical forests.