<b>Substrate-specific camouflage and its underlying metabolic pathway genetic basis maintain color polymorphism in a the wandering wolf spider Pardosa pseudoannulata</b>

Integrating these data with avian visual modeling demonstrated that each morph achieves precise spectral matching with its respective microhabitat substrate, whereas mismatched pairings result in elevated visual contrast that makes them highly conspicuous to avian predators. To empirically validate this, we further conducted choice-predation trials using naïve domestic chicks. The result showed a severe survival disadvantage for mismatched spider: grey spiders suffered significantly higher attack rates upon on black soil, whereas black spiders were preferentially preyed upon on grey soil.

Thus, our study provides direct empirical evidence that substrate-specific background matching confers a profound survival advantage against visually oriented predators.</p><p><br></p><p dir="ltr">3. Thirdly, to elucidate the physiological underpinnings this phenotype, we assessed epidermal pigmentation and biochemical activity of spiders in the two color morphs. Fontana–Masson histochemical staining revealed significantly greater area of fraction of cuticular melanin deposition in the black morph, which correlated with significantly elevated levels of epidermal tyrosinase, the rate-limiting enzyme of melanogenesis.

In addition, by <a href="" target="_blank">conducting epidermal transcriptomic profiling analysis, we identified a core network of four candidate genes (</a><i>JHAMT</i>, <i>NIM1</i>, <i>SRF</i>, and <i>TYR</i>) that associated with cuticle development and melanization. All these four genes exhibited marked upregulation in the black morph across both sexes, with <i>TYR</i> (tyrosinase) emerging as the central regulatory node.</p><p><br></p><p dir="ltr">4.

Finally, functional validation via RNA interference (RNAi) confirmed the epistatic role of <i>TYR</i>, as targeted gene silencing successfully depleted epidermal mRNA transcripts and disrupted cuticular darkening in vivo. Take together, this study elucidates the core metabolic pathway and regulatory network driving substrate-specific coloration, highlighting the conserved role of melanin biosynthesis in animal body coloration.</p><p><br></p>