Minimally toxic tetrodotoxin analogs function as rapid chemical warning signals in pufferfish larvae.

Early developmental stages are exceptionally vulnerable to predation, prompting many animals to rely on maternally provisioned chemical defenses. In pufferfish (Takifugu spp.), the potent neurotoxin tetrodotoxin (TTX) has long been assumed to be the primary deterrent. Here, we demonstrate that predator avoidance is robustly elicited by the minimally toxic TTX analog, 5,6,11-trideoxytetrodotoxin (TDT). Crucially, we show that while specific predators ingest TTX-laden larvae, they consistently reject those containing TDT upon first contact. Through sequential predation assays controlling for social learning, we reveal that larvae are actively captured but elicit immediate regurgitation by multiple predator species when TDT is present. Although both compounds are localized within the larval epidermis, TDT provides near-absolute protection against predators that bypass actual toxicity. These findings suggest that TDT functions as a high-efficiency chemical warning signal that prompts rapid unlearned aversion. Our study highlights a complementary survival strategy during vulnerable early life stages, where lethal toxicity and sensory-mediated deterrence complement each other to maximize protection.