Regulation of venom composition by the Australian funnel-web spider Hadronyche infensa is achieved via compartmentalised toxin production and venom metering.

The ability to regulate the composition of secreted venom has been demonstrated in an increasing number of taxa, including scorpions, centipedes, and assassin bugs. However, the extent to which this regulation occurs across venomous lineages, and how it is achieved remains largely unknown. Here, we investigate the ability of the lethal Australian funnel-web spider Hadronyche infensa to regulate the biochemical composition of its venom. Using a combination of transcriptome-guided mass spectrometry-based proteomic and peptidomic analyses of serial defensive venom secretions from multiple spiders from the same population, we show that venom composition changes both qualitatively and quantitatively across a series of secretions. We also find large intrapopulation variation in venom composition, with high conservation of only a few abundant toxins across all individuals, including two of the most potent insecticidal toxins. Secretion of these insecticidal toxins tends to be preceded by the secretion of non-insecticidal, presumably defensive, toxins. Using mass spectrometry imaging and histological techniques, we show that changes in toxin secretion is facilitated by differential storage of toxins in a morphologically differentiated venom gland. We propose that the spatial segregation of functionally distinct toxins in H. infensa is an adaptation that reduces the cost of defensive venom use. Our results highlight the importance of considering behavioural aspects of natural venom secretions in understanding toxin function and evolution.