Interactions between physiology and behaviour provide insights into the ecological role of venom in Australian funnel-web spiders: Interspecies comparison.

Australian funnel-web spiders are iconic species, characterized as being the most venomous spiders in the world. They are also valued for the therapeutics and natural bioinsecticides potentially hidden in their venom molecules. Although numerous biochemical and molecular structural approaches have tried to determine the factors driving venom complexity, these approaches have not considered behaviour, physiology and environmental conditions collectively, which can play a role in the evolution, complexity, and function of venom components in funnel-webs. This study used a novel interdisciplinary approach to understand the relationships between different behaviours (assessed in different ecological contexts) and morphophysiological variables (body condition, heart rate) that may affect venom composition in four species of Australian funnel-web spiders. We tested defensiveness, huddling behaviour, frequency of climbing, and activity for all species in three ecological contexts: i) predation using both indirect (puff of air) and direct (prodding) stimuli; ii) conspecific tolerance; and iii) exploration of a new territory. We also assessed morphophysiological variables and venom composition of all species. For Hadronyche valida, the expression of some venom components was associated with heart rate and defensiveness during the predation context. However, we did not find any associations between behavioural traits and morphophysiological variables in the other species, suggesting that particular associations may be species-specific. When we assessed differences between species, we found that the species separated out based on the venom profiles, while activity and heart rate are likely more affected by individual responses and microhabitat conditions. This study demonstrates how behavioural and morphophysiological traits are correlated with venom composition and contributes to a broader understanding of the function and evolution of venoms in funnel-web spiders.

Behaviour of the Sydney funnel-web spider Atrax robustus over different contexts, time, and stimuli.

Atrax robustus is an iconic Australian spider because the venom can be lethal to humans. Moreover, some of the venom biomolecules have promise as therapeutic and bioinsecticidal leads. Nonetheless, aspects related to the life history and behaviour of this species, which might influence changes in venom components, have been overlooked. We assessed different behavioural traits (antipredator behaviour, defensiveness and activity) of juveniles and adult females across different contexts (predation, conspecific tolerance and exploration of a new territory) and stimuli (puff of air versus prod) over time. Adults responded to a puff of air faster than juveniles, but in response to a prod, both juveniles and adults become more defensive over time. No differences were observed between adults and juveniles for conspecific tolerance and exploration. Understanding behaviour of venomous species is important because behaviours may affect physiological traits, such as venom, and the ability of spiders to adapt to different conditions.

Variation in venom composition in the Australian funnel-web spiders Hadronyche valida.

Mygalomorph venom properties and active components, which have importance in medicine, agronomy, venomics, ecology and evolution, have been widely studied, but only a small fraction have been characterised. Several studies have shown inter-individual variation in the composition of venom peptides based on ontogeny, sexual dimorphism, season and diet. However, intra-individual variation in venom composition, which could play a key role in the evolution, diversification and function of toxins, is poorly understood. In this study, we demonstrate significant intra- and inter-individual variation in venom composition in the Australian funnel-web spider Hadronyche valida, highlighting that individuals show different venom profiles over time. Fourteen (four juvenile and ten adult females) funnel-web spiders, maintained under the same environmental conditions and diet, were milked a total of four times, one month apart. We then used reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry to generate venom fingerprints containing the retention time and molecular weights of the different toxin components in the venom. Across all individuals, we documented a combined total of 83 individual venom components. Only 20% of these components were shared between individuals. Individuals showed variation in the composition of venom peptides, with some components consistently present over time, while others were only present at specific times. When individuals were grouped using the Jaccard clustering index and Kernel Principal Component Analysis, spiders formed two distinct clusters, most likely due to their origin or time of collection. This study contributes to the understanding of variation in venom composition at different levels (intra-individual, and intra- and inter-specific) and considers some of the mechanisms of selection that may contribute to venom diversification within arachnids. In addition, inter-specific variation in venom composition can be highly useful as a chemotaxonomic marker to identify funnel-web species.