Structural Identification, Synthesis and Biological Activity of Two Volatile Cyclic Dipeptides in a Terrestrial Vertebrate

Abstract

Single substances within complex vertebrate chemical signals could be physiologically or behaviourally active. However, the vast diversity in chemical structure, physical properties and molecular size of semiochemicals makes identifying pheromonally active compounds no easy task. Here, we identified two volatile cyclic dipeptides, cyclo(L-Leu-L-Pro) and cyclo(L-Pro-L-Pro), from the complex mixture of a chemical signal in terrestrial vertebrates (lizard genus Sceloporus), synthesised one of them and investigated their biological activity in male intra-specific communication. In a series of behavioural trials, lizards performed more chemosensory behaviour (tongue flicks, lip smacks and substrate lickings) when presented with the synthesised cyclo(L-Pro-L-Pro) chemical blend, compared to the controls, the cyclo(L-Leu-L-Pro) blend, or a combined blend with both cyclic dipeptides. The results suggest a potential semiochemical role of cyclo(L-Pro-L-Pro) and a modulating effect of cyclo(L-Leu-L-Pro) that may depend on the relative concentration of both compounds in the chemical signal. In addition, our results stress how minor compounds in complex mixtures can produce a meaningful behavioural response, how small differences in structural design are crucial for biological activity, and highlight the need for more studies to determine the complete functional landscape of biologically relevant compounds. Chemical signals of terrestrial vertebrates tend to be complex mixtures of compounds 1. However, this does not necessarily mean that numerous compounds are always needed for recognition by a signal receiver (e.g. 2-4). Single compounds, or even a selected profile from all mixture components, could be physiologically or behaviourally active in different contexts 5-8. Intra-specific chemical signals, often liberally referred to as "pheromones" in the extensive literature, can vary considerably in their chemical structure, physical properties and molecular size 9 , and there is currently no simple way to rule out the biological roles of additional mixture components. For example, even in an extensively studied model system such as the house mouse, the biological roles of volatile ligands, compared to the lipocalin proteins that are involved in different chemosensory functions 10-13 , are relatively unknown. Using an interdisciplinary approach, here we characterise two volatile cyclic dipeptides from the complex mixture of a chemical signal in terrestrial vertebrates (lizard genus Sceloporus) and investigate their biological activity in intra-specific communication. The structural diversity of compounds documented in terrestrial vertebrates is enormous 14 , and it has been difficult to associate specific structural designs or features with chemical signalling in general 1. It has been somewhat useful to divide potential chemosignals according to their volatility: while volatile pheromones can act in longer distance signalling, protein-like molecules and other highly polar substances with very low vapour pressure (e.g. polypept...

Evolutionary loss of a signalling colour is linked to increased response to conspecific chemicals

“…Chemical acts and visual displays are typical Sceloporus responses in playback studies to chemical (e.g. [28,29]) and visual stimuli (e.g. [35]), respectively.…”

“…Motion displays also expose ventrolateral and throat colour patches, most frequently found in males and typically blue, which are used during territorial disputes between males to signal aggression and fighting ability [22,23,25,26]. In addition, lizards actively and/or passively deposit scent marks secreted from femoral glands onto the substrate that serve as cues of physiological condition [27], and potentially convey individual and sex identity information [28,29]. Lizards respond to conspecific scents by adjusting aggression [30,31], altering their activity levels and space use within territories [32].…”

Protein–lipid Association in Lizard Chemical Signals

“…Conversely, proteins from femoral glands secretions have been far less studied ( Martín and López 2014 ; Mayerl et al 2015 ; Mangiacotti et al 2017 ; Romero-Diaz et al 2020 ), although they may represent most of the secretion mass ( Alberts 1990 ; Mangiacotti et al 2019b ), they are homogeneously mixed with lipid blend ( Alberts 1990 ; Mangiacotti et al 2017 ), and can be detected by lizards ( Alberts and Werner 1993a ; Cooper et al 2002 ; ). On this basis, two main, not exclusive, functions have been theorized for secretion proteins: On the one hand, the support-to-lipid hypothesis, which states that proteins represent an inert structural matrix that holds and protects lipids in the secretion from fading ( Cole 1966 ); and on the other hand, the semiochemical hypothesis, which considers the proteinaceous fraction to be directly involved in signaling ( Alberts 1990 ; Alberts and Werner 1993a ).…”

Population differences in multimodal lizard communication are not well explained by habitat or history

“…Although these signals are used widely across the genus, different species and populations often vary in display structure and use. For example, syntopic populations of two species, S. virgatus and S. jarrovii , were shown to differ in their basal frequency of chemosensory behavior (Romero‐Diaz et al, 2020; Romero‐Diaz, Campos, et al, 2021) and three populations of S. graciosus differed in the type of body postures and the number of up‐and‐down motions used to produce the visual display (Martins et al, 1998). A more holistic and informative approach would be to test differences in signal use across the two sensory modalities, among populations occupying different habitat types.…”