Proteomics of Galápagos Marine Iguanas Links Function of Femoral Gland Proteins to the Immune System.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

Functional Protein Composition in Femoral Glands of Sand Lizards (Lacerta agilis).

Proteins are ubiquitous macromolecules that display a vast repertoire of chemical and enzymatic functions, making them suitable candidates for chemosignals, used in intraspecific communication. Proteins are present in the skin gland secretions of vertebrates but their identity, and especially, their functions, remain largely unknown. Many lizard species possess femoral glands, i.e., epidermal organs primarily involved in the production and secretion of chemosignals, playing a pivotal role in mate choice and intrasexual communication. The lipophilic fraction of femoral glands has been well studied in lizards. In contrast, proteins have been the focus of only a handful of investigations. Here, we identify and describe inter-individual expression patterns and the functionality of proteins present in femoral glands of male sand lizards (Lacerta agilis) by applying mass spectrometry-based proteomics. Our results show that the total number of proteins varied substantially among individuals. None of the identified femoral gland proteins could be directly linked to chemical communication in lizards, although this result hinges on protein annotation in databases in which squamate semiochemicals are poorly represented. In contrast to our expectations, the proteins consistently expressed across individuals were related to the immune system, antioxidant activity and lipid metabolism as their main functions, showing that proteins in reptilian epidermal glands may have other functions besides chemical communication. Interestingly, we found expression of the Major Histocompatibility Complex (MHC) among the multiple and diverse biological processes enriched in FGs, tentatively supporting a previous hypothesis that MHC was coopted for semiochemical function in sand lizards, specifically in mate recognition. Our study shows that mass spectrometry-based proteomics are a powerful tool for characterizing and deciphering the role of proteins secreted by skin glands in non-model vertebrates.

Seasonal Variations in Femoral Gland Secretions Reveals some Unexpected Correlations Between Protein and Lipid Components in a Lacertid Lizard.

Animals modulate intraspecific signal shape and intensity, notably during reproductive periods. Signal variability typically follows a seasonal scheme, traceable through the expression of visual, acoustic, chemical and behavioral patterns. The chemical channel is particularly important in lizards, as demonstrated by well-developed epidermal glands in the cloacal region that secrete lipids and proteins recognized by conspecifics. In males, the seasonal pattern of gland activity is underpinned by variation of circulating androgens. Changes in the composition of lipid secretions convey information about the signaler's quality (e.g., size, immunity). Presumably, individual identity is associated with a protein signature present in the femoral secretions, but this has been poorly investigated. For the first time, we assessed the seasonal variability of the protein signal in relation to plasma testosterone level (T), glandular activity and the concentration of provitamin D3 in the lipid fraction. We sampled 174 male common wall lizards (Podarcis muralis) over the entire activity season. An elevation of T was observed one to two months before the secretion peak of lipids during the mating season; such expected delay between hormonal fluctuation and maximal physiological response fits well with the assumption that provitamin D3 indicates individual quality. One-dimensional electrophoretic analysis of proteins showed that gel bands were preserved over the season with an invariant region; a result in agreement with the hypothesis that proteins are stable identity signals. However, the relative intensity of bands varied markedly, synchronously with that of lipid secretion pattern. These variations of protein secretion suggest additional roles of proteins, an issue that requires further studies.

Evolutionary Interactions Between Visual and Chemical Signals: Chemosignals Compensate for the Loss of a Visual Signal in Male Sceloporus Lizards.

Animals rely on multimodal signals to obtain information from conspecifics through alternative sensory systems, and the evolutionary loss of a signal in one modality may lead to compensation through increased use of signals in an alternative modality. We investigated associations between chemical signaling and evolutionary loss of abdominal color patches in males of four species (two plain-bellied and two colorful-bellied) of Sceloporus lizards. We conducted field trials to compare behavioral responses of male lizards to swabs with femoral gland (FG) secretions from conspecific males and control swabs (clean paper). We also analyzed the volatile organic compound (VOC) composition of male FG secretions by stir bar extraction and gas chromatography-mass spectrometry (GC-MS) to test the hypothesis that loss of the visual signal is associated with elaboration of the chemical signal. Males of plain-bellied, but not colorful-bellied species exhibited different rates of visual displays when exposed to swabs of conspecific FG secretions relative to control swabs. The VOC composition of male Sceloporus FG secretions was similar across all four species, and no clear association between relative abundances of VOCs and evolutionary loss of abdominal color patches was observed. The emerging pattern is that behavioral responses to conspecific chemical signals are species- and context-specific in male Sceloporus, and compensatory changes in receivers, but not signalers may be involved in mediating increased responsiveness to chemical signals in males of plain-bellied species.

Inter- and intra-population variability of the protein content of femoral gland secretions from a lacertid lizard.

Femoral glands of male lizards produce waxy secretions that are involved in inter- and intraspecific chemical communication. The main components of these secretions are proteins and lipids, the latter having been extensively studied and already associated to male quality. On the opposite, the composition and role of proteins are nearly unknown, the only available information coming from few studies on iguanids. These studies got the conclusion that proteins might have a communicative function, notably they could signal individual identity. A generalization of these findings requires the extension of protein analysis to other lizard families, and the primary detection of some patterns of individual variability. Using the common wall lizard Podarcis muralis as a model species, the protein fraction of the femoral pore secretions was investigated to provide the first characterization of this component in a lacertid lizard and to explore its source of variability, as a first step to support the hypothesized communicative role. Samples of proteins from femoral secretions were collected from 6 Italian populations and subjected to 1-dimensional electrophoresis. The binary vector of the band presence/absence was used to define the individual profiles. Protein fraction is found to have a structured pattern, with both an individual and a population component. Although the former supports the potential communicative role of proteins, the latter offers a double interpretation, phylogenetic or environmental, even though the phylogenetic effect seems more likely given the climatic resemblance of the considered sites. Further studies are necessary to shed light on both these issues.

Diversity of compounds in femoral secretions of Galápagos iguanas (genera: Amblyrhynchus and Conolophus), and their potential role in sexual communication in lek-mating marine iguanas (Amblyrhynchus cristatus).

BACKGROUND: Chemical signals are widely used in the animal kingdom, enabling communication in various social contexts, including mate selection and the establishment of dominance. Femoral glands, which produce and release waxy secretions into the environment, are organs of central importance in lizard chemical communication. The Galápagos marine iguana (Amblyrhynchus cristatus) is a squamate reptile with a lek-mating system. Although the lekking behaviour of marine iguanas has been well-studied, their potential for sexual communication via chemical cues has not yet been investigated. Here we describe the diversity of the lipophilic fraction of males' femoral gland secretions among 11 island populations of marine iguanas, and compare it with the composition of its sister species, the Galápagos land iguana (Conolophus subcristatus). We also conducted behavioural observations in marine iguana territorial males in order to explore the possible function of these substances in the context of male dominance in leks. METHODS: Femoral secretions were analysed by gas chromatography coupled to mass spectrometry (GC-MS), and chromatography with a flame ionisation detector (GC-FID) in order to characterise the lipophilic composition. To understand the potential role of femoral secretions in marine iguana intraspecific communication, territorial males were sampled for their femoral glands and monitored to record their head bob rate-a territorial display behaviour in males-as well as the number of females present in their leks. RESULTS: We found that the gland secretions were composed of ten saturated and unsaturated carboxylic acids ranging in chain length between C16 and C24, as well as three sterols. Cholesterol was the main compound found. Intriguingly, land iguanas have a higher diversity of lipophilic compounds, with structural group of lipids (i.e. aldehydes) entirely absent in marine iguanas; overall the chemical signals of both species were strongly differentiated. Lipid profiles also differed among populations of marine iguanas from different islands, with some islands demonstrating a high diversity of lipophilic compounds (i.e. full spectra of compounds), and others lacking one or more compounds. Among the compounds most frequently found missing were 11- and 13-eicosenoic acids. Gland secretions of males with a better body condition and with a higher dominance status (i.e. those accompanied by females and with higher head bob display) were proportionately richer in C20-unsaturated fatty acids (11-eicosenoic acid). DISCUSSION: Land and marine iguanas strongly diverged in their chemical composition of the femoral glands likely due to ecological differences between both species. Despite that marine iguana populations varied in their femoral gland composition that was not related to their genetic structure. Our results indicated that 11-eicosenoic acid may play an important role in intraspecific chemical communication in marine iguanas.