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

Abstract

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.

Protein–lipid Association in Lizard Chemical Signals

“…Empirical and theoretical work suggest that the proteinaceous and lipid fraction of lizard gland secretions may be intertwined, and thereby contribute (even with potentially different roles) to the functionality of lizard chemical display ( Alberts 1990 ; Alberts and Werner 1993a ; Mangiacotti et al 2017 , 2019a , 2020 ; Tellkamp et al 2020 ; Ibáñez et al 2022 ). Considering the Lacertid family, the quantitative phylogenetically-informed analysis confirm such a co-variation between proteins and lipids to occur.…”

“…Notably, proteins may contribute to enhance signal efficacy ( sensu Endler 1993 ) by setting a “chemical environment” that inhibits more labile molecules (such as lipids) from oxidation, fast evaporation, or degradation ( Alberts 1992 , 1993 ; Wyatt 2010 ; Martín et al 2016 ). In the iguanid Amblyrhynchus cristatus ( Tellkamp et al 2020 ) and the lacertid Lacerta agilis ( Ibáñez et al 2022 ), femoral gland secretions hold proteins with chemical properties that may serve in pH regulation (e.g., carbonic anhydrase), anti-oxidant function (e.g., thioredoxin), bacterial resistance (e.g., oncocin) or lipid-binding (e.g., fatty acid-binding protein 9), and not only in structural roles (e.g., keratin). This opens to the idea that proteins may be active in the mixture, and, by their quantitative and qualitative modulation, be involved in the very fine regulation of the efficacy, stability, and durability of the signal ( Alberts 1992 ).…”

“…Indeed, behavioral studies on the green iguana ( Iguana iguana ; Alberts and Werner 1993a ), the common wall lizard ( Podarcis muralis ; Mangiacotti et al 2019a ), and Cyren's Rock Lizard ( Iberolacerta cyreni ; Mangiacotti et al 2020 ) showed that proteins could play an active role in lizard chemical communication, at least conveying identity-related information. Recently, using mass-spectrometry coupled with database-searching, proteins from femoral glands have been preliminary characterized in the marine iguana, Amblyrhynchus cristatus ( Tellkamp et al 2020 ), and the sand lizard, Lacerta agilis ( Ibáñez et al 2022 ). Although both studies failed in finding proteins immediately relatable to semiochemical functions, they underscored their role in supporting the lipophilic fraction, with potential binding, regulatory, anti-oxidant, antibacterial, and immune functions ( Tellkamp et al 2020 ; Ibáñez et al 2022 ).…”

“…Recently, using mass-spectrometry coupled with database-searching, proteins from femoral glands have been preliminary characterized in the marine iguana, Amblyrhynchus cristatus ( Tellkamp et al 2020 ), and the sand lizard, Lacerta agilis ( Ibáñez et al 2022 ). Although both studies failed in finding proteins immediately relatable to semiochemical functions, they underscored their role in supporting the lipophilic fraction, with potential binding, regulatory, anti-oxidant, antibacterial, and immune functions ( Tellkamp et al 2020 ; Ibáñez et al 2022 ).…”

Effect of MHC and inbreeding on disassortative reproduction: A data revisit, extension and inclusion of fertilization in sand lizards

“…Females prefer MHC dissimilar males in staged mating trials (Olsson et al, 2003), but this may not necessarily imply that they exert mate choice with respect to MHC genotypes in the wild, as this could be exerted by males; male body mass was negatively correlated with partner band sharing, suggesting that dominant, larger males may choose, or spend more time with, more MHCdissimilar females (Olsson et al, 2003). That said, further support on the importance of our previous work on the role of female choice on MHC odors in male femoral pore secretions come from proteomics research by Ibánez et al (2022). The Ibánez work…”