Analyzing Chemical Secretions in Lizards Using GC–MS/MS

The chemical messages that animals use to communicate can trigger a range of responses in members of the same species. The Column spoke to Jorge Saiz from the Centre of Metabolomics and Bioanalysis (CEMBIO) at the University San Pablo CEU, Spain, about his research into the chemical secretions of lizards and the role of gas chromatography–tandem mass spectrometry (GC–MS/MS) in his work.

Q. You recently analyzed the chemical secretions from femoral glands in lizards using GC–MS/MS (1). How did this project come about and why did you decide to study these 12 compounds in particular?

A: This project was a collaboration between two groups: the group of Instrumental Analysis, Environment, Food, and Health (Institute of Organic Chemistry, Spain), headed by Belén Gómara, in which I worked, and the group of Evolutionary Ecology (National Museum of Natural Sciences, Spain), headed by José Martín, in which Roberto García-Roa worked. We decided to join our efforts and apply new analytical technologies to the latest research in chemical ecology. This collaboration is still producing very interesting data that will be published about chemical secretions and chemical communication in lizards.

To date, chemical secretions in lizards have primarily been analyzed in an untargeted mode, screening for as many compounds as possible. This is the natural procedure when finding new compounds in different matrices. However, the composition of these secretions is already well known for some lizard species (especially Lacertid lizards) and so we decided to use more selective and more sensitive analytical tools to study these secretions in more depth. We focused this research in two species, Psammodromus algirus and Iberolacerta cyreni, and the aim was to develop a new gas chromatography–tandem mass spectrometry (GC–MS/MS) method to evaluate how different environmental and physiological factors might affect the composition of chemical secretions in lizards. Among all the compounds reported to be present in chemical secretions, we selected 12 relevant compounds often found in lizard secretions that have been suggested to have a role as potential signals and cues in their chemical communication (2–4). Then we tested the method with individuals of different species, from different populations, and with different diets.

Q. What can the chemical secretions of lizards reveal?

A: Chemical communication is now considered the oldest channel of communication in animals. It is based on scents that might convey different information in lizards, such as prey and predator recognition (5,6), territoriality (7), social recognition (8,9), and mate choice (10–12). When lizards crawl over a surface, they leave a scent from these secretions, which is a reflection of their physiological status and can be followed by other individuals. For example, a lizard can recognize a conspecific individual by using these secretions, which obviously has important implications to avoid false mates among heterospecific individuals. Our results showed that individuals of different species, from different populations, or with different diets had different chemical secretions in terms of their composition. This reflects how different physiological and environmental factors affect the expression of compounds in the chemical secretions and, therefore, it determines how other individuals will interact with the carrier of the secretion.

Q.  What does your method offer over other published methods?

A: The method that we have proposed uses the advantages that tandem mass spectrometry offers in terms of selectivity and sensitivity. It can be used for the target analysis of chemical secretions and for the quantitation of specific compounds. Since it is more sensitive and selective than other methods used for the screening of compounds, it could reveal the presence of certain compounds that have not been determined before in certain chemical secretions. This is the case for a-tocopherol (vitamin E), whose expression in chemical secretions of I. cyreni had not been described before in natural conditions. However, with this method, we could demonstrate that I. cyreni actually expresses this compound in chemical secretions of wild populations. This information might have been hindered before because the methods used previously were too generic for certain purposes. In these regards, it is very important to carefully adjust the MS parameters as well as selecting proper MS transitions to obtain the maximum selectivity and sensitivity. This can be done manually by inspecting the scan spectrum for potential ion precursors and the product ion scan spectrum for product ions. It is also possible to make use of the tools available that automatically select precursor and product ions, as well as the voltage conditions for the collision cell.

Q. What were the other main analytical challenges and how did you overcome them?

A: The chemical femoral secretions were extracted by pressing the femoral pores against glass vial necks; the animals were then successfully released. Chemical secretions in lizards can be very tiny samples. For the quantitation of compounds in these samples, it is necessary to measure their weights. We had the chance to use an ultra-microbalance (±0.1 mg), which was kindly provided by the Mass Spectrometry Service at the Institute of Organic Chemistry (Madrid, Spain). We strongly suggest the use of these microbalances for weighing chemical secretion of small lizards. This will avoid errors in the quantitation results.

Q. What are the next stages of this project?

A: The published method is not a closed method at all. More compounds of interest can be added to this method to study their functions in chemical communication and their variations after specific physiological and biological variations. We hope the use of tandem mass spectrometry for the analysis of chemical secretions will provide new information about the chemical ecology of lizards, which is an important source of information to help understand the chemical communication and chemical ecology in other animal groups as well.

References

1. J. Sáiz, R. García-Roa, J. Martín, and B. Gómara, Journal of Chromatography A1514, 110–119 (2017).
2. P.J. Weldon, B. Flachsbarth, and S. Schulz, Nat. Prod. Rep.25, 738–756 (2008).
3. J. Martín, and P. López, Horm. Behav.68, 14–24 (2015).
4. J. Martín, and P. López, Pheromones and chemical communication in lizards, in: The Reproductive Biology and Phylogeny of Lizards and Tuatara, CRC Press, 2014.
5. A. Labra and H.M. Niemeyer, Ethology110, 649–662 (2004). 

6. A. Labra, Chemoecology17, 103–108 (2007). 

7. P. Carazo, E. Font, and E. Desfilis, Anim. Behav. 74, 895–902 (2007). 

8. J. Martín and P. López, Chemoecology16, 31–38 (2006).
9. P. Carazo, E. Font, and E. Desfilis, Anim. Behav.76, 1953–1963 (2008). 

10. J. Martín and P. López, Funct. Ecol. 20, 1087–1096 (2006). 

11. J. Martín and P. López, Proc. Biol. Sci. 273, 2619–2624 (2006).
12. C. Mayerl, S. Baeckens, and R. Van Damme, Amphib.-reptil.36, 185–206 (2015).

Jorge Sáiz obtained his Ph.D. in 2013 at the University of Alcalá, Spain. In 2014 he started a postdoctoral project for the development of new portable capillary electrophoresis systems at the University of Alcalá. In 2015 and 2016 he was a postdoctoral researcher in the group of Instrumental Analysis, Environment, Food and Health (Institute of Organic Chemistry, Spain, Spain) establishing a collaboration with the Department of Evolutionary Ecology (National Museum of Natural Sciences, Spain) for the study of the chemical communication of reptiles. In 2017, he joined the Centre of Metabolomics and Bioanalysis (CEMBIO) at the University San Pablo CEU, Spain. His current research activity is focused on the discovery and validation of new biomarkers for the diagnosis of diseases and the evaluation of the metabolomic state in a pathological condition using a metabolomic platform comprised of liquid chromatography, gas chromatography, and capillary electrophoresis with mass spectrometry for the untargeted and targeted analysis of biological samples.

E-mail: jorge.saizgalindo@ceu.es