The project will develop new research topics at the applicants’ institution – neuroethology and behavioral genetics of vertebrates, no other group at the applicants’ Institution systematically investigates the neurochemical basis of normal behavior in wild animals nor behavior genetics of vertebrates.

The question this project aims to answer is what is the relationship between dopamine and aggressiveness, which after many research efforts is still not entirely clear.

For this purpose, we propose a simple non-standard model of interspecies interaction. On the mainland, populations of both Podarcis species coexist, probably because of the lack of constraints that are present in small islands where P. siculus competitively excludes P. melisellensis.

We have recently started investigating the relationship between these two species and observed higher levels of dopamine in the brains of P. siculus in comparison to P. melisellensis. This observation could be linked to the previously noted greater aggression of P. siculus. Using this simple yet informative model, we hope to further the understanding of the function of dopamine in the regulation of aggression.

The proposed project deals with the internationally relevant research topics of neuroethology and genomics in behavioral ecology as well as the use of a comparative approach in neuroscience. There is a need for well-established models that will allow for a thorough investigation of the many aspects affecting the expression of behavioral traits. Specifically, BOLDer will investigate the mechanisms involved in aggressiveness that are still far from elucidated.

The biochemical and behavioral differences between P. siculus and P. melisellensis as reptilian animal models is an advantage because studying reptilian behavioral traits and their neuroendocrine and genetic background gives us insight into the origin of different behavioral traits in terrestrial vertebrates.

The methodology to reach the aim of the project is to elucidate the relationship between the regulation of dopamine neurotransmission and aggressive behavior in two species of lizards that cohabitate in competition. In order to achieve this goal, lizards will be sampled from 3 locations, some will be tested onsite and transported to a laboratory setting to test behavior and analyze several aspects of dopamine regulation.

The field study will be conducted on three mainland locations (around towns of Knin, Sinj, and Pag) where populations of P. melisellensis and P. siculus coexist. The main criteria for population inclusion are that they are distinct non-communicating populations (at least 50 km apart) yet all in the same geographical region, Splitsko Dalmatinska County.

Sampling is planned on all populations for two consecutive years. We aim to capture 10 male and 10 female lizards of each species per location per year, a total of 240 lizards (120 per species).

The first two days on each location are planned for measuring
a) anti-predator behavior (flight initiation distance, FID), and the other two days for
b) lizard sampling and
c) morphological measurement.

The total of animals sampled during the duration of the project is 240, the number of animals is elevated because of the behavioral tests that require the pairing of animals. Collected lizards will be placed individually in lizard bags, and transported to the animal facility of the Faculty of Science, University of Zagreb, which will be equipped for successful maintenance of lizards (materials acquired through this proposal) and where behavioral studies will be done.

A principal part of the project consists of the establishment of a high-throughput behavioral testing facility to allow for the simultaneous testing of several animals in specialized behavioral tests. Behavioral tests will be filmed and analyzed with the Ethovision software (also upgraded through the project). Behavioral testing will be done in the Open field test apparatus.

The first set of tests will provide information on normal behavior in an open space. The second set of tests will measure aggression in dyadic encounters between lizards of the same sex, with only one heat source. The presence of only one heat source will motivate lizards to compete over this valuable resource for cold-blooded animals. Both intra and interspecies tests will be conducted.

After the completion of behavioral testing animals will be anesthetized and euthanized. Blood and brain tissue will be collected. Brains will be snap-frozen on dry ice and then cut in half along the midsagittal plane. One-half of the brain will be used for the measurement of brain levels of dopamine and its metabolites (HPLC). The other half of the brain will be used for gene expression analyses and histological analyses.

In order to be able to efficiently analyze gene expression and design quality primers and probes for our genes of interest, we will outsource the NGS and analysis to generate transcriptome assemblies of brain tissue for both species. We will principally analyze the expression of dopamine-related genes (NAT, MAO, COMT, VMAT, DBH, AAAH, DRD4) in the brains of animals from both species and sexes, with multiplex TaqMan probes, and analyzed using the delta-delta Ct method. In order to confirm both HPLC and gene expression results, immunohistochemical techniques will be used to detect dopamine and dopamine receptor content/localization in the brain. This part of the research will be done as a service provided by the Department of histology of the School of medicine.

Predictions that behavioral (boldness, aggressiveness, and dominance) and physiological (brain dopamine concentrations) traits in P. siculus and P. melisellensis are correlated will be tested using the generalized linear mixed models (GLMM), testing the dopamine level effect on behavioral traits.

Further on, multivariate relations between the studied variables will be interpreted using Principal component analysis (PCA). The prediction that specific biochemically differential traits in P. siculus and P. melisellensis are controlled through gene expression, will be tested by GLMM, including their interactions.

Partial least square analyses will be conducted to explore how each predictive variable (genetic and neurochemical traits) may be related to the dependent behavioral variables. All values will be tested for normality and transformed if required.

BOLDer will contribute several insights into the field of neuroethology and behavioral genetics.

First, results from behavioral testing of two coexisting and competitive lizard species living in the same climatic region (eumediterranean) allow us to identify discreet yet significant differences. Moreover, behavioral research on these species and genus is scarce.

Second, relative to mammals, a limited number of neurotransmitters in the brain of lower vertebrates have been analyzed to date and relatively few studies have linked those parameters with specific behavioral traits.

Third, the expression of dopaminergic genes expressed in the brain has only rarely been correlated with behavioral traits in reptiles. Such a comparison may elucidate the neuronal basis of differences in behavior, and those results can aid the broader understanding of behavioral differences in other species.

Fourth, conclusions derived from this project will help clarify the contribution of neurobiological, physiological, and genetic mechanisms to variation in behavior and establish a behavior-neurochemical-genetic link, driven by environmental influences.

Finally, this project will contribute to conservation biology. Understanding the role of specific behaviors and molecular markers will help further elucidate the basis of P. siculus invasion success. On the other hand, P. melisellensis is endemic, under special natural protection in Croatia, and listed in the Red book of amphibians and reptiles of Croatia. Therefore, data gained by this project will contribute to the knowledge of the biology of this species and help in the assessment of future conservation efforts.