A guide to incubate eggs of Tropidurus lizards under laboratory conditions

Abstract

Studies in Evo-Devo benefit from the use of a variety of organisms, as comparative approaches provide a better understanding of Biodiversity and Evolution. Standardized protocols to incubate eggs and manipulate embryo development enable postulation of additional species as suitable biological systems for research in the field.In the past decades, vertebrate lineages such as Squamata (lizards, snakes, and amphisbaenians) emerged as crucial study systems for addressing topics as diverse as phenotypic evolution and climate change. However, protocols for maintaining gravid females and incubating eggs in the lab under experimental conditions are available to only a few squamate species. This resource article presents a simple incubation guide that standardizes conditions to maintain embryos of Tropidurus catalanensis (Squamata: Tropiduridae) under different experimental conditions, manipulating relevant environmental factors like temperature and humidity. We identified associated effects relating the egg incubation condition to developmental stage, incubation time, hatching success, and resulting morphotypes. Temperature and humidity play a key role in development and require attention when establishing the experimental design. Current literature comprises information for Tropidurus lizards that ponders how general in Squamata are the ecomorphs originally described for Anolis. Studies evaluating phenotypic effects of developmental environments suggest plasticity in some of the traits that characterize the ecomorphological associations described for this family. We expect that this incubation guide encourages future studies using Tropidurus lizards to address Evo-Devo questions.

Native Lizards Living in Brazilian Cities: Effects of Developmental Environments on Thermal Sensitivity and Morpho-Functional Associations of Locomotion

“…From the 20 clutches obtained (one from each female), we distributed 80 eggs into small plastic cylindric containers of 145 ml (7.5 cm × 5.0 cm) filled with moist vermiculite. We perforated the lids to enable air renovation and fixed the water potential at -150 cmkPa, replacing eventual evaporated water (based on Steele et al, 2018 and following;Rossigalli-Costa et al, 2021). To avoid genetic bias, we separated the clutches and equally distributed the eggs between two developmental environments: 24 °C (cold, N = 40) and 30 °C (warm, N = 40); we did not consider the thermal regime of 36 °C (overheat) as a third developmental condition because this temperature seems unfeasible for the development of T. catalanensis embryos (see Rossigalli-Costa et al, 2021).…”

“…We perforated the lids to enable air renovation and fixed the water potential at -150 cmkPa, replacing eventual evaporated water (based on Steele et al, 2018 and following;Rossigalli-Costa et al, 2021). To avoid genetic bias, we separated the clutches and equally distributed the eggs between two developmental environments: 24 °C (cold, N = 40) and 30 °C (warm, N = 40); we did not consider the thermal regime of 36 °C (overheat) as a third developmental condition because this temperature seems unfeasible for the development of T. catalanensis embryos (see Rossigalli-Costa et al, 2021). Due to the lack of information for nest temperatures in T. catalanensis, we chose developmental temperatures within the range of air, substrate and body temperatures recorded for Tropidurus lizards at São Simão and other Brazilian cities where this species is found (see Kohlsdorf and Navas, 2006;Leirião et al, 2019;Maia-Carneiro and Navas, 2021), which also agree with the range of preferred temperatures recorded for Tropidurus lizards in thermal gradients (31 °C to 37 °C, see Kohlsdorf and Navas, 2006).…”

“…Eggs were daily inspected for monitoring the development of the embryos, being maintained in the incubators until hatching. These procedures followed Rossigalli-Costa et al (2021).…”

“…Among environmental parameters that usually affect development, temperature is probably one of the most relevant variables shaping the phenotype in ectotherm vertebrates (Angilletta, 2009). Thermal responses during ontogeny may produce different phenotypes (e.g., large or small neonate lizards; see Rossigalli-Costa et al, 2021) and can be addressed in the context of developmental plasticity and reaction norms (see Angilletta, 2009;Refsnider et al, 2019;Singh et al, 2020). When the thermal regimes change, species that exhibit behavioral flexibility or quickly respond to new conditions through plasticity in morphological or physiological traits tend to prosper in the novel environmental conditions (Winchell et al, 2016;Lapiedra et al, 2017;Noble et al, 2021).…”

“…The genus Tropidurus comprises habitat specialists but also generalist species (see Werneck et al, 2015), and records suggest that some species are relatively prone to occupy new niches and persist despite the surrounding urban growth (Rand and Rand, 1966). Another positive aspect of Tropidurus lizards for studies focusing on phenotypic aspects of species found in urban sites is the extensive literature available for eco-morphology (e.g., Kohlsdorf et al, 2001;Kohlsdorf et al, 2008;Grizante et al, 2010;Tulli et al, 2016), behavior (e.g., Leirião et al, 2019;Rodrigues and Kohlsdorf, 2019), locomotion (Kohlsdorf and Navas, 2012;Brandt et al, 2015) and physiology (Longhini et al, 2019) in this group, including information for development (Py-Daniel et al, 2017;Rossigalli-Costa et al, 2021) and thermal relationships (Kohlsdorf and Navas, 2006;Leirião et al, 2019;Piantoni et al, 2019) of species found in urban areas. In this study, we isolated temperature effects from other environmental parameters likely affected by urbanization (e.g., hydrological regimes or pollutants) and evaluate developmental plasticity in offspring of a Tropidurus species commonly found in Brazilian cities.…”