Brown anole eggs in the field. Photo by Jenna Pruett.

Most oviparous reptiles (excluding birds) bury their eggs in the ground. Usually, after laying, females abandon the eggs and provide no parental care thereafter. As such, non-avian reptiles (henceforth “reptiles”) have often served as model organisms to understand how the environment influences embryo development. Environmental factors of interest are usually temperature and moisture. Indeed, nest temperature can have large effects on development. For example, warm incubation temperatures often result in hatchlings that can run relatively fast while cool temperatures result in hatchlings that run slow. Moisture is also important during development since relatively wet incubation conditions improve the conversion of yolk to body mass resulting in larger hatchlings compared to dry conditions. This process by which the environment has lasting effects on development is known as developmental plasticity. Despite decades of research concerning developmental plasticity in reptiles, there are still many aspects of natural nest environments that are understudied.

One example of such an understudied environmental factor is the type of substrate (i.e. soil) in which females bury eggs. Although many field studies demonstrate that females lay eggs in a diversity of substrates, very few studies have considered exactly how these different substrates might influence development. These few existing studies have focused on turtles. For example, Mitchell and Janzen (2019) buried turtle eggs in three types of substrates in the field: loam, sand, and gravel. Despite all nests experiencing the same prevailing weather conditions, important aspects of the nest environment like moisture available to eggs and temperature differed among the substrates. This resulted in important differences among the hatchling turtles. Indeed, because this species exhibits temperature-dependent sex determination (i.e. the egg temperature determines if hatchlings are male or female), the sex ratios of the hatchlings differed according to the type of substrate in which the eggs were buried.

No study has rigorously considered how substrate types influence development of squamates (lizards and snakes). Therefore, my research associates and I decided to conduct a lab experiment using our good friend the brown anole (Anolis sagrei). This study was recently published in the journal Integrative Zoology (Hall et al. 2021). At our field site in Florida, female anoles lay eggs in two main types of substrates: sand/crushed sea shells and organic debris (Figure 1). We collected male and female lizards from one of our study islands and brought them back to our lab at Auburn University. We also collected a few buckets of the two substrates in which females commonly nest. We collected eggs from the breeding colony and incubated them in each substrate at 4 different moisture concentrations. The goal was to understand if these two substrates had any important effects on development. Moreover, using different moisture concentrations in each substrate allowed us to see if the two substrates might have similar effects on development given particular moisture concentrations.

Figure 1. Representative photos of (a) a female brown anole (Anolis sagrei), (b) aerial view of the substrate collection island, (c) ground view of substrate collection island, (d) organic substrate, and (e) sand/shell substrate. In panel (b), the area inside the red circle is the portion of the island that is most densely populated with lizards. The area within the black line is an example of open canopy habitat where substrate is primarily sand and crushed shell. The area inside the white line is an example of closed canopy habitat with dark, organic substrate. Panel (c) shows the ground view of the same open and closed canopy sites outlined in panel (b).

We measured a variety of traits including water uptake by eggs (eggs absorb water during development), developmental rates of embryos, egg survival, hatchling body size, and hatchling performance (i.e. endurance). The amount of moisture available to eggs provided expected results: greater moisture content resulted in greater water absorption by eggs and larger hatchling body size. We found that the two substrates had little effect on most traits; however, egg survival and developmental rate differed between the substrates: eggs were more likely to die and developed more slowly in the organic substrate than in the sand/crushed shell. Although statistically significant, these effects were not large. The difference in egg survival was about 6% and the difference in developmental rates between the substrates resulted in a one-day difference in the incubation period (i.e. the number of days it takes for the egg to hatch).

It isn’t completely obvious why we observed these differences in egg survival and physiology (i.e. developmental rate). We think the organic substrate might support a greater load of microbes (i.e. fungal spores and bacteria) than the sand/shell substrate. Thus, in the organic substrate, eggs may compete with microorganisms for resources like oxygen during development. Additionally, when exposed to an abundance of microorganisms, eggs may expend energy to fight infection which could slow development and reduce survival. Regardless, other studies have also found that developmental rate can be influenced by the type of incubation substrate, but no mechanism has yet been rigorously tested. Thus, there is still much to learn about how reptile embryos interact with natural nest environments!

In conclusion, the type of incubation substrate can have important effects on embryo physiology and survival, but only a few studies have explored these relationships. What would be most helpful now is a series of studies that consider how microbial communities differ among substrates and how these communities might interact with eggs. Perhaps this work will rest on the shoulders of Kaitlyn Murphy who is currently using microbiology techniques to understand effects of the microbiome on embryo development using brown anoles. If so, the future of this unexplored area of research is in capable hands.

You can read the full article here: http://doi.org/10.1111/1749-4877.12553

Hall, J. M., Miracle, J., Scruggs, C. D., & Warner, D. A. (2021). Natural nest substrates influence squamate embryo physiology but have little effect on hatchling phenotypes. Integrative Zoology.

Mitchell, T. S., & Janzen, F. J. (2019). Substrate influences turtle nest temperature, incubation period, and offspring sex ratio in the field. Herpetologica, 75(1), 57-62.

Joshua Hall
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