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The embryos of most species of oviparous reptiles spend the majority of their total developmental period (typically, the latter two-thirds) outside the mother’s body, in external nests (e.g., Shine, 1983). As a result, embryos of many species are exposed to a wide range in both the average values and temporal variance of abiotic factors such as temperature and moisture (e.g., Singh et al., 2020). Experimental studies confirm that even minor variation in such factors can significantly modify fitness-relevant traits such as incubation duration, and the phenotypic traits (morphological, physiological, and behavioral) of hatchlings (e.g., Georges et al., 2005; Amiel and Shine, 2012; Bodensteiner et al., 2015). Importantly, embryos in external nests are also exposed to biotic challenges including predation and infection by pathogens (Moreira and Barata, 2005), potentially inducing adaptive responses (e.g., immediate hatching of eggs induced by physical disturbance: Doody, 2011).

In the current article, we explore a neglected aspect of embryo-environment interactions in reptiles: the possibility that microbiota (bacteria, viruses, fungi, etc.) in the soil within a nest may penetrate the eggshell and affect the embryo. Although we are not aware of any studies that have looked for such penetration, it is feasible on physical grounds because soil microbiota are diverse and abundant (e.g., Wolmarans and Swart, 2014), pores within the squamate eggshell are large enough to permit ingress of micro-organisms (Packard and DeMarco, 1991), and eggs take up substantial water from the incubation substrate during development (thereby providing a straightforward means of entry for microbiota: Warner et al., 2011).

Eggs might also be exposed to potentially harmful microbiota even before oviposition. Pathogens could be present as the eggs pass through the oviduct (e.g., vertical transmission of Salmonella) and through the cloaca (fecal contamination) (D’Alba and Shawkey, 2015; Zając et al., 2021). Hydric and thermal conditions inside nests also may expose eggs to high concentrations of saprophytic microbes (e.g., Löwenborg et al., 2011).

The most critical factor thus may not be whether or not microbiota can find their way into developing eggs (surely, they can) but the effects of such exposure on embryos. Studies on later life stages (free-living reptiles) document many effects of infection, including immune responses and stress reactions (e.g., Wellehan and Johnson, 2005; Jacobson, 2007). By analogy, embryos might exhibit such responses as well (as they do in other vertebrates: e.g., Ygberg and Nilsson, 2012). The selective benefit of responding to microbial invasion might include adjusting immune-system development to better match the pathogens likely to be encountered after hatching (Grindstaff et al., 2006). Plausibly, natural selection might fine-tune embryonic responses to microbial exposure either directly, or via facultative manipulation of maternal allocation of resources to the egg (e.g., transfer of immune compounds such as antibodies) in response to the mother’s own history of encounters with pathogens.

The potential impacts of soil microbiota on reptilian embryogenesis are relevant to anthropogenic disturbances to the nest environment. For example, human activities such as agriculture and pollution greatly modify microbial communities within the soil (Wolmarans and Swart, 2014), humans affect nest-site selection of reptiles by destroying traditional nest-sites and/or creating new options (Francis et al., 2019; Doherty et al., 2020), and conservation-focused management frequently involves incubation of eggs in places other than where they were laid (e.g., Jarvie et al., 2014; Mitchell et al., 2016) or in substrates that contain very different microbiota than are found in natural nest-sites. In the extreme, captive breeding of oviparous reptiles often includes incubation of eggs in artificial media such as vermiculite, which may be virtually sterile (Köhler, 2005; Tefera and Vidal, 2009). In humans, artificial elimination of contact with the vaginal microbiota in surgically delivered (cesarean) babies can have deleterious long-term effects on infant health (Dominguez-Bello et al., 2016).

As a first step toward addressing this gap in knowledge, we conducted experimental incubation trials with the eggs of an oviparous tropical snake. In the first experiment, we incubated eggs in soil that either contained or did not contain the natural microbiota (i.e., was or was not autoclaved prior to use). Because any null result from such a study may be due to lack of penetration of the shell by local micro-organisms, we then ran another study in which we directly introduced lipopolysaccharide (LPS, a component of bacterial cell walls) into the egg to stimulate an immune response by the embryo. This second experiment allowed us to assess the effects of chemically signaling the presence of pathogenic microbiota without introducing any confounding deleterious affects (such as propagation) that would arise from actually introducing harmful bacteria. To quantify the effects of these manipulations, we measured aspects of developmental rate (incubation period), hatchling morphology, and circulating immune cells (leucocytes). Because the ratio of certain leucocytes (heterophils: lymphocytes; H:L) provides an index of stress (Davis et al., 2008), we looked specifically at levels of those cell types. Specifically, we predicted that the presence of microbiota (or their chemical cues) could impose developmental costs on embryos that would be manifested in altered morphological or immunological measures.

In both experiments, incubation treatments generated significant differences in hatchling body size. Although much of the variation among clutches in hatchling size is driven by heritable (genetic) factors in this species (Brown and Shine, 2007b), considerable variation also is induced by the conditions under which eggs are incubated. For example, incubation on dry substrates substantially reduces offspring size in keelbacks (Shine and Brown, 2002; Brown and Shine, 2005a) as in many other species of squamate reptiles (Bodensteiner et al., 2015). Longer incubation also can allow squamate embryos to develop more fully, affecting body size at the time of hatching (Shine and Olsson, 2003). These complicating factors cannot explain the effect of treatment on offspring size in the present study, however, because neither rates of water uptake nor incubation periods were affected by our experimental treatments (above). Thus, exposure to microbiota (Experiment #1) or to cues associated with bacterial infection (Experiment #2) invoked morphological changes in offspring that were independent of egg size, incubation period or moisture balance. Our data thus add yet another variable to the long list of external factors that are known to affect developmental trajectories in embryonic reptiles [e.g., see reviews by Noble et al. (2018) and While et al. (2018)].

In Experiment #1, autoclaving the soil used for incubation resulted in a small but statistically significant reduction in hatchling head and body length but did not affect body mass (Table 1). The mechanism behind this shift in hatchling morphology is unknown. Soil is difficult to sterilize (Nowak and Wronkowska, 1987) so our single bout of autoclaving might only have resulted in a temporary decrease or alteration of the microbiotic community. Further, the high temperature (131°C) and pressure (2.2 Bar) associated with autoclaving might also have altered physical or chemical (e.g., pH) characteristics of the soil. Changes in the substrate characteristics such as these could have caused subtle developmental shifts in embryos.

In contrast, WBC profiles of hatchling snakes were not significantly affected by exposure to the immune challenges that were manipulated in the present study. The lack of a significant impact of microbiota presence on hatchling immune function in Experiment #1 could be attributed to the eggshell blocking signals regarding external bacterial levels from reaching the embryo. If this were the case, then studies on other systems (different focal species, different soil microbiota, etc.) might generate different results. To test that possibility, our second experiment inoculated pathogen signals directly into the egg, bypassing any shielding provided by the shell. Again, however, we saw no significant immune response in WBC counts of hatchlings.

That lack of response in Experiment #2 is surprising, because administration of LPS into an animal typically initiates a strong immune reaction (Lind et al., 2020; de Figueiredo et al., 2021). By exposing the individual to a component of bacterial cell walls, injection of LPS signals the presence of a pathogen, and thereby triggers a range of immunological and behavioral responses. In particular, WBC profiles often change in response to LPS injection, as specific cell types are recruited to fight the apparent challenge (Keller et al., 2006; Bowen et al., 2009). Why, then, did we find no difference in WBC profiles between treatment and control hatchlings in either of our experiments?

One possible explanation is that both egg yolk and albumin contain compounds with immune functions. Because reptile eggs are in close contact with soil microbiota, and typically without parental care (i.e., cleaning) of incubating eggs, shells and egg contents may have been under strong selection to develop antimicrobial capability (Gayen et al., 1977; Araki et al., 1998; Zimmerman et al., 2010; van Hoek, 2014). Compounds with antimicrobial abilities (e.g., lysozyme, defensin, ovotransferrin) have been isolated from reptile eggs and others likely remain to be identified (van Hoek, 2014). Furthermore, embryos may be chemically protected by specific maternally provisioned immune compounds (such as antibodies) based on the mother’s previous exposure to pathogens or her general health (Hasselquist et al., 2012). Such maternal effects on offspring immunocompetence have been documented in reptiles, but identifying the precise mechanisms through which such effects operate remains a nascent field (Uller et al., 2006; Itonaga et al., 2011; Brown and Shine, 2016). LPS may possibly be destroyed or altered by these compounds before reaching the embryo, thereby preventing any immune response involving WBC populations.

It remains possible that other components of the immune system were activated, without altering WBC profiles. Reptiles in general and snakes in particular are known to possess several extracellular immune mechanisms (Zimmerman et al., 2010). For example, natural antibodies and complement provide rapid non-specific protection against general common pathogens (Madsen et al., 2007; Brusch et al., 2019, 2020). Notably, exposing embryos to LPS produced significantly smaller offspring, consistent with energy allocation trade-offs. The nature of these trade-offs, and the specific components involved, remain to be identified.

Although our experimental treatments did not affect the WBC profiles of hatchling snakes, we did identify strong correlates of WBC variation among individuals. Notably, both the sex and body size of a hatchling snake affected its WBC profile, consistent with our previous research in keelbacks. Two earlier studies reported a negative correlation between H:L ratio and SVL in this species (Brown and Shine, 2016, 2018). This congruence among disparate studies suggest that the association is a consistent feature of keelback biology. The H:L ratio is often described as a stress leukogram because the number of heterophils relative to lymphocytes increases after exposure to known stressors (Davis et al., 2008; Davis and Maney, 2018). However, constitutive differences in H:L ratios among hatchlings might also reflect different allocation to “cheap” vs. “expensive” immune mechanisms (Brown and Shine, 2016).

If H:L ratio does indeed represent a stress response, our results suggest that the treatments we imposed in the present study were not stressful. The lack of stress response following injection of bacterial-derived compound (LPS) directly into the egg suggests that either the embryo’s immune system is not yet sufficiently well-developed to respond to such a challenge; or that as-yet-unknown defense mechanisms protect a growing embryo either by shielding it from these hostile incursions, or by buffering the effects of infective agents by changes other than those revealed by WBC counts.

Lastly, we address the implications of our results for anthropogenic disruptions to microbiota in the soil. As noted in the Introduction to this article, humans may modify the incubation-associated microbiota either inadvertently (e.g., through pollution) or intentionally (e.g., by translocating eggs to new nest sites, or by incubating eggs in sterile media). Our results suggest that (contrary to our a priori hypotheses), such manipulations do not affect immune-system function in hatchlings; but do affect hatchling body sizes, which in turn are strongly linked to immune-function parameters. In keelbacks (as is likely the case in many reptile populations), hatchling size is under strong directional selection in the field (Brown and Shine, 2004); and thus, managers need to evaluate the impacts of their manipulations on such traits in order to maximize the viability of hatchlings produced either in the field or the laboratory.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by The University of Sydney Animal Ethics Committee.

GB devised the study, gathered data, and conducted statistical analyses. Both authors drafted the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.