Vertically transmitted microbiome protects eggs from fungal infection and egg failure

Background Beneficial microbes can be vertically transmitted from mother to offspring in many organisms. In oviparous animals, bacterial transfer to eggs may improve egg success by inhibiting fungal attachment and infection from pathogenic microbes in the nest environment. Vertical transfer of these egg-protective bacteria may be facilitated through behavioral mechanisms such as egg-tending, but many species do not provide parental care. Thus, an important mechanism of vertical transfer may be the passage of the egg through the maternal cloaca during oviposition itself. In this study, we examined how oviposition affects eggshell microbial communities, fungal attachment, hatch success, and offspring phenotype in the striped plateau lizard, Sceloporus virgatus, a species with no post-oviposition parental care. Results Relative to dissected eggs that did not pass through the cloaca, oviposited eggs had more bacteria and fewer fungal hyphae when examined with a scanning electron microscope. Using high throughput Illumina sequencing, we also found a difference in the bacterial communities of eggshells that did and did not pass through the cloaca, and the diversity of eggshell communities tended to correlate with maternal cloacal diversity only for oviposited eggs, and not for dissected eggs, indicating that vertical transmission of microbes is occurring. Further, we found that oviposited eggs had greater hatch success and led to larger offspring than those that were dissected. Conclusions Overall, our results indicate that female S. virgatus lizards transfer beneficial microbes from their cloaca onto their eggs during oviposition, and that these microbes reduce fungal colonization and infection of eggs during incubation and increase female fitness. Cloacal transfer of egg-protective bacteria may be common among oviparous species, and may be especially advantageous to species that lack parental care.

Recovered microbiome of an oviparous lizard differs across gut and reproductive tissues, cloacal swabs, and feces

Microbial diversity and community function are related, and can be highly specialized in different gut regions. The cloacal microbiome of Sceloporus virgatus provides antifungal protection to eggshells during oviposition – a specialized function that suggests a specialized microbial composition. Here, we describe the S. virgatus cloacal microbiome from tissue and swab samples, and compare it to tissue samples from the gastrointestinal (GI) tract and oviduct, adding to the growing body of evidence of microbiome localization in reptiles. We further assessed whether common methods of microbial sampling – cloacal swabs and feces – provide accurate representations of these microbial communities and whether feces might “seed” the cloacal microbiome or impact the accuracy of cloacal swab sampling. We found that different regions of the gut had unique microbial community structures. The cloacal community, in particular, showed extreme specialization averaging 99% Proteobacteria (Phylum) and 83% Enterobacteriacaea (Family). Cloacal swabs recovered communities similar to that of lower intestine and cloacal tissues, but fecal samples had much higher diversity and a distinct composition (62% Firmicutes and 39% Lachnospiraceae) relative to all gut regions. Finally, we found that feces and cloacal swabs recover different communities, but cloacal swabs may be contaminated with fecal matter if taken immediately after defecation. These results serve as a caution against the assumption that fecal samples provide an accurate representation of the gut, and that although cloacal swabs can reflect a portion of the lower GI tract microbiome, they may also result in a mixed community of gut and fecal microbes.

Specialization of the cloacal microbiome relative to gut, oviduct, and feces of an oviparous lizard

Microbial diversity and community function are related, and both can be highly specialized in different regions of the gut. The cloacal microbiome of Sceloporus virgatus lizards has low diversity, suggesting a specialized function, and is known to transfer antifungal microbes to eggshells during oviposition. We hypothesize that the cloacal microbiome is distinct from other parts of the digestive and reproductive systems. Here, we compare the microbiome of tissue samples from the cloaca, lower intestine, upper intestine, and oviduct. We further assessed whether common methods of microbial sampling – cloacal swabs and feces – provide accurate representations of these tissues, and whether feces might “seed” the cloacal microbiome. We found that the upper intestine and oviduct had unique microbial communities, while the lower intestine and cloaca had similar communities with lower diversity indicative of regional specialization. The cloacal community, in particular, showed extreme specialization averaging 99% Proteobacteria (Phylum) and 83% Enterobacteriacaea (Family). Cloacal swabs recovered communities similar to that of lower intestine and cloacal tissues, but fecal samples had much higher diversity and a distinct composition (62% Firmicutes and 39% Lachnospiraceae) relative to all gut regions. This result serves as a caution against the frequent assumption that fecal samples provide an accurate representation of the gut. Finally, we found that defecation did not alter the cloacal microbiome, suggesting that community is robust to perturbations from transient microbiota.

Experimental removal reveals only weak interspecific competition between two coexisting lizards

Competition for resources is an important mechanism that shapes ecological communities. Interspecific competition can affect habitat selection, fitness, and abundance in animals. We used a removal experiment and mark–recapture to test the hypothesis that competition with the larger and more abundant Striped Plateau Lizard (Sceloporus virgatus H.M. Smith, 1938) limits habitat selection, fitness, and abundance in Ornate Tree Lizards (Urosaurus ornatus (Baird in Baird and Girard, 1852)). Ornate Tree Lizards in the plots where Striped Plateau Lizards were removed switched between habitat types more frequently and moved farther than Ornate Tree Lizards in control plots. However, there were no significant changes in the relative densities of Ornate Tree Lizards in each habitat type or in microhabitat use. We also found no changes in growth rates, survival, or abundance of Ornate Tree Lizards in response to the removal of Striped Plateau Lizards. Our results suggest that interspecific competition was not strong enough to limit habitat use or abundance of Ornate Tree Lizards. Perhaps interspecific competition is weak between coexisting species when resource levels are not severely depleted. Therefore, it is important to consider environmental conditions when assessing the importance of interspecific competition.

Directional escape strategy by the striped plateau lizard (Sceloporus virgatus): turning to direct escape away from predators at variable escape angles

A prey’s orientation to a predator’s approach path affects risk of fleeing straight ahead. By turning to flee closer to straight away from the predator before fleeing, prey can reduce risk. Laboratory studies suggest that escape angles should lead away from predators and be unpredictable. I studied orientation, turn, and escape angles and in a study of striped plateau lizards,Sceloporus virgatus. Lizards fled away from a predator, but often not straight away. Escape angles were variable and bimodally distributed: one mode was straight away for distancing prey from predator and one was near 90°, which maintains ability to monitor the predator or requires turning by the predator. Turn angles increased as orientation shifted toward the predator. Escape angle was closer to straight away when turn angle was larger, but turning did not fully compensate for degree of orientation toward the predator. Directional escape strategies of diverse prey are compared.

Stop-and-go approach by a predator: a novel predation risk factor for the phrynosomatid lizards Sceloporus virgatus and Callisaurus draconoides

As an immobile prey monitors an approaching predator, the predator may move at a constant speed directly toward the prey or on a path that bypasses the prey. These scenarios have been studied extensively. Economic escape theory successfully predicts flight initiation distance (FID = predator-prey distance when escape begins). However, predators often alter their speed and may exhibit stops and starts during approaches. Empirical studies have shown that prey rapidly adjust assessed risk to a predator’s changes in approach speed and direction, but effects of interrupted (stop-start) approach are unknown. Because a prey is likely to assess that a nearby predator that resumes approaching has detected it and is attacking, escape theory predicts that assessed risk is greater at a given predator-prey distance when approach resumes than is continuous. Therefore, we predicted that FID is longer when a predator approaches, stops nearby, and renews its approach than when it approaches continuously. Second, although assessed risk increases as duration of the predator’s stop nearby increases, as indicated by latency to flee, we predicted that pause duration does not affect FID because prey interpret resumed approach as attack. Field experiments with two lizards, Sceloporus virgatus and Callisaurus draconoides, verified the predictions: FID was longer for discontinuous than continuous approaches and pause duration did not affect FID. We also observed distance fled and probability of entering refuge, escape behaviors for which theory is undeveloped. Distance fled was unrelated to continuity of approach in both species, as was refuge entry in S. virgatus.

FEAR and DREAD: starting distance, escape decisions and time hiding in refuge

Flight initiation distance (FID = predator–prey distance when escape begins) increases as starting distance (predator–prey distance when approach begins) increases. The flush early and avoid the rush (FEAR) hypothesis proposes that this relationship exists because monitoring an approach is costly. Hypothesized causes are increase in assessed risk and decrease in obtainable benefits while monitoring as starting distance increases. We propose the delay risking emergence and avoid dying (DREAD) hypothesis: hiding time in refuge increases as starting distance increases because prey use risk assessed during approach to estimate risk upon emerging. In the lizard Callisaurus draconoides, FID increased as standardized starting distance increased at faster approach speeds, supporting the FEAR hypothesis. In its first test, the DREAD hypothesis was supported: hiding time in the lizard Sceloporus virgatus increased as standardized starting distance increased. No large benefits were attainable, suggesting that dynamic increase in assessed risk accounts for these findings.