Transcending sea turtles: First report of hatching failure in eggs of an Amazonian freshwater turtle with symptoms of the fungal emerging disease fusariosis.

During the last few decades, fungal pathogens have caused devastating population declines across a broad range of taxa. A newly emerging fungal disease, sea turtle egg fusariosis, caused by members of the Fusarium solani species complex (FSSC), has been reported to be responsible for hatching failure in sea turtles worldwide. However, this has not been detected in fresh water turtle species. Here, using relocated clutches and artificial incubation, we report high hatching failure in eggs symptomatic of fusariosis in the yellow-spotted Amazon River turtle (Podocnemis unifilis) inhabiting a pristine environment in the Ecuadorian Amazon. In 2020, we screened 680 eggs of the yellow-spotted Amazon River turtle, relocated from wild nesting areas to artificial nests, for visual symptoms of fusariosis and to estimate hatchability despite infection. We selected 68 eggs sampled in 2019 to confirm Fusarium infection by PCR amplification of the TEF-1α gene and sequenced seven of those amplicons on an Illumina Miseq to assess FSSC membership. We observed fusariosis symptoms in 42% of the 680 eggs. The proportion of symptomatic eggs within nests was negatively linked to the proportion of eggs that hatched. Hatchability was 8% for symptomatic eggs compared with 72% of asymptomatic eggs. Through PCR testing, 58% of symptomatic and 8% of asymptomatic eggs sampled in 2019 tested positive for Fusarium spp., and sequencing revealed that nine sequence variants from three asymptomatic and four symptomatic eggs corresponded to F. keratoplasticum, F. solani and F. falciforme, the three major FSSC pathogens reported in sea turtle egg fusariosis. Our study suggests that hatching failure in eggs linked to symptoms of fusariosis appears to be partially caused by Fusarium pathogens within FSSC in a freshwater turtle. Thus, fusariosis is more widespread among the Testudines than previously reported and is not limited to sea environments, findings of particular conservation concern.

Egg microbiota is the starting point of hatchling gut microbiota in the endangered yellow-spotted Amazon river turtle.

Establishment and development of gut microbiota during vertebrates' early life are likely to be important predictors of health and fitness. Host-parental and host-environment interactions are essential to these processes. In oviparous reptiles whose nests represent a source of the parent's microbial inocula, the relative role of host-selection and stochastic environmental factors during gut microbial assemblage remains unknown. We sampled eggs incubated in artificial nests as well as hatchlings and juveniles (up to 30 days old) of the yellow-spotted Amazon river turtle (Podocnemis unifilis) developing in tubs filled with river water. We examined the relative role of the internal egg microbiota and the abiotic environment on hatchling and juvenile turtle's cloacal microbiota assemblages during the first 30 days of development. A mean of 71% of ASVs in hatched eggs could be traced to the nest environmental microbiota and in turn a mean of 77% of hatchlings' cloacal ASVs were traced to hatched eggs. Between day 5 and 20 of juvenile turtle's development, the river water environment plays a key role in the establishment of the gut microbiota (accounting for a mean of 13%-34.6% of cloacal ASVs) and strongly influences shifts in microbial diversity and abundance. After day 20, shifts in gut microbiota composition were mainly driven by host-selection processes. Therefore, colonization by environmental microbiota is key in the initial stages of establishing the host's gut microbiota which is subsequently shaped by host-selection processes. Our study provides a novel quantitative understanding of the host-environment interactions during gut microbial assemblage of oviparous reptiles.