Beyond Sea Turtles: Fusarium keratoplasticum in Eggshells of Podocnemis unifilis, a Threatened Amazonian Freshwater Turtle.

The endangered yellow-spotted river turtle (Podocnemis unifilis) has experienced a dramatic population decline in the Ecuadorian Amazonia, mainly due to overexploitation of its eggs. To reverse this trend, the Wildlife Conservation Society has developed a head-start program in Yasuní National Park since 2008, but the potential risk that microbes associated with its eggs might represent for hatching success has not been evaluated yet. Members of the Fusarium solani species complex (FSSC) are involved in egg failure in sea turtles under natural and hatchery conditions, but their role in infecting the eggs of P. unifilis is unknown. In this study, we collected eggshells of P. unifilis and obtained 50 fungal and bacterial isolates. Some potentially pathogenic fungi of the genera Fusarium, Penicillium and Rhizopus were identified based on molecular data. Most importantly, the sea turtle pathogenic species F. keratoplasticum not only was present, but it was the most frequently found. Conversely, we have also isolated other microorganisms, such as Pseudomonas or Phoma-like species, producing a wide spectrum of antifungal compounds that may have a protective role against fungal diseases. Our survey provides useful information on potential pathogens found in P. unifilis eggshells, upon which the success of conservation programs may depend.

Unravelling the microbiome of eggs of the endangered sea turtle Eretmochelys imbricata identifies bacteria with activity against the emerging pathogen Fusarium falciforme.

Habitat bioaugmentation and introduction of protective microbiota have been proposed as potential conservation strategies to rescue endangered mammals and amphibians from emerging diseases. For both strategies, insight into the microbiomes of the endangered species and their habitats is essential. Here, we sampled nests of the endangered sea turtle species Eretmochelys imbricata that were infected with the fungal pathogen Fusarium falciforme. Metagenomic analysis of the bacterial communities associated with the shells of the sea turtle eggs revealed approximately 16,664 operational taxonomic units, with Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes as the most dominant phyla. Subsequent isolation of Actinobacteria from the eggshells led to the identification of several genera (Streptomyces, Amycolaptosis, Micromomospora Plantactinospora and Solwaraspora) that inhibit hyphal growth of the pathogen F. falciforme. These bacterial genera constitute a first set of microbial indicators to evaluate the potential role of microbiota in conservation of endangered sea turtle species.

Global distribution of two fungal pathogens threatening endangered sea turtles.

Nascent fungal infections are currently considered as one of the main threats for biodiversity and ecosystem health, and have driven several animal species into critical risk of extinction. Sea turtles are one of the most endangered groups of animals and only seven species have survived to date. Here, we described two pathogenic species, i.e., Fusarium falciforme and Fusarium keratoplasticum, that are globally distributed in major turtle nesting areas for six sea turtle species and that are implicated in low hatch success. These two fungi possess key biological features that are similar to emerging pathogens leading to host extinction, e.g., high virulence, and a broad host range style of life. Their optimal growth temperature overlap with the optimal incubation temperature for eggs, and they are able to kill up to 90% of the embryos. Environmental forcing, e.g., tidal inundation and clay/silt content of nests, were correlated to disease development. Thus, these Fusarium species constitute a major threat to sea turtle nests, especially to those experiencing environmental stressors. These findings have serious implications for the survival of endangered sea turtle populations and the success of conservation programs worldwide.

Fusarium solani is responsible for mass mortalities in nests of loggerhead sea turtle, Caretta caretta, in Boavista, Cape Verde.

The fungus Fusarium solani (Mart.) Saccardo (1881) was found to be the cause of infections in the eggs of the sea turtle species Caretta caretta in Boavista Island, Cape Verde. Egg shells with early and severe symptoms of infection, as well as diseased embryos were sampled from infected nests. Twenty-five isolates with similar morphological characteristics were obtained. Their ITS rRNA gene sequences were similar to the GenBank sequences corresponding to F. solani and their maximum identity ranged from 95% to 100%. Phylogenetic parsimony and Bayesian analyses of these isolates showed that they belong to a single F. solani clade and that they are distributed in two subclades named A and C (the latter containing 23 out of 25). A representative isolate of subclade C was used in challenge inoculation experiments to test Koch postulates. Mortality rates were c. 83.3% in challenged eggs and 8.3% in the control. Inoculated challenged eggs exhibited the same symptoms as infected eggs found in the field. Thus, this work demonstrates that a group of strains of F. solani are responsible for the symptoms observed on turtle-nesting beaches, and that they represent a risk for the survival of this endangered species.