The crayfish plague caused by the pathogen Aphanomyces astaci has decimated the European and Asian populations of freshwater crayfish and represents an important threat to the other highly susceptible crayfish species in the world, such as the Australian, Madagascar, and South American species. The development and application of molecular methods addressed to the identification of A. astaci has increased exponentially during the last decades in contrast to a slow trend of the pathogen biology and host interaction. There is still a need for a better comprehension of the A. astaci-crayfish interactions, specifically the resistance and tolerance immune mechanism. These types of studies required a robust basic knowledge on the developmental biology of the pathogen in order to reproduce life stages and to perform infection experiments. A great piece of work in this area was carried out during the 1960s to 80s in University of Uppsala. Thus, the purpose of this work was to update previous protocols as well as to generate new guidelines to reproduce key developmental biology stages of A. astaci, to eventually identify crayfish populations with higher resistance and tolerance to this pathogen. This work also refers to other methodologies and guidelines for the diagnosis of crayfish plague, the pathogen isolation, and the in vitro production of zoospores.

Crayfish plague, caused by the oomycete pathogen Aphanomyces astaci, is one of the most devastating of the emerging infectious diseases. This disease is responsible for the decline of native European and Asian freshwater crayfish populations. Over the last few decades, some European crayfish populations were reported to display partial to total resistance to the disease. The immune response in these cases was similar to that exhibited by the natural carriers of the pathogen, North American freshwater crayfish, e.g., weak-to-strong melanization of colonizing hyphae. We tested the degree of resistance displayed by 29 native Iberian populations of Austropotamobius pallipes that were challenged by zoospores of the pathogen. We measured the following parameters: (i) mean survival time, (ii) cumulative mortality, and (iii) immune response, and found that the total cumulative mortality of all the challenged populations was 100%. The integration of the results from these parameters did not allow us to find differences in resistance towards A. astaci among the northern and central populations of the Iberian Peninsula. However, in the southern populations, we could identify four distinct population responses based on an evaluation of a GLM analysis. In the first case, the similar response could be explained by the effect of a pathogen strain with a lower-than-expected virulence, and/or an actual increase in resistance. In the Southern populations, these differences appear to be the consequence of either whole population or individual resistance. Individuals that survived for a longer period than the others showed a stronger immune response, i.e., presence of partially or fully melanized hyphae, which is similar to that of North American crayfish species. This might be the consequence of different mechanisms of resistance or/and tolerance towards A. astaci.

The fungal pathogens Fusarium keratoplasticum and Fusarium falciforme are responsible for the emerging infectious disease named sea turtle egg fusariosis (STEF). This disease affects all sea turtle species throughout the world, causing low hatching success and mass mortalities. In this study, we investigated the potential use of widely available and easy-to-handle eggs of the invasive alien red-eared slider turtle, Trachemys scripta, as part of an in vivo host model to improve our knowledge of the biological properties of the pathogens responsible of the STEF. Specifically, we performed in vivo experiments, in which T. scripta eggs were challenged with conidia of F. keratoplasticum isolated from diseased sea turtle eggs. We found that the pathogen could colonize and develop similar signs to those observed in nature and fulfill Koch’s postulates. The pathogen showed high virulence properties (e.g., high disease incidence, severity, and low hatching success) and its ability to modify the pH in both the egg surface and culture media, confirming previously described fungal pathogen models. These results support the use of T. scripta as an experimental in vivo host model for studying the biological characteristics of STEF, thus providing valuable insights into the mechanisms underlying the emergence of this fungal disease.

The global rise of fungal pathogens presents an emerging threat to biodiversity, with significant risks to species such as endangered sea turtles. The fungal disease known as sea turtle egg fusariosis (STEF) is associated with high embryo mortality rates and represents a substantial conservation challenge. This disease is caused by two fungal species, namely Fusarium falciforme (Ff) and Fusarium keratoplasticum (Fk), and their identification is essential for guiding future efforts to address potential fungal infections, particularly on important nesting beaches such as those in Costa Rica. In this study, we conducted fungal isolations from sea turtle eggshells and nest sand at four key nesting beaches along the Pacific and Caribbean coasts of Costa Rica to evaluate the presence of STEF-causing species. For accurate identification, we employed a multilocus sequence typing (MLST) approach, analyzing three genetic loci. We obtained 147 axenic cultures, of which 32% belonged to the STEF-causing species Ff (n = 32) and Fk (n = 15). Fusarium falciforme was found across all study locations on both coasts of Costa Rica, whereas Fk was only detected at one beach on the Caribbean coast. This study represents the first survey to accurately identify STEF-causing species in Costa Rica, revealing a widespread presence on the main nesting beaches. Currently, STEF is not severely affecting sea turtles in Costa Rica; however, various factors, such as changes in the nesting beach environment and sand composition, could increase the incidence and severity of the disease, posing a threatening risk to embryonic development. Therefore, a better understanding of the presence and distribution of these pathogens is critical for preventing the development of this emerging disease.