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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.

European crayfish species are a clear example of the drastic decline that freshwater species are experiencing. In particular, the native species of the Iberian Peninsula, the white clawed-crayfish (WCC) Austropotamobius pallipes, is listed as "endangered" by the IUCN and included in Annex II of the EU Habitat Directive and requires especially attention. Currently, implemented conservation management strategies require a better understanding of the genetic diversity and phylogeographic patterns, as well as of its evolutionary history. For this purpose, we have generated the largest datasets of two informative ribosomal mitochondrial DNA regions, i.e., cytochrome oxidase subunit I and 16S, from selected populations of the WCC covering its geographical distribution. These datasets allowed us to analyze in detail the (i) genetic diversity and structure of WCC populations, and (ii) divergence times for Iberian populations by testing three evolutionary scenarios with different mtDNA substitution rates (low, intermediate, and high rates). The results indicate high levels of haplotype diversity and a complex geographical structure for WCC in the Iberian Peninsula. The diversity found includes new unique haplotypes from the Iberian Peninsula and reveals that most of the WCC genetic variability is concentrated in the northern and central-eastern regions. Despite the fact that molecular dating analyses provided divergence times that were not statistically supported, the proposed scenarios were congruent with previous studies, which related the origin of these populations with paleogeographic events during the Pleistocene, which suggests an Iberian origin for these WCC. All results generated in this study, indicate that the alternative hypothesis of an introduced origin of the Iberian WCC is highly improbable. The result of this study, therefore, has allowed us to better understand of the genetic diversity, structure patterns, and evolutionary history of the WCC in the Iberian Peninsula, which is crucial for the management and conservation needs of this endangered species.

Abstract The oomycete Aphanomyces astaci is an emerging infectious pathogen affecting freshwater crayfish worldwide and is responsible for one of the most severe wildlife pandemics ever reported. The pathogen has caused mass mortalities of freshwater crayfish species in Europe and Asia, and threatens other susceptible species in Madagascar, Oceania and South America. The pathogen naturally coexists with some North American crayfish species that are its chronic carriers. Presumptions that A. astaci originated in North America are based on disease outbreaks that followed translocations of North American crayfish and on the identification of the pathogen mainly in Europe. We studied A. astaci in the southeastern US, a center of freshwater crayfish diversity. In order to decipher the origin of the pathogen, we investigated (1) the distribution and haplotype diversity of A. astaci, and (2) whether there are crayfish species-specificities and/or geographical restrictions for A. astaci haplotypes. A total of 132 individuals, corresponding to 19 crayfish species and one shrimp species from 23 locations, tested positive for A. astaci. Mitochondrial rnnS and rnnL sequences indicated that A. astaci from the southeastern US exhibited the highest genetic diversity so far described for the pathogen (eight haplotypes, six of which we newly describe). Our findings that A. astaci is widely distributed and genetically diverse in the region supports the hypothesis that the pathogen originated in the southeastern US. In contrast to previous assumptions, however, the pathogen exhibited no clear species-specificity or geographical patterns.

Several European freshwater crayfish species are currently included in one of the IUCN Red list categories. In the Iberian Peninsula, the native Austropotamobius pallipes species complex (the white clawed crayfish, WCC) has experienced a drastic decline since 1973. Implementing conservation management strategies for this species requires a better understanding of the patterns and structure of its genetic diversity. In this study, we assessed the levels and patterns of genetic variation in 71 populations along the whole distributional range of the WCC in the Iberian Peninsula. The two mitochondrial markers analyzed (Cytochrome Oxidase subunit I and 16S rRNA genes) indicated high levels of genetic diversity, which are significantly geographically structured in three main genetic groups, two corresponding to Northern and one to Central-Eastern and the westernmost Iberian Peninsula. The diversity found included new private haplotypes, and revealed the potential effect of paleogeographic barriers and last glaciations in the population structure observed. Current conservation and management programs for the WCC in the Iberian Peninsula should consider these three phylogeographic groups as essential management units in order to preserve the remaining genetic diversity in the species.

Native European crayfish conservation was triggered by invasion of crayfish plague disease agent, Aphanomyces astaci, starting 1860s in Northern Italy. Resulting crayfish plague epidemics quickly spread over Continental Europe, then to Finland, Sweden and finally, after running amok around Europe, A. astaci was discovered also in Iberian Peninsula, Norway, Ireland, and United Kingdom in 1970s and 1980s. By that time significant proportion of native crayfish stocks had been lost, and while crayfish plague epidemics were still recorded, also industrialization and waterways construction were causing damage to remaining native crayfish stocks. While alien crayfish introductions, at least Faxonius limosus, already gave rise to first wave of crayfish plague epidemics in late 19th century, later in 1960s it was decided that introductions of alien Pacifastacus leniusculus should be initiated to replace native European crayfish populations. Decisions were based on presumed advantages for fishery, suitable habitat requirements and supposed immunity against A. astaci. Furthermore, conservation of native European crayfish species was sidelined and focus shifted toward alien crayfish stocking routine and consumption. Alien crayfish species introductions resulted in repeated waves of crayfish plague epidemics among remaining native crayfish stocks. It was soon discovered that alien crayfish of North American origin were, as suspected, permanent reservoirs for A. astaci, that some of those alien species were losing their resistance against selected strains of A. astaci and struggled in European aquatic ecosystems. In this article, we introduce numerous motives behind grand mistake of introducing alien crayfish species to Europe and then promoting their stocks instead of focusing on conservation of native crayfish species. We outline how false economical, biological and ecologic assumptions were used to justify a hasty introduction of alien crayfish, which has further devastated native crayfish and also permanently changed European aquatic ecosystems, both with disastrous consequences. Lesson to be learnt is that science-based warnings about alien species damage to native ecosystems and native crayfish must be taken with utmost caution. Protection of native European crayfish should be core issue, not commercial activities. Finally, we summarize main threats and actions needed to protect remaining native freshwater crayfish fauna in Europe.

The crayfish plague pathogen Aphanomyces astaci is one of the main factors responsible for the decline in European and Asian native crayfish species. This pathogen was transported to these regions through its natural carriers, North American crayfish species, which were introduced during the last century. Since then, the carrier species and the pathogen have spread worldwide due to globalization and the highly invasive nature of these species. In Europe, five carrier species have been categorized as high-risk as they are responsible for the loss of provisioning services, which endangers freshwater ecosystems. The red swamp crayfish Procambarus clarkii, in particular, is currently one of the most concerning species as its spread threatens crayfish biodiversity and freshwater ecosystems worldwide. In this study, we describe the first detection of A. astaci in an introduced population of P. clarkii in Central America, specifically in Costa Rica. Using molecular approaches, we analyzed 48 crayfish samples collected from Reservoir Cachí and detected the presence of A. astaci in four of these samples. The introduction of P. clarkii and the incorrect management of the species (related to its fishery and the commercialization of live specimens) over the past decades in Europe are mistakes that should not be repeated elsewhere. The detection of the pathogen is a warning sign about the dangerous impact that the introduction of this invasive crayfish may have, not only as a carrier of an emerging disease but also as a direct risk to the invaded ecosystems. Our results may serve to (1) assess current and future consequences, and (2) direct future research activities, such as determining the potential impacts of A. astaci on native decapod species, or on other introduced crayfish species that are used for aquaculture purposes, such as Cherax quadricarinatus.

Procambarus clarkii is a worldwide freshwater invasive crustacean from North America and was first introduced into Europe the 1970s. Along with P. clarkii, another invasive alien species was also spreading: Aphanomyces astaci. This pathogen is listed among the 100 World’s worst invasive species and involved in the European native crayfish decline. Although both species live in freshwater ecosystems, P. clarkii can withstand brackish waters and inhabit estuarine habitats. However, the presence of A. astaci associated to North American crayfishes has never been described in brackish waters. In this study, we have investigated the presence of A. astaci in a P. clarkii population of a Mediterranean coastal lagoon in the Albufera Natural Park, Valencia, Spain introduced in 1976. Our study confirmed the presence of this pathogen, and suggests that A. astaci has been spreading for more than four decades in the mentioned estuarine environment. Mitochondrial ribosomal rnnS and rnnL indicated that the isolated pathogen belongs to d1-haplotype (i.e., D-haplogroup) typically hosted by P. clarkii. The presence of this pathogen in a brackish environment may suggest a better adaptation than other A. astaci strains to adverse conditions, such as high salinity levels. This is a matter of concern for the conservation of European native freshwater crayfish and highlights once more the risk of introducing invasive alien crustaceans.

The pathogenic oomycete Saprolegnia parasitica secretes effector proteins that translocate into host cells through unclear mechanisms. Here, Trusch et al. show that the uptake of effector protein SpHtp3, resulting in RNA degradation, depends on a gp96-like host receptor and a second effector protein.

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 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.

Crayfish plague, caused by the pathogen Aphanomyces astaci, is one of the main factors responsible for the decimation of the native European crayfish species Austropotamobius pallipes. In Spain, two North American freshwater crayfish species, Procambarus clarkii and Pacifastacus leniusculus, were intentionally introduced during the 1970s for aquaculture and fishery purposes. Since then, incidences of crayfish plague have been continually reported. In this work, we evaluated more than 50 diagnosed cases of crayfish plague that have occurred in the Iberian Peninsula since 2004 by performing a microscopic examination of infected specimens and by molecularly identifying and haplotyping the pathogen. Our results showed that (i) the pathogen A. astaci has been active 45 years since the first introductions of the invasive North American crayfish species in the Iberian Peninsula, and (ii) P. clarkii and P. leniusculus are chronic reservoirs of the crayfish plague pathogen. Moreover, our data confirmed a correspondence between pathogen origin and spread and the specific haplotypes carried by the North American invasive crayfish located in the vicinity of each outbreak. We generated a crayfish plague incidence map of the Iberian Peninsula that shows (i) a northern area, mainly inhabited by alien P. leniusculus, where crayfish plague cases are associated with the b-haplotype specific to P. leniusculus, and (ii) southern, central and eastern areas that are basically inhabited by alien P. clarkii, where crayfish plague cases are associated with the d1- and d2-haplotypes specific to P. clarkii. The results presented here are evidence of the long standing and negative impact of the two invasive crayfish species on the native species, indicating the need for more extensive control measures.

The secondary cysts of the fish pathogen oomycete Saprolegnia parasitica possess bundles of long hooked hairs that are characteristic to this economically important pathogenic species. Few studies have been carried out on elucidating their specific role in the S. parasitica life cycle and the role they may have in the infection process. We show here their function by employing several strategies that focus on descriptive, developmental and predictive approaches. The strength of attachment of the secondary cysts of this pathogen was compared to other closely related species where bundles of long hooked hairs are absent. We found that the attachment of the S. parasitica cysts was around three times stronger than that of other species. The time sequence and influence of selected factors on morphology and the number of the bundles of long hooked hairs conducted by scanning electron microscopy study revealed that these are dynamic structures. They are deployed early after encystment, i.e., within 30 sec of zoospore encystment, and the length, but not the number, of the bundles steadily increased over the encystment period. We also observed that the number and length of the bundles was influenced by the type of substrate and encystment treatment applied, suggesting that these structures can adapt to different substrates (glass or fish scales) and can be modulated by different signals (i.e., protein media, 50 mM CaCl2 concentrations, carbon particles). Immunolocalization studies evidenced the presence of an adhesive extracellular matrix. The bioinformatic analyses of the S. parasitica secreted proteins showed that there is a high expression of genes encoding domains of putative proteins related to the attachment process and cell adhesion (fibronectin and thrombospondin) coinciding with the deployment stage of the bundles of long hooked hairs formation. This suggests that the bundles are structures that might contribute to the adhesion of the cysts to the host because they are composed of these adhesive proteins and/or by increasing the surface of attachment of this extracellular matrix.

Global introductions of aquatic species and their associated pathogens are threatening worldwide biodiversity. The introduction of two North American crayfish species, Procambarus clarkii and Pacifastacus leniusculus, into Japan in 1927 seems to have negatively affected native Japanese crayfish populations of Cambaroides japonicus. Several studies have shown the decline of these native populations due to competition, predation and habitat colonization by the two invasive North American crayfish species. Here, we identify an additional factor contributing to this decline. We report the first crayfish plague outbreaks in C. japonicus populations in Japan, which were diagnosed using both histological and molecular approaches (analyses of the internal transcribed spacer region). Subsequent analyses of the mitochondrial ribosomal rnnS and rnnL regions of diseased specimens indicate that these outbreaks originated from a P. clarkii population and identify a novel haplotype of Aphanomyces astaci, d3-haplotype, hosted by P. clarkii. Overall, our findings demonstrate the first two cases of crayfish plague in Japan, and the first case in a non-European native crayfish species, which originated from the red swamp crayfish P. clarkii. This finding is a matter of concern for the conservation of the native freshwater species of Japan and also highlights the risk of introducing crayfish carrier species into biogeographic regions harboring species susceptible to the crayfish plague.

The pathogen Aphanomyces astaci Schikora 1906 is responsible for the decline of the native crayfish species of Europe, and their current endangered status. This pathogenic species is native to North America and only colonizes aquatic decapods. The North American crayfish species have a high resistance to this pathogen, while species from other regions are highly susceptible. However, recent field and laboratory observations indicate that there might exist some populations with resistance against this disease. The objective of this study was to test the susceptibility of 8 selected native European crayfish populations of Austropotamobius pallipes Lereboullet 1858 from the Pyrenees. We challenged them against the genome sequenced strain AP03 of A. astaci isolated from a North American red swamp crayfish, Procambarus clarkii Girard 1852, in the Garrotxa Natural Park, Girona. The results showed that there are significant differences (P

Emerging fungal diseases are threatening ecosystems and have increased in recent decades. In corals, the prevalence and consequences of these infections have also increased in frequency and severity. Coral reefs are affected by an emerging fungal disease named aspergillosis, caused by Aspergillus sydowii. This disease and its pathogen have been reported along the Caribbean and Pacific coasts of Colombia. Despite this, an important number of coral reefs worldwide have not been investigated for the presence of this pathogen. In this work, we carried out the surveillance of the main coral reef of the Ecuadorian Pacific with a focus on the two most abundant and cosmopolitan species of this ecosystem, Leptogorgia sp. and Leptogorgia obscura. We collected 59 isolates and obtained the corresponding sequences of the Internal Transcribed Spacers (ITS) of the ribosomal DNA. These were phylogenetically analyzed using MrBayes, which indicated the presence of two isolates of the coral reef pathogen A. sydowii, as well as 16 additional species that are potentially pathogenic to corals. Although the analyzed gorgonian specimens appeared healthy, the presence of these pathogens, especially of A. sydowii, alert us to the potential risk to the health and future survival of the Pacific Ecuadorian coral ecosystem under the current scenario of increasing threats and stressors to coral reefs, such as habitat alterations by humans and global climate change.

Fungal morphogenesis is an exciting field of cell biology and several mathematical models have been developed to describe it. These models require experimental evidences to be corroborated and, therefore, there is a continuous search for new microscopy and image analysis techniques. In this work, we have used a Canny-edge-detector based technique to automate the generation of hyphal profiles and calculation of morphogenetic parameters such as diameter, elongation rates and hyphoid fitness. The results show that the data obtained with this technique are similar to published data generated with manualbased tracing techniques and that have been carried out on the same species or genus. Thus, we show that application of edge detector-based technique to hyphal growth represents an efficient and accurate method to study hyphal morphogenesis. This represents the first step towards an automated analysis of videomicroscopy images of fungal morphogenesis.

Colletotrichum acutatum is major pathogen of fruit crops, causing economically important losses of temperate, subtropical and tropical fruits worldwide. However, few studies have been carried out on key aspects of its biology. This is mainly because traditionally isolates of C. acutatum were often wrongly identified as C. gloeosporioides. Effective separation of the two species was not possible until the introduction of molecular tools for taxonomy. The life cycle of C. acutatum comprises a sexual and an asexual stage and much remains to be resolved regarding the genetics of sexuality and the effects of the sexual stage on population structure. Colletotrichum acutatum exhibits both infection strategies described for Colletotrichum species, i.e. intracellular hemibiotrophy and subcuticular-intramural necrotrophy, and may also undergo a period of quiescence in order to overcome resistance mechanisms in immature fruit such as pre-formed toxic compounds and phytoalexins, or due to the unsuitability of unripe fruit to fulfill the nutritional and energy requirements of the pathogen. Colletotrichum acutatum may overwinter as mycelium and/or appressoria in or on different parts of the host. Conidia are water-born and spread by rain episodes so infections are usually highest during the wettest periods of the growing season. Current management strategies for this fungus comprise the exploitation of cultivar resistance, cultural, chemical, and biological control methods, and preventive strategies such as disease-forecasting models. This review focuses on the current knowledge of biological aspects of C. acutatum and related Colletotrichum species and includes a discussion of the progress towards their control.

The description, identification and classification of organisms are the pillar in biodiversity and evolutionary studies. The fungal-like organism Saprolegnia contains important animal pathogens. However, its taxonomy is weak, making it difficult to perform further studies. This problem mainly arises from the unavailability of suitable holotypes. We propose a standardized protocol for describing Saprolegnia spp. that includes good cultural practices and proper holotype preservation. In order to illustrate this new proposal, we describe two species, Saprolegnia aenigmatica sp. nov. and Saprolegnia racemosa sp. nov., based on the recently described molecular operational taxonomic units (MOTUs), phylogenetic relationships, and the analyses of morphological features. We show that they belong to two different MOTUs that are grouped into two sister clades. Morphologically, we find that S. racemosa exhibits a species-specific character, i.e., aggrupation of oogonia in racemes, while S. aenigmatica does not have any specific characters. Analyses of a combined set of characters, i.e., length and breadth of sporangia, length/breadth ratio (l/b) of oogonia, cyst and oospore diameter, and the number of oospores per oogomium, allow distinguishing these two species. To improve Saprolegnia taxonomy, we propose to incorporate into the protologue: (i) several isolates of the new species; (ii) the rDNA sequences to compare them to data-bases of Saprolegnia sequences of reference; (iii) a phylogenetic analysis to check relationships with other species; (iv) to preserve holotypes in absolute ethanol and to include lyophilized material from holotype; and (v) the ex-type as a pure culture from single-spore isolates stored in at least two different collections.

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.

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.