Protocols for studying the crayfish plague pathogen, Aphanomyces astaci, and its host-pathogen interactions.

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 1960 s to 80 s 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.

eDNA monitoring as a tool for evaluating the reintroduction of Austropotamobius pallipes after a crayfish plague outbreak.

The crayfish plague, a severe disease caused by the oomycete Aphanomyces astaci, is responsible for most population declines of susceptible crayfish in Europe. This pathogen has been devastating native populations of Austropotamobius pallipes since the 1970s in the Iberian Peninsula. In this study, we report a massive mortality event in one of the most important Spanish populations of A. pallipes. We aimed to: (i) identify the cause of the mortality, and (ii) evaluate the reintroduction viability of the species. Over the course of six months, we used environmental DNA (eDNA) and traditional trap-based methods to detect the presence of A. astaci or of native or invasive crayfish in order to evaluate the reintroduction viability of A. pallipes to the affected population. We did not capture any live crayfish or detect the presence of A. astaci in the reservoir water during the six months following the mass mortality event. Our analyses indicated that it was feasible to initiate a reintroduction program at the site, which will continue to be monitored for three to five years and will help improve the conservation status of A. pallipes.

On the conservation of white-clawed crayfish in the Iberian Peninsula: Unraveling its genetic diversity and structure, and origin.

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.

Aphanomyces astaci in Mexico: A new haplotype from dwarf crayfish Cambarellus montezumae.

The crayfish plague is an emerging infectious disease caused by the pathogen Aphanomyces astaci (Oomycota), which is responsible for the decimation of Eurasian freshwater crayfish. This pathogen can coexist with the North American crayfish. These are chronic carriers of the disease as consequence of an immune response that can contain the growth of the pathogen without killing it. The origin of A. astaci locates in the southeastern United States and coincides with the origin of the family Cambaridae. This diverse family of decapods is distributed in North America from southern Canada to Honduras. However, only the native crayfish species from Canada and the USA have been examined for the presence of A. astaci. In this study, we describe for the first time the presence of A. astaci in Mexico in a population of the native species Cambarellus montezumae. By analyzing the small (rrnS) and large (rrnL) mitochondrial ribosomal regions, we showed the presence of two haplotypes of A. astaci within the same population (d1-haplotype and, a novel haplotype that was named, mex1-haplotype). The finding of A. astaci in Mexico confirms the occurrence of this pathogen within the range of the family Cambaridae. The individuals of C. montezumae appear to be chronic carriers of A. astaci, indicated by the lack of documented crayfish plague outbreaks in this population, similar to the pattern observed in other North American species. Thus, the results are of special concern to susceptible species of southern regions of America, i.e., Parastacidae. Therefore, this work emphasizes the need to better understand the distribution and genetic diversity of A. astaci within the distribution range of the natural carriers, i.e., North American species, especially the unexplored area of the family Cambaridae.

Austropotamobius pallipes can be infected by two haplotypes of Aphanomyces astaci: A key example from an outbreak at an ex-situ conservation facility.

The crayfish plague, caused by the pathogen Aphanomyces astaci, is a pandemic disease endemic to North America that has been devastating susceptible crayfish populations in Europe since the 19th century. In Spain, this disease has decimated populations of the native crayfish species Austropotamobius pallipes due to introductions of North American crayfish, which act as vectors of the pathogen. To combat against these losses, several regional governments have established ex-situ breeding programs to restock wild populations of the species. In this study, we report on an outbreak of A. astaci that occurred in one of the most important A. pallipes aquaculture centers in Spain. Using a variety of detection methods, we analyzed affected crayfish and environmental samples from the facilities over a period of six months and determined that the outbreak was caused by two haplotypes of A. astaci, d1 and d2, which are both associated with the North American crayfish species Procambarus clarkii. To our knowledge, this is the first report of a two-haplotype coinfection of A. astaci outside the native range of this pathogen.

Fungal signatures of oral disease reflect environmental degradation in a facultative avian scavenger.

Degradation of natural ecosystems increases the risk of infections in wildlife due to microbiota dysbiosis. However, little is known about its influence on the development of fungal communities in predators and facultative avian scavengers. We evaluated the incidence of oral disease in wild nestling black kites (Milvus migrans) under contrasting environmental degradation conditions, and explored their oral fungal patterns using molecular methods and multivariate analysis. Oral lesions were found in 36.8% of the 38 nestlings examined in an anthropogenically altered habitat (southeastern Madrid, Spain), but in none of the 105 nestlings examined in a well-conserved natural area (Doñana National Park, Spain). In a subsample of 48 black kites, the composition of the oral fungal community differed among symptomatic nestlings from Madrid (SM) and asymptomatic nestlings from Madrid (AM) and Doñana (AD). Opportunistic fungal pathogens (e.g., Fusarium incarnatum-equiseti species complex, Mucor spp., Rhizopus oryzae) were more prevalent in SM and AM than in AD. Hierarchical clustering and principal component analyses revealed that fungal patterns were distinct between both study areas, and that anthropogenic and natural environmental factors had a greater impact on them than oral disease. Fungal signatures associated with anthropogenic and natural stresses harbored some taxa that could be used to flag oral infection (F. incarnatum-equiseti species complex and Alternaria), indicate environmental degradation (Alternaria) or provide protective benefits in degraded environments (Trichoderma, Epicoccum nigrum and Sordaria). Co-occurrence associations between potentially beneficial and pathogenic fungi were typical of AM and AD, hinting at a possible role in host health. This study shows that early-life exposure to highly degraded environments induces a shift towards a higher prevalence of pathogenic species in the oral cavity of black kites, favoring oral disease. Furthermore, our findings suggest potential ecological applications of the monitoring of oral mycobiome as a bioindication of oral disease and environmental degradation.

Resistance to Crayfish Plague: Assessing the Response of Native Iberian Populations of the White-Clawed Freshwater Crayfish.

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 invasive alien red-eared slider turtle, Trachemys scripta, as a carrier of STEF-disease pathogens.

The fungal pathogens Fusarium falciforme and Fusarium keratoplasticum are responsible for the sea turtle egg fusariosis (STEF) throughout main nesting areas of the world. In this study, we investigated whether eggs of the invasive alien red-eared slider turtle, Trachemys scripta, can carry these fungal pathogens. Using multilocus sequence typing of four nuclear DNA regions, we found that eggs of T. scripta naturally can carry these two Fusarium pathogenic species, as well as other Fusarium species belonging to the Fusarium solani species complex. Physiological studies on F. falciforme and F. keratoplasticum isolates revealed that their optimal growth temperature coincided with the pivotal temperature for T. scripta embryos, ca 29.5 ± 0.5 °C, providing an evidence of a potential advantageous biological property for host colonization and virulence. A host-pathogen interaction network analysis of species of the FSSC and their hosts confirmed that F. falciforme and F. keratoplasticum are generalist pathogens in a wide range of animal hosts of worldwide geographical distribution. Finally, we show that nesting areas of this invasive turtle T. scripta in the Mediterranean freshwater marshes can act as chronic reservoirs of these STEF pathogens, and this invasive species can act as a potential vector for the spread of STEF among wild native species and even to humans.

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.

Evaluation of Potential Transfer of the Pathogen Saprolegnia parasitica between Farmed Salmonids and Wild Fish.

Saprolegnia infections are among the main parasitic diseases affecting farmed salmonids. The distribution and potential transfer of Saprolegnia spp. between farms and the natural environment has been scarcely investigated. Therefore, this work aimed to study the diversity and abundance of oomycete species in salmonid farms, tributary water, and effluent water systems. Four trout farms in Italy and two Atlantic salmon farms in Scotland were considered. In Italian farms, 532 isolates of oomycetes were obtained from fish and water, at upstream, inside, and downstream the farms. In Scottish farms, 201 oomycetes isolates were obtained from water outside the farm and from fish and water inside the farming units. Isolates were identified to the species level through amplification and sequencing of the ITS rDNA region. In Italy, S. parasitica was significantly more present in farmed than in wild fish, while in water it was more frequently isolated from the wild, particularly in effluent systems, not associated with more frequent isolation of S. parasitica in wild fish downstream the farm. In Scotland, S. parasitica was the most prevalent species isolated from fish, while isolates from water were mostly Pythium spp. with few S. parasitica isolates from upstream and downstream the farms.

Tracing the origin of the crayfish plague pathogen, Aphanomyces astaci, to the Southeastern United States.

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.

Aphanomyces astaci mtDNA: insights into the pathogen's differentiation and its genetic diversity from other closely related oomycetes.

The causative agent of crayfish plague, Aphanomyces astaci (Saprolegniales, Oomycota), is one of the 100 world's worst invasive alien species and represents a major threat to freshwater crayfish species worldwide. A better understanding of the biology and epidemiology of A. astaci relies on the application of efficient tools to detect the pathogen and assess its genetic diversity. In this study, we validated the specificity of two recently developed PCR-based approaches used to detect A. astaci groups. The first relies on the analysis of mitochondrial ribosomal rnnS (small) and rnnL (large) subunit sequences and the second, of sequences obtained by using genotype-specific primers designed from A. astaci whole genome sequencing. For this purpose, we tested the specificity against 76 selected isolates, including other oomycete species and the recently described species Aphanomyces fennicus, which, when used in nrITS-based specific tests for A. astaci, is known to result in a false positive. Under both approaches, we were able to efficiently and accurately identify A. astaci and its genetic groups in both pure cultures and clinical samples. We report that sequence analysis of the rnnS region alone is sufficient for the identification of A. astaci and a partial characterization of haplogroups. In contrast, the rnnL region alone is not sufficiently informative for A. astaci identification as other oomycete species present sequences identical to those of A. astaci.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Mapping 15 years of crayfish plague in the Iberian Peninsula: The impact of two invasive species on the endangered native crayfish.

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.

Cell entry of a host-targeting protein of oomycetes requires gp96.

The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants.

Hidden sites in the distribution of the crayfish plague pathogen Aphanomyces astaci in Eastern Europe: Relicts of genetic groups from older outbreaks?

The crayfish plague agent Aphanomyces astaci is one of the world's most threatening invasive species. Originally from North America, the pathogen is being imported alongside American crayfish species, which are used for various purposes. In this study, we investigated the marginal, currently known distribution area of the pathogen in Eastern Europe by sampling narrow-clawed crayfish (Astacus leptodactylus) and spiny-cheek crayfish (Orconectes limosus) populations. In addition, using specific real-time PCR, we tested several marine decapod species, which also occur in brackish waters of the Danube at the West coast of the Black Sea and the Dniester River basin. By sequencing the nuclear chitinase gene, mitochondrial rnnS/rnnL DNA and by genotyping using microsatellite markers, we identified the A. astaci haplogroups of highly infected specimens. The A. astaci DNA was detected in 9% of the investigated A. leptodactylus samples, both in invaded and non-invaded sectors, and in 8% of the studied O. limosus samples. None of the marine decapods tested positive for A. astaci. The results revealed that narrow-clawed crayfish from the Dniester River carried the A. astaci B-haplogroup, while A. astaci from the Danube Delta belonged to the A- and B-haplogroups. In the invaded sector of the Danube, we also identified the A-haplogroup. Microsatellite analysis revealed a genotype identical to the genotype Up. It might be that some of the detected A. astaci haplogroups are relics from older outbreaks in the late 19th century, which may have persisted as a chronic infection for several decades in crayfish populations.

Crayfish plague in Japan: A real threat to the endemic Cambaroides japonicus.

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.

MtDNA allows the sensitive detection and haplotyping of the crayfish plague disease agent Aphanomyces astaci showing clues about its origin and migration.

The oomycete Aphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogen A. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studied A. astaci strains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating from Orconectes sp., Pacifastacus leniusculus and Astacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from the P. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating from Procambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from the Orconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of the A. astaci haplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.

Specialized attachment structure of the fish pathogenic oomycete Saprolegnia parasitica.

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.