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.


Results
Aphanomyces astaci detection. We obtained a total of 391 crayfish from 30 locations in five states (Kansas, Kentucky, Louisiana, Mississippi and South Carolina) (Fig. 2) Table 1). Additionally, one species of freshwater shrimp (Palaemon kadiakensis) was sampled and analyzed for the presence of A. astaci.
From 392 individuals, 132 crayfish and one shrimp tested positive for the A. astaci ITS region, 102 tested negative and 158 were not analyzed (i.e., the crayfish did not molt). Aphanomyces astaci-positive samples came from 23 locations and included 19 crayfish species: C. shufeldtii, C. latimanus, C. striatus, C. fodiens, C. oryktes, F. etnieri species complex, Faxonius sp., F. tricuspis, F. wrighti, P. ablusus, P. acutus, P. clarkii, P. hayi, P. hybus, P. pubescens, P. raneyi, P. troglodytes, P. viaeviridis and P. vioscai) and one species of shrimp (Palaemon kadiakensis) ( Fig. 2 and Supplementary Table 1). The ITS sequences (specific primers 42 and 640) for the 132 clinical samples were 99.82% identical to sequences of A. astaci available in GenBank (e.g., sequence FM999249-isolate SAP302) and identical to each other.   67 with 476 and 355 bp fragments of rnnS and rnnL amplicons, respectively. The phylogenetic approximations (BI and ML) supported the differentiation of the two lineages previously described 52 (Fig. 4). The genetic diversity analysis confirmed and supported the phylogenetic analysis. Although both mitochondrial ribosomal rnnS and rnnL regions were informative, there were differences between them (Fig. 5). We obtained five haplotypes for the rnnS subunit (Fig. 5a), represented by four segregating sites (S), with a haplotype diversity (Hd) of 0.703, a nucleotide diversity (π) of 0.0022 and 1.018 average nucleotide differences (k). On the other hand, we obtained ten haplotypes for the rnnL subunit (Fig. 5b), represented by13 segregating sites (S), with a haplotype diversity (Hd) of 0.786, a nucleotide diversity (π) of 0.009 and 3.16 average nucleotide differences (k). However, concatenating rnnS and rnnL regions we confirmed a total of 12 haplotypes represented by 17 segregating sites (S), where 13 of them were parsimony informative (Fig. 5c). The concatenated sequences presented a haplotype diversity (Hd) of 0.801 with a nucleotide diversity (π) of 0.005 and 4.178 average nucleotide differences (k).
The phylogenetic approximations and the haplotype network confirmed the presence of eight haplotypes for the rrnS and rrnL concatenated regions among the analyzed samples: a, d2, usa1, usa2, usa3, usa4, usa5 and usa6haplotypes (Figs. 4, 5) ( Table 1). Six of these eight haplotypes (usa1, usa2, usa3, usa4, usa5 and usa6-haplotypes) are described and reported here for the first time, bringing the total number of known A. astaci haplotypes to  Table 1) in North America. Haplotype frequencies indicated by relative proportion of the pie graph. Each color of the legend represents a different haplotype based on concatenated mitochondrial rnnS and rnnL regions. Crayfish species hosting different haplotypes are indicated at each location (also in Table 1). Location code LP corresponds to Lake Pitt 50 , LT to Lake Tahoe 63 , BL to Big Lake 62 , TL to Trout Lake 62     We found five scenarios relative to the distribution of A. astaci haplotypes among crayfish species at various locations: (1) one haplotype from one crayfish species (Locations 1, 4, 7 and 9), (2) two haplotypes from one crayfish species (Locations 3 and 23) (see below), (3) one haplotype from two crayfish species (Location 13), (4) two haplotypes from two crayfish species (Location 6) (see below), and (5) three haplotypes, one from each of three crayfish species (Location 11). At Location 23, the two haplotypes were recovered from one P. raneyi molt (i.e., we isolated the pathogen in two different pure cultures) and at Location 6, one haplotype was recovered from both species, and the second haplotype from only one of the species (Fig. 3) (Table 1).

Discussion
We report and describe for the first time the presence, distribution and genetic diversity of the crayfish plague pathogen, A. astaci, in its potential center of origin, the southeastern US 68 . Previous studies regarding the origin, diversity, and distribution of A. astaci have addressed different questions. In the current study, we have explored several of them, including (1) the distribution and diversity of A. astaci in the southeastern US, and (2) whether A. astaci haplotypes are crayfish species-specific 50,54,55 and/or restricted to a narrow geographic region of the pathogen's native range 52,62,63 . Our results indicated that A. astaci is present and widely distributed in the southeastern US (e.g., it was present in 21 out of 23 sampling sites across the nine river basins we investigated) Table 1. Location and samples ID of the of the crayfish species analyzed for Aphanomyces astaci haplotypes. North American crayfish species from each of the analyzed locations (Location code:1-30), including the corresponding collection codes, state, sample type (clinical sample/culture), DNA isolation code (CE19/), BLAST species ID, result of the mitochondrial rnnS/rnnL haplotype and GenBank references for the rnnS and rnnL regions. NA: not applicable. Each unique collection code refers to an individual crayfish. *Different pieces from the same molt.  [62][63][64] . However, in the southeastern US, we examined 21 North American crayfish species and found a total of eight haplotypes, six of which are previously unreported. This represents almost 70% of the A. astaci haplotype diversity known globally. The genetic diversity we describe is comparable to that of other pathogenic oomycetes. For example, in the US, 13 Phytopthtora infestans haplotypes were recently described using five mitochondrial loci 69 . Moreover, we showed that A. astaci can chronically colonize 19 additional North American crayfish species and also one species of freshwater shrimp (Palaemon kadiakensis). Previously, the only known report of a wild shrimp carrying A. astaci was of a Macrobrachium lanchesteri population from Indonesia that co-occurred with P. clarkii 70 . Only two (Cambarus tenebrosus and Lacunicambarus ludovicianus) of the 21 crayfish species that we sampled did not test positive for A. astaci. Thus, we confirm the presence of A. astaci in a wider distribution (Fig. 2) than previously described in North America [62][63][64] , confirming the origin of this pathogenic disease in North America.
Although the presence of the crayfish plague in North America was previously reported 62-64 , those studies examined a limited number of crayfish species and found haplotypes that had been previously isolated in Europe and Japan (i.e., a or b-haplotypes) 33,62-64 . Likewise, we confirmed the presence in North America of two haplotypes (a-and d2-haplotypes) first described in Europe. In the case of the a-haplotype, it was described as the first haplotype introduced to Europe in the nineteenth century 43,52 , yet no North American carrier has been described in the literature for this introduction. Two recent studies described F. rusticus and F. obscurus as the only North American crayfish known to host the a-haplotype 62,64 . We expanded the known range of this haplotype to include the southeastern US and added three additional native taxa as hosts: the F. etnieri species complex, P. acutus and P. hybus. In the case of the d2-haplotype, the only two North American crayfish described as carriers so far in Europe are P. clarkii and Procambarus fallax virginalis. As we expected, our study shows for the first time the presence of A. astaci in native populations of P. clarkii 54 , which carried the d2-haplotype and the newly discovered usa6-haplotype.
We confirmed that at least some A. astaci haplotypes are neither host species-specific nor narrowly distributed. Broadening and deepening our knowledge of A. astaci haplotype diversity and distribution, we recovered the d2-haplotype from two species in different genera (Cambarellus and Procambarus) in one river basin (Locations 3, 4, 6 and 7) and from a third genus (Cambarus) in a different river basin (Location 11) ( Table 1). Although the a-haplotype was previously thought to be restricted to F. rusticus and F. obscurus 62,64 , we found it not only in another congener, but also in another genus (Procambarus). In addition, we documented the a-haplotype in two river basins in the southeastern US, even though it was previously thought to be restricted to the northern US. We also documented the d2-haplotype from two river basins. Our results indicated that A. astaci haplotypes tend to be neither host species-specific nor restricted to small geographic areas.
We also found instances of multiple haplotypes of A. astaci occurring in one species, in one individual, and in one location. We isolated two pure cultures of two different, but closely related, A. astaci haplotypes (usa3 and usa5) from a single individual (SC32 MOLT in Location 23). Also, multiple A. astaci haplotypes often co-occurred in a location. Within Location 11, we found three haplotypes from two lineages: usa2-haplotype from Lineage 1 and d2-haplotype and usa4-haplotype from Lineage 2. We also recovered two closely related haplotypes (d2 and usa6) from Locations 3 and 6 and two others (usa3 and usa5) from Location 23. None of the phenomena described have been documented previously. Our analysis showed no clear haplotype distributional patterns with respect to crayfish host species or geography. Thus, the biogeographic distribution of A. astaci genetic diversity within North America needs further investigation.
The pathogen's genetic diversity and observed lack of species-specificity may have implications within North America as well as on continents where A. astaci is introduced. Crayfish have been frequently translocated within North America. For example, although P. clarkii and F. virilis are native in parts of North America, both are invasive beyond their native range, including west of the Great Divide where all native crayfish belong to the family Astacidae 71,72 . The genetic diversity of A. astaci suggests potential for intracontinental impacts from translocations of haplotypes. Presumably all North American crayfish are resistant to all haplotypes of A. astaci, considering that no mass mortalities have been observed after crayfish translocations. However, more subtle effects of translocated haplotypes with differing virulence from native haplotypes would not likely have been detected. Further understanding of the geographical distribution of A. astaci genetic diversity, its virulence, and the immune responses of crayfish to novel haplotypes would be beneficial for managing crayfish translocations and conserving native species in North America.
Our new approach of obtaining A. astaci from crayfish molts produced larger amounts of the pathogen for identification and isolation in pure cultures. This resulted in detection of A. astaci in 56.17% of the analyzed clinical samples. Instead of analyzing parts of the crayfish most susceptible to infection (i.e., soft abdominal cuticle, telson or walking legs) 73 , we waited until the molting period. Thus, we avoided the unnecessary killing of crayfish and maximized the amount of pathogen grown within the original host. Moreover, by incubating crayfish molts in distilled water for three days, we allowed the pathogen to continue growing both within and outside of the cuticle. By controlling the incubation temperature at 4 °C, we reduced bacterial blooms during the first stages of the pathogen isolation. Bacteria commonly surround the A. astaci hyphae, and antibiotics are often added to the PGA medium to inhibit bacterial growth. However, because bacteria rapidly develop resistance to these antibiotics, their addition becomes less effective over time. By reducing the incubation temperature and introducing a physical barrier 74 , we controlled bacterial blooms 75 and obtained many clean isolates of the pathogen.
By using this new approach, we more readily detected and isolated A. astaci. Moreover, we strongly recommend isolating the pathogen in an axenic culture to assure an optimal concentration of the pathogen for analysis. In this study, we combined the sequencing of the ITS region (for identification of the Aphanomyces species) 76  www.nature.com/scientificreports/ the mtDNA (for identification of the A. astaci haplotype) 52 in both clinical samples and pure cultures. Several studies have examined the genetic diversity of A. astaci using diverse methodologies (i.e., RAPD-PCR, the chitinase gene, AFLP, microsatellites) 50,52,56,[58][59][60]77 . Although the mtDNA approximation 52 requires a large concentration of the pathogen, it provides reliable results 67 and enables detection of new diversity within A. astaci that might go undetected with other approximations 59,60 . Other approaches, such as eDNA monitoring 78 , could also be used to detect the presence of A. astaci in water samples and potentially to detect additional genetic diversity. The uniquely high diversity of A. astaci haplotypes found in this study are an important step toward confirming the host-pathogen co-evolution between A. astaci and North American crayfish species. Our results suggest that further sampling in North America will reveal additional undiscovered A. astaci haplotype diversity vital to answering new host-pathogen co-evolutionary questions. Further, long-term monitoring might reveal emerging A. astaci diversity, depending on the rate of pathogen evolution.

Methods
Sample collection. We sampled 25 locations in five states between February and May 2019. Additionally, we included nine crayfish samples previously collected from five more locations and preserved in 95% ethanol. The specimens from Kansas were from an introduced population of Procambarus clarkii (Supplementary Table 1). Crayfish were captured by kick-seining and trapping and then held, separated by species and collection location, in the laboratory (US Forest Service, Southern Research Station, Center for Bottomland Hardwoods Research, in Oxford [Mississippi, USA]) until processed. Crayfish were kept individually in labeled, individual, plastic containers with chlorine-free water, aerators, gravel and medium size rocks. Each crayfish was kept alive until it molted and was subsequently preserved in 95% ethanol.

Microscopic examination and Aphanomyces astaci isolation.
For the microscopic examination, the molts were carefully removed and handled individually due to the fragility of the samples. Molts were kept in individual petri dishes with distilled water at 4 °C in order to reduce bacterial growth and optimize the growth of potential mycelium 75 . After three days, each molt was examined with an inverted microscope to check for the presence of growing hyphae. Each sample was divided into two parts: one for molecular identification and one for the pathogen isolation.
Pieces of the molt intended for molecular identification were transferred into 1.5-ml tubes and were frozen at − 80 °C until the DNA extraction was carried out in the laboratory at the Department of Biology at the University of Mississippi (UM). Samples were subsequently homogenized by manual mechanical disruption. A DNeasy Blood & Tissue Kit (Qiagen, Valencia, California, USA) was used to isolate genomic DNA.
Pieces of the molt intended for culture isolation were grown in Peptone Glucose Agar (PGA) at 4 °C. A selected agar plug was cut out from the resulting mycelia and inserted within an aluminum ring placed on a new PGA plate to protect the growing isolate from bacterial growth 74 . Plates were incubated at 4 °C for seven days, and each isolate was transferred into new PGA media once the hyphae spread under the metal ring. This process was repeated until no bacterial growth was observed using an inverted microscope. Additionally, a selected agar plug containing mycelia was placed in a 9 mm Petri dish containing 10 mL of liquid Peptone Glucose (PG-1) and incubated at room temperature for 48 h in order to obtain material for molecular identification. The obtained mycelium was transferred into 1.5-ml tubes and frozen at − 20 °C until the DNA extraction was carried out in the laboratory at UM. Samples were subsequently homogenized by manual mechanical disruption, and DNA extractions were carried out using an E.Z.N.A. Fungal DNA Mini Kit (Omega Biotek, Norcross, Atlanta, USA).

Aphanomyces astaci detection and haplotyping.
To test for the presence of the A. astaci pathogen, a fragment of the internal transcribed spacer (ITS) region was amplified using the diagnostic primers 42 (5′-GCT TGT GCT GAG GAT GTT CT-3′) 73 and 640 (5′-CTA TCC GAC TCC GCA TTC TG-3′) 79 (which amplify ITS1, the 5.8S rDNA and ITS2) in a single round of amplification according to the assay described by 73 . DNA extracted from a pure culture of A. astaci was used as the positive control; sterile Milli-Q water was used as the negative control. Amplified products (3 μL of each reaction) were analyzed by electrophoresis in 2% agarose SB gels stained with ethidium bromide and then purified using magnetic beads. Sequencing of both strands of positive products was performed using an automated sequencer (Applied Biosystems 3730xl DNA Analyzer, DNA Analysis Facility at Yale, USA and Applied Biosystems 3730xl DNA, Macrogen, The Netherlands). Sequences were aligned and edited using the program Geneious 10.0.2 80 . A BLAST search (NCBI database) was performed to verify the identity of each sequence.
Genomic DNA samples that tested positive for the presence of A. astaci with diagnostic primers 42 73 and 640 79 were used to characterize the phylogenetic relationships and haplotypes. The mitochondrial ribosomal small (rnnS) and large (rnnL) subunits were amplified using the primer pairs AphSSUF/AphSSUR (5′-AGC ACT CCG CCT GAA GAG TA-3′ and 5′-GGG CGG TGT GTA CAA AGT CT-3′) and AphLSUF/AphLSUR (5-AGG CGA AAG CTT ACT ATG ATGG-3′ and 5′-CCA ATT CTG TGC CAC CTT CT-3′), respectively, as described by 52 . Positive and negative controls were included. Amplified products were analyzed by electrophoresis, purified, sequenced and aligned as described above.
Phylogenetic approximations based on Bayesian inference (BI) and maximum likelihood (ML) were used to reconstruct relationships. The BI analysis was performed in MrBayes v.3.2.6 81 using the MCMC method with 10 million generations, three runs (8 chains per run) with a burn-in of 25% and a standard deviation of split frequencies < 0.01. Nodes with posterior probability (pp) values ≥ 0.95 were considered supported. The ML analysis was performed using RAxML v.8 82 , as implemented in raxmlGUI v1.5b1 83