Transcriptome of Sphaerospora molnari (Cnidaria, Myxosporea) blood stages provides proteolytic arsenal as potential therapeutic targets against sphaerosporosis in common carp.

BACKGROUND: Parasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. Myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. Their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. Some myxozoans are important aquaculture pathogens such as Sphaerospora molnari replicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. Proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. In the absence of a S. molnari genome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identify S. molnari proteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. Furthermore, a subset of orthologs was used to characterize 3D structures and putative druggable targets. RESULTS: An assembled and host filtered transcriptome containing 9436 proteins, mapping to 29,560 contigs was mined for protease virulence factors and revealed that cysteine proteases were most common (38%), at a higher percentage than other myxozoans or cnidarians (25-30%). Two cathepsin Ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. We also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qPCR). In total, 235 transcripts were identified as putative proteases using a MEROPS database. In silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development. CONCLUSIONS: In silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. The present study of S. molnari's protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. The pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change.

Kudoa sp. (Myxozoa, Multivalvulida): first report in five commercial fish species from the Canary Islands-FAO 34 (Macaronesia-Spain).

Kudoid myxozoans have been reported causing serious chronic problems in marine fisheries, by reducing the market value of infected fish through pathological damage to the host musculature. We report here the overall prevalence of a Kudoa species in 84/277 (30.3%) fishes from 20 different species of high commercial value captured between October 2011 and December 2013 from the United Nations Food and Agriculture Organization (FAO) 34 commercial fishing area, near the coast of the Canary Islands (Spain). Macroscopic examination showed myxozoan-like cysts in skeletal muscle from 5 of the 20 fish species examined, with the following prevalences: Pagellus acarne (86.7%), Pagellus erythrinus (46.5%), Serranus cabrilla (27.8%), Spondyliosoma cantharus (19.4%), and Sarpa salpa (28.6%). Infection intensity was determined based on spore counts following muscle tissue digestion. Morphometric studies to characterize the species and DNA sequence analysis results suggest that these infections are attributable to a Kudoa species closely related to Kudoa trachuri. This paper reports the first study on a multivalvulidan species to be identified from the Canary Islands. Furthermore, this is the first report of Kudoa parasites in all of the hosts mentioned above, with the exception of P. acarne.

[Henneguya wolinensis (Myxosporea: Myxobolidae), a new for Russian fauna parasite from the perch Perca fluviatilis L.].

The infection of the perch Perea fluviatilis L. with myxosporean Henneguya wolinensis Romuk-Wodoracki, 1990 has been detected. This is the second finding of this parasite after its original descriptin and the first for Russia. Plasmodium of this species develops in the epidermis under scales throughout the body causing the formation of white cysts up to 1 mm. Spores are fusiform, large, their average length constitutes 25.5 µm without the caudal appendages and 62 µm with them. Slight morphological differences in spore structure comparing to original description have been revealed.

The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi.

Thelohanellus kitauei is a freshwater myxosporean parasite causing intestinal giant cystic disease of common carp. To clarify the life cycle of T. kitauei, we investigated the oligochaete populations in China and Hungary. This study confirms two distinct aurantiactinomyxon morphotypes (Aurantiactinomyxon type 1 and Aurantiactinomyxon type 2) from Branchiura sowerbyi as developmental stages of the life cycle of T. kitauei. The morphological characteristics and DNA sequences of these two types are described here. Based on 18S rDNA sequence analysis, Aurantiactinomyxon type 1 (2048 bp) and Aurantiactinomyxon type 2 (2031 bp) share 99.2-99.4 %, 99.8-100 % similarity to the published sequences of T. kitauei, respectively. The 18S rDNA sequences of these two aurantiactinomyxon morphotypes share 99.4 % similarity, suggesting intraspecific variation within the taxon, possibly due to geographic origin. Phylogenetic analyses demonstrate the two aurantiactinomyxon types clustered with T. kitauei. Regardless, based on 18S rDNA synonymy, it is likely that Aurantiactinomyxon type 1 and 2 are conspecific with T. kitauei. This is the fourth elucidated two-host life cycle of Thelohanellus species and the first record of T. kitauei in Europe.

Small subunit ribosomal RNA sequence links the myxospore stage of Henneguya mississippiensis n. sp. from channel catfish Ictalurus punctatus to an actinospore released by the benthic oligochaete Dero digitata.

There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 µm (range = 40.7-61.6 µm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 µm (14.4-19.3 µm) in length and 5.0 ± 0.3 µm (4.5-5.5 µm) in width. The two polar capsules were 6.2 ± 0.4 µm (5.8-7.0 µm) long and 5.0 ± 0.3 µm (4.5-5.5 µm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 µm (22.9-40.6 µm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4% similarity (100% coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.