Morphology and phylogeny of Ameson portunus n. sp. (Microsporidia) infecting the swimming crab Portunus trituberculatus from China

Accepted Manuscript Title: Morphology and phylogeny of Ameson portunus n. sp. (Microsporidia) infecting the swimming crab Portunus trituberculatus from China Authors: Yuan Wang, Xin-Cang Li, Guihong Fu, Shu Zhao, Yuange Chen, Hao Wang, Tiantian Chen, Junfang Zhou, Wenhong Fang PII: DOI: Reference:

S0932-4739(17)30075-5 https://doi.org/10.1016/j.ejop.2017.09.008 EJOP 25535

To appear in: Received date: Revised date: Accepted date:

31-3-2017 16-9-2017 18-9-2017

Please cite this article as: Wang, Yuan, Li, Xin-Cang, Fu, Guihong, Zhao, Shu, Chen, Yuange, Wang, Hao, Chen, Tiantian, Zhou, Junfang, Fang, Wenhong, Morphology and phylogeny of Ameson portunus n.sp.(Microsporidia) infecting the swimming crab Portunus trituberculatus from China.European Journal of Protistology https://doi.org/10.1016/j.ejop.2017.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Morphology and phylogeny of Ameson portunus n. sp. (Microsporidia) infecting the swimming crab Portunus trituberculatus from China Yuan Wanga, Xin-Cang Lia, Guihong Fua, Shu Zhaoa,Yuange Chena, Hao Wangb, Tiantian Chenc, Junfang Zhoua, Wenhong Fanga a

East China Sea Fisheries Research Institute, China Academy of Fishery Sciences, Shanghai 200090, China

b

College of Ocean and Earth Sciences, Jimei University, Xiamen 361021, China

c

College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

_______________________ 

Corresponding authors: Tel.: +86 021 65699301

E-mail addresses: [email protected] (J. Zhou), [email protected] (W. Fang)

Abstract Ameson portunus n. sp. is a new microsporidian species that infects the skeletal muscle of Portunus trituberculatus, a pond-reared swimming crab from China. This parasite was characterized using morphological and molecular phylogenetic data. Light and transmission electron microscopy revealed that this microsporidian experienced disporogonic and polysporogonic (chain-like) life cycles. Mature uninucleate spores appeared ovoid, measured 1.4 ± 0.06 × 1.0 ± 0.07 μm on ultrathin sections, and exhibited no dimorphism. The isofilar polar filament was coiled in 8–9 turns. Of these coils, 5–9 were arranged in large regular outer layers; the remaining coils (0–3 coils) were situated internally. According to phylogenetic analyses based on the small subunit (SSU) rDNA gene, A. portunus n. sp. belonged to the group comprising Ameson spp. and Nadelspora canceri. The result of comprehensive analysis of ultrastructural features, molecular phylogenetic data, host and geographical differences among known species supports the establishment of a new Ameson species for this parasite. Ameson portunus n. sp. is the first Ameson species described from the coasts of East Asia. Page 1 of 20

Keywords: Ameson; Microsporidiosis; Parasite; Taxonomy; Toothpaste crab disease; Ultrastructure

Introduction Microsporidia are obligate intracellular fungal parasites (Capella-Gutiérrez et al., 2012). Recent advances indicate that they emerge from the Rozellomycota (Cryptomycota) (Corsaro et al., 2016; Corsaro et al., 2014; James et al., 2013). Microsporidia infect a wide range of vertebrate and invertebrate species (Franzen, 2008) and cause various clinical symptoms, such as muscular damage, reduced productivity, growth retardation, and diarrhea. In recent years, some newly identified microsporidian pathogens, such as Enterospora nucleophila (Palenzuela et al., 2014), Enterocytozoon hepatopenaei (Rajendran et al., 2016; Tourtip et al., 2009), and a microsporidian from groupers (Xu et al., 2017), have resulted in significant economic losses to the aquaculture industry in different geographical areas. To date, more than 40 microsporidian genera have been found in crustaceans (Wang et al., 2013). Approximately nine of these genera, namely, Abelspora, Ameson, Areospora, Enterospora, Hepatospora, Nadelspora, Nosema, Ormieresia, and Thelohania, have been described in crabs, at least based on their ultrastructural features (Azevedo, 1987; Olson et al., 1994; Ryazanova and Eliseikina, 2010; Sprague, 1977; Stentiford et al., 2007; Stentiford et al., 2011; Stentiford et al., 2014; Vivarès, 1980; Vivarès et al., 1977; Walker and Hinsch, 1975). Among these genera, Ameson, Nadelspora, and Ormieresia target the musculature tissues of the hosts, and Ameson and Areospora possess hair-like ornamentations covering their spores. These characters have been considered as the basis of classification to differentiate Ameson from Perezia, another genus infecting decapods but lacking the hair-like appendages, and reclassify Ameson nelsoni as Perezia nelsoni (Vivarès and Sprague, 1979). Widely distributed in the coastal waters of Asia-Pacific countries, Portunus trituberculatus, supports a large commercial fishery in China. This crab farming in east China has expanded rapidly over the last decades (Wang et al., 2014). Several new P. trituberculatus diseases have emerged, and some causative agents, such as white spot syndrome virus (Wang et al., 2008), Vibrio alginolyticus (Wang et al., 2006), Vibrio harveyi (Zhou et al., 2013), Metschnikowia bicuspidate (Wang et al., 2007), and Hematodinium (Li et al., 2013; Xu et al., 2007), have been characterized. In 2000, crab dealers and aquaculturists in China discovered the swimming crabs with white opaque musculature and joints. These crabs are termed as ‘toothpaste crab’ because their musculature can be secreted from their appendages by compression and resemble white toothpaste. In 2012, we initially investigated toothpaste crab disease through light and electron microscopy examination and revealed the discolored musculature of P. Page 2 of 20

trituberculatus is caused by a microsporidian. In the current study, the ultrastructure and life cycle of this parasite were examined and the SSU rDNA sequence was phylogenetically analyzed to determine its relationship with other microsporidians. Our results supported the classification of this microsporidian as a novel Ameson species, for which the name Ameson portunus n. sp. was proposed.

Material and Methods Source of specimens A total of 205 swimming crabs (body weight = 208 ± 48 g, ranging from 100 g to 350 g; carapace width = 154 ± 10 mm, ranging from 121 mm to 163 mm) with toothpaste-like appearance were collected from aquaculture ponds from September 2012 to December 2014. Water salinity ranged from 20 to 27 practical salinity units. These crabs were transported to our laboratory in water at approximately 15 °C–20 °C. Smears of skeletal muscle, heart muscle, gill, hepatopancreas, and gonad of each crab were examined to confirm the presence of microsporidian spores through light microscopy (LM). The infected samples were preserved for histology, transmission electron microscopy (TEM), and DNA analyses. LM and TEM For LM, the smears of fresh spores excised from infected muscles were observed under a light microscope (Olympus-BX51). For histology, dissected skeletal musculature were fixed in Bouin’s fixative for 24 hours and then dehydrated through a graded ethanol series. Samples were embedded in paraffin and sectioned at 5 μm. Paraffin sections were stained with haematoxylin and eosin (H&E). For TEM, small fragments of the muscle and heart were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C for 24 h. The tissues were rinsed overnight in the same buffer at 4 °C and post-fixed in 1.0% osmium tetroxide for 1 h. After washing three times for 15 min each with the same buffer, specimens were dehydrated in an increasing ethanol series (50% to 100%), embedded in Epoxy Resin 812, and polymerized at 60 °C for 48 h. Semi-thin sections (1μm) were cut on the ultramicrotome with a glass knife, to target the infected area. Ultrathin sections (60–80 nm) were mounted on uncoated copper grids and stained with uranyl acetate and lead citrate, and subsequently examined by using an electron microscope (JEM-1230) at 80 kV. DNA extraction, cloning, and sequencing Small pieces of the infected muscle tissues were preserved in 100% ethanol. The DNA extraction method was Page 3 of 20

modified on the basis of Stentiford et al. (2010). Briefly, a mixture containing 0.1 g of tissue samples, 0.2 g of 0.6 mm glass beads, and 300 μl of GA buffer (Tiangen) was shaken in a fast prep FP120 homogenizer (Thermo Savant) for 3 rounds of 20 s at maximum speed. The mixtures were centrifuged at 12,000 × g for 1 min, and genomic DNA was recovered from the supernatants by using a tissue extraction kit (Tiangen) according to the manufacturer’s protocol. DNA was eluted in 50 µl of TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0) and stored at −20 °C. The SSU rDNA fragment of the microsporidian was amplified with the primer set V1f (5´CACCAGGTTGATTCTGCC-3´) and 1492r (5´-GGTTACCTTGTTACGACTT-3´) (Nilsen, 2000; Vossbrinck et al., 1993). Amplifications were performed with an initial denaturation step at 94 ℃ for 5 min, followed by 35 cycles of 94 ℃ for 1 min, 52 ℃ for 35 s, and 72 ℃ for 90 s, with a final extension at 72 ℃ for 5 min. PCR products, approximately 1300bp in length, were purified by using a DNA fragment purification kit (Takara) and cloned into pMD 18-T vector plasmid (Takara). The constructed plasmids were separately transformed into competent Escherichia coli DH5α cells. Transformants were selected on Luria–Bertani (LB) plates containing 100 μg/ml ampicillin. Positive clones were checked in terms of their correct insertion size through PCR with a pair of universal M13 primers and re-grown in 3 ml of standard LB medium containing 100 μg/ml ampicillin. Ten clone products were sequenced in an ABI 3730xl DNA analyzer (Applied Biosystems). Phylogenetic analyses Phylogenetic analyses were performed using the partial SSU rDNA gene sequences from 24 representative microsporidian species, including the closest matches in a Basic Local Alignment Search Tool search and other microsporidian sequences. Basidiobolus ranarum (AY635841) was used as the outgroup for phylogenetic reconstructions. Multiple alignments were performed in Clustal W of MEGA 7 (Kumar et al., 2016). The 5´- and 3´ends of SSU rDNA were trimmed. As a result, the alignment comprised 794 informative positions in the final dataset. Pairwise genetic distances were calculated using the options p-distance and complete deletion of position with gaps or missing data. Maximum-likelihood (ML) phylogenetic analyses were carried out in Tamura–Nei and discrete gamma distribution (TN93+G) model, as suggested by Modeltest in MEGA 7. Neighbor-joining (NJ) and maximum parsimony (MP) analyses were also conducted. For NJ, Kimura’s two-parameter substitution model with gamma distribution (shape parameter = 1) was used. For MP, a min-mini heuristic algorithm with a search factor of 1 was applied. The robustness of the bootstrap consensus tree clades was determined through bootstrap analyses with 1000 replications. A Bayesian tree was constructed by using MrBayes (v3·2) to verify the reliability of the phylogenetic relationships estimated by the ML/NJ/MP analyses (Ronquist et al., 2012). Bayesian inference was performed by Page 4 of 20

using the GTR + G model. MrBayes was operated for 1,000,000 generations, and every 1000th generation was sampled. The first 25% of the samples were discarded as burn-in, and the standard deviation of the split frequencies was less than 0.01 after 1,000,000 generations.

Results Signs and symptoms, LM, and histopathology The infected swimming crabs could ingest the flesh of fish until the cheliped was unable to bring food to its mouthparts. Heavily infected crabs sluggishly stayed in shallow parts of the pond and died soon after collection. Severe infection could be recognized by the opaque white-to-gray ventral sternum and two white stripes in the propodus of the swimming leg of the crab (Fig. 1C). Among 205 crab samples, there are 202 showing white opaque skeletal musculature (Figs. 1A and 1B) after dissection and 3 showing pink opaque skeletal muscle, which might be caused by individual difference. Hemolymph from heavily infected crabs failed to clot. The gonads of the affected female crabs (sampled in November) did not develop after the mating season (from September to October) (Fig. 1A). By contrast, the normal gonads with orange appearance in unaffected female crabs were common within the same period (Fig. 1A). Microscopic examinations revealed that ovoid microsporidian spores (Fig. 2A) were widely localized in the skeletal musculature (Fig. 2C) and there was no sporophorous vesicles. Some binucleate sporonts and chain-like sporonts were also observed (Fig. 2B). In the paraffin sections of heavily infected muscle tissues, numerous spores were embedded among the myofibrils, and the musculature were almost replaced by spores (Fig. 2D). Merogony and sporogony TEM results revealed no other pathogens in the musculature tissue. The merogonial and sporogonial life stages of the microsporidian developed in direct contact with the cytoplasm of host muscle cells. No sporophorous vesicle was present at any point in the developmental cycle of the parasite. The observed earliest life stages were apparently uninucleate and diplokaryotic meronts limited by a simple plasma membrane (Fig. 3A). Two kinds of the earliest meronts appeared round to ovoid and characterized by the highest nucleus-to-cytoplasm ratio (Figs. 3A and 4A). Diplokaryons exhibiting a tight contact between two nuclear counterparts were likely formed by the incomplete division of the nucleus of the uninucleate meronts (Fig. 3A). No diplokaryotic stage was observed during sporogony. Ultrastructural details showed two different pathways in the multiplication of life cycle stages. Page 5 of 20

Pathway I: Ovoidal diplokaryotic meronts were elongated by increasing the vacuolated cytoplasm (Figs. 3A and 3B). The elongated meronts surrounded by a plasma membrane presumably developed into the chain-like meronts through diplokaryon division. The transition from late meront chains to early sporont chains was characterized by the acquisition of a thick and dense cell coat located on the outer surface of the plasma membrane (Fig. 3C inset). Most of the early sporont chains were observed in cytokinesis. The sporont chains contained three or more isolated nuclei, which were generally connected by invaginated plasma membrane. The connection was caused by the delayed cytokinesis and sometimes persisted to the mature sporoblast stage. Hair-like projections began to develop on the thickened plasma membrane. In the same stage, the polar filament formed (Fig. 3D). After the sporogonial divisions were completed, the sporoblasts with one nucleus were discretely formed. Pathway II: In the initial stage, ovoidal diplokaryotic meronts were enlarged by increasing the vacuolated cytoplasm (Figs. 4A and 4B). The division of diplokaryon resulted in the formation of a tetranucleate (two diplokaryons) meront (Fig. 4C). The tetranucleate meront underwent cytokinesis (presumably) and, two new diplokaryotic meronts with the highly vacuolated cytoplasm were produced (Fig. 4D). The diplokaryon underwent dissociation by separating two nuclear envelopes (Figs. 4E and 4F) and transformed into an early binucleate sporont. The presporont experienced a thickening plasma membrane. As membrane structure thickening was gradually completed (Figs. 5A, 5B and 5C), two isolated nuclei separated from the center of the cell, the electron-lucent cytoplasm was reduced, the precursors of the extrusion apparatus including the anchoring disk and polar filament formed, and the hair-like projections appeared. The division then occurred. The invagination of the completely thickened wall separated the binucleate sporont into two connected parts (Figs. 5D and 5E). The subsequent growth of each part formed two largely similar presporoblasts, which were characterized by electron-lucent vacuole and posterior vacuole (Fig. 5F). The fission of the two connected presporoblasts produced two uninucleate sporoblasts. The sporoblasts could not divide further (Fig. 6A). Spores The sporoblasts developed into immature spores by the shrinkage of the posterior vacuole, condensation of their cytoplasm, disappearance of electron-lucent vacuole, preformation of the anchoring disk and polaroplast, and elongation of the spore shape (Figs. 6A, 6B and 6C). The immature spores were characterized by the gradual arrangement of spore organelles. The maturation of these cells was marked by the preformation of an electron-lucent endospore and the disappearance of the posterior vacuole (Fig. 6D). The mature spores were monomorphic and ovoid and possessed distinct hair-like projections (Fig. 7A). The spores in ultrathin sections measured 1.4 ± 0.06 × 1.0 ± 0.07 μm (n = 85), which ranged from 1.3−1.5 × 0.8−1.1 μm. Page 6 of 20

The single large nucleus was located closely to the posterior of the polaroplast (Fig. 7C). The polar filament formed 8−9 isofilar turns with an average diameter of 80 nm, usually about 5−7 turns lay in the large outer coil and the remaining coils (0–3 coils) situated internally (Figs. 7A and 7B). These coils were also arranged in a single-line row (Fig. 7C). The spore wall consisted of a thin exospore and a thick electron-lucent endospore (Figs. 7B and 7E). The endospore was greatly thinned over the mushroom-like anchoring disc (Fig. 7D). The polaroplast contained two distinct parts: an electron dense outer part and a laminar internal part composed of 5–6 layers. The electron dense body was infrequently observed in the sporoplasm of immature and mature spores (Figs. 6D and 7A). The hair-like projections covering the surface of the exospore consisted of a short stubby root and a relatively long thin extension (Figs. 7F and 7G). Molecular characterization and phylogeny The amplification of SSU rDNA sequence was 1282bp in length. A portion (1194bp) of this sequence was deposited in GenBank under the accession number KC915038. The pairwise distance analyses of the alignment of 25 nucleotide sequences revealed that A. portunus n. sp. exhibited the highest similarity to N. canceri infecting Cancer magister from USA (99.1%), A. pulvis infecting Carcinus maenas from United Kingdom (98.6%), Ameson michaelis infecting Callinectes sapidus from USA (96.0%), and Ameson metacarcini infecting Metacarcinus magister from USA (96.0%) (Table 1). The percentage similarity to all of the other species was lower than 96.0%. Phylogenetic analyses with ML, NJ, and MP method produced three trees with similar topology (Fig.8 left side). The large clade composed of Ameson spp. and the monotypic Nadelspora yielded a high bootstrap support (100%, 100%, and 100%, respectively). Ameson portunus n. sp. clustered with a clade consisting of N. canceri and A. pulvis (UK) (bootstrap 42%, 61%, and 73%). In another phylogenetic tree constructed with Bayesian inference, Ameson, Nadelspora, and Perezia were placed in one group (Fig. 8 right side). Ameson spp. and N. canceri were clustered in a well-supported clade. In contrast to the ML tree, A. portunus n. sp. was clustered together with A. michaelis and A. metacarcini within a subclade. A. pulvis (UK) was clustered with N. canceri within another subclade.

Discussion Life cycle of Ameson portunus n. sp. On the basis of our ultrastructural observations, we propose a putative life cycle of A. portunus n. sp. The two different pathways of development are depicted in Fig. 9. In pathway I: The ovoidal diplokaryotic meront elongates Page 7 of 20

and presumably develops into chain-like meront, which contains three or more isolate nuclei. The membrane thickens to achieve the transition from chain-like meront to chain-like sporont. The initial organelles, such as polar filament, are recognized. Finally, the sporont divides into three or more sporoblasts. In pathway II: the ovoidal diplokaryotic meront enlarges and forms a tetranucleate meront by the diplokaryon division. Then, the tetranucleate meront undergoes cytokinesis and forms two diplokaryotic meronts, the diplokaryon of this kind meront undergoes dissociation and transforms into a binucleate sporont marked by the thickened wall. The organelles begin to form. Finally, the late sporont divides into two sporoblasts. All sporoblasts produced by two pathways develop into immature spores and further into mature spores. Five Ameson species, including newly discovered A. metacarcini from Metacarcinus magister in British Columbia (Small et al., 2014), have been described from different marine crabs (Table 2). The chain-like sporont and hair-like projections are the important characters of the genus Ameson, as first described in 1977 by Sprague and amended in 1979 by Vivarès and Sprague. Four developing sporoblasts contained in one sporont were commonly found among Ameson species, such as A. atlanticum, A. michaelis, A. metacarcini, A. pulvis (Fr), and A. pulvis (UK). Stentiford et al. (2013a) inferred that the chain-like sporont containing four isolated nuclei was formed by uninucleate meronts division. However, Small et al. (2014) thought that the formation of this kind sporont was the result of the mitosis and diplokaryon division of the diplokaryotic meront. In this study, we could not determine the source of chain-like sporont, but according to morphological characteristics, the chain-like sporont was more consistent with elongated diplokaryotic meront. The morphological features of cells in various stages of the parasite are necessary to reconstruct the life cycle of microsporidian. Stentiford et al. (2013a) found that a microsporidian presumed to be A. pulvis isolated from the United Kingdom likely experienced a remarkable shift in its life cycle, and this shift consequently induced an extreme spore dimorphism with needle Nadelspora-like and ovoid Ameson-like spores. But according to our observation, no elongated Nadelspora-like spores of A. portunus n. sp. were detected in all infected crab muscles, heart, gill, hepatopancreas, and gonad. For this reason, we believed that A. portunus n. sp. did not have the characteristics of extreme spore dimorphism. Those authors also observed that tetranucleate meront (two diplokaryon) is present in the merogony of Ameson-like lineage and Nadelspora-like lineage, but is not involved in Ameson-like lineage at their concluded lifecycle figure. However, this phenomenon should be further investigated. Binucleate sporont, considered as an aberrant cells of A. pulvis (UK), was commonly observed in this study, therefore, we believed that it is an important feature of the multiplication. Comparison of Ameson portunus n. sp. with similar species Page 8 of 20

Traditionally, microsporidian parasites were mostly described on the basis of their structural characteristics, nuclear configuration, life cycle, and host specificity (Sprague et al., 1992). With current molecular data, the reliability of traditional approaches should be re-examined, and molecular phylogeny with SSU rDNA is proposed as the main discriminator of the relatedness among microsporidia (Gillett et al., 2016; Stentiford et al., 2013b). However, the main use of SSU rDNA for sequence analysis, which is limited in one genome region, can resolve taxonomic relationships to the genus level only (Bateman et al., 2016). The most reliable method to identify a new microsporidian is the comprehensive utilization of a range of features, including host type, infection site, ecology, pathology, ultrastructural morphology, and phylogenetics (Stentiford et al., 2013b; Vossbrinck et al., 2005), because accurate identification method has yet to be developed to distinguish closely related species, especially those with sequences exhibiting high similarity (approximately 99%). The spore morphology, host specificity, and locality were compared among Ameson spp. (Table 2). The spore sizes of listed Ameson species were all measured in the fixed condition. Ameson portunus n. sp. could be easily differentiated from A. michaelis, A. metacarcini, and A. atlanticum because of the smaller spore size and fewer polar filament coils of the former than those of the latter. Based on the length-to-width (l/w) ratio, the spore size of A. portunus n. sp. was slightly longer than those of the two other A. pulvis isolates. The thickness of the spore wall was also different (57 nm vs. 40 nm). Although A. portunus n. sp. and two A. pulvis isolates possessed an isofilar polar filament with 8–9 coils, the diameter of the polar filament of A. portunus n. sp. was smaller than that of the two other isolates (80 nm vs. 100 nm). Their polar filament arrangements also differed slightly. In particular, 8−9 coils of A. portunus n. sp. could appear in one layer close to the spore wall, but the same arrangement was not reported in the two other A. pulvis isolates. These structural dissimilarities implied that this microsporidian could be a new Ameson species. Genetic distance analysis among A. portunus, A. pulvis (UK), and N. canceri suggested that they were closely related at the SSU gene level (Table 1). For comparison, a low pairwise distance (0.009) was observed between Potaspora morhaphis and its congener Potaspora aequidens (Casal et al., 2008; Videira et al., 2015), and lower genetic distances (0.001) were found between Glugea arabica and Glugea nagelia (Abdel-Baki et al., 2015; Azevedo et al., 2016). The fine structure of N. canceri is largely different from A. portunus, which forms needle-like spores measuring 7.1–11.8 × 0.2–0.3 μm (Olson et al., 1994). By comparison, the elongated spores of A. pulvis (UK) reached approximately 6 × 0.3 μm. Although A. pulvis (UK) is classified into two spore types, these spore types were different from A. portunus n. sp. and N. canceri. Thus, the sequence divergence and morphological differences suggested that these species did not belong to the same taxon. The molecular comparison of the gene sequence data of A. pulvis (Fr) Page 9 of 20

is not feasible because only its morphological data have been published. Therefore, further studies should verify whether A. pulvis (Fr) and A. pulvis (UK) truly represent the same species, although they display a strong morphological similarity in terms of the host skeletal musculature (Stentiford et al., 2013a; Vivarès and Sprague, 1979). Phylogenetic analyses confirmed that A. portunus n. sp. was a new member of the genus Ameson. This species was not placed into the branch containing A. pulvis and N. canceri in Bayesian or ML/NJ/MP tree, although the SSU rDNA sequence of A. portunus n. sp. was more similar to that of N. canceri (99.1%) than to that of A. pulvis (UK) (98.6%). Our Bayesian result implied that A. portunus, A. michaelis, and A. metacarcini forming only ovoid spores were closely related to one another. By comparison, A. pulvis and N. canceri producing needle-like spores were closely associated with each other. Taxonomic summary Type species: Ameson portunus n. sp. Species description: Ovoid spores, monokaryotic, measuring 1.8 × 1.4 μm in fresh smears for light microscopy and 1.4 × 1.0 μm in specimens fixed for TEM. The spore wall was approximately 57 nm thick. The polar filament isofilar contained 8–9 coils. Of these coils, nearly 5–7 form a large outer layer and the remaining coils are situated internally. In some instances, 9 coils are arranged in a single-line row. The coiled PF was approximately 80 nm in diameter except the anterior part (manubrium), which was approximately 96 nm in diameter. The polaroplast comprises two distinct parts. The laminar internal part composed of 5 to 6 layers with thickness of approximately 30 nm for each layer. The nucleus was located posterior to the polaroplast. The electron dense body and posterior vacuole were infrequently observed. The hair-like projections possessed a short stubby root and a relatively long thin extension (about 560 nm longest). Type host: Portunus trituberculatus (Miers, 1876) Infection site: Skeletal muscle cells Type locality: Jiangsu Province, Eastern Coast of China Etymology: The specific name of the novel microsporidian is based on the genus name of its host species. Type of material: Histological sections and TEM resin blocks containing infected tissues of the specimens are deposited in the East China Sea Fisheries Research Institute, China Academy of Fishery Sciences, Shanghai. The SSU rDNA gene sequence was deposited at GenBank with the Acc. No. KC915038.

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Conclusions We compared the morphological and ultrastructural characteristics, host specificity, life cycle and available SSU rDNA sequences of the five recognized Ameson species and found that these species did not exhibit gross similarity to the novel microsporidian. Therefore, we identified the novel organism as A. portunus n. sp. This study demonstrated that A. portunus n. sp. is the etiological agent responsible for toothpaste crab disease of pond-reared P. trituberculatus. The infected wild swimming crabs were also found in coastal waters in China (unpublished data). To our knowledge, this study is the first to report an Ameson species from the coasts of East Asia. With the ecological and economic importance of the crab host, the discovery of this pathogen has been considered a relevant aspect in crab nursery and aquaculture sites. Further studies on the transmission mechanisms and prevention and control techniques should be developed.

Acknowledgments This work was supported by the Central-Level Non-profit Scientific Research Institutes Special Funds (East China Sea Fisheries Research Institute) (No. 2014T03) and the National Special Research Fund for Non-Profit Sector (Agriculture) (No. 201303047). The authors would like to thank Mr. Guofa Mao, Mr. Weixing Zhao, and Mr. Mingming Wang for their assistance in swimming crab sample collection. The authors would also like to express their gratitude to Mr. Yonghong Shi and Mrs. Yu Kong for their technical assistance in electron microscopy.

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References Abdel-Baki, A.S., Al-Quraishy, S., Rocha, S., Dkhil, M.A., Casal, G., Azevedo, C., 2015. Ultrastructure and phylogeny of Glugea nagelia sp. n. (Microsporidia: Glugeidae), infecting the intestinal wall of the yellowfin hind, Cephalopholis hemistiktos (Actinopterygii: Serranidae), from the Red Sea. Folia Parasitol. 62, 007. Azevedo, C., 1987. Fine structure of the microsporidan Abelspora portucalensis gen. n., sp. n. (Microsporida) parasite of the hepatopancreas of Carcinus maenas (Crustacea, Decapoda). J. Invertebr. Pathol. 49, 83-92. Azevedo, C., Abdel-Baki, A.S., Rocha, S., Al-Quraishy, S., Casal, G., 2016. Ultrastructure and phylogeny of Glugea arabica n. sp. (Microsporidia), infecting the marine fish Epinephelus polyphekadion from the Red Sea. Eur. J. Protistol. 52, 11-21. Bateman, K.S., Wiredu-Boakye, D., Kerr, R., WilliamsI, B., Stentiford, G.D., 2016. Single and multi-gene phylogeny of Hepatospora (Microsporidia)-a generalist pathogen of farmed and wild crustacean hosts. Parasitology 143, 971-982. Capella-Gutiérrez, S., Marcet-Houben, M., Gabaldón, T., 2012. Phylogenomics supports microsporidia as the earliest diverging clade of sequenced fungi. Bmc Biol. 10, 47. Casal, G., Matos, E., Teles-Grilo, M.L., Azevedo, C., 2008. A new microsporidian parasite, Potaspora morhaphis n. gen., n. sp. (Microsporidia) infecting the teleostean fish, Potamorhaphis guianensis from the river Amazon. Morphological, ultrastructural and molecular characterization. Parasitology 135, 1053-1064. Corsaro, D., Michel, R., Walochnik, J., Venditti, D., Muller, K.D., Hauroder, B., Wylezich, C., 2016. Molecular identification of Nucleophaga terricolae sp. nov. (Rozellomycota), and new insights on the origin of the Microsporidia. Parasitol. Res. 115, 3003-3011. Corsaro, D., Walochnik, J., Venditti, D., Steinmann, J., Muller, K.D., Michel, R., 2014. Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota. Parasitol. Res. 113, 1909-1918. Franzen, C., 2008. Microsporidia: A review of 150 years of research. Open Parasitol. J. 2, 1-34. Gillett, A.K., Ploeg, R., O Donoghue, P.J., Chapman, P.A., Webb, R.I., Flint, M., Mills, P.C., 2016. Ultrastructural and molecular characterisation of an Heterosporis-like microsporidian in Australian sea snakes (Hydrophiinae). Plos One 11, e150724. James, T.Y., Pelin, A., Bonen, L., Ahrendt, S., Sain, D., Corradi, N., Stajich, J.E., 2013. Shared signatures of parasitism and phylogenomics unite cryptomycota and microsporidia. Curr. Biol. 23, 1548-1553. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., w54. Li, C., Song, S., Liu, Y., Chen, T., 2013. Hematodinium infections in cultured Chinese swimming crab, Portunus trituberculatus, in northern China. Aquaculture 396, 59-65. Nilsen, F., 2000. Small subunit ribosomal DNA phylogeny of microsporidia with particular reference to genera that infect fish. J. Parasitol. 86, 128-133. Olson, R.E., Tiekotter, K.L., Reno, P.W., 1994. Nadelspora canceri n. g., n. sp., an unusual microsporidian parasite of the Dungeness crab, Cancer magister. J. Eukaryot. Microbiol. 41, 349-359. Palenzuela, O., Redondo, M.J., Cali, A., Takvorian, P.M., Alonso-Naveiro, M., Alvarez-Pellitero, P., Sitjà-Bobadilla, A., 2014. A new intranuclear microsporidium, Enterospora nucleophila n. sp., causing an emaciative syndrome in a piscine host (Sparus aurata), prompts the redescription of the family Enterocytozoonidae. Int. J. Parasitol. 44, 189203. Rajendran, K.V., Shivam, S., Praveena, P.E., Rajan, J.J.S., Kumar, T.S., Avunje, S., Jagadeesan, V., Babu, S.P., Pande, A., Krishnan, A.N., 2016. Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus (Litopenaeus) vannamei in India. Aquaculture 454, 272-280. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Page 12 of 20

Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539-542. Ryazanova, T.V., Eliseikina, M.G., 2010. Microsporidia of the genera Thelohania (Thelohaniidae) and Ameson (Pereziidae) in two species of lithodid crabs from the Sea of Okhotsk. Russ. J. Mar. Biol. 36, 435-442. Small, H.J., Meyer, G.R., Stentiford, G.D., Dunham, J.S., Bateman, K., Shields, J.D., 2014. Ameson metacarcini sp. nov. (Microsporidia) infecting the muscles of Dungeness crabs Metacarcinus magister from British Columbia, Canada. Dis. Aquat. Org. 110, 213-225. Sprague, V., 1977. Comparative pathobiology: volume 2 systematics of the Microsporidia. Plenum Press, New York. Sprague, V., Becnel, J.J., Hazard, E.I., 1992. Taxonomy of phylum Microspora. Crit. Rev. Microbiol. 18, 285-395. Sprague, V., Vernick, S.H., Lloyd, B.J., 1968. The fine structure of Nosema sp. Sprague, 1965 (Microsporida, Nosematidae) with particular reference to stages in sporogony. J. Invertebr. Pathol. 12, 105-117. Stentiford, G.D., Bateman, K.S., Dubuffet, A., Chambers, E., Stone, D.M., 2011. Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe. J. Invertebr. Pathol. 108, 156-166. Stentiford, G.D., Bateman, K.S., Feist, S.W., Chambers, E., Stone, D.M., 2013a. Plastic parasites: extreme dimorphism creates a taxonomic conundrum in the phylum Microsporidia. Int. J. Parasitol. 43, 339-352. Stentiford, G.D., Bateman, K.S., Feist, S.W., Oyarzún, S., Uribe, J.C., Palacios, M., Stone, D.M., 2014. Areospora rohanae n. gen. n. sp. (Microsporidia; Areosporiidae n. fam.) elicits multi-nucleate giant-cell formation in southern king crab (Lithodes santolla). J. Invertebr. Pathol. 118, 1-11. Stentiford, G.D., Bateman, K.S., Longshaw, M., Feist, S.W., 2007. Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus. Dis. Aquat. Org. 75, 61-72. Stentiford, G.D., Bateman, K.S., Small, H.J., Moss, J., Shields, J.D., Reece, K.S., Tuck, I., 2010. Myospora metanephrops (n. g., n. sp.) from marine lobsters and a proposal for erection of a new order and family (Crustaceacida; Myosporidae) in the Class Marinosporidia (Phylum Microsporidia). Int. J. Parasitol. 40, 1433-1446. Stentiford, G.D., Feist, S.W., Stone, D.M., Bateman, K.S., Dunn, A.M., 2013b. Microsporidia: diverse, dynamic, and emergent pathogens in aquatic systems. Trends Parasitol. 29, 567-578. Tourtip, S., Wongtripop, S., Stentiford, G.D., Bateman, K.S., Sriurairatana, S., Chavadej, J., Sritunyalucksana, K., Withyachumnarnkul, B., 2009. Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships. J. Invertebr. Pathol. 102, 21-29. Videira, M., Casal, G., Rocha, S., Gonçalves, E., Azevedo, C., Velasco, M., Matos, E.R., 2015. Potaspora aequidens n. sp. (Microsporidia, Tetramicridae), a parasite infecting the freshwater fish Aequidens plagiozonatus (Teleostei, Cichlidae) from Brazil. Parasitol. Res. 114, 2435-2442. Vivarès, C.P., 1980. An ultrastructural study of Thelohania maenadis (Microspora, Microsporidia) and new facts on the genus Thelohania. Arch. Protistenk. 123, 44-60. Vivarès, C.P., Azevedo, C., 1988. Ultrastructural observations of the life cycle stages of Ameson atlanticum sp. nov., a microsporidan parasitizing Cancer pagurus L. J. Fish Dis. 11, 379-387. Vivarès, C.P., Bouix, G., Manier, J.F., 1977. Ormieresia carcini gen. n., sp. n., Microsporidie du Crabe Méditerranéan, Carcinus mediterraneus Czerniavsky, 1884: Cycle Évolutif et Étude Ultrastructurale. J. Protozool. 24, 83-94. Vivarès, C.P., Sprague, V., 1979. The fine structure of Ameson pulvis (Microspora, Microsporida) and its implications regarding classification and chromosome cycle. J. Invertebr. Pathol. 33, 40-52. Vossbrinck, C.R., Baker, M.D., Didier, E.S., Debrunner-Vossbrinck, B.A., Shadduck, J.A., 1993. Ribosomal DNA sequences of Encephalitozoon hellem and Encephalitozoon cuniculi: species identification and phylogenetic construction. J. Eukaryot. Microbiol. 40, 354-362. Page 13 of 20

Vossbrinck, C.R., Debrunner-Vossbrinck, B.A., 2005. Molecular phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations. Folia Parasitol. 52, 131-142. Walker, M.H., Hinsch, G.W., 1975. Ultrastructural observations of a microsporidian protozoan parasite in Libinia dubia (Decapoda). II. Structure of the mature spore. J. Parasitol. 61, 1074-1080. Wang, G.L., Jin, S., Chen, Y.E., Li, Z., 2006. Study on pathogens and pathogenesis of emulsification disease of Portunus trituberculatus. Adv. Mar. Sci. 24, 526-531. Wang, T.C., Nai, Y.S., Wang, C.Y., Solter, L.F., Hsu, H.C., Wang, C.H., Lo, C.F., 2013. A new microsporidium, Triwangia caridinae gen. nov., sp. nov. parasitizing fresh water shrimp, Caridina formosae (Decapoda: Atyidae) in Taiwan. J. Invertebr. Pathol. 112, 281-293. Wang, W., Wu, X., Liu, Z., Zheng, H., Cheng, Y., 2014. Insights into hepatopancreatic functions for nutrition metabolism and ovarian development in the crab Portunus trituberculatus: gene discovery in the comparative transcriptome of different hepatopancreas stages. Plos One 9, e84921. Wang, X., Chi, Z., Yue, L., Li, J., Li, M., Wu, L., 2007. A marine killer yeast against the pathogenic yeast strain in crab (Portunus trituberculatus) and an optimization of the toxin production. Microbiol. Res. 162, 77-85. Wang, Z.F., Wang, Z.Z., Xu, W.J., He, W.X., Gu, S.Y., 2008. Quantitative study of lethal effect of WSSV on Portunus trituberculatus from mix-culture ponds of prawns and crabs. Oceanol. Et Limnol. Sin. 39, 184-189. Xu, L.W., Liu, X.H., Zhang, J.Y., Liu, G.F., Feng, J., 2017. Outbreak of enteric microsporidiosis of hatchery-bred juvenile groupers, Epinephelus spp., associated with a new intranuclear microporidian in China. J. Fish Dis. 40, 183-189. Xu, W.J., Shi, H., Xu, H., Small, H., 2007. Preliminary study on the Hematodinium infection in cultured Portunus trituberculatus. Acta Hydrobiol. Sin. 31, 637-642. Zhou, Y.Q., Li, D.F., Peng, J., Shikongo, E.J., Chen, J., Liu, L.G., Gu, X.Y., 2013. Isolation and identification of pathogenic Vibrio harveyi from swimming crab, Portunus trituberculatus. J. Appl. Oceanogr. 32, 215-221.

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Figure1. Gross observations of infected and uninfected crabs, Portunus trituberculatus. (A) Diseased crab muscles were opaque with chalky white appearance, compared to an unaffected crab muscle tissue. Scale bar = 5 cm. (B) The morphologic aspects of infected (arrow) and uninfected muscles of swimming legs. Scale bar = 1 cm. (C) Two white stripes (arrows) in the propodus of swimming leg, a marker used by dealers and aquaculturists to distinguish “toothpaste crab” from the normal crab. Scale bar = 1 cm.

Page 15 of 20

Figure 2. Light microscopy of Ameson portunus infecting Portunus trituberculatus. (A) Free fresh ovoid spores in wet mount preparation. (B) Binucleate sporont (black arrow) and chain-like multinucleated sporonts. (C) Skeletal muscle smears showing numerous spores. (D) Paraffin section of skeletal muscles infected with microsporidian spores (black arrow), H&E stain. All scale bar = 5 μm.

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Figure 3. Transmission electron micrographs of pathway I: the merogony and sporogony of Ameson portunus in Portunus trituberculatus. (A) Uninucleate and diplokaryotic meronts with the highest nuclear−cytoplasm ratio. Monokaryon (N) and diplokaryon (2n) were shown. Insert shows the plasma membranes (white arrow) and nuclear envelopes (black arrow) of two different meronts. (B) The elongated diplokaryotic meront surrounded by a plasma membrane, preparing for nuclear division to produce a chain-like meront. Scale bar = 1 μm. (C) Late trinucleate meront chain/early trinucleate sporont chain in the process of cytoplasmic division began by the invagination of the plasma membrane (black arrows). Scale bar = 1 μm. Insert shows the detail of the thickening wall of the meront (white arrowhead). Insert scale bar = 0.2 μm. (D) Late trinucleate sporont chain with polar filaments (PF), prior to division into three sporoblasts. Scale bar = 1 μm.

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Figure 4. Transmission electron micrographs of pathway II: the merogony of Ameson portunus in Portunus trituberculatus. (A) Early meront containing a diplokaryon (2n). In the area of contact between the two nuclei, the nuclear envelopes are closely adjacent to each other. (B) Diplokaryotic meront with vacuolated cytoplasm (black asterisk). Insert showing the detail of the four membranes of the two nuclear envelopes (black arrow and white arrow). Insert scale bar = 0.2μm. (C) Tetranucleate meront (2×2n) containing large vacuoles. Two pairs of diplokaryotic nuclear located at opposing cellular borders. (D) Diplokaryotic meront with highly vacuolated cytoplasm (black asterisk). (E) Late diplokaryotic meront. The closely oppressed membranes between the two nuclei (n) began to separate from the margins. Scale bar = 0.6 μm. (F) Late diplokaryotic meront. Two nuclei slightly separated from each other, but still in pairs (black arrow). Scale bar = 0.6 μm.

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Figure 5. Transmission electron micrographs of pathway II: the sporogony of Ameson portunus in Portunus trituberculatus. (A) Early sporont showing two isolated nuclei and a plasma membrane that began to thicken (white arrowhead). (B) The binucleate sporont with a thickening plasma membrane (one nucleus unshown) (white arrowheads). (C) The new layer formed external to the plasma membrane was not yet completed (black arrowheads). The sporont contained two polar filament precursors (PF). (D) Cytokinesis of the binucleate sporont. The sporont with preformed anchoring disk (AD) and rough endoplasmic reticulum (RER). Two distinct nuclei migrated towards opposite poles of the cell. The thickened wall began to invaginate (black arrows). (E) The dividing binucleate sporont containing two developing anchoring disk (AD). Insert showing the detail of the preformed anchoring disk. Insert scale bar = 0.2μm. (F) Late sporont at the point of splitting into two uninucleate sporoblasts. Each part of the cell contained one nucleus, six polar filament coils, one electron lucent vacuole, and one posterior vacuole. A and B, Scale bar = 0.6 μm. C-E, Scale bar = 0.4 μm. F, Scale bar = 0.5 μm.

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Figure 6. Transmission electron micrographs showing the ultrastructure of sporoblast and immature spore of Ameson portunus in Portunus trituberculatus. (A) Individual suborbicular sporoblast with high electron dense, decrescent posterior vacuole (PV), removed the electron lucent vacuole, growing hair-like projections (white arrow). (B) Late sporoblast showing the anchoring disc (AD), polaroplast (PP), and polar filament (PF). (C) Egg-shaped immature spore showing a single nucleus, anchoring disc (AD), electron dense polaroplast (PP), polar filament (PF), rough endoplasmic reticulum (RER), smaller posterior vacuole (PV), and thickened dense wall (black arrowhead). (D) Late immature spore with a dense body (white asterisk) and preformed electron lucent endospore (white arrowhead). AD, Scale bar = 0.2 μm.

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Figure 7. Transmission electron micrographs showing the ultrastructure of mature spores of Ameson portunus in Portunus trituberculatus. (A) Many ovoid spores in host skeletal musculature tissue. (B) Mature spore with thin electron dense exospore (EX), thick electron lucent endospore (EN), laminar polaroplast (PP), and a polar filament (PF) making 7-8 turns. (C) Mature spore with a single nucleus (black asterisk) and a dense body (white asterisk). The polar filament (PF) has 9 turns in a linear arrangement. (D) Details of terminal anchoring disk (AD) of a mature spore. (E) Details of the spore wall (W) and polar filament coils (PF) of a mature spore. (F) Spore with long hair-like projections (white arrow). (G) Details of the hair-like projections with short stubby roots (white arrow) and long thin extensions (black arrow). A, Scale bar = 1 μm. B-G, Scale bar = 100 nm.

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Figure 8. Maximum likelihood tree (left side) and Bayesian tree (right side) of SSU rDNA sequences of Ameson portunus and other microsporidian species. Basidiobolus ranarum was the source of the outgroup sequence. ML/NJ/MP bootstrap values are shown on the left tree branches. Bayesian posterior probabilities are shown on the right tree branches. Scale bar corresponds to 0.2 substitutions per site. There were total of 794 positions in the final dataset. The species names follow behind GenBank accession numbers.

Page 22 of 20

Figure 9. Putative life cycle of Ameson portunus inferred from ultrastructural observations of life stages in skeletal musculature of Portunus trituberculatus. 1. Uninucleate meront. 2. Diplokaryotic meront. 3. Enlarged diplokaryotic meront. 4. Tetranucleate meront. 5. Late diplokaryotic meront. 6. Early binucleate sporont with two isolated nuclei and thickening membrane. 7. Binucleate sporont with thickened wall and preformed anchoring disk. 8. Late binucleate sporont prepare to divide into two sporoblast. 9. Elongated diplokaryotic meront. 10. Chain-like meront with three or more isolate nuclei. 11. Early chain-like sporont. 12. Late chain-like sporont. 13. Sporoblast. 14. Immature spore. 15. Mature spore.

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Table 1. Evolutionary distances based on some SSU rDNA sequences: Similarity percentage (upper diagonal) and pairwise distance (lower diagonal) obtained by p-distance analysis. 1 -

2

3

4

5

6

7

8

9

10

11

99.1

98.6

96.0

96.0

69.4

69.3

72.5

72.9

68.3

68.1

1

Ameson portunus n. sp.

2

Nadelspora canceri

0.009

-

98.9

95.1

95.6

69.0

68.9

72.4

72.8

68.3

68.1

3

Ameson pulvis (UK)

0.014

0.011

-

94.8

95.1

69.3

69.1

72.9

73.4

68.4

68.3

4

Ameson michaelis

0.039

0.048

0.050

-

92.7

67.6

67.5

71.3

71.7

67.3

67.1

5

Ameson metacarcini

0.039

0.043

0.048

0.071

-

68.9

68.8

70.5

71.5

67.3

67.1

6

Potaspora morhaphis

0.301

0.305

0.303

0.319

0.306

-

99.1

74.3

74.9

77.3

77.2

7

Potaspora aequidens

0.303

0.306

0.304

0.320

0.308

0.009

-

74.7

75.2

77.5

77.3

8

Perezia sp.

0.271

0.272

0.267

0.283

0.290

0.255

0.251

-

96.0

77.5

77.3

9

Perezia nelsoni

0.267

0.268

0.262

0.279

0.281

0.249

0.246

0.039

-

78.6

78.5

10

Glugea arabica

0.310

0.310

0.309

0.320

0.320

0.224

0.223

0.224

0.213

-

99.9

11

Glugea nagelia

0.311

0.311

0.310

0.321

0.321

0.225

0.224

0.225

0.214

0.001

-

Page 24 of 20

Table 2 Comparison of spore morphology for the known Ameson spp.

Spore Spore size (fixed) Spore

PF diameter/coils

Species shape and L/W Ratio

A. atlanticum

wall

number/arrangement

1.8 × 1.4μm

No

metacarcin ovoid

No data/9−12coils/8−9 of

data

i

coil

1.6 × 1.2μm ovoid

michaelis

Metacarcinus

Skeletal

magister

musculature

Callinectes

Skeletal

which form a linear outer (TEM), 1.3

A.

80nm/8−9 coils/5−7

1.4 × 1.0μm ovoid

form a linear outer coil,

n. sp.

Azevedo, 1988)

(Sprague et al., USA

sapidus

musculature

Portunus

Skeletal

trituberculatus

musculature

57nm (sometimes all) of which (TEM), 1.4

(Vivarès and France

USA (Small et al., 2014)

70nm 70nm/11coils/ irregularly (TEM), 1.3

References ty

(TEM), 1.3

A.

A.

Infection site

140n 170nm/11−12coils/9−10 of Skeletal which form a linear outer Cancer pagurus m musculature coil

2.0 × 1.5μm ovoid

portunus

Locali Hosts

1968)

China Present study

(Vivarès and A. pulvis

100nm/8−9coils/6−7 of

1.3 × 1.0μm (LM), ovoid

(Fr)

40nm which form a linear outer 1.3

coil

Carcinus maenas Skeletal

FranceSprague, 1979)

musculature

France(Vivarès and

Carcinus mediterraneus Azevedo, 1988)

1.2 ×1.0μm

No data/8−9coils/5−6 of

ovoid A. pulvis

(TEM), 1.2

No

needl (UK)

6 × 0.3μm (TEM), data e

Skeletal and

which form a linear outer coil, No data/uncoiled/

Carcinus maenas heart musculature

United(Stentiford et al., Kingd om 2013a)

20

Fr, the France isolate; UK, the United Kingdom isolate; L/W, length/width; LM, light microscopy; TEM, transmission electron microscopy.

Page 25 of 20