Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria.

The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores; Actinobacteria, forming exospores; Cyanobacteria, forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.

Morphological and genetic analysis of Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea.

We describe Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea. C. saurida was found in the gallbladders of 3/13 specimens of its type host, Saurida elongata Temminck and Schlegel 1846 (Aulopiformes). Myxospore characters were consistent with the original description to which we have added small subunit (SSU) rRNA gene data. C. mai sp. nov. was found in gallbladders of 3/13 specimens of S. elongata and 5/13 specimens of Neobythites sivicola Jordan and Snyder 1901 (Ophidiiformes). Mature myxospores of C. mai sp. nov. were crescentic in sutural view, with a deeply concave posterior angle 142.2±8.2° (125.8‒158.2°) and an arched anterior side. Shell valves were smooth and equal, 20.9±1.9 (17.3‒24.7) µm thick and 9.2±0.5 (8.1‒9.9) µm long, and joined at a straight, thin sutural plane passing between two nematocysts (polar capsules). The nematocysts were equal-sized, pyriform, 2.6±0.2 (2.4‒2.9) µm long and 2.7±0.2 (2.4‒3.3) µm wide, with their tapered ends pointed toward each other, located in the anterior third of the spore. Sequences of the SSU rRNA gene and internal transcribed spacer 1 showed that the isolates of C. mai sp. nov. obtained from S. elongata and N. sivicola were identical. The SSU rRNA gene sequence of C. mai sp. nov. was distinct from all known myxosporeans and clustered with C. saurida, and then with Ceratomyxa filamentosi Kalatzis, Kokkari and Katharios 2013, both of which also infect Aulopiformes fishes.

Ceratonova shasta: a cnidarian parasite of annelids and salmonids.

The myxozoan Ceratonova shasta was described from hatchery rainbow trout over 70 years ago. The parasite continues to cause severe disease in salmon and trout, and is recognized as a barrier to salmon recovery in some rivers. This review incorporates changes in our knowledge of the parasite's life cycle, taxonomy and biology and examines how this information has expanded our understanding of the interactions between C. shasta and its salmonid and annelid hosts, and how overarching environmental factors affect this host­parasite system. Development of molecular diagnostic techniques has allowed discrimination of differences in parasite genotypes, which have differing host affinities, and enabled the measurement of the spatio-temporal abundance of these different genotypes. Establishment of the C. shasta life cycle in the laboratory has enabled studies on host­parasite interactions and the availability of transcriptomic data has informed our understanding of parasite virulence factors and host defences. Together, these advances have informed the development of models and management actions to mitigate disease.

Insights into myxozoan composition and physiology revealed by histochemical properties of myxospores.

Myxozoa (phylum Cnidaria) are a diverse group of metazoan parasites that predominately infect fish. Little is known regarding the composition and physiology of their myxospore life stage. The objective of this work was to investigate the composition of myxospores and extrasporogonic stages of nine myxozoan species infecting various teleost fish using histochemical staining techniques. Thirty histochemical stains were applied to formalin-fixed, paraffin-embedded tissues processed routinely for light microscopic evaluation. The polar capsules were the most consistent stain target across the taxa examined. Polar capsule staining with Alizarin red, von Kossa and methyl green-pyronin suggests the presence of intracapsular calcium and phosphate, which may contribute to polar filament discharge or pathogenesis of host invasion. The shell valves and suture lines of most myxozoans were stained with Luna and phosphotungstic acid haematoxylin stains, consistent with the presence of chitin and microfibrils, respectively. Vacuoles were consistently highlighted by diastase-susceptible periodic acid-Schiff and Grocott's methenamine silver staining, indicating glycogen. Other histochemical stains exhibited inconsistent staining across the taxa, suggesting differences in myxospore composition potentially reflective of physiologic variations and tissue tropisms. This work provides some information on conserved features and taxa-associated composition of myxospores and lends insight into myxozoan physiology and host-parasite interactions.

Simulacricoccus ruber gen. nov., sp. nov., a microaerotolerant, non-fruiting, myxospore-forming soil myxobacterium and emended description of the family Myxococcaceae.

A non-fruiting group of myxobacteria was previously speculated to exist in nature based on metagenomics data containing uncultured members of the order Myxococcales. Here, we describe a myxobacterial strain, designated MCy10636T, which was isolated from a German soil sample collected in 2013. It exhibits swarming characteristics but atypically produces myxospores in the absence of fruiting bodies. The novel strain stains Gram-negative and Congo-red-negative and is characterized mesophilic, neutrophilic, chemoheterotrophic and microaerotolerant. Branched-chain fatty acids are the predominant cellular fatty acids over the straight-chain type, and contain the major fatty acids iso-C17 : 0 2-OH, C16 : 1, iso-C17 : 0 and iso-C15 : 0. Based on blastn results, the 16S rRNA gene sequence reveals similarity (97 %) to Aggregicoccus edonensis MCy1366T, (97 %) Myxococcus macrosporus DSM 14697T, (96 %) Corallococcus coralloides DSM2259T and Corallococcus exiguus Cc e167T. Phylogenetic analysis showed a novel lineage of MCy10636T in the family Myxococcaceae, suborder Cystobacterineae. Based on polyphasic taxonomic characterization, we propose that this unusual, non-fruiting, myxospore-forming and microaerotolerant myxobacterial strain, MCy10636T, represents a novel genus and species, Simulacricoccus ruber gen. nov., sp. nov. (DSM 106554T=NCCB 100651T).

Description, life cycle, and development of the myxozoan Myxobolus rasmusseni n. sp. in fathead minnows, Pimephales promelas: A possible emerging pathogen in southern Alberta, Canada.

Morphological, gene sequence, host tissue tropism, and life cycle characteristics were utilized to describe the myxozoan, Myxobolus rasmusseni n. sp. from fathead minnow, Pimephales promelas, collected from reservoirs in southern Alberta. Results from serial histological sections of whole heads showed that myxospores were contained within irregular-shaped and sized coelozoic capsules (=plasmodia). Clusters of membrane-bound, myxospore-filled plasmodia filled the head cavities of juvenile fathead minnows, leading to the development of large, white, disfiguring lesions in mid to late summer. Bilateral exopthalmia (pop-eye disease) was a common outcome of M. rasmusseni n. sp. development. BLASTn search of a 1974 bp sequence of the 18S rDNA gene isolated from myxospores indicated that M. rasmusseni n. sp. was distinct from other coelozoic and histozoic Myxobolus spp. cataloged in GenBank. 18S rDNA gene sequences from triactinomyxon spores released from the oligochaete Tubifex were 100% identical to sequences from myxospores collected from syntopic fathead minnows. Results from a longitudinal survey of the 2020 cohort of fathead minnows showed that young-of-the-year are exposed at 1-5 mo and that 60-90% of these had developed myxospore-filled lesions approximately one year later. Data regarding potential sources and timing of M. rasmusseni n. sp. emergence in fathead minnow populations are needed.

Morphological and Molecular Characterization of Myxobolus planilizae n. sp. (Cnidaria; Myxosporea; Myxobolidae) Infecting the Largescale Mullet Planiliza macrolepis (Smith, 1846) Collected From Cochin Backwaters, India.

PURPOSE: Myxobolus planilizae n. sp. is described from the intestinal muscles of the largescale mullet Planiliza macrolepis from Cochin backwaters, Kerala, India. METHODS: Host fishes inhabiting Cochin backwaters were collected using Chinese nets/gill nets. The morphometry and morphological studies were carried out using Nomarski differential interference contrast (DIC) optics, followed by molecular and phylogenetic analyses of the small subunit ribosomal DNA gene (SSU rDNA). RESULTS: Plasmodia small, pale white, and infect the muscles of the intestine; measured 0.13-0.22 (0.17) × 0.09-0.14 (0.13) mm. Mature myxospores pyriform in valvular view, and biconvex in sutural and apical views with a short anterior extension, and measured 7.45-8.75 (8.40) × 6.04-6.86 (6.25) µm. Shell valves with sutural ornamentations. Polar capsules two, equal, pyriform, measured 3.96-4.54 (4.45) × 2.22-2.94 (2.52) µm. Polar filament arranged in five coils, measured 24.41-34.44 (28.52) µm when extruded. In morphological and morphometric analysis, the present species exhibit remarkable variations from other species of the genus Myxobolus. In molecular analysis, the present species revealed the highest identity of 91.85% and divergence of 9.95% with related species, underlining its molecular uniqueness. In phylogenetic analysis, species of Myxobolus infecting mullets appeared as a separate clade and the present species was positioned distinctly with a high bootstrap value. CONCLUSIONS: Based on morphology, morphometry, and molecular and phylogenetic analyses, along with tissue/host specificities and geographic location, the present parasite is treated as new and is reported here as M. planilizae n. sp.

Four carangid fish species as new host records for Kudoa trachuri Matsukane, Sato, Tanaka, Kamata et Sugita-Konishi, 2011 (Myxozoa: Multivalvulida), and description of a new species, Kudoa longichorda sp. n., forming pseudocysts in the muscle of Decapterus tabl Berry.

Multivalvulid myxosporeans of the genera Kudoa Meglitsch, 1947 and Unicapsula Davis, 1924 (Cnidaria: Myxozoa) are often the cause of unsightly cyst formation or postmortem myoliquefaction in the trunk muscle of commercial marine fish, which reduces the market value of infected individuals. Twenty species (18 Kudoa spp. and two Unicapsula spp.) have been recorded from carangid fish, although the majority of them, excluding polyxenous species, such as K. amamiensis Egusa et Nakajima, 1980, K. iwatai Egusa et Shiomitsu, 1983, K. nova Naidenova, 1975, K. quadratum (Thélohan, 1895) and K. yasunagai (Hsieh et Chen, 1984), are limited to a single or a few fish species. We report the occurrence of macroscopic cysts of Kudoa trachuri Matsukane, Sato, Tanaka, Kamata et Sugita-Konishi, 2011 in the trunk muscle of four new host fish species, i.e., Pseudocaranx dentex (Bloch et Schneider), Decapterus akaadsi Abe, D. muroadsi (Temminck et Schlegel) and Decapterus tabl Berry, fished from the Philippine Sea (Northwest Pacific Ocean), off southwestern of Japan. Myxospore morphology and genetic characteristics of the ribosomal RNA gene (rDNA) of these isolates were consistent with previous records of K. trachuri from Trachurus japonicus (Temminck et Schlegel) from around Japan. In addition, a new species of Kudoa that forms long filamentous pseudocysts in trunk myofibres was found in four of the six D. tabl collected in this study. We describe Kudoa longichorda sp. n. for this new isolate, based on its morphology of subquadrate myxospores with four shell valves and polar capsules and with small dimensions (length 4.3-5.5 µm, width 6.0-6.8 µm, thickness 4.8-6.3 µm, polar capsule length 2.3-3.1 µm, polar capsule width 1.1-1.7 µm), as well as 18S and 28S rDNA sequences distinct from those of known species.

The cnidarian parasite Ceratonova shasta utilizes inherited and recruited venom-like compounds during infection.

BACKGROUND: Cnidarians are the most ancient venomous organisms. They store a cocktail of venom proteins inside unique stinging organelles called nematocysts. When a cnidarian encounters chemical and physical cues from a potential threat or prey animal, the nematocyst is triggered and fires a harpoon-like tubule to penetrate and inject venom into the prey. Nematocysts are present in all Cnidaria, including the morphologically simple Myxozoa, which are a speciose group of microscopic, spore-forming, obligate parasites of fish and invertebrates. Rather than predation or defense, myxozoans use nematocysts for adhesion to hosts, but the involvement of venom in this process is poorly understood. Recent work shows some myxozoans have a reduced repertoire of venom-like compounds (VLCs) relative to free-living cnidarians, however the function of these proteins is not known. METHODS: We searched for VLCs in the nematocyst proteome and a time-series infection transcriptome of Ceratonova shasta, a myxozoan parasite of salmonid fish. We used four parallel approaches to detect VLCs: BLAST and HMMER searches to preexisting cnidarian venom datasets, the machine learning tool ToxClassifier, and structural modeling of nematocyst proteomes. Sequences that scored positive by at least three methods were considered VLCs. We then mapped their time-series expressions in the fish host and analyzed their phylogenetic relatedness to sequences from other venomous animals. RESULTS: We identified eight VLCs, all of which have closely related sequences in other myxozoan datasets, suggesting a conserved venom profile across Myxozoa, and an overall reduction in venom diversity relative to free-living cnidarians. Expression of the VLCs over the 3-week fish infection varied considerably: three sequences were most expressed at one day post-exposure in the fish's gills; whereas expression of the other five VLCs peaked at 21 days post-exposure in the intestines, coinciding with the formation of mature parasite spores with nematocysts. Expression of VLC genes early in infection, prior to the development of nematocysts, suggests venoms in C. shasta have been repurposed to facilitate parasite invasion and proliferation within the host. Molecular phylogenetics suggested some VLCs were inherited from a cnidarian ancestor, whereas others were more closely related to sequences from venomous non-Cnidarian organisms and thus may have gained qualities of venom components via convergent evolution. The presence of VLCs and their differential expression during parasite infection enrich the concept of what functions a "venom" can have and represent targets for designing therapeutics against myxozoan infections.

New data on Thelohanellus nikolskii Achmerov, 1955 (Myxosporea, Myxobolidae) a parasite of the common carp (Cyprinus carpio, L.): The actinospore stage, intrapiscine tissue preference and molecular sequence.

Thelohanellus nikolskii, Achmerov, 1955 is a well-known myxozoan parasite of the common carp (Cyprinus carpio L.). Infection regularly manifests in numerous macroscopic cysts on the fins of two to three month-old pond-cultured carp fingerlings in July and August. However, a Thelohanellus infection is also common on the scales of two to three year-old common carp in ponds and natural waters in May and June. Based on myxospore morphology and tissue specificity, infection at both sites seems to be caused by the same species, namely T. nikolskii. This presumption was tested with molecular biological methods: SSU rDNA sequences of myxospores from fins of fingerlings and scales of older common carp were analysed and compared with each other and with related species available in GenBank. Sequence data revealed that the spores from the fins and scales represent the same species, T. nikolskii. Our study revealed a dichotomy in both infection site and time in T. nikolskii-infections: the fins of young carp are infected in Summer and Autumn, whereas the scales of older carp are infected in Spring. Myxosporean development of the species is well studied, little is known, however about the actinosporean stage of T. nikolskii. A previous experimental study suggests that aurantiactinomyxon actinospores of this species develop in Tubifex tubifex, Müller, 1774. The description included spore morphology but no genetic sequence data (Székely et al., 1998). We examined >9000 oligochaetes from Lake Balaton and Kis-Balaton Water Reservoire searching for the intraoligochaete developmental stage of myxozoans. Five oligochaete species were examined, Isochaetides michaelseni Lastochin, 1936, Branchiura sowerbyi Beddard, 1892, Nais sp., Müller, 1774, Dero sp. Müller, 1774 and Aelosoma sp. Ehrenberg, 1828. Morphometrics and SSU rDNA sequences were obtained for the released actinospores. Among them, from a single Nais sp., the sequence of an aurantiactinomyxon isolate corresponded to the myxospore sequences of T. nikolskii.

Morphological and molecular description of a new species of Myxobolus (Myxosporea: Myxobolidae) infecting Planiliza macrolepis (Smith, 1846) from India.

The present paper describes a novel species of Myxobolus parasitizing the gill filaments of the largescale mullet, Planiliza macrolepis from Cochin backwaters, Kerala, India. The parasite develops in the gill filaments; plasmodia elongated, milky white, measured 1.37-2.18 (1.78 ± 0.35) mm × 0.07-0.12 (0.10 ± 0.02) mm in size. Mature myxospores ovoid in valvular view, biconvex in sutural view with smooth shell valves and measured 6.24-7.02 (6.63 ± 0.23) × 5.01-6.18 (5.68 ± 0.25) µm in size. Polar capsules equal, oval with pointed anterior ends, 3.07-3.58 (3.33 ± 0.12) × 1.68-2.42 (2.09 ± 0.18) µm in size. Polar filaments with 4 coils, measured 29.61 ± 4.75 µm in length when extruded. Sporoplasm binucleate with a rudimentary nucleus and a vacuole. A comparison with related Myxobolus species revealed significant morphological and morphometric differences. In BLASTN and genetic distance analysis, the present parasite showed high divergence with other myxosporean sequences, indicating its molecular uniqueness. In Maximum Likelihood and Bayesian Inference analysis, the present species stands out with M. ramadus as sister branch within the Myxobolus clade. In infected gill filaments, the plasmodia caused swelling/deformation, compression of lamellae and reduction in respiratory surface area. Three of 222 P. macrolepis screened were infected, indicating a prevalence of 1.3%. Considering the morphological, morphometric, molecular and phylogenetic differences with the previously described species of myxosporeans, along with the dissimilarities in host and geographical locations, the present parasite is treated as a new species and the name Myxobolus cochinensis n. sp. is proposed.

Novel and known myxobolids (Cnidaria, Myxozoa) infecting Chondrostoma angorense (Cypriniformes: Leuciscidae) in Turkey.

Turkey has more than 200 endemic freshwater fish species, one of which is the Ankara nase, Chondrostoma angorense Elvira, 1987 (Cypriniformes: Leuciscidae), a food fish in northern Turkey. Like most endemic fish species in Turkey, its myxosporean parasite fauna (Cnidaria: Myxosporea) are not yet described. We surveyed twenty C. angorense from Lâdik Lake in northern Turkey, and identified two myxosporean parasites from gills of these fish: Myxobolus arrabonensis Cech, Borzák, Molnár, Székely, 2015, and a co-infection of a novel species, Myxobolus polati sp. nov. We characterized both infections based on myxospore morphology, morphometry, tissue tropism, small subunit ribosomal DNA sequence and phylogenetic analysis. Plasmodia of both species were observed in gills, but had distinct tropism: M. arrabonensis is an intrafilamental vascular type, and M. polati sp. nov. is an intralamellar vascular type. We identified M. arrabonensis on the basis of myxospore characters and 100% similarity to the type DNA sequence from the closely-related host C. nasus. The small subunit ribosomal DNA sequence of M. polati sp. nov. (1946 base pairs; GenBank Accession number MH392318) had a maximum similarity of 98% with any Myxobolus sp. from other Eurasian cypriniforms. Phylogenetic analysis revealed that M. polati sp. nov. is most closely related to gill-infecting Myxobolus diversicapsularis from Rutilus rutilus (L.). The present study is the first record of myxosporean species infecting C. angorense comprising a novel species, M. polati sp. nov. and a known species M. arrabonensis.

Description of myxosporeans (Cnidaria: Myxozoa) infecting the popular food fish Notopterus notopterus (Pisces: Notopteridae) in Malaysia and India.

This study was a co-operative investigation of myxosporean infections of Notopterus notopterus, the bronze featherback, which is a popular food fish in the South Asian region. We examined fish from Lake Kenyir, Malaysia and the River Ganga, Hastinapur, Uttar Pradesh, India, and observed infections with two myxosporeans: Myxidium cf. notopterum (Myxidiidae) and Henneguya ganapatiae (Myxobolidae), respectively. These species were identified by myxospore morphology, morphometry and host tissue affinity, and the original descriptions supplemented with small-subunit ribosomal DNA sequences and phylogenetic analysis. Free myxospores of M. cf. notopterum were found in the gallbladder, and measured 14.7 ±â€¯0.6 µm long and 6.3 ±â€¯0.6 µm wide; host, tissue and myxospore dimensions overlapped with the type, but differed in morphological details (spore shape, valve cell ridges) and locality (Malaysia versus India). Plasmodia and spores of H. ganapatiae were observed in gills, and myxospores had a spore body 9.7 ±â€¯0.4 µm long, 4.5 ±â€¯0.5 µm wide; sample locality, host, tissue, spore morphology and morphometry matched the original description. Small-subunit ribosomal DNA sequences were deposited in GenBank (M. cf. notopterum MT365527, H. ganapatiae MT365528) and both differed by >7% from congeneric species. Although the pathogenicity and clinical manifestation of myxozoan in humans are poorly understood, consumption of raw fish meat with myxozoan infection was reported to be associated with diarrhea. Identification of current parasite fauna from N. notopterus is an essential first step in assessing pathogen risks to stocks of this important food fish.

Henneguyosis in gills of Metynnis hypsauchen: an Amazon freshwater fish.

This study describes aspects of infection caused by Myxozoa of the genus Henneguya sp. in gills of fish belonging to the species Metynnis hypsauchen. Two sampling were made in the Capim river, close to the Ribeira community, in the municipality of Ipixuna do Pará, State of Pará, Brazil, during the months of August 2018 and March 2019. The animals were captured and transported live to the Laboratório de Pesquisa Carlos Azevedo, at the Universidade Federal Rural da Amazônia (UFRA), in Belém, Pará, Brazil. The animals were examined, and after parasitism was confirmed, Differential Interference Contrast Microscopes were used, to evaluate the parasite spores. Ziehl-Neelsen stain techniques were used in histology. Necroscopic analyses of Metynnis hypsauchen specimens found parasites in 80% of the hosts (16/20), with whitish-colored cysts in the branchial filaments, containing mature spores of the genus Henneguya. The histopathological analysis indicated large areas with cystic lesions with associated ischemic necrosis. The descriptions from this study indicate that the parasite drastically compromises the host's respiratory system. Additionally, it is worth noting that parasite fauna studies of fishes in the Capim river are still a vast topic for research; this is the first record of infection by Henneguya sp. in Metynnis hypsauchen, captured in the Capim river in Ipixuna do Pará.