Genome and Evolutionary Analysis of Nosema ceranae: A Microsporidian Parasite of Honey Bees

Microsporidia comprise a phylum of single cell, intracellular parasites and represent the earliest diverging branch in the fungal kingdom. The microsporidian parasite Nosema ceranae primarily infects honey bee gut epithelial cells, leading to impaired memory, suppressed host immune responses and colony collapse under certain circumstances. As the genome of N. ceranae is challenging to assembly due to very high genetic diversity and repetitive region, the genome was re-sequenced using long reads. We present a robust 8.8 Mbp genome assembly of 2,280 protein coding genes, including a high number of genes involved in transporting nutrients and energy, as well as drug resistance when compared with sister species Nosema apis. We also describe the loss of the critical protein Dicer in approximately half of the microsporidian species, giving new insights into the availability of RNA interference pathway in this group. Our results provided new insights into the pathogenesis of N. ceranae and a blueprint for treatment strategies that target this parasite without harming honey bees. The unique infectious apparatus polar filament and transportation pathway members can help to identify treatments to control this parasite.

A Severe Microsporidian Disease in Cultured Atlantic Bluefin Tuna (Thunnus thynnus L.)

One of the most promising aquaculture species is the Atlantic bluefin tuna ( Thunnus thynnus ) with high market value; disease control is crucial to prevent and reduce mortality and monetary losses. Microsporidia Balbiani, 1882 (Fungi) are a potential source of damage to bluefin tuna aquaculture. A new microsporidian species is described from farmed bluefin tunas from the Spanish Mediterranean. This new pathogen is described in a juvenile associated with a highly severe pathology of the visceral cavity. Whitish xenomes from this microsporidian species were mostly located at the caecal mass and ranged from 0.2 to 7.5 mm. Light and transmission electron microscopy of the spores revealed mature spores with an average size of 2.2 × 3.9 μm in size and a polar filament with 13–14 coils arranged in one single layer. Phylogenetic analysis clustered this species with the Glugea spp. clade. The morphological characteristics and molecular comparison confirm that this is a novel microsporidian species, Glugea thunni sp. nov. The direct life cycle and the severe pathologies observed makes this parasite a hard risk for bluefin tuna cultures.

Isolation of the Parasite Enterocytospora artemiae From Chinese Grass Shrimp (Palaemonetes sinensis)—First Report in Asia

Chinese grass shrimp (Palaemonetes sinensis) is an economically important crustacean in Chinese aquaculture. Recently, we found that shrimp in Panjin city were infected with microsporidia, a group of fungi. The hepatopancreas of several infected shrimp showed white turbidity and pathological changes that negatively affected the health and appearance of the shrimp. Histopathology and transmission electron microscopy were used to examine the development of the parasite within its parasitophorous vacuole. Our results indicated that microsporidia developed asynchronously within the same parasitophorous vacuole. The spores were predominantly small, and rod or oval-shaped. The sizes of fresh spores were approximately 3.1 × 2.4 μm and fixed spores were 1.9 × 1.1 μm. The polar filament was isofilar with 5–6 coils and the thickness was 103.2 nm. Merogonial divisions occurred by binary fission and sporogonial division occurred by plasmotomy. The small subunit ribosomal DNA sequence (1295 bp) from the parasite was highly similar to the previously reported parasite Enterocytospora artemiae (99% nucleotide identity, JX915760). Using maximum likelihood to analyze the phylogenetic relationships, we found that this microsporidian should be grouped within Clade IV, an Enterocytospora-like clade, of the Microsporidia phylum. Based on this parasite’s life cycle characteristics, morphology, and small subunit ribosomal DNA sequence, the parasite described here is likely E. artemiae, which has previously only been described in Europe and North America. Thus, this is the first report of E. artemiae both in Asia and economically important shrimp.

A new isolate of Nosema fumiferanae (Microsporidia: Nosematidae) from the date moth Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839 (Lepidoptera: Pyralidae)

In this study, a microsporidian pathogen of the date moth (Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839) also known as the carob moth, is described based on light microscopy, ultrastructural characteristics and comparative molecular analysis. The pathogen infects the gut and hemolymph of A. ceratoniae. All development stages are in direct contact with the host cell cytoplasm. Fresh spores with nuclei arranged in a diplokaryon are oval and measured 3.29 ± 0.23 μm (4.18–3.03 μm, n = 200) in length and 1.91 ± 0.23 μm (2.98–1.66 μm, n = 200) in width. Spores stained with Giemsa's stain measured 3.11 ± 0.31 μm (3.72–2.41 μm, n = 150) in length and 1.76 ± 0.23 μm (2.16–1.25 μm, n = 150) in width. Spores have an isofilar polar filament with 10-12 coils. An 1110 bp long alignment of the current microsporidium showed an SSU rRNA gene difference of only 0.0009, corresponding to >99.91% sequence similarity with Nosema fumiferanae, while RPB1 gene sequences were 98.03% similar within an alignment of 969 bp. All morphological, ultrastructural and molecular features indicate that the microsporidian pathogen of A. ceratoniae is the new isolate of the N. fumiferanae and is named here as Nosema fumiferanae TY61.

A new Myxobolus (Cnidaria: Myxosporea) infecting the ornamental catfish Corydoras schwartzi from the Purus River in Brazil

A new microscopic cnidarian is described, infecting five of thirty (16.6%) specimens of Corydoras schwartzi caught in the Purus River, State of Amazonas, Brazil. Histological analysis showed that cyst development occurred in the serosa layer of the intestine. Mature myxospores are ovoid in body shape in frontal view, 22.4 ± 0.3 μm in total length and 16.3 ± 0.1 μm in width. Internally, two aubergine-shaped, elongate symmetrical polar capsules occupy more than half the length of the spore, 14.3 ± 0.2 μm in length and 6.5 ± 0.1 μm in width. Ultrastructural analysis provided evidence of five polar filament coils inside the polar capsule and binucleated sporoplasm containing a moderate number of sporoplasmosomes. The valvogenic cells abutting each other form a sutural ridge and frequently a thin layer of homogeneous material separates the cells. The outer surface of the myxospore valves is smooth, with no evidence of formation of ridges in the valves. Immature myxospores in various stages of development were observed. This study is the first report of a myxosporean parasitizing C. schwartzi and the first report of a myxosporean infection in the intestine of an ornamental fish from South America.

Henneguya sacacaensis n. sp. (Myxozoa: Myxosporea) parasitizing gills of the acará bicudo Satanoperca jurupari (Osteichthyes: Cichlidae) in eastern Amazon

This study describes Henneguya sacacaensis n. sp. in specimens of the Osteichthyes Satanoperca jurupari (Heckel, 1840), collected in the Rio Curiaú Environmental Protection Area in the city of Macapá, state of Amapá Brazil. Using optical microscopy and molecular analysis, these cyst-shaped parasites were analyzed. The gills of 57.14% of the analyzed S. jurupari contained hundreds of spores. The cysts found on the gill lamellae were oval-shaped and whitish. The Henneguya spores had an average length of 46.5 (41.3-56.92) µm. The fusiform body of the Henneguya measured 16.5 (13.16-20.01) µm long and 5.1 (3.91-6.12) µm in width, the two polar capsules had a taper of 3.83 (3.4-4.32) µm and a width of 1.68 (1.4-1.99) µm, and the tail measured 30 (22.47-41.67) µm in length, containing a polar filament coiled seven to nine times. Morphogical and phylogenetic analysis allowed the preposition of a new species, Henneguya sacacaensis n. sp, that belongs to the family Myxobolidae and the genus Henneguya.

Single and multi-gene phylogeny ofHepatospora(Microsporidia) – a generalist pathogen of farmed and wild crustacean hosts

SUMMARYAlmost half of all known microsporidian taxa infect aquatic animals. Of these, many cause disease in arthropods.Hepatospora, a recently erected genus, infects epithelial cells of the hepatopancreas of wild and farmed decapod crustaceans. We isolatedHepatosporaspp. from three different crustacean hosts, inhabiting different habitats and niches; marine edible crab (Cancer pagurus), estuarine and freshwater Chinese mitten crab (Eriocheir sinensis) and the marine mussel symbiont pea crab (Pinnotheres pisum). Isolates were initially compared using histology and electron microscopy revealing variation in size, polar filament arrangement and nuclear development. However, sequence analysis of the partial SSU rDNA gene could not distinguish between the isolates (~99% similarity). In an attempt to resolve the relationship betweenHepatosporaisolated fromE. sinensisandC. pagurus, six additional gene sequences were mined from on-going unpublished genome projects (RNA polymerase, arginyl tRNA synthetase, prolyl tRNA synthetase, chitin synthase, beta tubulin and heat shock protein 70). Primers were designed based on the above gene sequences to analyseHepatosporaisolated from pea crab. Despite application of gene sequences to concatenated phylogenies, we were unable to discriminateHepatosporaisolates obtained from these hosts and concluded that they likely represent a single species or, at least subspecies thereof. In this instance, concatenated phylogenetic analysis supported the SSU-based phylogeny, and further, demonstrated that microsporidian taxonomies based upon morphology alone are unreliable, even at the level of the species. Our data, together with description ofH. eriocheirin Asian crab farms, reveal a preponderance for microvariants of this parasite to infect the gut of a wide array of decapods crustacean hosts and the potential forHepatosporato exist as a cline across wide geographies and habitats.

Ultrastructure and molecular phylogeny of Pleistophora hyphessobryconis (Microsporidia) infecting hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a Brazilian aquaculture facility

SUMMARYA microsporidian infecting the skeletal muscle of hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a commercial aquaculture facility in Brazil is described. Affected fish exhibited massive infections in the skeletal muscle that were characterized by large opaque foci throughout the affected fillets. Histologically, skeletal muscle was replaced by inflammatory cells and masses of microsporidial developmental stages. Generally pyriform spores had a wrinkled bi-layer spore wall and measured 4·0 × 6·0 µm. Multinucleate meronts surrounded by a simple plasma membrane were observed. The polar filament had an external membrane and a central electron dense mass. The development of sporoblasts within a sporophorous vesicle appeared synchronized. Ultrastructural observations and molecular analysis of 16S rDNA sequences revealed that the microsporidian was Pleistophora hyphessobryconis. This study is the first report of a P. hyphessobryconis infection in a non-ornamental fish.

Nosema sp. PM-1, a new isolate of microsporidian from infected Papilio machaon Linnaeus, based on ultrastructure and molecular identification

A new microsporidium, Nosema sp. PM-1, was first isolated from Papilio machaon Linnaeus. The spore shape of the PM-1 isolate was a long oval with an average size of 3.22 μm × 1.96 μm. Ultrastructure observation showed that PM-1 had a typical Nosema common diplokaryotic nuclei structure with 10-13 polar filament coils, spore wall, plasma membrane, and anchoring disk. The complete rRNA gene sequences were obtained by polymerase chain reaction amplification and each rRNA unit was arrayed as follows: 5′-LSU (2497 bp)-ITS (179 bp)-SSU (1232 bp)-IGS (278 bp)-5S (115 bp)-3′, which was the same as typical Nosema. The phylogenetic trees of rRNA, DNA-directed RNA polymerase II subunit, and tubulin genes all show that PM-1 was a sister to the clade comprising Nosema bombycis, Nosema spodopterae, and Nosema sp. PX1. The spore morphology, ultrastructure, and complete rRNA structure indicate that this isolate assigned to the ˝true˝ Nosema group, can parasitized in Papilio machaon Linnaeus, which provides a wider host range for Nosema.

Ultrastructural characterization of Acarispora falculifera n.gen., n.sp., a new microsporidium (Opisthokonta: Chytridiopsida) from the feather mite Falculifer rostratus (Astigmata: Pterolichoidea)

Only about 20 species of microsporidia have been described from mites. All except one species produce typical spores with a long polar filament and a polaroplast. This paper is the first study of an atypical microsporidium infection in a feather mite (Falculifer rostratus). The infection of the pigeon feather mite is restricted to the colon epithelium where it leads to hypertrophy of the concerned cells. During sporogony, a multinucleate plasmodial aggregate is formed within a sporont (endogenous sporogony resulting in a polysporophorous vesicle). The cisterns delimiting the single sporoblasts later form the spore walls. Sporogonial stages are in direct contact to the host cell cytoplasm. Merogonial stages were not present. Spores are tiny (3.6 μm × 2.6 μm), broad oval in form and monokaryotic. The spore wall of mature spores consists of a three-layered endospore and a thin, electron-dense, wavy exospore. The polar filament is anisofilar and completely coiled in 3-4 turns. In cross-sections, it has a star-like appearance because the electron-dense core forms rounded compartments of lucent material at its surface. In superficial sections, this results in a honeycomb-like pattern. A polaroplast is missing. The polar filament arises subapically at a polar sac that lacks an internal anchoring disk. These atypical spore structures clearly classify the species from the feather mite as a member of the order Chytridiopsida. It could not be clearly affiliated to one of the known genera, so we created a new genus, Acarispora, with the species A. falculifera.