Enterocytozoon salmonis n. sp.: an intranuclear microsporidium from salmonid fish

The developmental stages of a recently described microsporidian from the nucleus of hematopoietic cells of salmonid fish were found to be unique among the Microsporida. All observed stages, including meronts, sporonts, and spores were in direct contact with the host cell nucleus (principally hematopoietic cells) of chinook salmon (Oncorhynchus tshawytscha). There is no parasitophorous vacuole and sporogony does not involve formation of a pansporoblastic membrane as with other members of the suborder Apansporoblastina. The extrusion apparatus differentiates prior to division of sporogonial plasmodia. The spores are ovoid (1 x 2 microns) and uninucleate, and possess a coiled polar tube with 8-12 turns. Developmental stages of the salmonid microsporidian are similar to those described for Enterocytozoon bieneusi as found in the intestinal mucosa of human AIDS patients. However, the intranuclear development, different cell types, and host infected clearly separate the salmonid and human parasites. Accordingly, the intranuclear parasite of salmonids is given the name Enterocytozoon salmonis n. sp. within the suborder Apansporoblastina.     

Amazonspora hassar n. gen. and n. sp. (phylum Microsporidia,fam. Glugeidae), a parasite of the Amazonian teleost Hassar orestis (fam. Doradidae)

We describe the microsporidian Amazonspora hassar n. gen., n. sp. from the gill xenomas of the teleost Hassar orestis (Doradidae) collected in the estuarine region of the Amazon River. The parasite appeared as a small whitish xenoma located in the gill filaments near the blood vessels. Each xenoma consisted of a single hypertrophic host cell (HHC) in the cytoplasm of which the microsporidian developed and proliferated. The xenoma wall was composed of up to approximately 22 juxtaposed crossed layers of collagen fibers. The plasmalemma of the HHC presented numerous anastomosed, microvilli-like structures projecting outward through the 1-3 first internal layers of the collagen fibrils. The parasite was in direct contact with host cell cytoplasm in all stages of the cycle (merogony and sporogony). Sporogony appears to divide by plasmotomy, giving rise to 4 uninucleate sporoblasts, which develop into uninucleate spores. The ellipsoidal spores measured 2.69 +/- 0.45 x 1.78 +/- 0.18 microm, and the wall measured approximately 75 nm. The anchoring disk of the polar filament was subterminal, being shifted laterally from the anterior pole. The polar filament was arranged into 7-8 coils in a single layer in the posterior half of the spore, surrounding the posterior vacuole. The polaroplast surrounded the uncoiled portion of the polar filament, and it was exclusively lamellar. The spores and different life-cycle stages were intermingled within the cytoplasm of the HHC, surrounding the central hypertrophic deeply branched nucleus. The ultrastructural morphology of this microsporidian parasite suggests the erection of a new genus and species.     

An Intranuclear Microsporidium Associated with Acute Anemia in the Chinook Salmon,Oncorhynchus tshawytscha1

An intranuclear microsporidium is described from hemoblastic cells of the chinook salmon, Oncorhynchus tshawytscha. The infection is associated with an acute anemia in the fish. Up to 47% of the hemoblast nuclei were infected in anemic fish. The organisms, found only in spleen and kidney tissues, were 1–2 μm in diameter and consisted of vegetative and early sporulation forms. This microsporidium differs from known species which parasitize fish in its tissue location; however, the absence of mature spores and other life cycle stages precludes determination of its precise taxonomic identity.     

Unikaryon phyllotretae sp. n. (Protista,Microspora), a new microsporidian pathogen of Phyllotreta undulata (Coleoptera; Chrysomelidae)

The microsporidium Unikaryon phyllotretae sp. n., a new pathogen of Phyllotreta undulata, is described based on light microscopic and ultrastructural characteristics. Microscopic examination of parasitized individuals revealed two types of spores. The majority of the spores were of the first type, which are oval and measured 2.74±0.17×1.93±0.17 μm when fresh. Fresh spores of the second type (very rare) are elongated and measured 4.39±0.18×1.61±0.20 μm. All life stages have single nuclei. Sporogony ends with uninucleate single sporoblasts and spores. The spores were only observed in Malpighian tubules. The isofilar polar filament of the parasite has six to eight coils, and a well-developed polaroplast was of the lamellated type, with closely packed anterior lamellae and loosely packed posterior lamellae.     

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

A fish-infecting Microsporidia Potaspora morhaphis n. gen., n. sp. found adherent to the wall of the coelomic cavity of the freshwater fish, Potamorhaphis guianensis, from lower Amazon River is described, based on light microscope and ultrastructural characteristics. This microsporidian forms whitish xenomas distinguished by the numerous filiform and anastomosed microvilli. The xenoma was completely filled by several developmental stages. In all of these stages, the nuclei are monokaryotic and develop in direct contact with host cell cytoplasm. The merogonial plasmodium divides by binary fission and the disporoblastic pyriform spores of sporont origin measure 2.8+/-0.3 x 1.5+/-0.2 microm. In mature spores the polar filament was arranged into 9-10 coils in 2 layers. The polaroplast had 2 distinct regions around the manubrium and an electron-dense globule was observed. The small subunit, intergenic space and partial large subunit rRNA gene were sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Kabatana, Microgemma, Spraguea and Tetramicra. The ultrastructural morphology of the xenoma, and the developmental stages including the spores of this microsporidian parasite, as well as the phylogenetic analysis, suggest the erection of a new genus and species.     

Description of Chytridiopsis Trichopterae N. Sp. (Microspora, Chytridiopsidae), A Microsporidian Parasite of the Caddis Fly Polycentropus Flavomaculatus (Trichoptera, Polycentropodidae), With Comments On Relationships Between the Families Chytridiopsidae and Metchnikovellidae

ABSTRACT. The microsporidium Chytridiopsis trichopterae n. sp., a parasite of the midgut epithelium of larvae of the caddis fly Polycentropus flavomaculatus found in southern Sweden, is described based on light microscopic and ultrastructural characteristics. All life cycle stages have isolated nuclei. Merogonial reproduction was not observed. the sporogony comprises two sequences: one with free spores in parasitophorous vacuoles, the other in spherical, 5.6-6.8 μm wide, sporophorous vesicles which lie in the cytoplasm. the free sporogony yields more than 20 spores per sporont. the vesicle-bound sporogony produces 8, 12 or 16 spores. the envelope of the sporophorous vesicle is about 82 nm thick and layered. the internal layer is the plasma membrane of the sporont; the surface layer is electron dense with regularly arranged translucent components. Both spore types are spherical. They have an ～ 35-nm thick spore wall, with a plasma membrane, an electron-lucent endospore, and an ～ 14-nm thick electron-dense exospore. the polar sac is cup-like and lacks a layered anchoring disc. the polar filament is arranged in two to three isofilar coils in the half of the spore opposite the nucleus. the coupling between the polar sac and the polar filament is characteristic. the surface of the polar filament is covered with regularly arranged membraneous chambers resembling a honeycomb. There is no polaroplast of traditional type. the cytoplasm lacks polyribosomes. the nucleus has a prominent, wide nucleolus. the two spore types have identical construction, but differ in dimensions and electron density. Free living spores are about 3.2 μm wide, the diameter of the polar filament proper is 102-187 nm, the chambers of the honeycomb are 70-85 nm high, and the polar sac is up to 425 nm wide. Living spores in the vesicle-bound sporogony are about 2.1 μm wide, the polar filament measures 69-102 nm, the chambers of the honeycomb are about 45 nm high, and these spores are more electron dense. Comparisons of cytology (especially the construction of the spore wall and the polar filament and associated structures) and life cycles reveal prominent differences among the Chytridiopsis-like microsporidia, and close relationships between the families Chytridiopsidae and Metchnikovellidae.     

Ultrastructure of Triangulamyxa amazonica n. gen. and n. sp. (Myxozoa, Myxosporea), a parasite of the Amazonian freshwater fish, Sphoeroides testudineus (Teleostei, Tetrodontidae)

Triangulamyxa amazonica n. gen. and n. sp. (Myxozoa, Ortholineidae), found in the lumen of the intestine of the freshwater fish Sphoeroides testudineus, is described. The fish were collected from the Amazon River near the city of Algodoal, State of the Pará, Brazil. Numerous irregular plasmodia containing different stages of sporogony, including spores, were observed. The plasmodia were lying free in the lumen or had slender pseudopodia-like cytoplasmic processes in contact with intestinal epithelial cells with microvilli projections. Spores, which are equilaterally triangular in valvar view with rounded pointed ends and ellipsoidal in transverse section, are 8.5 μm long, 7.6 μm wide, and 3.8 μm thick. The anterior end of the spores contains two equal drop-shaped polar capsules measuring 2.6 μm in length, each having an isofilar polar filament with 5–6 turns. The characteristics of the spore shape, the spore wall structure and its ridge organization, the plasmodial characteristics and the identity of the host suggest that the parasite is a new genus and species, which is herein designated T. amazonica.     

Life history and virulence are linked in the ectoparasitic salmon louse Lepeophtheirus salmonis

Models of virulence evolution for horizontally transmitted parasites often assume that transmission rate (the probability that an infected host infects a susceptible host) and virulence (the increase in host mortality due to infection) are positively correlated, because higher rates of production of propagules may cause more damages to the host. However, empirical support for this assumption is scant and limited to microparasites. To fill this gap, we explored the relationships between parasite life history and virulence in the salmon louse, Lepeophtheirus salmonis, a horizontally transmitted copepod ectoparasite on Atlantic salmon Salmo salar. In the laboratory, we infected juvenile salmon hosts with equal doses of infective L. salmonis larvae and monitored parasite age at first reproduction, parasite fecundity, area of damage caused on the skin of the host, and host weight and length gain. We found that earlier onset of parasite reproduction was associated with higher parasite fecundity. Moreover, higher parasite fecundity (a proxy for transmission rate, as infection probability increases with higher numbers of parasite larvae released to the water) was associated with lower host weight gain (correlated with lower survival in juvenile salmon), supporting the presence of a virulence–transmission trade‐off. Our results are relevant in the context of increasing intensive farming, where frequent anti‐parasite drug use and increased host density may have selected for faster production of parasite transmission stages, via earlier reproduction and increased early fecundity. Our study highlights that salmon lice, therefore, are a good model for studying how human activity may affect the evolution of parasite virulence.     

Surveillance of the Sensitivity towards Antiparasitic Bath-Treatments in the Salmon Louse (Lepeophtheirus salmonis)

The evolution of drug resistant parasitic sea lice is of major concern to the salmon farming industry worldwide and challenges sustainable growth of this enterprise. To assess current status and development of L. salmonis sensitivity towards different pesticides used for parasite control in Norwegian salmon farming, a national surveillance programme was implemented in 2013. The programme aims to summarize data on the use of different pesticides applied to control L. salmonis and to test L. salmonis sensitivity to different pesticides in farms along the Norwegian coast. Here we analyse two years of test-data from biological assays designed to detect sensitivity-levels towards the pesticides azamethiphos and deltamethrin, both among the most common pesticides used in bath-treatments of farmed salmon in Norway in later years. The focus of the analysis is on how different variables predict the binomial outcome of the bioassay tests, being whether L. salmonis are immobilized/die or survive pesticide exposure. We found that local kernel densities of bath treatments, along with a spatial geographic index of test-farm locations, were significant predictors of the binomial outcome of the tests. Furthermore, the probability of L. salmonis being immobilized/dead after test-exposure was reduced by odds-ratios of 0.60 (95% CI: 0.42–0.86) for 2014 compared to 2013 and 0.39 (95% CI: 0.36–0.42) for low concentration compared to high concentration exposure. There were also significant but more marginal effects of parasite gender and developmental stage, and a relatively large random effect of test-farm. We conclude that the present data support an association between local intensities of bath treatments along the coast and the outcome of bioassay tests where salmon lice are exposed to azamethiphos or deltamethrin. Furthermore, there is a predictable structure of L. salmonis phenotypes along the coast in the data, characterized by high susceptibility to pesticides in the far north and far south, but low susceptibility in mid Norway. The study emphasizes the need to address local susceptibility to pesticides and the need for restrictive use of pesticides to preserve treatment efficacy.     

Morphology,Molecular Phylogeny,and Ecology of Binucleata daphniae n. g., n. sp. (Fungi: Microsporidia), a Parasite of Daphnia magna Straus, 1820 (Crustacea: Branchiopoda)

ABSTRACT. We describe a new microsporidian species Binucleata daphniae, n. g., n. sp., that infects the integument cells lining the hemocoele cavity of the carapace and the postabdomen of the cladoceran Daphnia magna Straus. Infected cells filled with spores accumulate as large clusters in the carapace cavity and heavily infected hosts are detected by their opaque appearance. Despite the parasite's presence, infected Daphnia grow and molt, but have a reduced fecundity. During the parasite's life cycle, chain‐like meronts with isolated nuclei are formed, giving rise to binucleate presporonts, the most frequently observed, characteristic developmental stage. In sporogony, the nuclei of the presporont separate, divide, and eight spores enclosed in a thin‐walled sporophorous vesicle are formed. Spores are 4.9 × 2.5 μm in size (fresh) and have an anisofilar polar filament with eight coils. DNA sequence analysis places B. daphniae in a clade of microsporidians that parasitize crustaceans and mosquitoes and have assumed complex life cycles. Binucleata daphniae, however, has a simple and direct life cycle and can be transferred to naïve hosts and maintained as persistent infections in populations of its host D. magna. We propose that B. daphniae has simplified its life cycle by losing its secondary host, rendering it unique in this clade.     

Ultrastructural Study and Description of Ovavesicula popilliae N. G., N. Sp. (Microsporida: Pleistophoridae) from the Japanese Beetle,Popillia japonica (Coleoptera: Scarabaeidae)1

A new genus and species of microsporidia, Ovavesicula popilliae n. g., n. sp., is described from the Japanese beetle, Popillia japonica, on the basis of studies by light and electron microscopy. Parasite development primarily occurs within the Malpighian tubules of larvae, and spores are formed in a sporophorous vesicle. Meronts have diplokaryotic nuclei, develop in direct contact with the host cell cytoplasm, and divide by binary fission. Sporonts have unpaired nuclei, develop within a thick sporophorous vesicle, and undergo synchronous nuclear divisions producing plasmodia with 2, 4, 8, 16, and 32 nuclei. Cytokinesis of sporogonial plasmodia does not occur until karyokinesis is complete with 32 nuclei. Intact sporophorous vesicles are ovoid, containing numerous secretory products, and are surrounded by a persistent two-layered wall. The uninucleate spores are regularly formed in groups of 32, and the polar tube in each has six coils.     

A new parvicapsulid (Myxosporea) species in adult pink salmon, Oncorhynchus gorbuscha, from the Quinsam River, British Columbia, Canada

Myxospores consistent with species of Parvicapsula were observed in kidney of 15 of 95 (15.8%) adult pink salmon, Oncorhynchus gorbuscha, collected from the Quinsam River, British Columbia, Canada. The spores were elongate and curved with unequal valves, and 2 spherical-to-subspherical polar capsules within a highly refractile capsular region. The spores were unlike those of P. minibicornis found in nearby populations of Pacific salmon, Oncorhynchus spp. The spore dimensions were similar to those of Parvicapsula pseudobranchicola from Atlantic salmon, Salmo salar, in Norway, and the spores seemed similar to an undescribed Parvicapsula sp. from several Oncorhynchus spp. in Puget Sound, Washington. A sequence of 1,480 base pairs (bp) of the small subunit ribosomal RNA gene (SSU rDNA) of the parasite from pink salmon was most similar to, but distinct from, that of other Parvicapsula spp. The parasite is described as a new species, Parvicapsula kabatai n. sp. Polymerase chain reactions amplified a 158-bp sequence, unique to P. kabatai n. sp., from 22 of 93 (23.7%) adult pink salmon kidney samples, from 3 of 3 juvenile pink salmon collected in the ocean 125 km north of the Quinsam River, and from 2 of 5 archival coho salmon, Oncorhynchus kisutch, samples from Puget Sound. The parasite occurs within the lumen and epithelium of renal tubules and ducts, and within the renal interstitium. Concurrent infections with extrasporogonic stages of the myxosporean CKX, the microsporidian Loma salmonae, and a Myxidium sp. also were observed in the adult pink salmon.     

Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus

Only 1 genus (Nucleospora) within 1 family (Enterocytozoonidae) of the Microsporidia contains species that are parasitic within the nuclei of their host cells; to date, all described intranuclear Nucleospora spp. parasitise fish. This study describes the first intranuclear microsporidian parasite of an invertebrate, the European edible crab Cancer pagurus L. (Decapoda: Cancridae). Infected crabs displayed no obvious external signs, and maximum apparent prevalence of infection within a monthly sample was 3.45%. Infected hepatopancreatic tubules were characterised by varying numbers of hypertrophic and eosinophilic nuclei within epithelial cells. Parasite stages appeared as eosinophilic granular accumulations causing margination of host chromatin. In advanced cases, the tubule epithelia degenerated, with parasites and sloughed epithelial cells appearing in tubule lumens. All life stages of the parasite were observed within host nuclei. Uninucleate meronts were not detected, although binucleate stages were observed. Multinucleate plasmodia (sporogonal plasmodia) contained up to 22 nuclei in section, and late-stage plasmodia contained multiple copies of apparatus resembling the polar filament and anchoring disk, apparently associated with individual plasmodial nuclei. As such, aggregation and early assembly of sporoblast components took place within the intact sporogonial plasmodium, a feature unique to the Enterocytozoonidae. Liberation of sporoblasts from plasmodia or the presence of liberated sporoblasts was not observed in this study. However, large numbers of maturing and mature spores (measuring 1.3 +/- 0.02 x 0.7 +/- 0.01 microm) were frequently observed in direct contact with the host nucleoplasm. Considering the shared features of this parasite with microsporidians of the family Enterocytozoonidae, and the unique presence of this parasite within the nucleoplasm of decapod crustacean hepatopancreatocytes, this parasite (Enterospora canceri) is proposed as the type species of a new genus (Enterospora) of microsporidian. Molecular taxonomic work is now required, comparing Enterospora to Enterocytozoon and Nucleospora, the 2 other genera within the Enterocytozoonidae.     

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7 × 1.1 μm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10 nm) and an electron-dense exospore (2 nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848 bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.     

Plasmodium (Huffia) hermani sp. n. from wild turkeys (Meleagris gallopavo) in Florida*

SYNOPSIS. Plasmodium (Huffia) hermani sp. n. is described from wild turkeys (Meleagris gallopavo Linnaeus) in Florida. It produces rounded schizonts with 6–14 nuclei arranged peripherally as a rosette and elongate, slender gametocytes with irregular margins. Asexual stages parasitize all cells in the erythrocyte series and, in heavy infections, predominantly occur in erythroblasts and their precursors. Presence and degree of pigmentation vary with maturity of the host cell. Gametocytes occupy erythrocytes only, with pigment dispersed in black granules throughout the cytoplasm. Cells containing schizonts are often rounded and enlarged and those parasitized by gametocytes may be somewhat distorted in shape by lateral hypertrophy. Host cell nuclei may be displaced, but are not distorted, except slightly by pressure from the parasite. Plasmodium hermani differs from P. (Giovannolaia) durae by producing low level (> 6%), nonlethal parasitemias in turkey poults, an absence of phanerozoites in capillary endothelium of the brain and viscera, and inability to infect chicks. Plasmodium hermani is more like P. (Huffia) elongatum in gametocyte morphology, schizogony in all types of erythrocyte precursors, with gametocytes occurring in erythrocytes only, and concentration of schizonts in heavy infections in bone marrow and spleen. It differs from P. elongatum by its lack of infectivity to passeriform and anseriform hosts and by a strong immune response which develops in infected birds.     

Sphaerospora epinepheli n. sp. (Myxosporea: Sphaerosporidae) observed in grouper (Epinephelus malabaricus)

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus, in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6-22.9 microns (length) x 8.4-21.6 microns (width). Spores were 7.8-10.0 microns (length) x 12.3-14.5 microns (thickness), and 7.0-9.5 microns (width) with two spherical polar capsules of equal size measuring 2.9-4.4 microns in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.     

Ceratomyxa hungarica n. sp. and Chloromyxum proterorhini n. sp. (Myxozoa: Myxosporea) from the freshwater goby Proterorhinus marmoratus (Pallas)

When studying the parasite fauna of freshwater gobies Proterorhinus marmoratus collected from the reaches of the River Danube around Budapest, two species of Myxosporea were recovered, a renal and a gall-bladder form. Previously no myxosporeans had been reported from this fish species. The spores and pseudoplasmodia of the parasite described as Ceratomyxa hungarica n. sp. were found in the convoluted tubules of the kidney and in the cavity of Bowman's capsule. The pseudoplasmodia were loosely attached to the wall of the tubules, causing their distention. Within each pseudoplasmodium two spores were formed. In the case of Chloromyxum proterorhini n. sp. only spores floating freely in the contents of the gall-bladder were found. Since Ceratomyxa species are typically marine fish parasites, Proterorhinus marmoratus, a fish species which has adapted to freshwater, appears to have retained some of its marine myxosporean fauna.