Hypothesis of parasitic stress in parasitized insects caused by microsporidia

Pathological alterations being similar to those that can be seen while hormonal dysbalance, particularly the increase of juvenile hormone (JH) titre, is one of the consequences of microsporidian infections. Though the increase of JH in insects infected with microsporidia has not been shown directly, there are many indirect proofs of this. It has been believed that JH is produced by microsporidia. But this has not been shown for microsporidia or for other endoparasites. In this article we want to propose another hypothesis. We suppose that during microsporidiosis the following events develop: exhaustion of host nutrition stores and other destructive consequences of microsporidian dwelling in host cells lead to the decrease of host biosynthetical and reparation activity in the infected cells and then to destructive alterations that can be seen by electro-microscopic methods. The infected cells are stressed and then the typical answer for many physiological stresses follows. Secretion of prothoracicotropic hormone by brain neurosecretory cells is inhibited and as a result the production and release of ecdysone is also inhibited and ecdysteroid titre decreases. The activity of JH-esterases is decreased and as a result the JH titre is increased. If microsporidian infection causes the stress in the host cells, the endocrine system will undoubtedly answer to this stress and this answer will definitely be the same as for all other stresses. Thus, in any case JH titre will be increased in infected insects independently of whether microsporidia produce JH or not. So, hormonal alterations in infected insects should be the consequence not of the microsporidian JH production but of the host response reaction to infection. We suppose that microsporidia do not differ from other parasites of insects and that they can not produce JH.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.     

Aquatic tetrasporoblastic microsporidia from caddis flies (Insecta, Trichoptera): characterisation, phylogeny and taxonomic reevaluation of the genera Episeptum Larsson, 1986, Pyrotheca Hesse, 1935 and Cougourdella Hesse, 1935

Seven microsporidian species infecting caddis fly larvae, corresponding to conventional genera Episeptum, Pyrotheca and Cougourdella were studied using light and electron microscopy. Parts of their small subunit, ITS and large subunit ribosomal RNA genes were sequenced and compared with sequences of rDNA obtained from syntype slides of Cougourdella polycentropi Weiser 1965 and Pyrotheca sp. from Hydropsyche pellucidula. All studied caddis fly microsporidia form a closely related group. Their developmental stages in trichopteran hosts are restricted to fat body cells and oenocytes and have isolated nuclei. In late merogony, uninucleate meronts and binucleate plasmodia are formed. In sporogony a sporogonial plasmodium with four nuclei gives rise by rosette-like budding to four sporoblasts within a non-persistent sporophorous vesicle. Sporoblasts mature into pyriform to lageniform spores. The shape and size of spores, the number of polar filament coils, the structure of the polaroplast and of the exospore, together with morphometric characters present a set of markers unique for respective species. Four new species are established. The new genus Paraepiseptum is proposed to replace the tetrasporoblastic Pyrotheca and Cougourdella species from caddis flies. The genus Episeptum is redefined. Field and laboratory examinations as well as the phylogenetic position within the aquatic clade of microsporidia suggest that the life cycle of trichopteran microsporidia probably involves an alternate (copepod?) host and (or) transovarial transmission.     

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae), in Vietnam

In order to evaluate the potential application of microsporidia as a microbial control agent against lepidopteran insect pests, microsporidian infection in a field population of the common cutworm, Spodoptera litura (Fabricius), was surveyed in vegetable crop fields in Can Tho City, Vietnam, in March 2007. The infection rate of microsporidia was 46.7% (99/212 individuals) in adult S. litura, and 16 samples of infected adults were used to characterize the microsporidia at the molecular level. Analysis of the small subunit ribosomal RNA (SSU rRNA) sequences indicated that microsporidian strains isolated from S. litura were closely related to Nosema bombycis from the silkworm, Bombyx mori (Linnaeus); however, phylogenetic analysis based on genome profiling produced a different result from the SSU rRNA sequences. Temperature gradient gel electrophoresis profiles of 12 microsporidian strains from S. litura were closely related to N. bombycis strains, while the profiles of three microsporidian strains formed a different cluster. The Vietnamese strains did not form a single group, but were classified into at least three groups. These results suggested that the microsporidia isolated from S. litura in the Mekong Delta, Vietnam, are genetically diverse.     

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.     

The Genome of Spraguea lophii and the Basis of Host-Microsporidian Interactions

Microsporidia are obligate intracellular parasites with the smallest known eukaryotic genomes. Although they are increasingly recognized as economically and medically important parasites, the molecular basis of microsporidian pathogenicity is almost completely unknown and no genetic manipulation system is currently available. The fish-infecting microsporidian Spraguea lophii shows one of the most striking host cell manipulations known for these parasites, converting host nervous tissue into swollen spore factories known as xenomas. In order to investigate the basis of these interactions between microsporidian and host, we sequenced and analyzed the S. lophii genome. Although, like other microsporidia, S. lophii has lost many of the protein families typical of model eukaryotes, we identified a number of gene family expansions including a family of leucine-rich repeat proteins that may represent pathogenicity factors. Building on our comparative genomic analyses, we exploited the large numbers of spores that can be obtained from xenomas to identify potential effector proteins experimentally. We used complex-mix proteomics to identify proteins released by the parasite upon germination, resulting in the first experimental isolation of putative secreted effector proteins in a microsporidian. Many of these proteins are not related to characterized pathogenicity factors or indeed any other sequences from outside the Microsporidia. However, two of the secreted proteins are members of a family of RICIN B-lectin-like proteins broadly conserved across the phylum. These proteins form syntenic clusters arising from tandem duplications in several microsporidian genomes and may represent a novel family of conserved effector proteins. These computational and experimental analyses establish S. lophii as an attractive model system for understanding the evolution of host-parasite interactions in microsporidia and suggest an important role for lineage-specific innovations and fast evolving proteins in the evolution of the parasitic microsporidian lifecycle.     

Observations on Ichthyosporidium giganteum (Microsporida) with particular reference to the host-parasite relations during merogony

Connective tissue cells, particularly fibroblasts, of the fish Leiostomus xanthurus Lacépède respond to the invading microsporidian parasite Ichthyosporidium sp. [assumed to be identical with Ichthyosporidium giganteum (Thélohan)] by proliferating themselves, coalescing into a syncytium, synthesizing copious amounts of cytoplasm around the parasites, and walling off the parasitized islands of cytoplasm with fibrous capsules. The resulting cysts are xenoparasitic complexes of the syncytial xenoma type, clearly different from the cell hypertrophy tumor (xenoma sensu Weissenberg) exemplified by the Glugea cyst. These findings involve a new concept of the structure and host-parasite relations of Ichthyosporidium. Formerly, the parasitized masses of cytoplasm were interpreted as extracellular plasmodial stages of the parasite (stages uncharacteristic of the microsporidia), while the parasites themselves were interpreted as nuclei of the "plasmodia." Actually, the parasite undergoes merogony in parasitophorous vacuoles which coalesce before sporogony begins. The nuclei of the mermonts are very small chromatin granules, becoming transformed into large basophilic diplokarya of the sporonts. Sporulation is diplokaryotic throughout, the diplokarya becoming reduced in size through 2 steps during sporogony.     

Fish microsporidia: fine structural diversity and phylogeny

Structural diversity of fish microsporidian life cycle stages and of the host-parasite interface is reviewed. In the infected cell of the fish host, microsporidia may either cause serious degradation of the cytoplasm and demise of the cell, or they may elicit host cell hypertrophy, producing a parasite-hypertrophic host cell complex, the xenoma. The structure of the xenoma and of its cell wall may differ according to the genus of the parasite, and seems to express properties of the parasite rather than those of the host. In merogony, the parasite cell surface interacts with the host cell in diverse ways, the most conspicuous being the production of thick envelopes of different types. Sporogony stages reveal different types of walls or membranes encasing the sporoblasts and later the spores and these envelopes may be of host or parasite origin. Nucleospora differs from all other fish microsporidia by its unique process of sporogony. Except for the formation of conspicuous xenomas, there are no essentially different structures in fish-infecting microsporidia compared with microsporidia from other hosts. Although the structures associated with the development of fish microsporidia cannot be attributed importance in tracing the phylogeny, they are relevant for practical determination and assessing the relation to the host. The possibility of the existence of an intermediate host is discussed. Higher-level classification of Microsporidia is briefly discussed and structure and evolutionary rates in microsporidian rDNA are reviewed. Discussion of rDNA molecular phylogeny of fish-infecting microsporidia is followed by classification of these parasites. Most form a rather cohesive clade. Outside this clade is the genus Nucleospora, separated at least at the level of Order. Within the main clade, however, there are six species infecting hosts other than fish. Based on data available for analysis, a tentative classification of fish-infecting microsporidia into five groups is proposed. Morphologically defined groups represent families, others are referred to as clades. Group 1, represented by family Pleistophoridae, includes Pleistophora, Ovipleistophora and Heterosporis; Vavraia and Trachipleistophora infect non-fish hosts. Group 2, represented by family Glugeidae, is restricted to genus Glugea and Tuzetia weidneri from crustaceans. Group 3 comprises three clades: Loma and a hyperparasitic microsporidian from a myxosporean; Ichthyosporidium and Pseudoloma clade and the Loma acerinae clade. For the latter species a new genus has to be established. Group 4 contains two families, Spragueidae with the genus Spraguea and Tetramicridae with genera Microgemma and Tetramicra, and the Kabatana and Microsporidium seriolae clade. Group 5 is represented by the family Enterocytozoonidae with the genus Nucleospora and mammal-infecting genus Enterocytozoon.     

甜菜夜蛾微孢子虫研究:Ⅲ.超微结构与致病机理

从甜菜夜蛾幼虫体内首次分离到一种侵染寄主脂肪体、马氏管和中肠的微孢子虫,该微孢子虫对甜菜夜蛾、菜青虫和棉铃虫幼虫有较强的致病力,并可经卵垂直传播;水平传播主要借助于病虫的粪便及虫尸。用电镜观察了该微孢子虫感染甜菜夜蛾幼虫后的超微结构变化。结果表明：寄主细胞被感染后,细胞核膨大并变形,由正常的圆球形被挤压成长条状,但核膜及核周间隙没有发生变化;线粒体体积变小,嵴增宽,嵴的排列方向发生改变,双层膜部分     

Encephalitozoon cuniculi (Microspora): characterization of a phospholipid metabolic pathway potentially linked to therapeutics

Phospholipid metabolism of the microsporidian Encephalitozoon cuniculi, an obligate intracellular parasite, has been investigated. Labeled precursor incorporation experiments have shown that phosphatidylserine decarboxylase and phosphatidylethanolamine N-methyltransferase are more active in cells infected by E. cuniculi than in uninfected cells. In contrast, no difference was observed in the activity of Kennedy pathway's enzymes, the mammalian pathway. This suggests the occurrence in microsporidia of a bacteria- and fungi-typical pathway for phospholipid synthesis, which is supported by the identification of two genes implicated in this pathway, the cds gene encoding the key enzyme CDP-diacylglycerol synthase (E.C. 2.7.7.41) and the pss gene for CDP-alcohol phosphatidyltransferase. The pss gene could encode phosphatidylserine synthase (E.C. 2.7.8.8.), which catalyses the de novo synthesis of phosphatidylserine in bacteria and fungi. The complete CDP-diacylglycerol synthase messenger has been isolated and shows very short 5' and 3' untranslated regions. This is strong evidence for the functionality of a metabolic pathway which could be a potential target against microsporidia which infect humans.     

Congruent evidence from alpha-tubulin and beta-tubulin gene phylogenies for a zygomycete origin of microsporidia

The origin of microsporidia and the evolutionary relationships among the major lineages of fungi have been examined by molecular phylogeny using alpha-tubulin and beta-tubulin. Chytrids, basidiomycetes, ascomycetes, and microsporidia were all recovered with high support, and the zygomycetes were consistently paraphyletic. The microsporidia were found to branch within zygomycetes, and showed relationships with members of the Entomophthorales and Zoopagales. This provides support for the microsporidia having evolved from within the fungi, however, the tubulin genes are difficult to interpret unambiguously since fungal and microsporidian tubulins are very divergent. Rapid evolutionary rates a characteristic of practically all microsporidian genes studied, so determining their evolutionary history will never be completely free of such difficulties. While the tubulin phylogenies do not provide a decisive conclusion, they do further narrow the probable origin of microsporidia to a zygomycete-like ancestor.     

Viral alkaline nuclease in intranuclear dense bodies induced by herpes simplex infection

The distribution of the herpes simplex virus alkaline nuclease (HSV DNase) in rabbit fibroblast cells infected with HSV type 1 or 2 (HSV-1 or HSV-2) has been examined with the aid of immunocytochemical techniques using gold particles as markers. HSV DNase was found to be accumulated within infected nuclei as early as 2.5 hr post-infection. Labeled antibody to HSV DNase subsequently increased in all nuclei after 7 hr and 17 hr. At all times post-infection the virus induced nuclear dense bodies were always the most intensely labeled structures. The association of HSV DNase with nucleoprotein containing structures was readily dissociated by hypotonic shock and detergent treatment, but its association with the dense bodies was not disrupted. Nucleolar 100 kDa protein was found to be simultaneously present in the dense bodies. This suggests that HSV DNase might interfere with ribosomal RNA synthesis and play a role in the degradation of the host-cell metabolism.     

Mechanisms of nascent fiber formation during avian skeletal muscle hypertrophy

This study examined two putative mechanisms of new fiber formation in postnatal skeletal muscle, namely longitudinal fragmentation of existing fibers and de novo formation. The relative contributions of these two mechanisms to fiber formation in hypertrophying anterior latissimus dorsi (ALD) muscle were assessed by quantitative analysis of their nuclear populations. Muscle hypertrophy was induced by wing-weighting for 1 week. All nuclei formed during the weighting period were labeled by continuous infusion of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, and embryonic-like fibers were identified using an antibody to ventricular-like embryonic (V-EMB) myosin. The number of BrdU-labeled and unlabeled nuclei in V-EMB-positive fibers were counted. Wing-weighting resulted in significant muscle enlargement and the appearance of many V-EMB+ fibers. The majority of V-EMB+ fibers were completely independent of mature fibers and had a nuclear density characteristics of developing fibers. Furthermore, nearly 100% of the nuclei in independent V-EMB+ fibers were labeled. These findings strongly suggest that most V-EMB+ fibers were nascent fibers formed de novo during the weighting period by satellite cell activation and fusion. Nascent fibers were found primarily in the space between fascicles where they formed a complex anastomosing network of fibers running at angles to one another. Although wing-weighting induced an increase in the number of branched fibers, there was no evidence that V-EMB+ fibers were formed by longitudinal fragmentation. The location of newly formed fibers in wing-weighted and regenerating ALD muscle was compared to determine whether satellite cells in the ALD muscle were unusual in that, if stimulated to divide, they would form fibers in the inter- and intrafascicular space. In contrast to wing-weighted muscle, nascent fibers were always found closely associated with necrotic fibers. These results suggest that wing-weighting is not simply another model of regeneration, but rather produces a unique environment which induces satellite cell migration and subsequent fiber formation in the interfascicular space. De novo fiber formation is apparently the principal mechanism for the hyperplasia reported to occur in the ALD muscle undergoing hypertrophy induced by wing-weighting.     

Histopathological survey of pathogens and commensals of white-clawed crayfish (Austropotamobius pallipes) in England and Wales

A histopathological survey of white-clawed crayfish (Austropotamobius pallipes) from six rivers in England and Wales was conducted between November 2007 and September 2011. A. pallipes bacilliform virus (ApBV) was present in four samples; infected crayfish showed pathological responses ranging from mild hypertrophy of infected nuclei and emargination of chromatin through to loss of architecture of the hepatopancreatic cells. Crayfish were found to be hosts to two different ciliate species, mites, nematodes, digeneans and the microsporidian Thelohania contejeani. The variation in disease presence between populations highlights the importance of conducting appropriate surveys of native crayfish prior to movement of animals to refugia sites for relocation, restocking or breeding programmes.     

Nosema ceranae Escapes Fumagillin Control in Honey Bees

Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2) in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.     

A microsporidium,Nosema pilicornis sp. n., of the purslane sawfly,Schizocerella pilicornis

A new microsporidian species, Nosema pilicornis, which infects the purslane sawfly, Schizocerella pilicornis, is described. This microsporidium infects most body tissues of the host. N. pilicornis was compared to other microsporidian species infecting Hymenoptera and to a group of similar microsporidia infecting Lepidoptera. N. pilicornis could be distinguished from all other microsporidian species on the basis of host range and ultrastructural characteristics of the spore. Spores were oval, containing 11 to 12 polar filament coils, and the polar filament had an angle of tilt of about 80°. N. pilicornis infected lepidopteran larvae, but only when heavy spore dosages were fed to early larval instars. S. pilicornis is a good but sporadic biological control agent of common purslane, Portulaca oleracea, a pernicious weed of vegetable, ornamental, and orchard crops. N. pilicornis, which is transovarially transmitted and causes high mortality in infected larvae, affects the performance of S. pilicornis as a biological control agent.     

Plasma Membrane-Located Purine Nucleotide Transport Proteins Are Key Components for Host Exploitation by Microsporidian Intracellular Parasites

Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.     

A Broad Distribution of the Alternative Oxidase in Microsporidian Parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.     

Cytological variation and pathogenicity of the bumble bee parasite Nosema bombi (Microspora, Nosematidae)

In three field seasons, 2003-2005, bumble bees were collected in southern Sweden and eastern Denmark in search of microsporidian parasites. Of the 16 bumble bee species studied, microsporidia were found in Bombus hortorum, Bombus hypnorum, Bombus lapidarius, Bombus lucorum, Bombus pascuorum, Bombus pratorum, Bombus ruderarius, Bombus subterraneus and Bombus terrestris. Only one microsporidian species, Nosema bombi, was recorded. A microsporidium found in B. pratorum differed cytologically from microsporidia of the other host species. In the most frequently infected host, B. terrestris, the prevalence was 20.6%. Totally 1049 specimens were dissected. The light microscopic and ultrastructural cytology and pathology of N. bombi is described with focus on the variation recorded. Variation was especially prominent in the shape, size and coupling of spores, and in the length and arrangement of the polar filament. In four host species microsporidian infection was restricted to peripheral fat cells.