Examining the Association of Rare Allelic Variants in Urate Transporters SLC22A11, SLC22A13, and SLC17A1 with Hyperuricemia and Gout.

Genetic variations in urate transporters play a significant role in determining human urate levels and have been implicated in developing hyperuricemia or gout. Polymorphism in the key urate transporters, such as ABCG2, URAT1, or GLUT9 was well-documented in the literature. Therefore in this study, our objective was to determine the frequency and effect of rare nonsynonymous allelic variants of SLC22A11, SLC22A13, and SLC17A1 on urate transport. In a cohort of 150 Czech patients with primary hyperuricemia and gout, we examined all coding regions and exon-intron boundaries of SLC22A11, SLC22A13, and SLC17A1 using PCR amplification and Sanger sequencing. For comparison, we used a control group consisting of 115 normouricemic subjects. To examine the effects of the rare allelic nonsynonymous variants on the expression, intracellular processing, and urate transporter protein function, we performed a functional characterization using the HEK293A cell line, immunoblotting, fluorescent microscopy, and site directed mutagenesis for preparing variants in vitro. Variants p.V202M (rs201209258), p.R343L (rs75933978), and p.P519L (rs144573306) were identified in the SLC22A11 gene (OAT4 transporter); variants p.R16H (rs72542450), and p.R102H (rs113229654) in the SLC22A13 gene (OAT10 transporter); and the p.W75C variant in the SLC17A1 gene (NPT1 transporter). All variants minimally affected protein levels and cytoplasmic/plasma membrane localization. The functional in vitro assay revealed that contrary to the native proteins, variants p.P519L in OAT4 (p ≤ 0.05), p.R16H in OAT10 (p ≤ 0.05), and p.W75C in the NPT1 transporter (p ≤ 0.01) significantly limited urate transport activity. Our findings contribute to a better understanding of (1) the risk of urate transporter-related hyperuricemia/gout and (2) uric acid handling in the kidneys.

Functional Characterization of Rare Variants in OAT1/SLC22A6 and OAT3/SLC22A8 Urate Transporters Identified in a Gout and Hyperuricemia Cohort.

The OAT1 (SLC22A6) and OAT3 (SLC22A8) urate transporters are located on the basolateral membrane of the proximal renal tubules, where they ensure the uptake of uric acid from the urine back into the body. In a cohort of 150 Czech patients with primary hyperuricemia and gout, we examined the coding regions of both genes using PCR amplification and Sanger sequencing. Variants p.P104L (rs11568627) and p.A190T (rs146282438) were identified in the gene for solute carrier family 22 member 6 (SLC22A6) and variants p.R149C (rs45566039), p.V448I (rs11568486) and p.R513Q (rs145474422) in the gene solute carrier family 22 member 8 (SLC22A8). We performed a functional study of these rare non-synonymous variants using the HEK293T cell line. We found that only p.R149C significantly reduced uric acid transport in vitro. Our results could deepen the understanding of uric acid handling in the kidneys and the molecular mechanism of uric acid transport by the OAT family of organic ion transporters.

Female calling, life cycle, and microstructures of the parasitic beetle Ripidius quadriceps Abeille de Perrin.

The precopulatory behaviour of the larviform females of Ripidius quadriceps Abeille de Perrin, 1872 is described. The calling posture of virgin females is documented. The cephalic morphology and microstructures are visualised using scanning electron microscopy, in particular the secretory pores in the cuticle of inflatable maxillary palps. An exhaustive overview of relevant publications revealed that the location of secretory pores on the head of females is unique within the order Coleoptera. Compared to other beetles with sedentary calling females, the calling phase of the short-lived and non-feeding female of Ripidius is exceptionally short. For bioassays, various traps using virgin females of Ripidius were tested. It is likely that the sedentary behaviour of the short-lived female combined with a unique morphology and priority for investing in reproduction is compensated for by the actively flying males with remarkably flabellate antennae. The life cycle of this species, including some of the exceptions recorded at the individual level, is discussed. Perspectives for a biological and morphological survey of this rarely collected western Palaearctic species are outlined. In addition, the calling behaviour, secretory sites and location of pheromone glands in females of Coleoptera producing long range pheromones is reviewed.

Establishment of a new microsporidian genus and species, Pseudoberwaldia daphniae (Microsporidia, Opisthosporidia), a common parasite of the Daphnia longispina complex in Europe.

Microsporidia are among the most common microparasites of cladocerans and have potentially significant impact on host populations. However, many of these pathogens are known only from molecular-based studies. We provide ultrastructural data supported by molecular phylogeny for a common microsporidium infecting the Daphnia longispina complex, important planktonic filter-feeders in reservoirs and ponds in the temperate Holarctic region. This parasite, previously characterized only by molecular means, infects adipose cells around the Daphnia midgut and eventually fills the centre of the host body with ovoid-shaped spores. A new microsporidian genus and species belonging to the Agglomeratidae superclade is described as Pseudoberwaldia daphniae gen. et sp. nov. Molecular data indicate its widespread presence in Central European reservoirs (reported as isolate "MIC1") but also in Swedish coastal rockpools ("Ängskärs-klubben"). The most closely related lineage was reported from a caddisfly larva; we thus speculate that this taxon may have an insect secondary host in its life cycle. Morphological characterization and differential diagnosis of most commonly encountered microsporidian taxa infecting hosts in the D. longispina complex in Europe opens new possibilities for studies of their ecological and evolutionary interactions.

Molecular and structural assessment of microsporidia infecting daphnids: The "obtusa-like" microsporidia, a branch of the monophyletic Agglomeratidae clade, with the establishment of a new genus Conglomerata.

Microsporidia (Opisthosporidia, Microsporidia) are frequent parasites of planktonic cladocerans, including Daphnia (Crustacea, Branchiopoda). Analysis of available molecular data (ITS region and partial ssu and lsu rDNA) of these parasites indicates that many microsporidia infecting daphnids have a common ancestor and represent a large clade, which splits during evolution into a number of well supported subclades. These subclades are cytologically different but may be most conveniently characterised by their specific ITS barcode. We have analysed one of these subclades and we describe a new microsporidian genus and species combination, and assemble a large group of structurally indistinguishable microsporidian parasites that infect adipose cells of their hosts and form pyriform spores of a certain type ("obtuse spores"). Obtuse spores are non-infectious by feeding to their crustacean hosts and it is plausible that microsporidia forming them actually are parasites of insects with aquatic larval stages, with an obligate two-host life cycle, analogous to the Amblyospora life cycle involving copepods and mosquitoes.

Protistology Conferences: The Beginnings. The First International Protozoology Conference (Prague 1961) and the Tribute to Otto Jírovec, its Spiritual Father.

Two events have helped to shape protozoology/protistology as a specific scientific discipline. The first such event was the creation of the Society of Protozoologists in the U.S. in 1947 (and of its Journal of Protozoology, first published in 1954), the second event was the First International Conference on Protozoology, held in 1961 in Prague. The history of the Society of Protozoologists was comprehensively treated by Corliss (1998); the history of the Prague Conference is presented here as reminiscences and personal interpretation of events of the author, who was one of the conference organizers and a member of the organization committee. Special attention is given to the personality and scientific accomplishments of Otto Jírovec, the 1961 conference spiritual father and president. It is concluded that the Prague Conference, while establishing the tradition of protistology meetings, helped protistology to attain its present status as a fundamental science discipline, which discovers and interprets the web of life at one of its, basic, "microbial" levels. Protists literally permeate the earth biosphere and in a way represent the "dark matter" of the living world, still awaiting many discoveries.

Microsporidian genus Berwaldia (Opisthosporidia, Microsporidia), infecting daphnids (Crustacea, Branchiopoda): Biology, structure, molecular phylogeny and description of two new species.

Structural, molecular and life cycle data are presented for two microsporidian species of the genus Berwaldia: B. singularis Larsson, 1981 (type species of the genus) and B. schaefernai Vávra and Larsson, 1994, parasites of Daphnia pulex Leydig, 1860 and Daphnia galeata Sars, 1863, respectively. Analysis of the SSU rDNA gene confirmed the species status of both species and showed that the GenBank sequence data submitted previously in GenBank for the genus Berwaldia, are from microsporidia that are not Berwaldia. Correct SSU rDNA gene sequences for B. schaefernai and B. singularis are now deposited in GenBank. The life cycle of these two species appears incomplete as the spores collected from their respective infected hosts will not infect the same host when fed per os. B. schaefernai appears as a frequent parasite of Daphnia longispina/galeata complex daphnids, influencing the behaviour of the infected host. In addition, two new species, of Berwaldia, one infecting fat body tissues of Daphnia longispina/galeata complex, and the other, infecting hypodermis and fat cells of Simocephalus vetulus (O. F. Müller, 1776) are described.

Occurrence, pathology, and ultrastructure of iridovirus and cytoplasmic polyhedrosis viruses in daphnids from the Czech Republic.

Iridescent (IVs, family Iridoviridae, genus Iridovirus) and cytoplasmic polyhedrosis viruses (CPVs; family Reoviridae, genus Cypovirus) are well known in insects, with thirteen IV species recognized from various orders, and sixteen CPV species known from lepidopterans. In 1975, an IV and CPV were reported in the daphnid, Simocehpalus expinosus, in Florida, but other reported daphnid virus infections seem to be rare. Here we report infected daphnids from woodland and carp ponds in the Czech Republic, Daphnia curvirostris with an IV, and D. pulex and D. ambigua, with CPVs. This suggests these viruses are more common in daphnids, the rarity of reports due to few surveys.

Globulispora mitoportans n. g., n. sp., (Opisthosporidia: Microsporidia) a microsporidian parasite of daphnids with unusual spore organization and prominent mitosome-like vesicles.

The microsporidian parasite Globulispora mitoportans, n. g., n. sp., infects the intestinal epithelium of two species of daphnids (Crustacea: Cladocera). Mature spores are thin-walled and possess a novel type of polaroplast with a conspicuous part consisting of globules that occupies a large part of the spore volume. Both developmental stages and the spores possess large, electron-lucent vesicles enveloped by a double membrane and filled with an internal web of filamentous material, corresponding structurally to microsporidian mitosomes. The SSU rRNA phylogeny places Globulispora into a specific "Enterocytospora-like" clade, part of a large "non-enterocytozoonidae" clade, grouping a heterogenous assemblage of microsporidia infecting almost exclusively insects and crustacea.

Description and phylogeny of Zelenkaia trichopterae gen. et sp. nov. (Microsporidia), an aquatic microsporidian parasite of caddisflies (Trichoptera) forming spore doublets.

Two novel microsporidia infecting the fat body tissues in larvae of two hosts, Halesus digitatus and Micropterna sequax (Trichoptera, Limnephilidae), were investigated using light and electron microscopy and rDNA sequence analyses. The molecular and morphological characters of these isolates warrant creation of a new microsporidian genus, Zelenkaia gen. n., with two species, one named herein. Developmental stages of Zelenkaia spp. have single nuclei. In sporogony, a plasmodium with four nuclei gives rise by rosette-like budding to two pairs of uninucleate sporoblasts, each within a thin-walled, subpersistent sporophorous vesicle. Sporoblasts and mature spores adhere temporary together, forming doublets oriented in parallel, within the sporophorous vesicle. Spores are long-oval and uninucleate, and those of the type species Z. trichopterae measure 10.3×3.5µm and have 24-25 polar filament coils. Phylogenetic analysis based on rDNA places Zelenkaia spp. within the aquatic clade of microsporidia and, more specifically, in the clade containing some microporidia from amphipod hosts.

Microsporidia and 'the art of living together'.

Parasitism, aptly defined as one of the 'living-together' strategies (Trager, 1986), presents a dynamic system in which the parasite and its host are under evolutionary pressure to evolve new and specific adaptations, thus enabling the coexistence of the two closely interacting partners. Microsporidia are very frequently encountered obligatory intracellular protistan parasites that can infect both animals and some protists and are a consummate example of various aspects of the 'living-together' strategy. Microsporidia, relatives of fungi in the superkingdom Opisthokonta, belong to the relatively small group of parasites for which the host cell cytoplasm is the site of both reproduction and maturation. The structural and physiological reduction of their vegetative stage, together with the manipulation of host cell physiology, enables microsporidia to live in the cytosolic environment for most of their life cycle in a way resembling endocytobionts. The ability to form structurally complex spores and the invention and assembly of a unique injection mechanism enable microsporidia to disperse within host tissues and between host organisms, resulting in long-lasting infections. Microsporidia have adapted their genomes to the intracellular way of life, evolved strategies how to obtain nutrients directly from the host and how to manipulate not only the infected cells, but also the hosts themselves. The enormous variability of host organisms and their tissues provide microsporidian parasites a virtually limitless terrain for diversification and ecological expansion. This review attempts to present a general overview of microsporidia, emphasising some less known and/or more recently discovered facets of their biology.

Opportunistic nature of the mammalian microsporidia: experimental transmission of Trachipleistophora extenrec (Fungi: Microsporidia) between mammalian and insect hosts.

Spores of Trachipleistophora extenrec, originally isolated from the muscles of the Madagascan insectivore Hemicentetes semispinosus and maintained by serial passage in severe combined immunodeficiency (SCID) mice, were fed to larvae of the Egyptian cotton leafworm Spodoptera littoralis. Extensive infection of larval tissues ensued and caused larval and pupal mortality. The development of T. extenrec in the insect host, studied both by light and electron microscopy, followed generally the same life cycle as in the mammalian host. However, some differences in the fine structure of the parasite grown in both types of hosts were found. Spores isolated from the insect host caused infection of SCID mice when injected intramuscularly. Our results suggest that T. extenrec may be originally an insect microsporidian. This likelihood is corroborated by its structural similarity and phylogenetic relationship to two other microsporidia having insects either as unique hosts (Vavraia culicis) or being able to infect both mammalian and insect host (Trachipleistophora hominis).

Morphology, molecular phylogeny, and ecology of Binucleata daphniae n. g., n. sp. (Fungi: Microsporidia), a parasite of Daphnia magna Straus, 1820 (Crustacea: Branchiopoda).

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 x 2.5 microm 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.

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris.

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.

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.

Vavraia culicis (Weiser, 1947) Weiser, 1977 revisited: cytological characterisation of a Vavraia culicis-like microsporidium isolated from mosquitoes in Florida and the establishment of Vavraia culicis floridensis subsp. n.

A brief nomenclatural history of Vavraia culicis (Weiser, 1947), the type species for the genus Vavraia Weiser, 1977, is presented together with a detailed description of the cytological and ultrastructural characteristics of a Vavraia culicis-like microsporidian species isolated from Aedes albopictus (Scuse) in Florida. This "Florida isolate", is the only known isolate of a species of the genus Vavraia from mosquitoes propagated in laboratory culture. Although the Florida isolate has been used under the name Vavraia culicis in several molecular phylogeny and host-parasite studies, it has not been structurally characterized and its relationship to the type species Vavraia culicis has never been examined. Structural data strongly support placement of the Florida isolate within the genus Vavraia and indicate its close relationship to both the type species of the genus and to other Vavraia-like mosquito microsporidia to which the name V. culicis has been applied. However, the identity of the Florida isolate with V. culicis (Weiser, 1947) Weiser, 1977 cannot be presently confirmed. Morphometric examination of spores of several Vavraia-like microsporidia isolates from mosquitoes, including the type material of Vavraia culicis, indicates that Vavraia culicis-like microsporidia probably represent not a single species, but a group of closely related organisms. Subspecies status is proposed for the Florida isolate.

Trachipleistophora extenrec n. sp. a new microsporidian (fungi: microsporidia) infecting mammals.

A new microsporidian Trachipleistophora extenrec n. sp. was isolated from a muscle lesion of the streaked tenrec Hemicentetes semispinosus Cuvier, 1798 (Mammalia, Tenrecidae), an insectivore endemic to Madagascar. The spores isolated from the tenrec were infectious to severe combined immunodeficient (SCID) mice by intramuscular injection. Material obtained from muscular lesions in mice was used for the parasite description. All developmental stages of the microsporidian were covered by a dense coat, which during sporogony changed into the sporophorous vesicle wall. Eight, 16, 32, or more spores were formed inside the sporophorous vesicle as the result of the division by plasmotomy and sequential fission of a multinucleate sporogonial plasmodium. Spores were ovoid, 4.7 x 2.8 microm in size, had a large posterior vacuole, and had an isofilar polar tube with 15-16 coils. Although the fine structure and the developmental pattern of the organism were in some respects similar to the genus Vavraia, molecular phylogeny based on the gene sequences of the small subunit rRNA and RNA polymerase subunit II indicated that the organism belongs to the genus Trachipleistophora. The diagnostic characters of the genera Trachipleistophora and Vavraia are discussed as well as the discrepancies between the phylogenies of these two microsporidian genera based on morphology and molecules.

Vairimorpha disparis n. comb. (Microsporidia: Burenellidae): a redescription and taxonomic revision of Thelohania disparis Timofejeva 1956, a microsporidian parasite of the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae).

Investigation of pathogens of populations of the gypsy moth, Lymantria dispar (L.) in Central and Eastern Europe revealed the existence of a microsporidium (Fungi: Microsporidia) of the genus Vairimorpha. The parasite produced three spore morphotypes. Internally infective spores are formed in the gut and adjacent muscle and connective tissue; single diplokaryotic spores and monokaryotic spores grouped by eight in sporophorous vesicles develop in the fat body tissues. The small subunit rDNA gene sequences of various isolates of the Vairimorpha microsporidia, obtained from L. dispar in various habitats in the investigated region, revealed their mutual identity. In phylogenetic analyses, the organism clustered with other L. dispar microsporidia that form only diplokaryotic spores in the sporogony cycle. The octospores of certain microsporidia infecting Lepidoptera that were previously described as Thelohania spp., have recently been shown to be one of the several spore morphotypes produced by species in the genus Vairimorpha. Because the description and drawings of a parasite described as Thelohania disparis by Timofejeva fit the characteristics of Vairimorpha, and all octospore-producing microsporidia collected from L. dispar since 1985 are genetically identical Vairimorpha species, it is believed that the parasite characterized here is identical to T. disparis Timofejeva 1956, and is herein redescribed, characterized, and transferred to the genus Vairimorpha as the new combination Vairimorpha disparis n. comb.

Nosema chrysorrhoeae n. sp. (Microsporidia), isolated from browntail moth (Euproctis chrysorrhoea L.) (Lepidoptera, Lymantriidae) in Bulgaria: characterization and phylogenetic relationships.

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth (Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Schädlingskunde, Pflanzen-Umweltschutz, 48, 11-12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.

Microsporidia in aquatic microcrustacea: the copepod microsporidium Marssoniella elegans Lemmermann, 1900 revisited.

Marssoniella elegans Lemmermann, 1900, a parasite of ovarial tissues of the copepod Cyclops vicinus Uljanin, 1875, was studied as a representative of aquatic-clade microsporidia which form "heteroinfectious spores" (spores not infective to the original host as opposed to "homoinfectious spores" which are infective for the original host) and which thus should require an alternate host. Several structural characters of this microsporidian are similar to those of copepod morphs of microsporidia infecting mosquitoes. However, small subunit ribosomal DNA phylogeny indicates that caddis flies (Insecta, Trichoptera) might be the alternate hosts of Marssoniella. Ultrastructural data obtained are used to redefine the genus Marssoniella Lemmermann, 1900 and its type species Marssoniella elegans.