Nosema parkeri sp. n., a Microsporidan from the Argasid Tick, Ornithodoros parkeri Cooley

SYNOPSIS. Nosema parkeri sp. n. is described from nymphs and adults of the argasid tick, Ornithodoros parkeri Cooley, from a laboratory colony. Schizogonic and sporogonic stages are described from various tick tissues. Spores are binucleate, measuring 3.2 (3–4) × 1.9 (1.8–2.5) μm. Transmission is transovarial and transstadial. The parasite does not appear to affect adversely the development or reproduction of the tick. Dermacentor andersoni Stiles was experimentally infected. Attempts to infect Swiss mice by tick feeding or by injection of infected tick suspensions were unsuccessful. The microsporidan differs in structure from Encephalitozoon ixodis Weiser) and Nosema slovaca Weiser & Reháček, the only other microsporidans known from ticks.     

Fine Structure of the Sporogonic Stages of Nosema parkeri

SYNOPSIS The fine structure of sporogonic stages of Nosema parkeri Krinsky, a microsporidan from the argasid tick, Ornithodoros parkeri Cooley, is described. Developmental changes in the spore coat and cytoplasmic organelles are discussed. As a sporoblast transforms into a spore, the organelles become more compact and the membranes surrounding them appear to become more taut. It is suggested that the polaroplast complex is involved in fluid transport during development of the spore. Organelles in the mature spore include 2 contiguous nuclei enveloped in a lattice containing ribosome-like particles, a polaroplast complex composed of laminar and saccular regions, and a coiled tubular polar filament attached to a polar sac. Sporogonic stages do not appear to have mitochondria, Golgi apparatus, or a posterior vacuole. The fine structure of the spore of N. parkeri is very similar to that of species of Nosema found in insects, crustaceans, and trematodes.     

Molecular identification of Rickettsia parkeri infecting Amblyomma triste ticks in an area of Argentina where cases of rickettsiosis were diagnosed

Specimens of the hard tick Amblyomma triste were found infected with Rickettsia parkeri in an area of Argentina (General Lavalle, Buenos Aires Province) where cases of human illness attributed to this microorganism have been reported. Molecular detection of R. parkeri was based on polymerase chain reactions that amplify a ca. 400-bp fragment of the 23S-5S intergenic spacer and a ca. 500-bp fragment of the gene encoding a 190-kDa outer membrane protein. Three (6.97%) of 43 A. triste ticks were determined to be positive for R. parkeri. These results provide strong evidence that A. triste is the vector of R. parkeri in the study area. The findings of this work have epidemiological relevance because human parasitism by A. triste ticks has been frequently recorded in some riparian areas of Argentina and Uruguay and new cases of R. parkeri rickettsiosis might arise in the South American localities where humans are exposed to the bites of this tick species.     

Absence of insect juvenile hormones in the American dog tick,Dermacentor variabilis (Say) (Acari:Ixodidae), and in Ornithodoros parkeri Cooley (Acari:Argasidae)

Synganglia, salivary gland, midgut, ovary, fat body and muscle alone and in combination from the ixodid tick, Dermacentor variabilis (Say), or the argasid tick, Ornithodoros parkeri Cooley, were incubated in vitro in separate experiments with L-[methyl-(3)H]methionine and farnesoic acid or with [1-(14)C]acetate. Life stages examined in D. variabilis were 3 and 72 h old (after ecdysis) unfed nymphs, partially fed nymphs (18 and 72 h after attachment to the host), fully engorged nymphs (2 d after detachment from host), 3 and 72 h old (after eclosion) unfed females, partially fed unmated females (12-168 h after attachment to host) and mated replete females (2 d after detachment from the host). Those from O. parkeri were third and fourth stadium nymphs and female O. parkeri, 1-2 d after detachment. Corpora allata from Diploptera punctata, Periplaneta americana and Gromphadorina portentosa were used as positive controls in these experiments. No farnesol, methyl farnesoate, JH I, JH II, JH III, or JHIII bisepoxide was detected by radio HPLC from any tick analysis while JH III, methyl farnesoate, and farnesol were detected in the positive controls. To examine further for the presence of a tick, insect-juvenilizing agent, Galleria pupal-cuticle bioassays were conducted on lipid extracts from 10 and 15 d old eggs, unfed larvae (1-5 d after ecdysis), unfed nymphs (1-7 d after ecdysis), and partially fed, unmated female adults (completed slow feeding phase) of D. variabilis. Whole body extracts of fourth stadium D. punctata and JH III standard were used as positive controls. No juvenilizing activity in any of the tick extracts could be detected. Electron impact, gas chromatography-mass spectrometry of hemolymph extracts from fed, virgin (forcibly detached 7 d after attachment) and mated, replete (allowed to drop naturally) D. variabilis and fully engorged (1-2 d after detachment) O. parkeri females also failed to identify the common insect juvenile hormones. The same procedures were successful in the identification of JH III in hemolymph of fourth stadium D. punctata. Last stadium nymphal (female) O. parkeri implanted with synganglia from second nymphal instars underwent normal eclosion to the adult. The above studies in toto suggest that D. variabilis and O. parkeri do not have the ability to make the common insect juvenile hormones, and these juvenile hormones do not regulate tick metamorphosis or reproduction as hypothesized in the literature.     

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 Sch?dlingskunde, 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.     

The Influence of Oxygen on the Development of Nanorana parkeri Tadpoles

Ectothermic animals are tolerant of variable oxygen availability, whether low-oxygen levels constrain the fitness of ectotherms remains unclear. Nanorana parkeri, an anuran endemic to the southern Tibetan plateau, is an excellent model with which to answer this question. In this study, we raised tadpoles of N. parkeri in oxygenated water(high-oxygen group) and deoxygenated unchlorinated tap water(low-oxygen group) and monitored their growth, mortality, and telomere length. The growth rate for body length and body weight was higher in the low-oxygen group than in the high-oxygen group. However, dissolved oxygen did not affect development time, mortality, and telomere length of the tadpoles. These results suggest that although the oxygen concentration influenced some phenotype traits of plateau tadpoles, but it didn't influence the telomere length and survival rate, potential explanations are the local adaptation and N. parkeri tadpoles' wide oxygen tolerance, and fluctuant toxic content that resulted in little oxidative stress on tadpoles. These results indicated that low oxygen was not a stress to N. parkeri tadpoles' fitness and survival. This study is helpful in understanding the adaptation mechanisms of Tibetan plateau amphibians.     

Nosema muris n. sp., a New Microsporidian Parasite of the White Mouse (Mus musculus L.)

SYNOPSIS. A microsporidian, Nosema muris n. sp., is described from laboratory white mice in Czechoslovakia. I t differs from Nosema cuniculi (Levaditi, Nicolau and Schoen, 1923) and other Microsporidia in mice in that it develops in the omentum and heart muscle, iorming large masses of schizonts and spores. The parasite is distributed to other tissues (liver, spleen, fat, peritoneal muscles, connective tissue and brain) by infected mononudear cells, and then forms localized cysts in them. Kidney, lungs, adrenal glands, pancreas and salivary glands are not infected. The parasite can be transmitted via the placenta, by feeding on infected tissues, or by experimental injection of ascitic fluid.     

高山倭蛙消化道结构初步观察

高山倭蛙食道粘膜上皮为复层柱状上皮,且固有膜中有丰富的复泡状腺;胃贲门部粘膜上皮无杯状细胞,PAS反应显示固有膜中有深红色颗粒分布,胃体中胃腺丰富;肠分为小肠、大肠、直肠3部分.小肠和直肠上皮中杯状细胞数量多.无尾类消化道结构与海拔高度无明显相关关系.     

Impact of a novel species of Nosema on the southwestern corn borer (Lepidoptera: Crambidae)

A study was undertaken to elucidate the impact of an undescribed Nosema sp. on the southwestern corn borer (SWCB; Diatraea grandiosella Dyar). The Nosema sp. (isolate 506) included in the study was isolated from an overwintering SWCB larva in Mississippi. It was highly infectious per os, with a median infective dose of 2.0 x 10(3) spores per larva. Even at the highest dosage tested (10(7) spores per larva), minimal mortality (< or = 3%) was observed in infected larvae, pupae, and adults reared in the laboratory on an artificial diet. However, infected pupae (0- and 7-d-old) were smaller, and the time to adult eclosion from pupation was slightly increased. Furthermore, the number of eggs produced by infected SWCB female moths substantially decreased (32%), and this effect was most pronounced on day 2, when the greatest number of eggs were oviposited by infected and noninfected moths. For eggs produced by infected females mated with infected males, hatch was slightly decreased by 16 and 15% for eggs laid on days 2 and 3, respectively. In addition, egg hatch was reduced in eggs oviposited by noninfected females mated with infected males on day 3. A low prevalence of infection (< 6%) was observed in the F1 generation originating from infected females mating with noninfected males, from noninfected females mating with infected males, and from infected females mating with infected males. Nosema 506 spores were observed in the proximity of reproductive tissues of infected female and male moths. Spores also were detected on the chorion surface and within eggs laid by infected females. Furthermore, 1-11% of larvae hatching from surface-sterilized eggs were infected by Nosema 506 indicating a transovarial mechanism of transmission.     

Brachiola vesicularum, N. G., N. Sp., a New Microsporidium Associated with AIDS and Myositis

Brachiola vesicularum, n. g., n. sp., is a new microsporidium associated with AIDS and myositis. Biopsied muscle tissue, examined by light and electron microscopy, revealed the presence of organisms developing in direct contact with muscle cell cytoplasm and fibers. No other tissue types were infected. All parasite stages contain diplokaryotic nuclei and all cell division is by binary fission. Sporogony is disporoblastic, producing 2.9 times 2 μm diplokaryotic spores containing 8-10 coils of the polar filament arranged in one to three rows, usually two. Additionally, this microsporidium produces electron-dense extracellular secretions and vesiculotubular appendages similar to Nosema algerae. However, the production of protoplasmic extensions which may branch and terminate in extensive vesiculotubular structures is unique to this parasite. Additionally, unlike Nosema algerae , its development occurred at warm blooded host temperature (37-38° C) and unlike Nosema connori , which disseminates to all tissue types, B. vesicularum infected only muscle cells. Thus, a new genus and species is proposed. Because of the similarities with the genus Nosema , this new genus is placed in the family Nosematidae. Successful clearing of this infection (both clinically and histologically) resulted from treatment with albendazole and itraconozole.     

Induction of egg maturation and oviposition in the tick Ornithodoros parkeri (Acari: Argasidae)

Hemocoelic injection and vaginal insertion of selected male reproductive and non-reproductive tissue homogenates into fed-virgin female Ornithodoros parkeri stimulated varying degrees of ovum maturation and/or oviposition during 14 and 30 day observation periods, respectively. Mean times for oviposition and mean numbers of eggs laid per ovipositing female receiving hemocoelic injections of male reproductive tissue homogenates did not differ significantly from fed-mated controls. In addition, hemocoelic injection of male salivary glandular homogenate induced oviposition, yet synganglial homogenate did not. Although vaginal insertion induced both ovum maturation and oviposition, the effect was not as pronounced as when similar doses were administered by hemocoelic injection. These results indicate that a complex interrelated series of precopulatory and copulatory stimuli are necessary for oviposition to occur in fed O. parkeri.     

Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway

Rickettsiae are obligate intracellular pathogens that are transmitted to humans by arthropod vectors and cause diseases such as spotted fever and typhus. Although rickettsiae require the host cell actin cytoskeleton for invasion, the cytoskeletal proteins that mediate this process have not been completely described. To identify the host factors important during cell invasion by Rickettsia parkeri, a member of the spotted fever group (SFG), we performed an RNAi screen targeting 105 proteins in Drosophila melanogaster S2R+ cells. The screen identified 21 core proteins important for invasion, including the GTPases Rac1 and Rac2, the WAVE nucleation-promoting factor complex and the Arp2/3 complex. In mammalian cells, including endothelial cells, the natural targets of R. parkeri, the Arp2/3 complex was also crucial for invasion, while requirements for WAVE2 as well as Rho GTPases depended on the particular cell type. We propose that R. parkeri invades S2R+ arthropod cells through a primary pathway leading to actin nucleation, whereas invasion of mammalian endothelial cells occurs via redundant pathways that converge on the host Arp2/3 complex. Our results reveal a key role for the WAVE and Arp2/3 complexes, as well as a higher degree of variation than previously appreciated in actin nucleation pathways activated during Rickettsia invasion.     

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.     

The impact of mixed infection of three species of microsporidia isolated from the gypsy moth,Lymantria dispar L. (Lepidoptera: Lymantriidae)

The outcome of mixed infection by three species of microsporidia in the genera Endoreticulatus, Nosema, and Vairimorpha, isolated from different populations of Lymantria dispar in Bulgaria, was evaluated in the laboratory. All possible combinations of two species were administered either simultaneously or sequentially to larvae, and mortality, duration of development, and larval weight at 20 days post-infection (simultaneous inoculation) or 23 days post-infection (sequential inoculation) were chosen as the outcome variables. Larvae were also dissected and the presence of each species of microsporidia and the tissues infected were recorded for each treatment. Effects of infection were dependent on both host sex and the type of exposure. Infected larvae were more likely to die than uninfected larvae, but there were no differences in mortality between single and mixed infections. Addition of Endoreticulatus to infections of Nosema or Vairimorpha significantly increased duration of development to the fourth ecdysis; this effect was additive. Addition of Nosema or Vairimorpha to an existing infection had no such effect. When Nosema was administered simultaneously with Endoreticulatus or Vairimorpha, infected larvae weighed more than larvae that had single infections with either pathogen. Nosema was displaced from the silk glands by Vairimorpha and Nosema suppressed octospore formation by Vairimorpha in fat body. The histological evidence combined with the data on larval weight supports the hypothesis that competition occurred in mixed infections.     

Rickettsia infection in five areas of the state of São Paulo, Brazil

This study investigated rickettsial infection in animals, humans, ticks, and fleas collected in five areas of the state of S?o Paulo. Eight flea species (Adoratopsylla antiquorum antiquorum, Ctenocephalides felis felis, Polygenis atopus, Polygenis rimatus, Polygenis roberti roberti, Polygenis tripus, Rhopalopsyllus lugubris, and Rhopalopsyllus lutzi lutzi), and five tick species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Ixodes loricatus, and Rhipicephalus sanguineus) were collected from dogs, cats, and opossums. Rickettsia felis was the only rickettsia found infecting fleas, whereas Rickettsia bellii was the only agent infecting ticks, but no animal or human blood was shown to contain rickettsial DNA. Testing animal and human sera by indirect immunofluorescence assay against four rickettsia antigens (R. rickettsii, R. parkeri, R. felis, and R. bellii), some opossum, dog, horse, and human sera reacted to R. rickettsii with titers at least four-fold higher than to the other three rickettsial antigens. These sera were considered to have a predominant antibody response to R. rickettsii. Using the same criteria, opossum, dog, and horse sera showed predominant antibody response to R. parkeri or a very closely related genotype. Our serological results suggest that both R. rickettsii and R. parkeri infected animals and/or humans in the studied areas.     

Diversity of Nosema associated with bumblebees (Bombus spp.) from China

Bumblebees (Bombus spp.) are important pollinators of many economically important crops and microsporidia are among the most important infections of these hosts. Using molecular markers, we screened a large sample (n=1,009 bees) of workers of 27 different Bombus spp. from China (Sichuan, Qinghai, Inner Mongolia, and Gansu provinces). The results showed that 62 individuals representing 12 Bombus spp. were infected by microsporidia with an overall prevalence of 6.1%. Based on the haplotypes (ssrRNA sequences), we confirmed the presence of Nosema bombi, Nosema ceranae and (likely) Nosema thomsoni. In addition, four new putatively novel taxa were identified by phylogenetic reconstruction: Nosema A, Nosema B-complex, Nosema C-complex and Nosema D-complex. In many cases, hosts were infected by more than one Nosema taxon. Possible caveats of sequence analyses are discussed.     

Developmental profile, isolation, and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari: Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari: Argasidae)

A novel lipoglycoheme-carrier protein (CP) in the American dog tick, Dermacentor variabilis (Say) has been purified and characterized. CP was purified by native-PAGE from partially fed virgin females. CP has a density of 1.25 g/ml with a molecular weight of 200 K by native-PAGE and 340 K by gel filtration chromatography. CP is comprised of two majour subunits, 98 K and 92 K in molecular weight by SDS-PAGE. Separate amino acid composition of the two subunits indicated high contents of As(x), Gl(x) and leucine. However, the N-terminal amino acid sequence of the two subunits was only 13% identical. The lower molecular weight subunit showed 61% identity to artemocyanin (biliprotein) in fairy shrimps, 46% identity to minor vitellogenin in chickens and 13% identity to vitellin of the black-legged tick. No similarity match was found for the other subunit. CP is a lipoglycoheme-protein as indicated by selective staining of native-PAGE gel for lipids, carbohydrates and heme. Lipid analysis by thin layer chromatography revealed the presence of cholesterol, phospholipids, monoacylglycerides, triacylglycerides and free fatty acids. Heme associated with purified CP demonstrated a lambda(max) of 397.5 nm while the lambda(max) of crude hemolymph plasma was 402.5 nm. The presence of CP in whole body homogenates of eggs, unfed and fed larvae and fed nymphs as well as in the plasma of unfed and fed adults including vitellogenic females was demonstrated by native-PAGE. Although a protein of analogous size was not found in the soft tick, Ornithodoros parkeri Cooley, a high molecular weight protein (500 K) is the predominant plasma protein in both unfed and fed male and female adults of that species as determined by native-PAGE. Also, CP appears to function as a biliprotein which sequesters heme.     

Effects of temperature and tick weight on expression of autogeny in the argasid tick Ornithodoros parkeri Cooley (acari: Argasidae)

The effects of different temperatures on the expression of autogenic development in unfed/mated Ornithodoros parkeri Cooley females were examined. High temperature (29 C) induced autogeny in lower, middle and higher weight classes (11-20, 21-30, 31-40 mg, respectively) with all females in the highest weight class and most in the middle weight class ovipositing within 51 days post-mate. At a lower temperature (21 C) autogenic development was not expressed by higher and middle weight females, whereas lower weight females exhibited similar degrees of autogeny at both temperatures. Results are discussed in relation to presumptive factors involved in the control and mediation of autogeny.