Species-specific oligonucleotide probes for macroalgae: molecular discrimination of two marine fouling species of Enteromorpha (Ulvophyceae)

The green seaweeds Enteromorpha intestinalis and E. compressa are important fouling organisms commonly found in polluted and nutrient-enriched marine and brackish water habitats, where they are used in environmental monitoring. Discrimination of the two species is extremely difficult because of overlapping morphological characters. In this study a quick molecular method for species identification was developed based on the nuclear rDNA ITS2 sequence data of 54 E. intestinalis samples and 20 E. compressa samples from a wide geographical range. Oligonucleotide probes were designed for species-specific hybridization to dot-blots of the PCR-amplified ITS1, 5.8S gene and ITS2 fragment of both E. intestinalis and E. compressa. Specificity of the oligonucleotide probes was confirmed by tests with taxonomically diverse species that could morphologically be confused with E. intestinalis or E. compressa. This is the first use of species-specific probes for macroalgae. The restriction endonuclease NruI digested specifically the amplified PCR product from E. compressa into two fragments detectable on agarose gels, but no suitable restriction sites were identifiable in the PCR product of E. intestinalis.     

Phylogeographical structure, distribution and genetic variation of the green algae Ulva intestinalis and U. compressa (Chlorophyta) in the Baltic Sea area

The marine algae Ulva intestinalis and U. compressa are morphologically plastic with many overlapping characters and are therefore difficult to distinguish from each other. The present distribution of U. intestinalis and U. compressa is investigated along the salinity gradient in the Baltic Sea area through analyses of internal transcribed spacer (ITS) sequence data. Also, the amount and distribution of intraspecific genetic polymorphism in the ITS region is studied allowing inferences on the phylogeographical pattern and postglacial recolonization of the Baltic Sea area. The data show that of the two species only U. intestinalis occurs in the Baltic Sea. The distribution of U. compressa is more restricted than previously reported, and it was not found in salinities lower than 15 ppt. All of Scandinavia and the Baltic Sea were covered with ice during the last ice age and the organisms in the Baltic Sea must have colonized the area after the ice had started to melt. The genetic diversity of U. intestinalis and U. compressa in the Baltic Sea and the neighbouring area was found to be reduced compared to that in the British Isles. This reduction may be the result of either a historical reduction of diversity or an adaptation of specific clones to the northern environmental conditions.     

渤海浒苔属绿藻调查

于2009年至2010年对渤海及其周边10个沿海城市和6个岛屿的浒苔属绿藻进行了调查,初步确定该海区缘管浒苔、肠浒苔、扁浒苔、浒苔、条浒苔5个种类的地理分布情况。并对生物量进行了评估。与历史资料相比,渤海中辽东湾、渤海湾和莱州湾三个湾内的浒苔多样性和生物量下降明显或保持较低水平,湾周边地区则维持较高水平。此外,在近海人工基质上,以浒苔属为主的绿藻相对于红藻、褐藻分布更为广泛。     

Abscessed bony areas on Acanthopagrus cuvieri (Day) as a substrate for epiphytic algae

Enteromorpha compressa (L.) Greville and E. clathrata (J. Agardh), widely distributed green algae in Arabian Gulf, were observed growing on Acanthopagrus cuvieri (Day). Abscessed areas on the operculum provided suitable sites for attachment of both species of algae; broken spine tips were preferred by E. compressa . This study showed that these algae preferred hard substrata, with no scales for attachment. E. compressa appeared earlier and grew faster. The condition of the fish was normal.     

First detection and genotyping of human-associated microsporidia in pigeons from urban parks

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.     

Encephalitozoon cuniculi: Light and Electron Microscopic Evidence for Di-, Tetra-, and Octosporous Sporogony and a Note on the Molecular Phylogeny of Encephalitozoonidae

We demonstrate, based on the light, electron microscopic, and immunofluorescence studies carried out on two isolates of Encephalitozoon cuniculi established in culture, that E. cuniculi exhibits di-, tri-, tetra- and octosporous sporogony. We therefore propose that the generic characters of Encephalitozoon should be amended to include tetra-sporous sporogony as generic features. Additionally, the molecular phylogenetic analysis indicates that E. cuniculi, E. hellem, and E. (Septata) intestinalis form a cohesive group.     

Patterns of ion excretion and survival in two stoloniferous arid zone grasses

Desert plants show specific mechanisms to thrive under prevailing harsh conditions. To study the survival mechanism(s) in native desert plant species, Lesser Cholistan desert in Pakistan was surveyed and two potential salt secretory grass species, Aeluropus lagopoides and Ochthochloa compressa , were selected from five saline sites. Both these grasses responded differentially to saline environments by showing specialized mechanisms of survival including excretion of toxic ions through trichomes, vesicular and glandular hairs through leaf surface. In A. lagopoides , salt tolerance was associated with excreted Na⁺ concentration through leaf surface and accumulation of useful ions like Ca²⁺ and K⁺ in the shoot. Contrarily, O. compressa excreted all the ions through leaves without discriminating among toxic or beneficial ions. Results suggested that A. lagopoides was more successfully adapted to saline desert environments than O. compressa by excretion of excessive toxic ions and retention of Ca²⁺ and K⁺ in the shoot. This appears to be an adaptive character of the former species to successfully thrive in harsh desert conditions.     

Identification and characterization of two subpopulations of Encephalitozoon intestinalis

Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% +/- 5.1%). The presence of two subpopulations was confirmed by microscopic examination and image analysis (P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included > or = 25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4',6'-diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or beta-tubulin genes and subsequent sequence analysis confirmed both subpopulations as E. intestinalis. Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in purified spore suspensions should be considered when designing disinfection and drug treatment studies.     

Chlorophyll and peptide compositions in the two photosystems of marine green algae.

The molar ratios of chlorophyll a to b in the thalli of marine green algae were between 1.5 and 2.2, being appreciably lower than the ratio between 2.8 and 3.4 found for the leaves of higher plants and the cells of fresh-water green algae. The ratio of chlorophylls to P-700 in these marine algae was also lower than that in higher plants. The a/b ratios in the pigment proteins of Photosystems 1 and 2 separated by polyacrylamide-gel electrophoresis from sodium dodecyl sulfate-solubilized chloroplasts of four species of marine green algae, Bryopsis maxima, Cheatomorpha spiralis, Enteromorpha compressa and Ulva conglobata, were approximately 5 and 1, which are considerably smaller than the ratios, 7 and 2, respectively, found for the pigment proteins of the two photosystems of higher plants separated by the same technique. The chloroplasts of Bryopsis maxima and Cheatomorpha spiralis lacked two of the peptides associated with Photosystem II, which are present in the chloroplasts of Spinacia oleracea and Taraxacum officinale.     

Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation

Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).     

Sponge white patch disease affecting the Caribbean sponge Amphimedon compressa

We report on a novel sponge disease, hereafter termed 'sponge white patch' (SWP), affecting the Caribbean sponge species Amphimedon compressa. SWP is characterized by distinctive white patches of variable size that are found irregularly on the branches of diseased sponges. Nearly 20% of the population of A. compressa at Dry Rocks Reef, Florida, USA, showed symptoms of SWP at the time of investigation (November 2007-July 2010). Approximately 21% of the biomass of SWP individuals was bleached, as determined by volume displacement. Scanning electron microscopy analysis showed severe degradation of bleached tissues. Transmission electron microscopy of the same tissues revealed the presence of a spongin-boring bacterial morphotype that had previously been implicated in sponge disease (Webster et al. 2002; Mar Ecol Prog Ser 232:305-309). This particular morphotype was identified in 8 of 9 diseased A. compressa individuals investigated in this study. A close relative of the aforementioned disease-causing alphaproteobacterium was also isolated from bleached tissues of A. compressa. However, whether the spongin-boring bacteria are true pathogens or merely opportunistic colonizers remains to be investigated. Molecular fingerprinting by denaturing gradient gel electrophoresis (DGGE) demonstrated a distinct shift from the microbiota of healthy A. compressa to a heterogeneous mixture of environmental bacteria, including several phylotypes previously implicated in sponge stress or coral disease. Nevertheless, tissue transplantation experiments conducted in the field failed to demonstrate infectivity from diseased to healthy sponges, leaving the cause of SWP in A. compressa to be identified.     

Aquaculture fouling: Efficacy of potassium monopersulphonate triple salt based disinfectant (Virkon® Aquatic) against Ciona intestinalis

With the increasing spread of invasive marine species and their detrimental effects on aquaculture operations globally, mitigation strategies need to be optimized to mitigate economic impacts. The efficacy of a potassium monopersulphonate triple salt based disinfectant used in the aquaculture industry (Virkon? Aquatic at 0.5-5%) was evaluated against the solitary tunicate Ciona intestinalis, as well as the susceptibility of three different age groups of C. intestinalis to the treatment and the effect of the disinfectant on mussel mortality. Younger C. intestinalis were most affected by all treatments, and almost all immersion applications significantly decreased the biomass of C. intestinalis compared to untreated plates. Disinfectant solutions of ≥ 1% reduced biomass below pre-treatment levels. Mussel mortality was low, especially for solutions <3%. C. intestinalis should be treated 4 weeks post-settlement to maximize antifouling treatment effects. Immersion in 3% disinfectant for 30 s reduced the biomass of C. intestinalis by up to 89% and would be feasible in field applications using existing treatment equipment.     

Human intestinal parasites in Subsaharan Africa. I. Eastern Boè and Canhabaque Island (Guinea-Bissau)

A coprological survey was carried out in 1982-1983 in East Boè and in Canhabaque Island, (Bijagòs Islands), where 289 and 288 specimens of stools, respectively, have been collected. The samples were taken from apparently healthy subjects, of both sexes and of different age groups. The tests were made according to the modified Ritchie technique. The following results were obtained: A) East Boè: 1) Protozoa: E. coli 68.9%, E. nana 24.6%, G. intestinalis 8.7%, I. buetschlii 5.9%, E. histolytica 1.7%, E. hartmanni 0.7%, T. intestinalis 0.7%. b) Helminths: Ancylostomatidae 69.2%, T. trichiura 38.4%, S. stercoralis 6.2%, Taenia sp. 1.7%, H. nana 0.7%, S. haematobium 0.7%, S. mansoni 0.7%, S. fuelleborni 0.7%. B) Canhabaque: a) Protozoa: E. coli 85.1%, I. buetschlii 14.9%, E. nana 12.5%, G. intestinalis 8.3%, C. mesnili 7.3%, E. hartmanni 1.4%, E. histolytica 1.0%, T. intestinalis 0.4%. b) Helminths: Ancylostomatidae 87.9%, T. trichiura 9.4%, S. stercoralis 7.9%, S. fuelleborni 2.8%, Trichostrongylus sp. 1.4%, A. lumbricoides 0.7%, H. nana 0.4%. Eggs of Capillaria sp. probably pseudoparasites, were found in 8.7% of samples. The high prevalence of Ancylostomatidae infections appears to be related to the heavy fecal pollution all over the examined territory. The utilization of antihelminthic drug of popular medicine, which has a specific action on A. lumbricoides, could explain the lack and the low prevalence, respectively, of A. lumbricoides infections in both examined areas. In the past, other authors emphasized the low prevalence of this parasite for other Guinea Bissau regions which have a different geomorfological constitution. The cases of S. fuelleborni and H. nana are the first reported for Guinea Bissau.     

Comparative Studies on the Ecophysiological Differences of Two Green Tide Macroalgae under Controlled Laboratory Conditions

Yellow Sea green tides have occurred in coastal China almost every year from 2007 to 2011. Ulva prolifera (Müller) J. Agardh has been identified as the causative macroalgal species. U. intestinalis, however, has been observed in the bloom areas, co-occurring with U. prolifera, but it has not been found to be causative. The Yellow Sea green tide has shown consistent phases of development that match corresponding environmental changes. U. prolifera, not U. intestinalis, is dominant. Our experimental design was based on these observed phenomena, and the results of our field investigation indicated a close relationship between changes in principal environmental factors (irradiance, temperature, and salinity) and the development of each phase of the bloom. These main environmental factors were simulated to allow estimation and comparison of the physiological responses of U. prolifera and U. intestinalis. Ecophysiological differences were found between these two species. (1) More photosynthetic activity and plasticity were detected in U. prolifera. (2) U. prolifera was found to be more sensitive to dynamic environments, especially harsh and changing environmental conditions. U. intestinalis was found to be more stable, probably due to the higher stress tolerance given by its antioxidant system. (3) Markedly higher nutrient absorption activity was observed in U. prolifera. Comparisons of the ecophysiological traits of these two species in this present study may foster understanding of their natural ecological processes. Specifically, U. prolifera seemed to be more engaged with the ephemeral blooms, while U. intestinalis seemed to be directed toward persistence. This also suggests that the ecological success of U. prolifera may be inextricably linked to its higher capacity for photosynthesis, nutrient absorption, and nutrient assimilation.     

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.     

Genome-Wide Analyses of Recombination Suggest That Giardia intestinalis Assemblages Represent Different Species

Giardia intestinalis is a major cause of waterborne enteric disease in humans. The species is divided into eight assemblages suggested to represent separate Giardia species based on host specificities and the genetic divergence of marker genes. We have investigated whether genome-wide recombination occurs between assemblages using the three available G. intestinalis genomes. First, the relative nonsynonymous substitution rates of the homologs were compared for 4,009 positional homologs. The vast majority of these comparisons indicate genetic isolation without interassemblage recombinations. Only a region of 6 kbp suggests genetic exchange between assemblages A and E, followed by gene conversion events. Second, recombination-detecting software fails to identify within-gene recombination between the different assemblages for most of the homologs. Our results indicate very low frequency of recombination between the syntenic core genes, suggesting that G. intestinalis assemblages are genetically isolated lineages and thus should be viewed as separated Giardia species.     

Antiparasitic activity of flavonoids and isoflavones against Cryptosporidium parvum and Encephalitozoon intestinalis

Flavonoids, polyphenolic compounds found in plants, have demonstrated activity against several parasites and can augment the efficacy of other drugs by either increasing the uptake or decreasing the efflux of these drugs. We evaluated 11 of these compounds alone or in combination in order to test the hypothesis that flavonoids are effective against Cryptosporidium parvum and Encephalitozoon intestinalis. Using in vitro cell culture assays, HCT-8 cells or E6 cells were infected with C. parvum and E. intestinalis, respectively, and treated with compounds at doses ranging from 1 to 200 microM. We found that six compounds were active against C. parvum. Naringenin and genistein had the greatest activities with EC(50) of 15 and 25 microM, respectively. Two compounds, quercetin and apigenin, had activity against E. intestinalis at EC(50) of 15 and 50 microM, respectively. The EC(50) of trifluralin, a dinitroaniline compound, was decreased significantly when combined with genistein in an in vitro assay, suggesting that compounds may be used alone on in combination with other moderately active drugs to increase efficacy. In addition, induction of apoptosis by these compounds was studied but not observed to be a significant mechanism of action.     

Molecular characterization of Giardia intestinalis haplotypes in marine animals: variation and zoonotic potential

Giardia intestinalis is a microbial eukaryotic parasite that causes diarrheal disease in humans and other vertebrates worldwide. The negative effect on quality of life and economics caused by G. intestinalis may be increased by its potential status as a zoonosis, or a disease that can be transmitted from animals to humans. The zoonotic potential of G. intestinalis has been implied for over 2 decades, with human-infecting genotypes (belonging to the 2 major subgroups, Assemblages A and B) occurring in wildlife and domesticated animals. There are recent reports of G. intestinalis in shellfish, seals, sea lions and whales, suggesting that marine animals are also potential reservoirs of human disease. However, the prevalence, genetic diversity and effect of G. intestinalis in marine environments and the role that marine animals play in transmission of this parasite to humans are relatively unexplored. Here, we provide the first thorough molecular characterization of G. intestinalis in marine vertebrates. Using a multi-locus sequencing approach, we identify human-infecting G. intestinalis haplotypes of both Assemblages A and B in the fecal material of dolphins, porpoises, seals, herring gulls Larus argentatus, common eiders Somateria mollissima and a thresher shark Alopias vulpinus. Our results indicate that G. intestinalis is prevalent in marine ecosystems, and a wide range of marine hosts capable of harboring zoonotic forms of this parasite exist. The presence of G. intestinalis in marine ecosystems raises concerns about how this disease might be transmitted among different host species.     

Infectivity of microsporidia spores stored in water at environmental temperatures

To determine how long waterborne spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis could survive at environmental temperatures, culture-derived spores were stored in water at 10, 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. intestinalis were still infective after 12 mo, whereas those of E. hellem and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, spores of the same species remained infective for 10, 6, and 2 mo, and at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intestinalis and E. hellem were infective for 3 mo, but those of E. cuniculi were infective for only 3 wk. At 30 C, the former 2 species were infective for 3 wk and 1 mo, respectively, and the latter species for only 1 wk. These findings indicate that spores of different species of Encephalitozoon differ in their longevity and temperature tolerance, but at temperatures from 10 to 30 C, all 3 have the potential to remain infective in the environment long enough to become widely dispersed.