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Effects of pulp and paper mill effluents on the microplankton and microbial self-purification capabilities of the Biobío River, Chile.

Most studies focus on the ecotoxicity of pulp and paper mill effluents, rather than on how they affect the physicochemical and biological structure and the intrinsic ecological capabilities of the receiving watercourses. We investigated the impact of such effluents on the water quality, microplankton system and microbial self-purification capacity (degradation of polymeric organic compounds via extracellular enzymes) of the Biobío River in Chile. The physicochemical impact on the water quality was indicated by raised conductivity, by the pollution of the water body with nitrate, nitrite and soluble reactive phosphorus, by the appearance of tannin and lignin, and by the steady accumulation of inorganic and organic suspended matter (SPM) along the river. From the biological structure of the microplankton system, very low and declining concentrations of chlorophyll a and heterotrophic flagellate densities were determined. The pulp and paper mill effluents introduced high bacterial abundances and biomass concentrations into the river water. This reflects the effective use made of the abundantly available inorganic and organic nutrients within this industrial and municipal process water by bacteria adapted to these extreme environments, additionally supported by concomitant low grazing pressure derivable from low heterotrophic flagellate abundances. Indeed, in one section of the river affected by a pulp mill, the plant was found to significantly contribute to the self-cleaning capacity of the river. However, this elevated degradation capacity was not enough to compensate for the additionally discharged organic material which, together with the toxic effects of the paper plant effluents, significantly interferes with the ecological status of the Biobío River.

Animals↗

Screening for unicellular algae as possible bioassay organisms for monitoring marine water samples.

ECOTOX is an automatic early warning system to monitor potential pollution of freshwater, municipal or industrial waste waters or aquatic ecosystems. It is based on a real time image analysis of the motility and orientation parameters of the unicellular, photosynthetic flagellate Euglena gracilis. In order to widen the use of the device to marine habitats and saline waters nine marine flagellates were evaluated as putative bioassay organisms, viz. Dunaliella salina, Dunaliella viridis, Dunaliella bardawil, Prorocentrum minimum Kattegat, P. minimum Lissabon, Tetraselmis suecica, Heterocapsa triquetra, Gyrodinium dorsum and Cryptomonas maculata. Because of their slow growth the last three strains were excluded from further evaluation. Selection criteria were ease of culture, density of cell suspension, stability of motility and gravitactic orientation. The sensitivity toward toxins was tested using copper(II) ions. The instrument allows the user to automatically determine effect-concentration (EC) curves from which the EC(50) values can be calculated. For the interpretation of the EC curves a sigmoid logistic model was proposed which proved to be satisfactory for all tested strains. The inhibition of the motility was considered as the most appropriate movement parameter as an endpoint. The Dunaliella species had the lowest sensitivity to copper with EC(50) values of 220, 198 and 176 mg/L for D. salina, D. bardawil and D. viridis, respectively, followed by T. suecica with an EC(50) value of 40 mg/L. The Prorocentrum species were found to be the most sensitive with an EC(50) value of 13.5 mg/L for P. minimum Lissabon and 7.5 mg/L for P. minimum Kattegat.

Biological Assay↗

High prevalence of Trypanosoma rangeli and Trypanosoma cruzi in opossums and triatomids in a formerly-endemic area of Chagas disease in Southeast Brazil.

In Brazil Trypanosoma rangeli has been detected in humans, sylvatic mammals and vectors in the Amazon Basin and in wild rodents in a Southern State. Here we report for the first time a high prevalence of T. rangeli in opossums and triatomids captured in peridomestic environments in a formerly-endemic area of Chagas disease in Southeast Brazil. Five molecular typing tools clearly indicate the presence of T. rangeli and Trypanosoma cruzi in mammalian reservoirs and triatomids. Twenty-one opossums (Didelphis albiventris) were captured and flagellates were detected in the blood of 57.1% (12/21) of the animals. Single infections with T. rangeli or T. cruzi were diagnosed, respectively, in 58.4 and 8.3% of the opossums. Mixed infections were observed in 33.3%. Forty-four triatomids (38 Rhodnius neglectus and 6 Panstrongylus megistus) were collected in palm trees within 50 m from human dwellings. Flagellates were observed in the digestive tract and feces of 50% of the insects. PCR assays performed in DNA samples obtained from 16 cultures of the intestinal tract revealed single infection with T. cruzi (68.7%) or T. rangeli (6.3%), as well as mixed infections (25%). T. rangeli was also detected in the hemolymph of two specimens. Genotyping revealed predominance of T. cruzi I. The data suggest that R. neglectus in conjunction with D. albiventris may be significant factors in the maintenance of the sylvatic and peridomestic cycles of T. rangeli in the region. The finding of T. cruzi and T. rangeli in triatomine species capable of domiciliation and therefore considered as alternative vectors for the parasite transmission opens up the possibility of re-establishment of Chagas disease following reinfestation of houses.

Animals↗

Modes of association of Trypanosoma cruzi with the intestinal tract of the vector Triatoma infestans.

The interface between Trypanosoma cruzi and two regions of the intestinal tract of reduviid bugs, the small intestine and the rectum, was investigated by electron microscopy. The mode of association of the trypanosomes with the midgut surface differs fundamentally from that in the rectum, the preferred site of colonisation by T. cruzi. The parasites caused no detrimental changes in the extracellular membrane layers, microvilli or epithelial cells. Parasites resided mainly at the border of the gut contents, also regularly showing parasite-parasite interdigitations. In regions in which the extracellular membrane layers were absent or only weakly developed, trypanosome bodies or flagella occasionally could be found inserted shallowly between the tips of the microvilli. Since there were usually no ultrastructural modifications of the cell body and/or flagellum associated with attachment, there is apparently no strong attachment of the flagellates to the wall of the midgut. In the rectal lumen the flagellates also interdigitated with each other and on the rectal wall T. cruzi was intimately attached to the rectal cuticle lining. At the attachment site flagella were enlarged and sometimes contained electron-dense, hemidesmosome-like material beneath the plasma membrane.

Animals↗

Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai.

A phosphatase activity of the trypanosomatid parasite Herpetomonas samuelpessoai was characterized using intact living cells. The effects of dimethyl sulfoxide (DMSO) on this activity were investigated. This phosphatase activity (2.53+/-0.01 nmol P(i)/mg protein x min) was linear with cell density and with time for at least 60 min. The optimum pH for the H. samuelpessoai phosphatase lies in the acid range. This phosphatase activity was inhibited by metal chelators and classical phosphatase inhibitors. A robust stimulation of the phosphatase activity was observed when the flagellates were grown in the presence of 4% DMSO, both when intact flagellates and when culture supernatant from those cells were assayed, as observed by biochemical and cytochemical analysis. We also demonstrate that DMSO induced the secretion and/or shedding of this phosphatase to the extracellular medium, with a possible involvement of protein kinase C in this process.

Animals↗

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus.

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.

Animals↗

Genes and susceptibility to leishmaniasis.

Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly where they grow as motile, flagellated promastigotes in the gut, and the mammalian macrophage where they grow intracellularly as non-flagellated amastigotes. Leishmaniasis is the outcome of an evolutionary 'arms race' between the host's immune system and the parasite's evasion mechanisms which ensure survival and transmission in the population. The spectrum of disease manifestations and severity reflects the interaction between the genome of the host and that of the parasite, and the pathology is caused by a combination of host and parasite molecules. This chapter examines the genetic basis of host susceptibility to disease in humans and animal models. It describes the genetic tools used to map and identify susceptibility genes, and the lessons learned from murine and human cutaneous leishmaniasis.

Animals↗

Cell biology of Leishmania.

Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly, where they grow as motile flagellated promastigotes in the gut, and the mammalian macrophage, where they survive and grow intracellularly as non-flagellated amastigotes in the phagolysosome. Leishmaniasis is the outcome of an evolutionary 'arms race' between the host's immune system and the parasite's evasion mechanisms, which ensure survival and transmission in the population. The diverse spectrum of patterns and severity of disease reflect the varying contributions of parasite virulence factors and host responses, some of which act in a host protective manner while others exacerbate disease. This chapter describes the interaction of the Leishmania with their hosts, with emphasis on the molecules and mechanisms evolved by the parasites to avoid, subvert or exploit the environments in the sandfly and the macrophage, and to move from one to the other.

Animals↗

Substrate depletion upregulates uptake of myo-inositol, glucose and adenosine in Leishmania.

Leishmania flagellates undergo a digenetic life cycle in the gut of the sandfly insect vector and in macrophage phagolysosomes of the mammalian host. This involves vast changes of the environment to which the parasite has to adapt, including temperature, pH and concentration of nutrients between different types of meals of the insect vector or within the enclosed intracellular environment of the phagolysosome. The regulation of transporters for important organic substrates in Leishmania donovani, Leishmania mexicana and Leishmania enriettii has been investigated. A pronounced upregulation of inositol (25-fold), adenosine (11-fold) or glucose (5-fold) uptake activities was found when cells were depleted of the respective substrates during culture. Inositol-depleted cells showed a half-maximal uptake rate at nanomolar inositol concentration. Depletion of inositol only affected inositol uptake but did not affect uptake of glucose analog or proline in control experiments, indicating the specificity of the mechanism(s) underlying transport regulation. Adenosine-depleted cells showed an approximately 10-fold increase in both adenosine and uridine uptake, both mediated by the L. donovani nucleoside transporter 1 (LdNT1), but no change in guanosine uptake, which is mediated by the L. donovani nucleoside transporter 2 (LdNT2). These results suggest that extracellular adenosine concentration specifically regulates LdNT1 transport activity and does not affect LdNT2. The data imply that upregulation of transport activities by substrate depletion is a general phenomenon in protozoan flagellates, which is in remarkable contrast to bacteria where upregulation typically follows an increase of extracellular organic substrate. Hence, the parasites can maximize the uptake of important nutrients from the host even under limiting conditions, whereas bacteria often have dormant stages (spores) to overcome unfavorable environmental conditions or are heterotrophic for organic substrates.

Adenosine↗

Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype.

We have shown that the growth, starvation and population heterogeneity of Salmonella typhimurium and its isogenic nuoG and cydA mutants can be monitored by flow cytometry. Bacterial cells were analysed unstained, and after staining with rhodamine 123, propidium iodide and acridine orange. In unstained cultures it was possible to distinguish flagellated and non-flagellated cells. nuoG and cydA mutants were less stained with rhodamine confirming their defects in generating membrane potential. Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains. Acridine orange staining showed that there was retarded development in stationary phase in nuoG and cydA mutants. Furthermore, up to day 28, a small portion of cells showed high RNA and DNA levels. To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts. Because the OD values increased throughout the whole study in both wild-type and mutant strains, while the viable counts gradually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication.

Acridine Orange↗

Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

Actins↗

Graviperception and gravitaxis in algae.

Photosynthetic flagellates are among the most intensely studied unicellular organisms in the field of graviperception and gravitaxis. While the phenomenon of graviorientation has been known for many decades, only recently was the molecular mechanism unveiled. Earlier hypotheses tried to explain the precise orientation by a passive buoy mechanism assuming the tail end to be heavier than the front. In the photosynthetic flagellate Euglena gracilis, the whole cell body is denser than the surrounding medium, pressing onto the lower cell membrane where it seems to activate mechanosensitive ion channels specific for calcium. The calcium entering the cells during reorientation can be visualized by the fluorescence probe, Calcium Crimson. Cyclic AMP is likewise involved in the molecular pathway. Inhibitors of calcium channels and ionophores impair gravitaxis while caffeine, a blocker of the phosphodiesterase, enhances the precision of orientation.

Animals↗

Isolation and characterisation of a linked cluster of genes from Agrobacterium tumefaciens encoding proteins involved in flagellar basal-body structure.

We report the DNA sequence of 7205 bp of the Agrobacterium tumefaciens chromosome. This contains a putative operon encoding homologues of the flagellar rod and associated proteins FlgBCG and FliE, the L and P ring proteins (FlgHI) a possible flagellum-specific export protein FliP, and two proteins of unknown function, FlgA and FliL. Several of these genes have overlapping stop and start codons. Three non-flagellate Tn5-induced mutations map to this operon: fla-11 to the first gene, encoding the rod protein FlgB; fla-15 to flgA; and fla-12 to fliL. A site-specific mutation introduced into the final gene in this cluster, fliP, also resulted in a non-flagellate phenotype. This indicates that the operon is expressed, and that at least FlgB, FlgA, FliL and FliP are required for flagellar assembly in A. tumefaciens. The bulk of this operon is conserved in the same order in Rhizobium meliloti.

Agrobacterium tumefaciens↗

Dual cellulose-digesting system of the wood-feeding termite, Coptotermes formosanus Shiraki.

The distribution of endo-beta-1,4-glucanase (EG) components in the digestive system of the wood-feeding termite, Coptotermes formosanus Shiraki, was investigated by zymogram analysis using polyacrylamide gel electrophoresis, followed by N-terminal protein sequencing. EG components similar to glycoside hydrolase family (GHF) 9 members were restricted to the salivary glands, the foregut, and the midgut, whereas components similar to GHF7 members were confined to the hindgut where numerous cellulolytic flagellates were harbored. RT-PCR experiments revealed that five GHF9 EG mRNAs (1348 bp) homologous to other termite EGs were expressed in the salivary glands and the midgut. The crude extract prepared from the midgut as well as that from the hindgut produced glucose from crystalline cellulose. These data suggest that C. formosanus has two independent cellulose-digesting systems: one in the midgut where cellulose digestion is accomplished by endogenous cellulases and the other in the hindgut which makes use of other cellulases possibly from symbiotic flagellates.

Acrylic Resins↗

Systematics of the enigmatic kathablepharids, including EM characterization of the type species, Kathablepharis phoenikoston, and new observations on K. remigera comb.nov.

The colorless flagellate Kathablepharis has consisted of five species based on light microscopic studies, and the ultrastructure of the type species, Kathablepharis phoenikoston, is described for the first time. The heterotrophic, marine flagellate Leucocryptos consisted of two species, but additional ultrastructural details for one of these, Kathablepharis remigera comb. nov. (= Leucocryptos remigera Vørs), indicates that it should be transferred to Kathablepharis. The cellular structure of these two species is similar to previously studied kathablepharids. However, there is variation in the feeding apparatus and cytoskeleton. The feeding apparatus of both species has a cytostome, a cytostomal ring, and cytopharyngeal rings. The cytoskeleton consists of inner microtubular arrays and outer or sub-pellicular microtubular arrays. In addition, several features of the flagellar apparatus are described for K. phoenikoston and K. remigera. The ultrastructure of these two species is compared with other kathablepharids to evaluate their taxonomy and phylogeny. We classify Kathablepharis and Leucocryptos in the family Kathablepharididae incertae sedis.

Animals↗

Virulence of Salmonella enterica serotype Enteritidis aflagellate and afimbriate mutants in a day-old chick model.

Certain fimbriae and the flagellae of Salmonella enterica serovar Typhimurium have been shown to contribute to attachment and invasion of gut epithelium in the murine typhoid infection model and to contribute to pathogenesis in the chick. However, little is known of the role these organelles play in Enteritidis poultry infections and, to study this, day-old chicks were dosed orally in separate experiments with defined multiply afimbriate and/or aflagellate mutant strains of Enteritidis. The colonization and invasion characteristics of each mutant were compared with those of the isogenic wild type strain by the determination of the number of bacteria recovered from livers and spleens at known time points post infection. Compared with wild type Enteritidis, a mutant unable to express flagella but retaining the genetic potential to express fimbriae was recovered post mortem from livers and spleens in significantly reduced numbers compared to the isogenic wild-type at all time points post infection (P < 0.001). Conversely, a flagellate but multiply afimbriate mutant (defective for the elaboration of five different fimbrial types) and a flagellate but non-motile 'paralysed' mutant were recovered from livers and spleens in similar numbers to the wild-type. The data suggested that Enteritidis flagella, but not fimbriae, played an important role in pathogenesis in the chick model and that the flagellar apparatus itself and not motility per se contributed significantly to this role.

Animals↗

Electrochemical system for the simultaneous monitoring of algal motility and phototaxis.

A compact electrochemical system equipped with dual electrodes was developed for the simultaneous monitoring of motility and phototaxis of flagellates. Photoinduced behavior of unicellular flagellate alga Chlamydomonas reinhardtii in the presence of diltiazem, azide, or ethanol was recorded as changes in the redox currents for a coexisiting redox marker. The system responded to the chemicals in significantly different ways; it allows qualitative and semiquantitative evaluation of influences of chemical species on the algal cells. Therefore, the present system is potentially applicable to not only aquatic risk assessment but also screening of newly found and synthesized compounds in terms of hazard probability and severity. Additionally, it unveiled some new and interesting behaviors of the algae.

Animals↗

Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant evolution.

Sequence comparisons suggest that all living green plants belong to one of two major phyla: Streptophyta (land plants and their closest green algal relatives, the charophytes); and Chlorophyta (the rest of green algae). Because no green algae are known that pre-date the Streptophyta/Chlorophyta split, and also because the earliest diverging green algae show considerable morphological variation, the nature of the unicellular flagellate ancestor of the two green plant phyla is unknown. Here we report that the flagellate Mesostigma viride belongs to the earliest diverging green plant lineage discovered to date. We have sequenced the entire chloroplast DNA (118,360 base pairs) of this green alga and have conducted phylogenetic analyses of sequences derived from this genome. Mesostigma represents a lineage that emerged before the divergence of the Streptophyta and Chlorophyta, a position that is supported by several features of its chloroplast DNA. The structure and gene organization of this genome indicate that chloroplast DNA architecture has been extremely well conserved in the line leading to land plants.

Chlorophyta↗