Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Bacterial diversity of the digestive gland of Sydney rock oysters,Saccostrea glomerata infected with the paramyxean parasite,Marteilia sydneyi

Aims: To determine whether the infestation by the protozoan paramyxean parasite, Marteilia sydneyi, changes the bacterial community of the digestive gland of Sydney rock oysters, Saccostrea glomerata. Methods and Results: Six 16S rDNA clone libraries were established from three M. sydneyi‐infected and three un‐infected oysters. Restriction enzyme analysis followed by sequencing representative clones revealed a total of 23 different operational taxonomic units (OTUs) in un‐infected oysters, comprising the major phyla: Firmicutes, Proteobacteria, Cyanobacteria and Spirocheates, where the clone distribution was 44, 36, 7 and 5%, respectively. Close to half of the OTUs are not closely related to any other hitherto determined sequence. In contrast, S. glomerata infected by M. sydneyi had only one OTU present in the digestive gland. Phylogenetic analysis of the 16S rDNA sequence reveals that this dominant OTU, belonging to the α‐Proteobacteria, is closely related to a Rickettsiales‐like prokaryote (RLP). Conclusions: The microbiota of the digestive gland of Sydney rock oysters is changed by infection by M. sydneyi, becoming dominated by a RLP, and generally less diverse. The bacterial community of un‐infected S. glomerata differs from previous studies in that we identified the dominant taxa as Firmicutes and α‐Proteobacteria, rather than heterotrophic γ‐Proteobacteria. Significance and Impact of the Study: This is the first culture‐independent study of the microbiota of the digestive glands of edible oysters to the species level. The commercial viability of the Sydney rock oyster industry in Australia is currently threatened by Queensland Unknown disease and the changes in the bacterial community of S. glomerata corresponding with infection by M. sydneyi sheds further light on the link between parasite infection and mortality in this economically damaging disease.     

Molecular characterisation of a haplosporidian parasite infecting rock oysters Saccostrea cuccullata in north Western Australia

A haplosporidian parasite was identified in rock oysters (Saccostrea cuccullata Born, 1778) from the Montebello Islands (latitude -20.4'S longitude 115.53'E) off the northern coast of Western Australia by histopathological examination, PCR amplification and DNA sequencing of a segment of the SSU region of the parasite's rRNA gene. An oligonucleotide probe was constructed from the parasite's SSU rRNA gene in order to confirm its presence by in situ hybridisation. The parasite was disseminated throughout the gonad follicles of the host and to a lesser extent in the gills. The only parasite life stages thus far observed in this study were a uninucleate naked cell assumed to be a precursor to multinucleate plasmodial stages and a binucleate plasmodial stage. Whilst no parasite spores were detected in affected rock oysters, a phylogenetic analysis of the SSU region of the parasite's rRNA gene indicates the parasite belongs to the genus Minchinia. A PCR and in situ hybridisation assay for the Minchinia sp. was used to identify haplosporidians described by Hine and Thorne [Hine, P.M.., Thorne, T., 2002. Haplosporidium sp. (Haplosporidia: Haplosporidiidae) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia. Dis. Aquat. Organ. 51, 123-13], in archived rock oyster tissues from the same coastline.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

First record of Perkinsus olseni, a protozoan parasite infecting the commercial clam Ruditapes decussatus in Spanish Mediterranean waters

We present the first record in Spanish Mediterranean waters of the protozoan parasite Perkinsus olseni infecting the clam Ruditapes decussatus. Perkinsus infection was detected all year around albeit at a low level of infection intensity. Histological analysis, induction of zoospores and in situ hybridisation assay confirmed the presence of Perkinsus sp. The identity of the parasite was determined by species-specific PCR assay in DNA samples obtained from infected clams. Sequencing of amplified fragments showed 100% identity to the ITS region of P. olseni. We confirmed for the first time the presence of P. olseni in Spanish Mediterranean waters.     

First report of three protozoan parasites (a haplosporidian, Marteilia sp. and Marteilioides sp.) from the Manila clam, Venerupis (=Ruditapes) philippinarum in Japan

Recently, natural stocks of the Manila clam, Venerupis (=Ruditapes) philippinarum, have been drastically reduced in Japan. To clarify the reason for this decline in number, clams were sampled monthly from Yamaguchi and processed for histological observations, during which three protozoan parasites were discovered. Transmission electron microscopy revealed that these parasites were unidentified haplosporidian in the connective tissues, Marteilia sp. in the digestive gland and Marteilioides sp. in the oocytes. Histopathological observations suggest that Marteilia sp. and Marteilioides sp. were not pathogenic to the host. However, infection with a haplosporidian may have a negative impact on the clams. The prevalence of these parasites was low and further investigations should be undertaken to clarify their taxonomic status and establish any pathogenicity to clams.     

Steinernema feltiae: ammonia triggers the emergence of their infective juveniles

Entomopathogenic nematodes complete their life cycles inside dead insects. The emergence of new infective juveniles from the cadaver has been attributed (but never demonstrated) to food depletion or to the accumulation of metabolites from the breakdown of the host's tissues. Here we give evidence that emergence is triggered by ammonia, a product of nematode defecation. We found that the emergence of Steinernemafeltiae infective juveniles from Galleriamellonella cadavers was stimulated by a particular level of ammonia. Emergence was delayed when ammonia in the cadaver was decreased and was prompted when increased. These findings will further improve the understanding of the nematode life cycle. Here we speculate that production of infective juveniles can be mediated by ammonia and work in a manner analogous to that of the dauer recovery inhibiting factor (DRIF) in Caenorhabditiselegans.     

In vitro effects of temperature and salinity on fatty acid synthesis in the oyster protozoan parasite Perkinsus marinus

The effects of temperature and salinity on fatty acid synthetic activities in the oyster protozoan parasite, Perkinsus marinus, were tested in vitro at 10, 18 and 28 °C in a salinity of 28 psu and 14, 20 and 28 psu at a temperature of 28 °C using ¹³C sodium acetate as a substrate. Salinity treatments exhibited few treatment effects, but temperature significantly affected cell proliferation, fatty acid content and fatty acid synthesis rates. Fatty acid synthesis rates increased approximately two-fold for every 10 °C increase in temperature; however, the predominant fatty acid synthesized differed between treatments. At 10 °C, the synthesis rate for 18:1(n−9) was not significantly different from the 18 °C treatment and weight percent of 18:1(n−9) was higher at 10 than 18 and 28 °C. In contrast, the synthesis rate for 20:4(n−6) was over five times lower at 10 than at 18 and 28 °C, and the percent fatty acid content of 20:4(n−6) was over two-fold lower at 10 than at 18 and 28 °C. Results suggest that further elongation and desaturation of 18:1(n−9) to 20 carbon polyunsaturated fatty acids may be inhibited at low temperatures. These findings may be relevant to field observations that disease progression and virulence of this parasite are correlated to high water temperatures.     

Involvement of nitric oxide in the gastroprotective effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen, Amaranthaceae, in rats

The plants belonging to Pfaffia genus are used in folk medicine to treat gastric disturbances. This study examined the effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen (AEP) on the gastrointestinal tract. Wistar rats were pretreated orally (p.o.) with the AEP (125, 250, 500 and 1000 mg.kg(-1)) before induction of ulcers by hypothermic restraint stress (HRS, 3 h restraint stress at 4 degrees C), ethanol (ET, 70%; 0.5 ml/animal; p.o.) or indomethacin (IND, 20 mg.kg(-1); s.c.). Control animals received water (C) or ranitidine (60 mg.kg(-1)) p.o. The AEP protected rats against HRS and ET-induced ulcers, but was not able to protect the gastric mucosa against IND-induced ulcers. When injected into the duodenal lumen, the AEP reduced total acidity and both basal and histamine-stimulated acid secretion in pylorus-ligated rats. In addition, gastric secretion from AEP-treated animals exhibited increased concentrations of nitrite and nitrate. Treatment of animals with L-NAME (120 mg.kg(-1), p.o.) prevented both the reduction of total acidity and the increase in NOx levels promoted by AEP treatment. In conclusion, AEP effectively protected the gastric mucosa and inhibited gastric acid secretion in rats, probably by involving the histaminergic pathway and an enhanced production of nitric oxide in the stomach.     

Developmental stages and molecular phylogeny of Hepatozoon tuatarae, a parasite infecting the New Zealand tuatara, Sphenodon punctatus and the tick, Amblyomma sphenodonti

The developmental stages of Hepatozoon tuatarae were elucidated in both the tuatara host, Sphenodon punctatus and the tick, Amblyomma sphenodonti. PCR amplicons from A. sphenodonti samples identified DNA matching H. tuatarae. Dissection of tick samples showed oogenesis and sporogony occurring in the haemocoel of A. sphenodonti with the average mature oocyst size being 236 × 228 μm. Partial sequence data of the parasite’s small subunit ribosomal gene, obtained by PCR, was used for phylogenetic comparison. Characterisation of the H. tuatarae lifecycle will help in conservation management of the tuatara.     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.     

Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.     

Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9^+/−;Msx1^+/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.     

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae

Neobenedenia girellae, a monogenean, is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. Aiming to establish a new control method by interfering with the gametogenesis of N. girellae, we focused on vasa (vas)-related genes that are expressed exclusively in the germline granules in Drosophila, Caenorhabditis elegans and other animals. Three vas-related genes (N. girellae vasa-like gene, Ngvlg1, Ngvlg2 and Ngvlg3) were isolated by PCR and Ngvlg1 and Ngvlg2 were shown to be expressed only in germ cells. We demonstrated that introduction of double-stranded Ngvlg1 or Ngvlg2 RNA by soaking resulted in partial or complete loss of germ cells. Moreover, the hatching rate of eggs from animals showing partial loss of germ cells decreased significantly. These results suggest that Ngvlg1 and Ngvlg2 are essential genes for germ cell quantity and quality. The possibility that a new control method can be developed by controlling gametogenesis of N. girellae was proven, because sterilised N. girellae could be produced.     

Isolation of putative type 2 metallothionein encoding sequences and spatial expression pattern in the seagrass Posidonia oceanica

Metallothioneins (MTs) are a group of proteins with low molecular mass and high cysteine content that bind to heavy metals and are thought to play a role in their metabolism and detoxification. Genes encoding MT-like proteins have been isolated in a number of plants. In this work we isolated nine MT-like sequences from copper- or cadmium-exposed plants of the seagrass Posidonia oceanica, a marine Angiosperm playing a major role in maintaining infralittoral ecosystems in the Mediterranean sea. These sequences, together with two other MT genes previously isolated from this species, show high similarities with genes encoding type 2 MTs. Neighbour-joining analysis, at both deduced protein and 3′-UTR sequence level, indicates that at least two subgroups occur within Posidonia type 2 MTs, showing, however, a strong sequence uniformity. Southern analysis of two type 2 MT-encoding sequences (Pomt2b and Pomt2f) belonging to the two different subgroups showed distinct hybridisation patterns. For both type 2 MTs, we have determined, by in situ technique, the expression domain in Posidonia plants. The members of these two MT subgroups show differences in their histological expression, with Pomt2b associated with proliferative tissues whereas Pomt2f is associated with lignified or suberized cell wall.     

Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica

NAD(H) kinase catalyzes the phosphorylation of NAD(H) to form NADP(H) using ATP or inorganic polyphosphate as a phosphoryl donor. While the enzyme is conserved throughout prokaryotes and eukaryotes, remarkable differences in kinetic parameters including substrate preference, cation dependence, and physiological roles exist among the organisms. In the present study, we biochemically characterized NAD(H) kinase from the anaerobic/microaerophilic fermentative protozoan parasite Entamoeba histolytica, which lacks the conventional mitochondria capable of oxidative phosphorylation, leading to ATP. The kinetic properties of E. histolytica NAD(H) kinase recombinantly produced in Escherichia coli showed remarkable differences from those in bacteria and higher eukaryotes. Entamoeba NAD(H) kinase preferred NADH to NAD+ as the phosphoryl acceptor, utilized nucleoside triphosphates including ATP, GTP and deoxyATP, but not nucleoside di-, mono-phosphates, or inorganic polyphosphates, as the phosphoryl donor. To further understand the physiological roles in E. histolytica, we generated a stable transformant overexpressing NAD(H) kinase. Overexpression of NAD(H) kinase resulted in a 1.6–2 fold increase in the NADPH and NADP+ concentrations, a 40% reduction of the intracellular concentration of reactive oxygen species, and also led to increased tolerance toward hydrogen peroxide. These data, together with the essentially of NAD(H) kinase gene, underscore its significance as an NADP(H)-producing enzyme in this organism, and should help in designing of drugs targeting this enzyme.