A Comparative Characterization of the Mitochondrial Genomes of Paramoeba aparasomata and Neoparamoeba pemaquidensis (Amoebozoa,Paramoebidae)

Marine amebae of the genus Paramoeba (Amoebozoa, Dactylopodida) normally contain a eukaryotic endosymbiont known as Perkinsela‐like organism (PLO). This is one of the characters to distinguish the genera Neoparamoeba and Paramoeba from other Dactylopodida. It is known that the PLO may be lost, but PLO‐free strains of paramoebians were never available for molecular studies. Recently, we have described the first species of the genus Paramoeba which has no parasome—Paramoeba aparasomata. In this study, we present a mitochondrial genome of this species, compare it with that of Neoparamoeba pemaquidensis, and analyze the evolutionary dynamics of gene sequences and gene order rearrangements between these species. The mitochondrial genome of P. aparasomata is 46,254 bp long and contains a set of 31 protein‐coding genes, 19 tRNAs, two rRNA genes, and 7 open reading frames. Our results suggest that these two mitochondrial genomes within the genus Paramoeba have rather similar organization and gene order, base composition, codon usage, the composition and structure of noncoding, and overlapping regions.     

Microheterogeneity and coevolution: an examination of rDNA sequence characteristics in Neoparamoeba pemaquidensis and its prokinetoplastid endosymbiont

Neoparamoeba pemaquidensis, the etiological agent of amoebic gill disease, has shown surprising sequence variability among different copies of the 18S ribosomal RNA gene within an isolate. This intra-genomic microheterogeneity was confirmed and extended to an analysis of the internal transcribed spacer (ITS) region. High levels of intra-genomic nucleotide diversity (Pi=0.0201-0.0313) were found among sequenced ITS regions from individual host amoeba isolates. In contrast, the ITS region of its endosymbiont revealed significantly lower levels of intra-genomic nucleotide diversity (Pi=0.0028-0.0056) compared with the host N. pemaquidensis. Phylogenetic and ParaFit coevolution analyses involving N. pemaquidensis isolates and their respective endosymbionts confirmed a significant coevolutionary relationship between the two protists. The observation of non-shared microheterogeneity and coevolution emphasizes the complexity of the interactions between N. pemaquidensis and its obligate endosymbiont.     

Characterization of Neoparamoeba pemaquidensis strains: PCR-RFLP of the internal transcribed spacer region from the amoeba and endosymbiont

Neoparamoeba pemaquidensis continues to be an ongoing problem for commercial finfish aquaculture and has also sporadically been associated with mass mortalities of commercially relevant marine invertebrates. Despite the ubiquity and importance of this amphizoic amoeba, our understanding of the biology as it applies to host range, pathogenicity, tissue tropism, and geographic distribution is severely lacking. This may stem from the inability of current diagnostic tests based on morphology, immunology, and molecular biology to differentiate strains at the subspecies level. In the present study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the internal transcribed spacer (ITS) region that can accurately differentiate amoeba strains of N. pemaquidensis. The investigation focused on the complications of the amoeba ITS microheterogeneity in the development of a subspecies marker and the use of the endosymbiont, Ichthyobodo necator related organism (IRO), ITS region as an alternative marker. The combination of host amoeba and endosymbiont ITS PCR-RFLP analyses was successfully used to correctly identify and characterize an N. pemaquidensis isolate from an outbreak of amoebic gill disease in Atlantic salmon Salmo salar from the west coast of North America (Washington State, USA).     

Neoparamoeba pemaquidensis引起的海水鱼阿米巴鳃病

海水养殖的鲑鱼及鲽鱼(Scophthalmusmaximus)的阿米巴鳃病是由Neoparamoebapemaquidensis引起的。在西班牙,该病对鲑鱼的海水养殖造成巨大损失,同时也正威胁着鲽鱼的养殖。组织病理损伤主要是鱼鳃上皮细胞的增生和肥大。该虫仅寄生在鱼鳃表面。现已有证据证明,非特异免疫参与鱼类抵御该病,但还没有证明特异性免疫在此过程中发挥作用的相似证据。对鲑鱼来说,治疗该病惟一有效的方法就是用淡水浸泡     

Cultured gill-derived Neoparamoeba pemaquidensis fails to elicit amoebic gill disease (AGD) in Atlantic salmon Salmo salar

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.     

Molecular characterization of Iranian Trichogrammatids (Hymenoptera: Trichogrammatidae) and their Wolbachia endosymbiont

During 2009–2010, a field survey of native Trichogramma species was carried out in six provinces of Iran, including Khorasan Razavi, Tehran, Mazandaran, Guilan, Golestan, and Qom. In this study, a molecular method for identifying Trichogramma and for determining the prevalence of Wolbachia in those species was used. Based on ITS2 (internal transcribed spacer 2) sequence, 14 populations were identified as the species T. embryophagum, T. evanescens, or T. brassicae. Wolbachia infection in these Trichogrammatids was detected using wsp gene sequencing. The highest infection rates in Trichogramma were found in Mazandaran and Golestan provinces. There was no evidence of infection in Trichogramma species in Guilan and Qom provinces. Of the three infected populations, two populations of T. evanescens were infected with only one Wolbachia strain from sib subgroup and one population was superinfected. Here, we report the first data on molecular characterization of Iranian Trichogrammatids and their Wolbachia-endosymbionts.     

18S ribosomal DNA-based PCR identification of Neoparamoeba pemaquidensis, the agent of amoebic gill disease in sea-farmed salmonids

Neoparamoeba pemaquidensis is a parasomal amoeboid protozoan identified as the agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar reared in sea-pens in Tasmania, Australia, and coho salmon Oncorhynchus kisutch farmed on the west coast of the USA. Outbreaks of AGD caused by immunologically cross-reactive paramoebae have also been reported in sea-farmed salmonids in several other countries. Complete 18S rDNA sequences were determined for respective paramoebae isolated from infected gills of salmon from Tasmania and Ireland, and N. pemaquidensis isolates from the USA and UK, including representative free-living isolates. Alignments over 2110 bp revealed 98.1 to 99.0% sequence similarities among isolates, confirming that paramoebae implicated in AGD in geographically distant countries were homologous and belonged to the same species, N. pemaquidensis. The results supported previous findings that N. pemaquidensis exists as a widely distributed, amphizoic marine protozoan. Partial 18S rDNA sequences were obtained for the ultrastructurally similar species, N. aestuarina, and for the morphologically similar but non-parasomal amoeba Pseudoparamoeba pagei. N. aestuarina had 95.3 to 95.7% sequence similarities with N. pemaquidensis strains, which distinguished 2 closely related but separate species. Neoparamoeba spp. were not analogous to P. pagei or to other marine Gymnamoebia. We designed 4 oligonucleotide primers based on elucidated 18S rDNA sequences and applied them to single-step and nested 2-step PCR protocols developed to identify N. pemaquidensis to the exclusion of apparently closely related and non-related protistan taxa. Nested PCR was able to detect the AGD parasite from non-purified, culture-enriched net microfouling samples from Atlantic salmon sea-pens in Tasmania, and confirmed that N. pemaquidensis was also responsible for AGD in chinook salmon O. tshawytscha in New Zealand. Our sequence and PCR analyses have now shown that AGD affecting 3 different salmonid species farmed in 4 countries are associated with N. pemaquidensis. A species-specific diagnostic PCR provides for the first time, a highly specific detection and identification assay for N. pemaquidensis that will facilitate future ecological and epidemiological studies of AGD.     

Phylogeny of the Highly Divergent Echinosteliales (Amoebozoa)

Myxomycetes or plasmodial slime molds are widespread and very common soil amoebae with the ability to form macroscopic fruiting bodies. Even if their phylogenetic position as a monophyletic group in Amoebozoa is well established, their internal relationships are still not entirely resolved. At the base of the most intensively studied dark‐spored clade lies the order Echinosteliales, whose highly divergent small subunit ribosomal (18S) RNA genes represent a challenge for phylogenetic reconstructions. This is because they are characterized by unusually long variable helices of unknown secondary structure and a high inter‐ and infraspecific divergence. Current classification recognizes two families: the monogeneric Echinosteliaceae and the Clastodermataceae with the genera Barbeyella and Clastoderma. To better resolve the phylogeny of the Echinosteliales, we obtained three new small subunit ribosomal (18S) RNA gene sequences of Clastoderma and Echinostelium corynophorum. Our phylogenetic analyses suggested the polyphyly of the family Clastodermataceae, as Barbeyella was more closely related to Echinostelium arboreum than to Clastoderma, while Clastoderma debaryanum was the earliest branching clade in Echinosteliales. We also found that E. corynophorum was the closest relative of the enigmatic Semimorula liquescens, a stalkless‐modified Echinosteliales. We discuss possible evolutionary pathways in dark‐spored Myxomycetes and propose a taxonomic update.     

Biochemical characterization of Recombinase A from Wolbachia endosymbiont of filarial nematode Brugia malayi (wBmRecA)

Lymphatic filariasis is a debilitating disease that affects over 890 million people in 49 countries. A lack of vaccines, non-availability of adulticidal drugs, the threat of emerging drug resistance against available chemotherapeutics and an incomplete understanding of the immunobiology of the disease have sustained the problem. Characterization of Wolbachia proteins, the bacterial endosymbiont which helps in the growth and development of filarial worms, regulates fecundity in female worms and mediates immunopathogenesis of Lymphatic Filariasis, is an important approach to gain insights into the immunopathogenesis of the disease. In this study, we carried out extensive biochemical characterization of Recombinase A from Wolbachia of the filarial nematode Brugia malayi (wBmRecA) using an Electrophoretic Mobility Shift Assay, an ATP binding and hydrolysis assay, DNA strand exchange reactions, DAPI displacement assay and confocal microscopy, and evaluated anti-filarial activity of RecA inhibitors. Confocal studies showed that wBmRecA was expressed and localised within B. malayi microfilariae (Mf) and uteri and lateral chord of adult females. Recombinant wBmRecA was biochemically active and showed intrinsic binding capacity towards both single-stranded DNA and double-stranded DNA that were enhanced by ATP, suggesting ATP-induced cooperativity. wBmRecA promoted ATP hydrolysis and DNA strand exchange reactions in a concentration-dependent manner, and its binding to DNA was sensitive to temperature, pH and salt concentration. Importantly, the anti-parasitic drug Suramin, and Phthalocyanine tetrasulfonate (PcTs)-based inhibitors Fe-PcTs and 3,4-Cu-PcTs, inhibited wBmRecA activity and affected the motility and viability of Mf. The addition of Doxycycline further enhanced microfilaricidal activity of wBmRecA, suggesting potential synergism. Taken together, the omnipresence of wBmRecA in B. malayi life stages and the potent microfilaricidal activity of RecA inhibitors suggest an important role of wBmRecA in filarial pathogenesis.     

Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000

RSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the RSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the RSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A RSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these RSA1 genes as 5′ TGTTGT-(X)₁₃-ACAACA. The genomic sequence similarity between RSA1 and related phages 52237 and CTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. RSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the RSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a RSA1-related prophage (designated RSX). RSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. RSX ORFs shared very high amino acid identity with their RSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.     

Reinvestigation of Phryganella paradoxa (Arcellinida,Amoebozoa) Penard 1902

A major drawback in testate amoeba research is a general lack of scientific studies combining molecular approaches and classical laboratory experiments. We isolated five yet uncultured testate amoebae of the genus Phryganella Penard, 1902 from three different rivers and one pond in Germany. Based on established cultures we show their morphology, which we studied by light and electron microscopy, and present their unique feeding mode on abundant and common pennate diatoms like Nitzschia spp. and Synedra spp., whose frustules are bent and frequently, but not always, broken during the feeding process. We further obtained the first SSU rDNA sequences of strains of the family Phryganellidae, all of which contain introns. We used the sequences to confirm the taxonomic placement of the Phryganellidae in the Arcellinida (Amoebozoa), branching as a sister group to the Cryptodifflugiidae.     

Genomic characterization of the human prion protein (PrP) gene locus

Prion protein (PrP) is intimately linked with a class of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). Employing bioinformatics and direct molecular analysis, we demonstrated that the human PrP gene (PRNP) locus, which is situated at Chromosome (Chr) position 20p12-ter, contains three genes within a 55-kb interval: PRNP; DOPPEL or PRND, located 20 kb 3? of PRNP; and a novel gene, designated PRNT, that maps 3 kb 3? to PRND and is transcribed to generate at least three alternatively spliced mRNAs. All three genes of this locus demonstrate low sequence homology, implying that, although they may be evolutionarily related, they are functionally distinct. Analysis of both adult and fetal human tissues confirmed the ubiquitous but variable expression profile of PRNP, with the highest levels observed in the CNS and testis. Contrastingly, although PRND shows a wide tissue expression pattern in fetal tissues, it is expressed exclusively in adult testis, whereas all three PRNT isoforms were detected only in adult testis, implying that PRND is developmentally regulated. An investigation of the regulatory mechanisms underlying this complex gene expression pattern from the PRNP locus should provide insight into the function of these genes and the possible involvement of the non-PrP proteins in the development of TSEs.     

New record of Arcella gandalfi (Amoebozoa: Arcellinida) from Colombia

The reports of testate amoebae species in Colombia are limited to a few studies in lentic and peat bog systems; however, the diversity in lotic systems has been poorly investigated. Zooplankton sampling was performed in lotic environments of the Piedemonte Llanero in Colombia. Ten individuals of the species Arcella gandalfi were identified to document the first record of A. gandalfi from the Piedemonte Llanero in Colombia, expanding its distribution from Brazil to Colombia. This finding corroborates the status of A. gandalfi as a flagship species from the South America continent.     

Morphological and molecular characterization of a new succulenticolous Physarum (Myxomycetes,Amoebozoa) with unique polygonal spores linked in chains

A new plasmodiocarpic and sporocarpic species of myxomycete in the genus Physarum is described and illustrated. This new species appeared on decayed leaves and remains of succulent plants (Agave, Opuntia, Yucca) growing in arid zones. It differs from all other species in the genus in having polyhedral spores linked in chains like a string of beads, a unique feature within all known myxomycetes. Apart from detailed morphological data, partial sequences of both the small-subunit ribosomal RNA and elongation factor 1-alpha genes, generated from four isolates collected in two distant regions, i.e., Mexico and Canary Islands, are also provided in this study. Combined evidence supports the identity of the specimens under study as a new species.     

Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane

A large sugarcane EST (expressed sequence tag) project recently gave us access to 261,609 EST sequences from sugarcane, assembled into 81,223 clusters. Among these, we identified 88 resistance gene analogs (RGAs) based on their homology to typical pathogen resistance genes, using a stringent BLAST search with a threshold e-value of e(-50). They included representatives of the three major groups of resistance genes with NBS/LRR, LRR or S/T KINASE domains. Fifty RGAs showed a total of 148 single-dose polymorphic RFLP markers, which could be located on the sugarcane reference genetic map (constructed in cultivar R570, 2n=approximately 115). Fifty-five SSR loci corresponding to 134 markers in R570 were also mapped to enable the classification of the various haplotypes into homology groups. Several RGA clusters were found. One cluster of two LRR-like loci mapped close to the only disease resistance gene known so far in sugarcane, which confers resistance to common rust. Detailed sequence comparison between two NBS/LRR RGA clusters in relation to their orthologs in rice and maize suggests their polyphyletic origins, and indicates that the degree of divergence between paralogous RGAs in sugarcane can be larger than that from an ortholog in a distant species.