Revised karyotyping and gene mapping of the Biomphalaria glabrata embryonic (Bge) cell line

The fresh water snail Biomphalaria glabrata (2n = 36) belongs to the taxonomic class Gastropoda (family Planorbidae) and is integral to the spread of the human parasitic disease schistosomiasis. The importance of this mollusc is such that it has been selected as a model molluscan organism for whole genome sequencing. In order to understand the structure and organisation of the B. glabrata’s genome it is important that gene mapping studies are established. Thus, we have studied the genomes of two B. glabrata embryonic (Bge) cell line isolates 1 and 2 grown in separate laboratories, but both derived from Eder L. Hansen’s original culture from the 1970s. This cell line continues to be an important tool and model system for schistosomiasis and B. glabrata. Using these cell line isolates, we have investigated the genome content and established a revised karyotype based on chromosome size and centromere position for these cells. Unlike the original karyotype (2n = 36) established for the cell line, our investigations now show the existence of extensive aneuploidy in both cell line isolates to the extent that the total complement of chromosomes in both greatly exceeds the original cell line’s diploid number of 36 chromosomes. The isolates, designated Bge 1 and 2, had modal chromosome complements of 64 and 67, respectively (calculated from 50 metaphases). We found that the aneuploidy was most pronounced, for both isolates, amongst chromosomes of medium metacentric morphology. We also report, to our knowledge for the first time using Bge cells, the mapping of single-copy genes peroxiredoxin (BgPrx4) and P-element induced wimpy testis (piwi) onto Bge chromosomes. These B. glabrata genes were mapped onto pairs of homologous chromosomes using fluorescence in situ hybridization (FISH). Thus, we have now established a FISH mapping technique that can eventually be utilized for physical mapping of the snail genome.     

Effect of amphotericin B on the infection success of Schistosoma mansoni in Biomphalaria glabrata

In the present study, we examined the effect of amphotericin B on larval stages (miracidia and primary sporocyst) of the helminth Schistosoma mansoni, the causative agent of human schistosomiasis. Amphotericin B (AmB) is a polyene macrolide that disturbs the function of the cell membrane; it is widely used as prophylactic antimycotic agent in in vitro culture. We show for the first time that S. mansoni miracidia infectivity is considerably reduced after AmB treatment. Moreover we demonstrate that AmB does not affect the development, growth, viability, and behavior of miracidia and primary sporocysts. Our data indicate that AmB effects on S. mansoni sporocyst prevalence are linked to the oxidative properties of AmB. These may alter the capacity of sporocysts to respond to the oxidative stress generated by the snail immune defence system.     

Characterization of the mannose 6-phosphate receptor (Mr 300 kDa) protein dependent pathway of lysosomal enzyme targeting in Biomphalaria glabrata mollusc cells

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, β-hexosaminidase and α-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of ∼20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of ∼13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, β-hexosaminidase and α-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.     

Female biased sex-ratio in Schistosoma mansoni after exposure to an allopatric intermediate host strain of Biomphalaria glabrata

For parasites that require multiple hosts to complete their development, the interaction with the intermediate host may have an impact on parasite transmission and development in the definitive host. The human parasite Schistosoma mansoni needs two different hosts to complete its life cycle: the freshwater snail Biomphalaria glabrata (in South America) as intermediate host and a human or rodents as final host. To investigate the influence of the host environment on life history traits in the absence of selection, we performed experimental infections of two B. glabrata strains of different geographic origin with the same clonal population of S. mansoni. One B. glabrata strain is the sympatric host and the other one the allopatric host. We measured prevalence in the snail, the cercarial infectivity, sex-ratio, immunopathology in the final host and microsatellite frequencies of individual larvae in three successive generations.     

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.     

Bursts of transposition from non-long terminal repeat retrotransposon families of the RTE clade in Schistosoma mansoni

The genus Schistosoma is composed of blood flukes that infect vertebrates, from which three species are major causative agents of human schistosomiasis, a tropical disease that affects more than 200 million people. Current models of the recent evolution of Schistosoma indicate multiple events of migration and speciation from an Asian ancestral species. Transposable elements are important drivers of genome evolution and have been hypothesised to have an important role in speciation. In this work, we describe a comprehensive inventory of Schistosoma mansoni and Schistosoma japonicum retrotransposons, based on their recently published genomic data. We find a considerable difference in retrotransposon representation between the two species (22% and 13%, respectively). A large part of this difference can be attributed to higher representation of two previously described families of S. mansoni retrotransposons (SR2 and Perere-3/SR3), compared with the representation of their closest relative families in S. japonicum. A more detailed analysis suggests that these two S. mansoni families were the subject of recent bursts of transposition that were not paralleled by their S. japonicum counterparts. We hypothesise that these bursts could be a consequence of the evolutionary pressure resulting from migration of Schistosoma from Asia to Africa and their establishment in this new environment, helping both speciation and adaptation.     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Evolutionary divergence of human chromosome 9 as revealed by the position of the ABL protooncogene in higher primates

Attempts to solve the fundamental questions regarding the descent of man are dogged by superstitions and unexamined orthodoxies. The origin of humans, established a decade ago based upon cytological analysis of ape chromosomes, continues to be called into question. Although molecular methods have provided a framework for tracing the paths of human evolution, conclusive evidence remains elusive. We have used a single ABL gene probe derived from human chromosome 9 to assess the direction of change in the equivalent ape chromosomes. This approach has resulted in a few surprises which again challenge the prevailing view of early primate evolution based solely on chromosome banding patterns. The ABL protooncogene is present on human chromosome 9 at band q34. Similar DNA sequences presumed to represent an ABL gene, are present on chromosome 11 in chimpanzee (Pan troglodytes) but at a different relative location, indicating that the mechanism of the origin of human chromosome 9 is far more complex than has previously been suggested. Nevertheless, in gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus), the equivalent to human chromosome band 9 q34 is apparently located on chromosome 13 at a putative telomeric position and no discernible differences could be established. Despite the presence of the ABL protooncogene on human equivalent ape chromosomes, molecular systematics will continue to generate enigmas in the evolutionary context until the entire genome is sequenced.     

Fluorescence in situ hybridization for intracellular localization of nifH mRNA

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.     

Development of Schistosoma mansoni in the laboratory rat analyzed by light and confocal laser scanning microscopy

The kinetic of maturation (schistogram) of Schistosoma mansoni worms grown in laboratory rats was studied by light and confocal laser scanning microscopy. Infected rats with the BH strain were weekly euthanized 3-9 weeks pi. Recovered flukes stained with hydrochloric carmine were preserved as whole-mounts and analyzed by confocal and brightfield microscopy. Worms displayed varying degrees of maturation of the reproductive system at weeks 3-6. Male worms showed complete maturation of the reproductive system at week 6, while female worms completed their maturation at week 7. Males presented few tubercles in tegument in all weeks. Despite the presence of a developing embryo within the ootype, no uterine egg was found. The schistogram in rats follows a pattern similar to that observed in mice hosts.     

Isolation and characterization of genome-specific markers in Oryza species with the BB genome

Wild species of rice with many valuable agronomic traits are an important genetic resource for improving cultivated rice by wide hybridization. Genome- or chromosome-specific markers are useful for monitoring genome introgression and for identifying genome components. From 47 random amplified polymorphic DNAs (RAPDs) of nine Oryza species, three bands (Ogla225, Opun225, and Opun246) were found to be genome specific with distinct sizes. Their specificities were further characterized by Southern hybridization, sequence analysis, and fluorescent in situ hybridization (FISH). Ogla225 is specifically amplified from the AA genome but homologous sequences were conserved among Oryza species. Opun225 occurs at a low copy number although is specifically amplified from Oryza punctata. There are estimated 2000-3300 repeats of Opun246 in each haploid genome of Oryza species with the BB or BBCC genome. Clusters of Opun246 repeats were detected at heterochromatic regions on almost all chromosomes of the BB genomes by FISH. Opun246 may be a useful marker for monitoring the introgression of BB genome or for identifying the conserved components of BB genome in genetic resource. The results from this study and our previous study both indicate that numerous unique repeats play role in the differentiation of the BB genome from other Oryza genomes.     

Schistosoma mansoni: alterations in total protein and hemoglobin in the hemolymph of infected Biomphalaria glabrata

The total protein concentrations in the hemolymph of noninfected Biomphalaria glabrata and those parasitized by Schistosoma mansoni have been compared. That of the former, averaging 1.42 g/100 ml, is greater than that of the latter which averages 0.96 g/100 ml. The hemoglobin concentrations in the two categories of snails have also been compared and that in noninfected specimens, averaging 0.956 g/100 ml, is higher than that in infected snails which average 0.650 g/100 ml. It has also been ascertained that both the total protein and hemoglobin concentrations in snails of different sizes vary; specimens measuring between 10 and 16 mm in diameter tend to include more hemoglobin than larger ones. On the other hand, larger snails tend to include greater total protein concentrations.     

Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: Differential expression during the miracidium-to-sporocyst transformation

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, α3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail–schistosome interactions. Interestingly, Fucα2Fucα3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.     

The complete mitochondrial genome sequence and gene organization of the mud crab (Scylla paramamosain) with phylogenetic consideration

The complete mitochondrial genome is of great importance for better understanding the genome-level characteristics and phylogenetic relationships among related species. In the present study, we determined the complete mitochondrial genome DNA sequence of the mud crab (Scylla paramamosain) by 454 deep sequencing and Sanger sequencing approaches. The complete genome DNA was 15,824 bp in length and contained a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). Of 37 genes, twenty-three were encoded by the heavy strand (H-strand), while the other ones were encoded by light strand (L-strand). The gene order in the mitochondrial genome was largely identical to those obtained in most arthropods, although the relative position of gene tRNA^His differed from other arthropods. Among 13 protein-coding genes, three (ATPase subunit 6 (ATP6), NADH dehydrogenase subunits 1 (ND1) and ND3) started with a rare start codon ATT, whereas, one gene cytochrome c oxidase subunit I (COI) ended with the incomplete stop codon TA. All 22 tRNAs could fold into a typical clover-leaf secondary structure, with the gene sizes ranging from 63 to 73 bp. The phylogenetic analysis based on 12 concatenated protein-coding genes showed that the molecular genetic relationship of 19 species of 11 genera was identical to the traditional taxonomy.