Multilocus patterns of genetic variation across Cryptosporidium species suggest balancing selection at the gp60 locus

Cryptosporidium is an apicomplexan protozoan that lives in most vertebrates, including humans. Its gp60 gene is functionally involved in its attachment to host cells, and its high level of genetic variation has made it the reference marker for sample typing in epidemiological studies. To understand the origin of such high diversity and to determine the extent to which this classification applies to the rest of the genome, we analysed the patterns of variation at gp60 and nine other nuclear loci in isolates of three Cryptosporidium species. Most loci showed low genetic polymorphism (π_S <1%) and similar levels of between‐species divergence. Contrastingly, gp60 exhibited very different characteristics: (i) it was nearly ten times more variable than the other loci; (ii) it displayed a significant excess of polymorphisms relative to between‐species differences in a maximum‐likelihood Hudson–Kreitman–Aguadé test; (iii) gp60 subtypes turned out to be much older than the species they were found in; and (iv) showed a significant excess of polymorphic variants shared across species from random expectations. These observations suggest that this locus evolves under balancing selection and specifically under negative frequency‐dependent selection (FDS). Interestingly, genetic variation at the other loci clusters very well within the groups of isolates defined by gp60 subtypes, which may provide new tools to understand the genome‐wide patterns of genetic variation of the parasite in the wild. These results suggest that gp60 plays an active and essential role in the life cycle of the parasite and that genetic variation at this locus might be essential for the parasite's long‐term success.     

Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos: Evaluation of critical steps of the procedure

Previous trials achieved extremely poor results when using the one-step warming method in a syringe in combination with non-surgical deep intrauterine transfer (NET) of superfine open pulled straw (SOPS)-vitrified embryos. This study aimed to assess the effect of the warming procedure on the in vitro and in vivo development of SOPS-vitrified embryos. The effect of the passage of the vitrified-warmed (VW) embryos through the NET catheter was also evaluated. Groups of 4 to 6 morulae and blastocysts, collected from weaned sows, were SOPS-vitrified in 1 μL of vitrification medium, warmed by the one-step warming method in a dish or in a 1-mL syringe and cultured in vitro for 48 h to evaluate the embryo survival (ES) and hatching rates (HR). Warming in syringe had a deleterious effect (P < 0.05) on the in vitro ES (60.5 ± 10.4%) and HR (39.6 ± 9.5%) of VW embryos in comparison with embryos warmed in a dish (85.4 ± 10.6% and 69.0 ± 8.4%, respectively). This decreased embryonic development was due to the increased time required between the removal of the straws from the liquid nitrogen and the contact of the embryos with the warming medium when the warming was performed in a syringe in comparison with that for the warming in a dish. After verifying that the passage of VW embryos through the NET catheter does not have a damaging effect on their further in vitro development, the negative effect of warming in a syringe was also confirmed after NET. Fifteen fresh and SOPS-vitrified embryos warmed in a syringe or in a dish were transferred to each recipient (n = 28) and recovered 24 h later to assess their developmental progression. All embryos from the syringe group were found to have degenerated at recovery. The in vivo ES and HR from the dish group (80.4 ± 3.4% and 14.2 ± 7.2%, respectively) were lower (P < 0.05) than those from the fresh group (94.0 ± 4.1% and 36.8 ± 7.8%, respectively). Combining the warming in a dish and the NET procedure, 35 VW embryos were transferred to each of 10 gilts. Five recipients farrowed an average of 10.4 ± 0.9 piglets. In conclusion, the method of one-step warming in a syringe has a negative effect on the in vitro and in vivo viability of SOPS-vitrified porcine embryos. In addition, NET of SOPS-vitrified embryos warmed by the one-step method in a dish showed promising reproductive performance of recipients. However, despite the great potential of this technology, further developments are required for large-scale commercial applications.     

Detection of a pederin-like compound using a dilution-to-extinction-based platform for the isolation of marine bacteria in drug discovery strategies

The continued development of culturing technologies for the discovery of new molecules from marine microbes is of paramount importance for drug discovery. Coupled with this, the use of the high-throughput approach shows promise for increasing the number of Gram-negative and non-filamentous bacteria cultures that can be surveyed, since they show a lower potential of bioactivity. In this work, we propose a new strategy of high-throughput cultivation of bacteria inspired by a dilution-to-extinction (DTE) methodology for the isolation of, and screening for, new cytotoxic compound producing marine bacteria. A marine sponge tissue was directly used as inoculum and the results were compared with the data obtained through the direct plating isolation method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) genomic fingerprinting indicated the isolation of four bioactive strains, three of them producers of a pederin-like compound, and the fourth one able to synthesize a different compound, still unidentified, rendered by the DTE approach, in comparison with one bioactive strain identified through the plating method. Analyses based on the 16S rRNA gene data showed the existence of two different species belonging to the genus Labrenzia. The efficiency and diversity ratio in the number of isolates and compounds are discussed. In view of the results, the proposed DTE approach proved to be efficient for the isolation of new cytotoxic compounds of marine origin and pave the way for future potential applications.     

The recent spread of a vertically transmitted virus through populations of Drosophila melanogaster

The sigma virus is a vertically transmitted pathogen that commonly infects natural populations of Drosophila melanogaster. This virus is the only known host-specific pathogen of D. melanogaster, and so offers a unique opportunity to study the genetics of Drosophila-viral interactions in a natural system. To elucidate the population genetic processes that operate in sigma virus populations, we collected D. melanogaster from 10 populations across three continents. We found that the sigma virus had a prevalence of 0-15% in these populations. Compared to other RNA viruses, we found that levels of viral genetic diversity are very low across Europe and North America. Based on laboratory measurements of the viral substitution rate, we estimate that most European and North American viral isolates shared a common ancestor approximately 200 years ago. We suggest two explanations for this: the first is that D. melanogaster has recently acquired the sigma virus; the second is that a single viral type has recently swept through D. melanogaster populations. Furthermore, in contrast to Drosophila populations, we find that the sigma viral populations are highly structured. This is surprising for a vertically transmitted pathogen that has a similar migration rate to its host. We suggest that the low structure in the viral populations can be explained by the smaller effective population size of the virus.     

The lack of recombination drives the fixation of transposable elements on the fourth chromosome of Drosophila melanogaster

In regions of suppressed recombination, where selection is expected to be less efficient in removing slightly deleterious mutations, transposable element (TE) insertions should be more likely to drift to higher frequencies, and even to reach fixation. In the absence of excision events, once a TE is fixed it cannot be eliminated from the population, and accumulation of elements thus should become an irreversible process. In the long term, this can drive the degeneration of large non-recombining fractions of the genomes. Chromosome 4 of Drosophila melanogaster has very low levels of recombination, if any, and this could be causing its degeneration. Here we report the results of a PCR-based analysis of the population frequencies of TE insertions in a sample from three African natural populations. We investigated 27 insertions from 12 TE families, located in regions of either suppressed or free recombination. Our results suggest that TE insertions tend to be fixed in the non-recombining regions, particularly on the fourth chromosome. We have also found that this involves all types of elements, and that fixed insertions are significantly shorter and more divergent from the canonical sequence than those segregating in the sample (28.1% vs 86.3% of the canonical length, and average nucleotide divergence (D(XY)) = 0.082 vs 0.008, respectively). Finally, DNA-based elements seem to show a greater tendency to reach fixation than retrotransposons. Implications of these findings for the population dynamics of TEs, and the evolutionary forces that shape the patterns of genetic variation in regions of reduced recombination, are discussed.     

Patterns of molecular variation and evolution in Drosophila americana and its relatives

We present the results of a survey of DNA sequence variability at X-linked and autosomal loci in Drosophila americana and of patterns of DNA sequence evolution among D. americana and four other related species in the virilis group of Drosophila. D. americana shows a typical level of silent polymorphism for a Drosophila species, but has an unusually low ratio of nonsynonymous to silent variation. Both D. virilis and D. americana also show a low ratio of nonsynonymous to synonymous substitutions along their respective lineages since the split from their common ancestor. The proportion of amino acid substitutions between D. americana and its relatives that are caused by positive selection, as estimated by extensions of the McDonald-Kreitman test, appears to be unusually high. We cannot, however, exclude the possibility that this reflects a recent increase in the intensity of selection on nonsynonymous mutations in D. americana and D. virilis. We also find that base composition at neutral sites appears to be in overall equilibrium among these species, but there is evidence for departure from equilibrium for codon usage in some lineages.     

A long-term study on seasonal changes of gametic disequilibrium between allozymes and inversions in Drosophila subobscura

Seasonal variation (spring, early summer, late summer, and autumn) of gametic disequilibrium between gene arrangements (OST and O3+4) of the O chromosome and Lap, Pept-1, and Acph allozyme loci, located inside these inversions, has been recorded in a natural population of Drosophila subobscura during seven years over a 15-year period. The length of the study allowed us to investigate the temporal variation of the allozyme-inversion associations by statistical methods of time series analysis. Cyclic seasonal changes of allozyme-inversion associations for both Lap and Pept-1 are detected in the natural population. In both cases, the patterns of seasonal change are due to the seasonal change of frequency of Lap and Pept-1 allozymes occurring exclusively within the OST gene arrangement. In contrast, the allozyme frequencies at these loci within the O3+4 gene arrangement are stable along seasons. The patterns of temporal variation of allozyme-inversion associations for Lap and Pept-1 in the natural population are contrasted with those previously published that correspond to gene arrangements of the O chromosome and nucleotide polymorphism at the rp49 region located inside these inversions, suggesting that natural selection is operating on these allozyme-inversion associations.     

Multilocus population analysis of <Emphasis Type=Italic>Gavia immer</Emphasis> (Aves: Gaviidae) mtDNA reveals low genetic diversity and lack of differentiation across the species breeding range

We analyzed the patterns of nucleotide sequence variation at three mitochondrial DNA loci, the noncoding mitochondrial control region and two genes (cytochrome b and cytochrome oxidase I) of Gavia immer in the largest collection of wintering individuals from Southern Europe to date. The sample consisted of 33 birds, oiled during the 2002/2003 Prestige tanker spill and washed ashore on the Galician coast (NW Iberian Peninsula). The aims of the study were to investigate the levels of standing genetic variation in the species, and to identify the geographic origin of these wintering birds. To do this, all available sequences of these loci, mostly from North American specimens collected from both the Atlantic and Pacific coasts, were retrieved from GenBank and included in the analysis. Overall, only 14 genetic variants were detected in the nearly 2 Kb surveyed, which reflects very low levels of nucleotide site diversity in this species. Interestingly, all variants were found at very low frequencies, and there was no indication of any clear subdivision in the G. immer population. This genetic profile is consistent with G. immer being a single panmictic population of small effective population size as compared with other seabirds. These circumstances preclude identification of the breeding regions of these wintering birds relying solely on genetic data. In the light of these results, possible causes, and the genetic and ecological consequences, of this demographic scenario are discussed.     

Widespread evidence for horizontal transfer of transposable elements across Drosophila genomes

Background  

Horizontal transfer (HT) could play an important role in the long-term persistence of transposable elements (TEs) because it provides them with the possibility to avoid the checking effects of host-silencing mechanisms and natural selection, which would eventually drive their elimination from the genome. However, despite the increasing evidence for HT of TEs, its rate of occurrence among the TE pools of model eukaryotic organisms is still unknown.     

Inferences on the evolutionary history of the S-element family of Drosophila melanogaster

The S-element family of transposable elements has been characterized in D. melanogaster. Attempts to find it in other Drosophila-related species have failed, suggesting that this element family may have recently invaded the D. melanogaster genome by horizontal transfer. In order to investigate its evolutionary history, we analyzed the patterns of DNA polymorphism among the S-element copies present in a sample genome (Drosophila Genome Project). The observed levels of nucleotide diversity are significantly lower than theoretical expectations based on the neutral model. This is consistent with evidence for ongoing gene conversion among copies and for purifying selection on the elements' sequences, particularly on the terminal inverted repeats. A phylogenetic analysis revealed that the members of the S-element family can be grouped into at least two genetically differentiated clusters. The level of divergence between these clusters suggests that the S elements invaded the genome of the ancestor of D. melanogaster before the speciation of the D. melanogaster complex. However, other relevant scenarios are also discussed.