Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing.

Aim: Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. Materials & methods: 104 pathogenic isolates of Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus were analyzed. Results: Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of N. meningitidis serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of S. pyogenes was similar by FT-IR and WGS, but not S. aureus or S. pneumoniae. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in S. pneumoniae and cell wall proteins (biofilm formation, antibiotic and complement permeability) in S. aureus via WGS. Conclusion: FT-IR warrants further clinical evaluation as a promising diagnostic tool.

We tested a technique (FT-IR) to identify four different, common bacteria from 104 children with serious infections and compared it to lab methods for diagnosis. FT-IR was more accurate. We tested if it could identify subtypes of bacteria, which is important in outbreaks. It was able to subtype two species, but not the two other species. However, it is a much faster and cheaper technique than the gold standard. It may be useful in certain outbreaks. We also investigated the trends between genes and the length of hospital stay. This can support further laboratory research. As a fast, low-cost test, FT-IR warrants further testing before it is applied to clinical labs.

Levels of P-element-induced hybrid dysgenesis in Drosophila simulans are uncorrelated with levels of P-element piRNAs.

Transposable elements (TEs) are genomic parasites that proliferate within host genomes, and which can also invade new species. The P-element, a DNA-based TE, recently invaded two Drosophila species: Drosophila melanogaster in the 20th century, and D. simulans in the 21st. In both species, lines collected before the invasion are susceptible to "hybrid dysgenesis", a syndrome of abnormal phenotypes apparently due to P-element-inflicted DNA damage. In D. melanogaster, lines collected after the invasion have evolved a maternally acting mechanism that suppresses hybrid dysgenesis, with extensive work showing that PIWI-interacting small RNAs (piRNAs) are a key factor in this suppression. Most of these studies use lines collected many generations after the initial P-element invasion. Here, we study D. simulans collected early, as well as late in the P-element invasion of this species. Like D. melanogaster, D. simulans from late in the invasion show strong resistance to hybrid dysgenesis and abundant P-element-derived piRNAs. Lines collected early in the invasion, however, show substantial variation in how much they suffer from hybrid dysgenesis, with some lines highly resistant. Surprisingly, although, these resistant lines do not show high levels of cognate maternal P-element piRNAs; in these lines, it may be that other mechanisms suppress hybrid dysgenesis.

Drosophila Interspecific Hybridization Causes A Deregulation of the piRNA Pathway Genes.

Almost all eukaryotes have transposable elements (TEs) against which they have developed defense mechanisms. In the Drosophila germline, the main transposable element (TE) regulation pathway is mediated by specific Piwi-interacting small RNAs (piRNAs). Nonetheless, for unknown reasons, TEs sometimes escape cellular control during interspecific hybridization processes. Because the piRNA pathway genes are involved in piRNA biogenesis and TE control, we sequenced and characterized nine key genes from this pathway in Drosophilabuzzatii and Drosophilakoepferae species and studied their expression pattern in ovaries of both species and their F1 hybrids. We found that gene structure is, in general, maintained between both species and that two genes-armitage and aubergine-are under positive selection. Three genes-krimper, methyltransferase 2, and zucchini-displayed higher expression values in hybrids than both parental species, while others had RNA levels similar to the parental species with the highest expression. This suggests that the overexpression of some piRNA pathway genes can be a primary response to hybrid stress. Therefore, these results reinforce the hypothesis that TE deregulation may be due to the protein incompatibility caused by the rapid evolution of these genes, leading to a TE silencing failure, rather than to an underexpression of piRNA pathway genes.

Evolutionary potential of thermal preference and heat tolerance in Drosophila subobscura.

Evolutionary change of thermal traits (i.e., heat tolerance and behavioural thermoregulation) is one of the most important mechanisms exhibited by organisms to respond to global warming. However, the evolutionary potential of heat tolerance, estimated as narrow-sense heritability, depends on the methodology employed. An alternative adaptive mechanism to buffer extreme temperatures is behavioural thermoregulation, although the association between heat tolerance and thermal preference is not clearly understood. We suspect that methodological effects associated with the duration of heat stress during thermal tolerance assays are responsible for missing this genetic association. To test this hypothesis, we estimated the heritabilities and genetic correlations for thermal traits in Drosophila subobscura, using high-temperature static and slow ramping assays. We found that heritability for heat tolerance was higher in static assays (h2  = 0.134) than in slow ramping assays (h2  = 0.084), suggesting that fast assays may provide a more precise estimation of the genetic variation of heat tolerance. In addition, thermal preference exhibited a low heritability (h2  = 0.066), suggesting a reduced evolutionary response for this trait. We also found that the different estimates of heat tolerance and thermal preference were not genetically correlated, regardless of how heat tolerance was estimated. In conclusion, our data suggest that these thermal traits can evolve independently in this species. In agreement with previous evidence, these results indicate that methodology may have an important impact on genetic estimates of heat tolerance and that fast assays are more likely to detect the genetic component of heat tolerance.

Transposable Element Misregulation Is Linked to the Divergence between Parental piRNA Pathways in Drosophila Hybrids.

Interspecific hybridization is a genomic stress condition that leads to the activation of transposable elements (TEs) in both animals and plants. In hybrids between Drosophila buzzatii and Drosophila koepferae, mobilization of at least 28 TEs has been described. However, the molecular mechanisms underlying this TE release remain poorly understood. To give insight on the causes of this TE activation, we performed a TE transcriptomic analysis in ovaries (notorious for playing a major role in TE silencing) of parental species and their F1 and backcrossed (BC) hybrids. We find that 15.2% and 10.6% of the expressed TEs are deregulated in F1 and BC1 ovaries, respectively, with a bias toward overexpression in both cases. Although differences between parental piRNA (Piwi-interacting RNA) populations explain only partially these results, we demonstrate that piRNA pathway proteins have divergent sequences and are differentially expressed between parental species. Thus, a functional divergence of the piRNA pathway between parental species, together with some differences between their piRNA pools, might be at the origin of hybrid instabilities and ultimately cause TE misregulation in ovaries. These analyses were complemented with the study of F1 testes, where TEs tend to be less expressed than in D. buzzatii. This can be explained by an increase in piRNA production, which probably acts as a defence mechanism against TE instability in the male germline. Hence, we describe a differential impact of interspecific hybridization in testes and ovaries, which reveals that TE expression and regulation are sex-biased.

Drosophila Females Undergo Genome Expansion after Interspecific Hybridization.

Genome size (or C-value) can present a wide range of values among eukaryotes. This variation has been attributed to differences in the amplification and deletion of different noncoding repetitive sequences, particularly transposable elements (TEs). TEs can be activated under different stress conditions such as interspecific hybridization events, as described for several species of animals and plants. These massive transposition episodes can lead to considerable genome expansions that could ultimately be involved in hybrid speciation processes. Here, we describe the effects of hybridization and introgression on genome size of Drosophila hybrids. We measured the genome size of two close Drosophila species, Drosophila buzzatii and Drosophila koepferae, their F1 offspring and the offspring from three generations of backcrossed hybrids; where mobilization of up to 28 different TEs was previously detected. We show that hybrid females indeed present a genome expansion, especially in the first backcross, which could likely be explained by transposition events. Hybrid males, which exhibit more variable C-values among individuals of the same generation, do not present an increased genome size. Thus, we demonstrate that the impact of hybridization on genome size can be detected through flow cytometry and is sex-dependent.

Expression of the Retrotransposon Helena Reveals a Complex Pattern of TE Deregulation in Drosophila Hybrids.

Transposable elements (TEs), repeated mobile sequences, are ubiquitous in the eukaryotic kingdom. Their mobilizing capacity confers on them a high mutagenic potential, which must be strongly regulated to guarantee genome stability. In the Drosophila germline, a small RNA-mediated silencing system, the piRNA (Piwi-interacting RNA) pathway, is the main responsible TE regulating mechanism, but some stressful conditions can destabilize it. For instance, during interspecific hybridization, genomic stress caused by the shock of two different genomes can lead, in both animals and plants, to higher transposition rates. A recent study in D. buzatii-D. koepferae hybrids detected mobilization of 28 TEs, yet little is known about the molecular mechanisms explaining this transposition release. We have characterized one of the mobilized TEs, the retrotransposon Helena, and used quantitative expression to assess whether its high transposition rates in hybrids are preceded by increased expression. We have also localized Helena expression in the gonads to see if cellular expression patterns have changed in the hybrids. To give more insight into changes in TE regulation in hybrids, we analysed Helena-specific piRNA populations of hybrids and parental species. Helena expression is not globally altered in somatic tissues, but male and female gonads have different patterns of deregulation. In testes, Helena is repressed in F1, increasing then its expression up to parental values. This is linked with a mislocation of Helena transcripts along with an increase of their specific piRNA levels. Ovaries have additive levels of Helena expression, but the ping-pong cycle efficiency seems to be reduced in F1 hybrids. This could be at the origin of new Helena insertions in hybrids, which would be transmitted to F1 hybrid female progeny.