Impact of Heat Stress on Transposable Element Expression and Derived Small RNAs in Drosophila subobscura.

Global warming is forcing insect populations to move and adapt, triggering adaptive genetic responses. Thermal stress is known to alter gene expression, repressing the transcription of active genes, and inducing others, such as those encoding heat shock proteins. It has also been related to the activation of some specific transposable element (TE) families. However, the actual magnitude of this stress on the whole genome and the factors involved in these genomic changes are still unclear. We studied mRNAs and small RNAs in gonads of two Drosophila subobscura populations, considered a good model to study adaptation to temperature changes. In control conditions, we found that a few genes and TE families were differentially expressed between populations, pointing out their putative involvement in the adaptation of populations to their different environments. Under heat stress, sex-specific changes in gene expression together with a trend toward overexpression, mainly of heat shock response-related genes, were observed. We did not observe large changes of TE expression nor small RNA production due to stress. Only population and sex-specific expression changes of some TE families (mainly retrotransposons), or the amounts of siRNAs and piRNAs, derived from specific TE families were observed, as well as the piRNA production from some piRNA clusters. Changes in small RNA amounts and TE expression could not be clearly correlated, indicating that other factors as chromatin modulation could also be involved. This work provides the first whole transcriptomic study including genes, TEs, and small RNAs after a heat stress in D. subobscura.

High Stability of the Epigenome in Drosophila Interspecific Hybrids.

Interspecific hybridization is often seen as a genomic stress that may lead to new gene expression patterns and deregulation of transposable elements (TEs). The understanding of expression changes in hybrids compared with parental species is essential to disentangle their putative role in speciation processes. However, to date we ignore the detailed mechanisms involved in genomic deregulation in hybrids. We studied the ovarian transcriptome and epigenome of the Drosophila buzzatii and Drosophila koepferae species together with their F1 hybrid females. We found a trend toward underexpression of genes and TE families in hybrids. The epigenome in hybrids was highly similar to the parental epigenomes and showed intermediate histone enrichments between parental species in most cases. Differential gene expression in hybrids was often associated only with changes in H3K4me3 enrichments, whereas differential TE family expression in hybrids may be associated with changes in H3K4me3, H3K9me3, or H3K27me3 enrichments. We identified specific genes and TE families, which their differential expression in comparison with the parental species was explained by their differential chromatin mark combination enrichment. Finally, cis-trans compensatory regulation could also contribute in some way to the hybrid deregulation. This work provides the first study of histone content in Drosophila interspecific hybrids and their effect on gene and TE expression deregulation.

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.

Basal hsp70 expression levels do not explain adaptive variation of the warm- and cold-climate O3 + 4 + 7 and OST gene arrangements of Drosophila subobscura.

BACKGROUND: Drosophila subobscura exhibits a rich inversion polymorphism, with some adaptive inversions showing repeatable spatiotemporal patterns in frequencies related to temperature. Previous studies reported increased basal HSP70 protein levels in homokaryotypic strains for a warm-climate arrangement compared to a cold-climate one. These findings do not match the similar hsp70 genomic organization between arrangements, where gene expression levels are expected to be similar. In order to test this hypothesis and understand the molecular basis for hsp70 expression, we compared basal hsp70 mRNA levels in males and females, and analysed the 5' and 3' regulatory regions of hsp70 genes in warm- and cold-climate isochromosomal O3 + 4 + 7 and OST lines of D. subobscura. RESULTS: We observed comparable mRNA levels between the two arrangements and a sex-biased hsp70 gene expression. The number of heat-shock elements (HSEs) and GAGA sites on the promoters were identical amongst the OST and O3 + 4 + 7 lines analysed. This is also true for 3' AU-rich elements where most A and B copies of hsp70 have, respectively, two and one element in both arrangements. Beyond the regulatory elements, the only notable difference between both arrangements is the presence in 3' UTR of a 14 bp additional fragment after the stop codon in the hsp70A copy in five O3 + 4 + 7 lines, which was not found in any of the six OST lines. CONCLUSIONS: The equivalent hsp70 mRNA amounts in OST and O3 + 4 + 7 arrangements provide the first evidence of a parallelism between gene expression and genetic organization in D. subobscura lines having these arrangements. This is reinforced by the lack of important differential features in the number and structure of regulatory elements between both arrangements, despite the genetic differentiation observed when the complete 5' and 3' regulatory regions were considered. Therefore, the basal levels of hsp70 mRNA cannot account, in principle, for the adaptive variation of the two arrangements studied. Consequently, further studies are necessary to understand the intricate molecular mechanisms of hsp70 gene regulation in D. subobscura.

Chromosomal inversions promote genomic islands of concerted evolution of Hsp70 genes in the Drosophila subobscura species subgroup.

Heat-shock (HS) assays to understand the connection between standing inversion variation and evolutionary response to climate change in Drosophila subobscura found that "warm-climate" inversion O3+4 exhibits non-HS levels of Hsp70 protein like those of "cold-climate" OST after HS induction. This was unexpected, as overexpression of Hsp70 can incur multiple fitness costs. To understand the genetic basis of this finding, we have determined the genomic sequence organization of the Hsp70 family in four different inversions, including OST , O3+4 , O3+4+8 and O3+4+16 , using as outgroups the remainder of the subobscura species subgroup, namely Drosophila madeirensis and Drosophila guanche. We found (i) in all the assayed lines, the Hsp70 family resides in cytological locus 94A and consists of only two genes, each with four HS elements (HSEs) and three GAGA sites on its promoter. Yet, in OST, the family is comparatively more compact; (ii) the two Hsp70 copies evolve in concert through gene conversion, except in D. guanche; (iii) within D. subobscura, the rate of concerted evolution is strongly structured by inversion, being higher in OST than in O3+4 ; and (iv) in D. guanche, the two copies accumulated multiple differences, including a newly evolved "gap-type" HSE2. The absence of concerted evolution in this species may be related to a long-gone-unnoticed observation that it lacks Hsp70 HS response, perhaps because it has evolved within a narrow thermal range in an oceanic island. Our results point to a previously unrealized link between inversions and concerted evolution, with potentially major implications for understanding genome evolution.

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.

Changes of Osvaldo expression patterns in germline of male hybrids between the species Drosophila buzzatii and Drosophila koepferae.

Hybridization between different genomes is a source of genomic instability, sometimes associated with transposable element (TE) mobilization. Previous work showed that hybridization between the species Drosophila buzzatii and Drosophila koepferae induced mobilization of different (TEs), the Osvaldo retrotransposon being the most unstable. However, we ignore the mechanisms involved in this transposition release in interspecific hybrids. In order to disentangle the mechanisms involved in this process, we performed Osvaldo expression studies in somatic and germinal tissues from hybrids and parental species. There was a trend towards increased Osvaldo expression in the somatic tissues of hybrid females and males, which was always significant in males compared to the parental species D. buzzatii but, not in females compared to maternal species D. koepferae. There were massive changes of Osvaldo expression in the testes, which varied depending on the hybrid generation and family. Moreover, Osvaldo hybridization signals, restricted to the apical and primary spermatocyte regions in parents, occupied broader region in the hybrids. In ovaries, there were no significant differences in Osvaldo expression rates between hybrids and the maternal species D. koepferae. The transcript location was restricted to ovarian nurse cells in both parents and hybrids, undetectable in some hybrids. This research highlights first, the existence of putative complex deregulation mechanisms different between sexes and cell types and second, disruption of Osvaldo activity particularly evident in testes from sterile hybrid males. Deeper studies of the total transcriptome in hybrids and parental species are necessary to gain a better knowledge of the TE deregulation pathways in hybrids.

A genome-wide survey of genetic instability by transposition in Drosophila hybrids.

Hybridization between species is a genomic instability factor involved in increasing mutation rate and new chromosomal rearrangements. Evidence of a relationship between interspecific hybridization and transposable element mobilization has been reported in different organisms, but most studies are usually performed with particular TEs and do not discuss the real effect of hybridization on the whole genome. We have therefore studied whole genome instability of Drosophila interspecific hybrids, looking for the presence of new AFLP markers in hybrids. A high percentage (27-90%) of the instability markers detected corresponds to TEs belonging to classes I and II. Moreover, three transposable elements (Osvaldo, Helena and Galileo) representative of different families, showed an overall increase of transposition rate in hybrids compared to parental species. This research confirms the hypothesis that hybridization induces genomic instability by transposition bursts and suggests that genomic stress by transposition could contribute to a relaxation of mechanisms controlling TEs in the Drosophila genome.

Distribution of the transposable elements bilbo and gypsy in original and colonizing populations of Drosophila subobscura.

BACKGROUND: Transposable elements (TEs) constitute a substantial amount of all eukaryotic genomes. They induce an important proportion of deleterious mutations by insertion into genes or gene regulatory regions. However, their mutational capabilities are not always adverse but can contribute to the genetic diversity and evolution of organisms. Knowledge of their distribution and activity in the genomes of populations under different environmental and demographic regimes, is important to understand their role in species evolution. In this work we study the chromosomal distribution of two TEs, gypsy and bilbo, in original and colonizing populations of Drosophila subobscura to reveal the putative effect of colonization on their insertion profile. RESULTS: Chromosomal frequency distribution of two TEs in one original and three colonizing populations of D. subobscura, is different. Whereas the original population shows a low insertion frequency in most TE sites, colonizing populations have a mixture of high (frequency > or = 10%) and low insertion sites for both TEs. Most highly occupied sites are coincident among colonizing populations and some of them are correlated to chromosomal arrangements. Comparisons of TE copy number between the X chromosome and autosomes show that gypsy occupancy seems to be controlled by negative selection, but bilbo one does not. CONCLUSION: These results are in accordance that TEs in Drosophila subobscura colonizing populations are submitted to a founder effect followed by genetic drift as a consequence of colonization. This would explain the high insertion frequencies of bilbo and gypsy in coincident sites of colonizing populations. High occupancy sites would represent insertion events prior to colonization. Sites of low frequency would be insertions that occurred after colonization and/or copies from the original population whose frequency is decreasing in colonizing populations. This work is a pioneer attempt to explain the chromosomal distribution of TEs in a colonizing species with high inversion polymorphism to reveal the putative effect of arrangements in TE insertion profiles. In general no associations between arrangements and TE have been found, except in a few cases where the association is very strong. Alternatively, founder drift effects, seem to play a leading role in TE genome distribution in colonizing populations.

The evolutionary history of Drosophila buzzatii. XXXVI. Molecular structural analysis of Osvaldo retrotransposon insertions in colonizing populations unveils drift effects in founder events.

Previous work on transposable element distribution in colonizing populations of Drosophila buzzatii revealed a high frequency of occupancy in several chromosomal sites. Two explanatory hypotheses were advanced: the founder hypothesis, by which founder genetic drift was responsible, and the unstable hypothesis that assigns this unusual distribution to bursts of transposition toward some chromosomal sites. Here, we study the molecular structure of three euchromatic Osvaldo clones isolated from sites occupied at high (A4 and B9) and low frequency (B4) in colonizing populations, to test these hypotheses. Large insertions, duplications, and indels in the Osvaldo coding region and LTR were detected in the A4 clone and a truncated Osvaldo with many substitutions was found in the B9 clone. These altered sequences indicate that the two copies of this retroelement are precolonization insertions. Interestingly, the LTR of the A4 clone and the reverse transcriptase region of B9 show identical sequences in all colonizing populations indicating, most probably, that they are identical by descent. Moreover, Osvaldo is inserted at the same nucleotide site in all colonizing populations. On the other hand an almost identical LTR sequence, except by 1 base deletion, was found in the B4 clone compared to the canonical active Osvaldo element. These results suggest that Osvaldo copies in highly occupied sites are, most probably, identical by descent and strongly favor the founder hypothesis. On the other hand, low-insertion-frequency sites could represent recent transposition events. This work emphasizes the importance of molecular population studies to disentangle the effects of genetic drift and transposition in colonization.