Characterization of the chemoreceptor repertoire of a highly specialized fly with comparisons to other Drosophila species.

To explore the diversity of scenarios in nature, animals have evolved tools to interact with different environmental conditions. Chemoreceptors are an important interface component and among them, olfactory receptors (ORs) and gustatory receptors (GRs) can be used to find food and detect healthy resources. Drosophila is a model organism in many scientific fields, in part due to the diversity of species and niches they occupy. The contrast between generalists and specialists Drosophila species provides an important model for studying the evolution of chemoreception. Here, we compare the repertoire of chemoreceptors of different species of Drosophila with that of D. incompta, a highly specialized species whose ecology is restricted to Cestrum flowers, after reporting the preferences of D. incompta to the odor of Cestrum flowers in olfactory tests. We found evidence that the chemoreceptor repertoire in D. incompta is smaller than that presented by species in the Sophophora subgenus. Similar patterns were found in other non-Sophophora species, suggesting the presence of underlying phylogenetic trends. Nevertheless, we also found autapomorphic gene losses and detected some genes that appear to be under positive selection in D. incompta, suggesting that the specific lifestyle of these flies may have shaped the evolution of individual genes in each of these gene families.

Transposable elements alter gene expression and may impact response to cisplatin therapy in ovarian cancer.

Cisplatin is widely employed for cancer treatment; therefore, understanding resistance to this drug is critical for therapeutic practice. While studies have delved into differential gene expression in the context of cisplatin resistance, findings remain somewhat scant. We performed a comprehensive investigation of transposable elements (TEs) expression and their impact in host genes in two cisplatin-treated ovarian cancer cell lines. RNA-seq, ATAC-seq, and in-depth bioinformatics analysis were used to compare cisplatin-sensitive and -resistant ovarian cancer cell lines. Our results reveal that cisplatin therapy alters not only the expression of protein-coding genes, but also key TEs, including LINE1, Alu, and endogenous retroviruses, in both cisplatin-sensitive and -resistant cell lines. By co-expressing with downstream genes or by creating chimeric transcripts with host genes at their insertion sites, these TEs seem to control the expression of protein-coding genes, including tumor-related genes. Our model uncovers TEs influencing the expression of cancer genes and cancer pathways. Collectively, our findings indicate that TE alterations associated with cisplatin treatment occur in critical cancer genes and cellular pathways synergically. This research highlights the importance of considering the entire spectrum of transcribed elements in the genome, especially TE expression, for a complete understanding of complex models like cancer response to treatment.

The good, the bad and the ugly of transposable elements annotation tools.

Transposable elements are repetitive and mobile DNA segments that can be found in virtually all organisms investigated to date. Their complex structure and variable nature are particularly challenging from the genomic annotation point of view. Many softwares have been developed to automate and facilitate TEs annotation at the genomic level, but they are highly heterogeneous regarding documentation, usability and methods. In this review, we revisited the existing software for TE genomic annotation, concentrating on the most often used ones, the methodologies they apply, and usability. Building on the state of the art of TE annotation software we propose best practices and highlight the strengths and weaknesses from the available solutions.

Horizontal Transposon Transfer and Their Ecological Drivers: The Case of Flower-breeding Drosophila.

Understanding the mechanisms that shape the architecture, diversity, and adaptations of genomes and their ecological and genetic interfaces is of utmost importance to understand biological evolution. Transposable elements (TEs) play an important role in genome evolution, due to their ability to transpose within and between genomes, providing sites of nonallelic recombination. Here we investigate patterns and processes of TE-driven genome evolution associated with niche diversification. Specifically, we compared TE content, TE landscapes, and frequency of horizontal transposon transfers (HTTs) across genomes of flower-breeding Drosophila (FBD) with different levels of specialization on flowers. Further, we investigated whether niche breadth and ecological and geographical overlaps are associated with a potential for HTT rates. Landscape analysis evidenced a general phylogenetic pattern, in which species of the D. bromeliae group presented L-shaped curves, indicating recent transposition bursts, whereas D. lutzii showed a bimodal pattern. The great frequency of highly similar sequences recovered for all FBD suggests that these species probably experienced similar ecological pressures and evolutionary histories that contributed to the diversification of their mobilomes. Likewise, the richness of TEs superfamilies also appears to be associated with ecological traits. Furthermore, the two more widespread species, the specialist D. incompta and the generalist D. lutzii, presented the highest frequency of HTT events. Our analyses also revealed that HTT opportunities are positively influenced by abiotic niche overlap but are not associated with phylogenetic relationships or niche breadth. This suggests the existence of intermediate vectors promoting HTTs between species that do not necessarily present overlapping biotic niches.

Chemotherapy Drugs Act Differently in the Expression and Somatic Mobilization of the mariner Transposable Element in Drosophila simulans.

Transposable elements (TEs) are abundant in genomes. Their mobilization can lead to genetic variability that is useful for evolution, but can also have deleterious biological effects. Somatic mobilization (SM) has been linked to degenerative diseases, such as Alzheimer's disease and cancer. We used a Drosophila simulans strain, in which SM can be measured by counting red spots in the eyes, to investigate how chemotherapeutic agents affect expression and SM of the mariner TE. Flies were treated with Cisplatin, Dacarbazine, and Daunorubicin. After acute exposure, relative expression of mariner was quantified by RT-qPCR and oxidative stress was measured by biochemical assays. Exposure to 50 and 100 µg/mL Cisplatin increased mariner expression and ROS levels; catalase activity increased at 100 µg/mL. With chronic exposure, the number of spots also increased, indicating higher mariner SM. Dacarbazine (50 and 100 µg/mL) did not significantly alter mariner expression or mobilization or ROS levels, but decreased catalase activity (100 µg/mL). Daunorubicin (25 and 50 µM) increased mariner expression, but decreased mariner SM. ROS and catalase activity were also reduced. Our data suggest that stress factors may differentially affect the expression and SM of TEs. The increase in mariner transposase gene expression is necessary, but not sufficient for mariner SM.

Stress does not induce a general transcription of transposable elements in Drosophila.

Transposable elements, also known as "jumping genes," have the ability to hop within the host genome. Nonetheless, this capacity is kept in check by the host cell defense systems to avoid unbridled TE mobilization. Different types of stressors can activate TEs in Drosophila, suggesting that TEs may play an adaptive role in the stress response, especially in generating genetic variability for adaptive evolution. TE activation by stressors may also lead to the notion, usually found in the literature, that any form of stress could activate all or the majority of TEs. In this review, we define what stress is. We then present and discuss RNA sequencing results from several studies demonstrating that stress does not trigger TE transcription broadly in Drosophila. An explanation for the LTR order of TEs being the most overexpressed is also proposed.

Multiple long-range host shifts of major Wolbachia supergroups infecting arthropods.

Wolbachia is a genus of intracellular bacterial endosymbionts found in 20-66% of all insect species and a range of other invertebrates. It is classified as a single species, Wolbachia pipientis, divided into supergroups A to U, with supergroups A and B infecting arthropods exclusively. Wolbachia is transmitted mainly via vertical transmission through female oocytes, but can also be transmitted across different taxa by host shift (HS): the direct transmission of Wolbachia cells between organisms without involving vertically transmitted gametic cells. To assess the HS contribution, we recovered 50 orthologous genes from over 1000 Wolbachia genomes, reconstructed their phylogeny and calculated gene similarity. Of 15 supergroup A Wolbachia lineages, 10 have similarities ranging from 95 to 99.9%, while their hosts' similarities are around 60 to 80%. For supergroup B, four out of eight lineages, which infect diverse and distantly-related organisms such as Acari, Hemiptera and Diptera, showed similarities from 93 to 97%. These results show that Wolbachia genomes have a much higher similarity when compared to their hosts' genes, which is a major indicator of HS. Our comparative genomic analysis suggests that, at least for supergroups A and B, HS is more frequent than expected, occurring even between distantly-related species.

Somatic Mobilization: High Somatic Insertion Rate of mariner Transposable Element in Drosophila simulans.

Although transposable elements (TEs) are usually silent in somatic tissues, they are sometimes mobilized in the soma and can potentially have biological consequences. The mariner element is one of the TEs involved in somatic mobilization (SM) in Drosophila and has a high rate of somatic excision. It is also known that temperature is an important factor in the increase of the mariner element SM in the fly. However, it is important to emphasize that excision is only one step of TE transposition, and the final step in this process is insertion. In the present study, we used an assay based on sequencing of the mariner flanking region and developed a pipeline to identify novel mariner insertions in Drosophila simulans at 20 and 28 °C. We found that flies carrying two mariner copies (one autonomous and one non-autonomous) had an average of 236.4 (±99.3) to 279 (±107.7) new somatic insertions at 20 °C and an average of 172.7 (±95.3) to 252.6 (±67.3) at 28 °C. In addition, we detected fragments containing mariner and others without mariner in the same regions with low-coverage long-read sequencing, indicating the process of excision and insertion. In conclusion, a low number of autonomous copies of the mariner transposon can promote a high rate of new somatic insertions during the developmental stages of Drosophila. Additionally, the developed method seems to be sensitive and adequate for the verification and estimation of somatic insertion.

Molecular and biological effects of Cisplatin in Drosophila.

Cisplatin is widely used in cancer treatment and is one of the best cytostatic agents available for antitumor therapy. Drosophila melanogaster has one of the best annotated genomes and one of the best characterized sets of transposable elements (TE) sequences. This model organism is useful for analyzing the mode of action of several compounds in vivo and evaluating the behavioral consequences of treatments. The aim of our study was to increase the knowledge about the effects of Cisplatin in Drosophila by joining RNA-seq and biological assays. RNA-seq was followed by analyses of differential expression of genes (DEGs) and TEs (DETEs), and of pathways and ontology terms. DETEs were confirmed by qPCR. Cisplatin was evaluated at 50 and 100 µg/mL in Drosophila culture medium for 24 h. The fly locomotor assay, survival analysis, oviposition and development were used as biological assays. Cisplatin induced DEGs in a dose-dependent fashion, and four TEs were up-regulated. Most DEGs are related to DNA damage and detoxification processes. Cisplatin increases Drosophila locomotor activity and interrupts development. Genes and processes related to the assays were also identified. This is the first study to evaluate the effects of Cisplatin in flies using RNA-seq. Gene alteration was almost limited to drug metabolism and DNA damage, and the drug did not vastly affect Drosophila on the molecular level. Contrary to the hypothesis that stress dramatically alters TEs mobilization, only four TEs were up-regulated. Our study, together with previous knowledge, asserts Drosophila as a valuable organism in the study of chemotherapy drugs.

Comparative mitogenomics of Drosophilidae and the evolution of the Zygothrica genus group (Diptera, Drosophilidae).

The Zygothrica genus group of Drosophilidae encompasses more than 437 species and five genera. Although knowledge regarding its diversity has increased, uncertainties about its monophyly and position within Drosophilidae remain. Genomic approaches have been widely used to address different phylogenetic questions and analyses involving the mitogenome have revealed a cost-efficient tool to these studies. Thus, this work aims to characterize mitogenomes of three species of the Zygothrica genus group (from the Hirtodrosophila, Paraliodrosophila and Zygothrica genera), while comparing them with orthologous sequences from other 23 Drosophilidae species and addressing their phylogenetic position. General content concerning gene order and overlap, nucleotide composition, start and stop codon, codon usage and tRNA structures were compared, and phylogenetic trees were constructed under different datasets. The complete mitogenomes characterized for H. subflavohalterata affinis H002 and P. antennta present the PanCrustacea gene order with 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, 13 protein coding genes and an A+T rich region with two T-stretched elements. Some peculiarities such as the almost complete overlap of genes tRNAH/ND4, tRNAF/ND5 and tRNAS2/ND1 are reported for different Drosophilidae species. Non-canonical secondary structures were encountered for tRNAS1 and tRNAY, revealing patterns that apply at different phylogenetic scales. According to the best depiction of the mitogenomes evolutionary history, the three Neotropical species of the Zygothrica genus group encompass a monophyletic lineage sister to Zaprionus, composing with this genus a clade that is sister to the Drosophila subgenus.

Transcriptomic and Proteomic Tools in the Study of Hg Toxicity: What Is Missing?

Mercury is a hazardous substance that has unique neurodevelopmental toxic effects in humans. However, the precise sequence of molecular events that culminate in Hg-induced neuropathology is still unknown. Though the omics studies have been generating an enormous amount of new data about Hg toxicity, our ability to interpret such a large quantity of information is still limited. In this opinion article, we will reinforce the necessity of new high throughput and accurate analytical proteomic methodologies, especially, thiol and selenol-proteome. Overall, we posit that improvements in thiol- and selenol-proteomic analyses will be pivotal in identifying the primary cellular targets of Hg. However, a better understanding of the complex cascades and molecular pathways involved in its toxicity will require extensive complementary studies in more complex systems.

Phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica (Diptera, Drosophilidae), with insights into the evolution of breeding sites usage.

The Neotropical region harbors an astonishing diversity of species, but still encompasses the least studied biogeographic region of the world. These properties apply for different taxonomic groups, and can be exemplified by drosophilids. In fact, high levels of cryptic diversity have recently been discovered for Neotropical species of the Zygothrica genus group, but relationships among these species, or them and other Drosophilidae species still remains to be addressed. Therefore, the aim of this study was to evaluate the phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica, which together with Paramycodrosophila and Paraliodrosophila compose the Zygothrica genus group. For this, fragments of the mitochondrial cytochrome oxidase subunits I (COI) and II (COII) genes, and the nuclear alpha methyldopa (Amd) and dopa decarboxylase (Ddc) genes were newly characterized for 43 Neotropical specimens of fungus-associated drosophilids, and analyzed in the context of 51 additional Drosophilinae sequences plus one Steganinae outgroup. Based on the resulting phylogeny, the evolution of breeding sites usage was also evaluated through ancestral character reconstructions. Our results revealed the Zygothrica genus group as a monophyletic lineage of Drosophila that branches after the subgenera Sophophora and Drosophila. Within this lineage, Mycodrosophila species seem to encompass the early offshoot, followed by a grade of Hirtodrosophila species, with derived branches mostly occupied by representatives of Zygothrica. This genus, in particular, was subdivided into five major clades, two of which include species of Hirtodrosophila, whose generic status needs to be reevatuated. According to our results, the use of fungi as breeding sites encompasses a symplesiomorphy for the Zygothrica genus group, since one of the recovered clades is currently specialized in using flowers as breeding sites whereas a sole species presents a reversal to the use of fruits of a plant of Gentianales. So, in general, this study supports the paraphyly of Drosophila in relation to fungus-associated Neotropical species of Drosophilidae, providing the first molecular insights into the phylogenetic patterns related to the evolution of this diverse group of species and some of its characteristic traits.

Cyclophosphamide in Drosophila promotes genes and transposable elements differential expression and mitochondrial dysfunction.

Cyclophosphamide (CPA) is an alkylating agent used for cancer chemotherapy, organ transplantation, and autoimmune disease treatment. Here, mRNA sequencing and high-resolution respirometry were performed to evaluate the alterations of Drosophila melanogaster gene expression fed with CPA under acute (0.1 mg/mL, for 24 h) and chronic (0.05 mg/mL, for 35 days) treatments. Differential expression analysis was performed using Cufflinks-Cuffdiff, DESeq2, and edgeR software. CPA affected genes are involved in several biological functions, including stress response and immune-related pathways, oxi-reduction and apoptotic processes, and cuticle and vitelline membrane formation. In particular, this is the first report of CPA-induced mitochondrial dysfunction caused by the downregulation of genes involved with mitochondria constituents. CPA treatment also changed the transcription pattern of transposable elements (TEs) from the gypsy and copia superfamilies. The results presented here provided evidence of CPA mitochondrial toxicity mechanisms and that CPA can modify TEs transcription in Drosophila flies.

The Catenulida flatworm can express genes from its microbiome or from the DNA it ingests.

Stenostomum are tiny planarians of the phylum Platyhelminthes that reproduce asexually. We transfected these worms using plasmids containing a gfp reporter gene. Here we show that they can express genes present in plasmids carried by bacteria and those that are encoded by naked DNA, such as plasmids or PCR fragments, transfected by electroporation; they can also express genes taken up during feeding. The microbiome associated with worm maintenance was evaluated, and the results indicated that when a plasmid is maintained in the microbiome, gfp gene expression is stable. When genes originate from naked DNA or bacteria not maintained in the microbiome, GFP expression is transient. Therefore, changes in the microbiome can modify the ability of worms to express foreign genes. In stable GFP-expressing worms, NSG showed that the gfp gene was maintained in the plasmid and was not integrated into the chromosome. These results suggest that, at least for some organisms such as flatworms, the expression of genes provided by the microbiome or the environment can be considered among the potential sources of phenotypic plasticity, which can have implications for evolvability.

The mobilome of Drosophila incompta, a flower-breeding species: comparison of transposable element landscapes among generalist and specialist flies.

The Drosophila genus is one of the main model organisms in evolutionary studies, including those investigating the role of transposable elements (TE) in genomic evolution both at the nucleotide and chromosome levels. D. incompta is a species with restricted ecology, using Cestrum (Solanaceae) flowers as unique sources for oviposition, feeding and development. In the present study, we deeply characterise the D. incompta mobilome and generate a curated dataset. A total of 277 elements were identified, corresponding to approximately 14% of the genome, and 164 of these elements are new, of which 32.62% are putatively autonomous and 8.9% are transcriptionally active in adult flies. The restricted ecology does not seem to influence the dynamics of TE in this fly, since the proportion and diversity of TEs in its genome are similar to that of other Drosophila species. This result is reinforced by the absence of a clear pattern when comparing the TE landscape between generalist and specialist flies. Using 32 available Drosophila genomes-24 ecologically generalist species and 8 specialist species-no difference was found between their TE landscape patterns. However, differences were found between species of the Sophophora and Drosophila subgenus, indicating there are lineage-specific factors shaping TE landscapes.

A re-annotation of the Anopheles darlingi mobilome.

The mobilome, portion of the genome composed of transposable elements (TEs), of Anopheles darlingi was described together with the genome of this species. Here, this mobilome was revised using similarity and de novo search approaches. A total of 5.6% of the A. darlingi genome is derived of TEs. Class I gypsy and copia were the most abundant superfamilies, corresponding to 22.36% of the mobilome. Non-LTR elements of the R1 and Jockey superfamilies account for 11% of the TEs. Among Class II TEs, the mariner superfamily is the most abundant (16.01%). Approximately 87% of the A. darlingi mobilome consist of short, truncated and/or degenerated copies of TEs. Only three retrotransposons, two belonging to gypsy and one to copia superfamilies, are putatively active elements. Only one Class II element, belonging to the mariner superfamily, is putatively active, having 12 copies in the genome. The TE landscape of A. darlingi is formed primarily by degenerated elements and, therefore, somewhat stable. Future applications of TE-based vectors for genetic transformation of A. darlingi should take into consideration mariner and piggyBac transposons, because full length and putatively active copies of these elements are present in its genome.

The somatic mobilization of transposable element mariner-Mos1 during the Drosophila lifespan and its biological consequences.

Transposable elements (TEs) are mobile DNA sequences on genomes. Some elements are able to transpose in somatic cells, a process known as somatic transposition (ST), which has been associated with detrimental biological effects. The mariner-Mos1 element of Drosophila promotes transposition in somatic and germline cells and is an excellent model for studies related to the biological consequence of somatic excision (SE). In this work, we used temperature stress to induce increasing transposition of mariner-Mos1 during different stages of the development of D. simulans, aiming to quantify SE during lifespan. Furthermore, strains of D. melanogaster exhibiting differential expression of mariner-Mos1 were employed for estimating some biological consequences of mariner mobilization. It is shown that SE of mariner-Mos1 was not constant during development; the larval phase had the highest rates while the pupal stage exhibited lower rates, and in the embryonic stage, no difference was detected. SE can be detrimental, as suggested by correlation in SE level and reduction in behavioral activities and embryonic viability. This study showed that mariner-Mos1 SE accumulates during the Drosophila life cycle, and can be involved in detrimental effects.

De novo transcriptome assembly of the lobster cockroach Nauphoeta cinerea (Blaberidae).

The use of Drosophila as a scientific model is well established, but the use of cockroaches as experimental organisms has been increasing, mainly in toxicology research. Nauphoeta cinerea is one of the species that has been studied, and among its advantages is its easy laboratory maintenance. However, a limited amount of genetic data about N. cinerea is available, impeding gene identification and expression analyses, genetic manipulation, and a deeper understanding of its functional biology. Here we describe the N. cinerea fat body and head transcriptome, in order to provide a database of genetic sequences to better understand the metabolic role of these tissues, and describe detoxification and stress response genes. After removing low-quality sequences, we obtained 62,121 transcripts, of which more than 50% had a length of 604 pb. The assembled sequences were annotated according to their genes ontology (GO). We identified 367 genes related to stress and detoxification; among these, the more frequent were p450 genes. The results presented here are the first large-scale sequencing of N. cinerea and will facilitate the genetic understanding of the species' biochemistry processes in future works.

Drosophila relics hobo and hobo-MITEs transposons as raw material for new regulatory networks.

Hypermutable strains of Drosophila simulans have been studied for 20 years. Several mutants were isolated and characterized, some of which had phenotypes associated with alteration in development; for example, showing ectopic legs with eyes being expressed in place of antennae. The causal agent of this hypermutability is a non-autonomous hobo-related sequence (hoboVA). Around 100 mobilizable copies of this element are present in the D. simulans genome, and these are likely mobilized by the autonomous and canonical hobo element. We have shown that hoboVA has transcription factor binding sites for the developmental genes, hunchback and even-skipped, and that this transposon is expressed in embryos, following the patterns of these genes. We suggest that hobo and hobo-related elements can be material for the emergence of new regulatory networks.

A Mos1 transposase in vivo assay to screen new HIV-1 integrase inhibitors.

The integrase and transposase enzymes of retrovirus and transposons, respectively, share the catalytic DDE domain. In vitro assays showed that inhibitors of HIV-1 integrase generally inhibit the mariner Mos1 transposase. Using a Drosophila strain in which the mobilisation of the mariner element can be quantified by mosaic eyes, we showed that flies maintained in medium containing 210 µM to 4 mM of raltegravir, or 1 or 2 mM of dolutegravir, which are HIV-1 integrase inhibitor used in AIDS treatment, have 23-33% less somatic mobilisation in mosaic eyes when treated with raltegravir and 28-32% when treated with dolutegravir. The gene expression of the mariner transposase gene, estimated by qPCR, is similar among treated and control flies. The results suggest that in vivo assays using Drosophila can be used as a primary screening of inhibitory drugs for transposase and retroviral integrase. The advantages of this assay are that it is easy, quick, cheap and is an in vivo test, meaning that the tested substance has to have been taken in by cells and has arrived at the target site, which is not the case when in vitro assays are applied.