Evidence of acrylamide-induced behavioral deficit, mitochondrial dysfunction and cell death in Drosophila melanogaster.

Acrylamide (ACR), a ubiquitous compound with diverse route of exposure, has been demonstrated to have detrimental effects on human and animal health. The mechanisms underlying its toxicity is multifaceted and not fully elucidated. This study aims to provide further insight into novel pathways underlying ACR toxicity by leveraging on Drosophila melanogaster as a model organism. The concentrations of acrylamide (25, 50 and 100 mg/kg) and period of exposure (7-days) used in this study was established through a concentration response curve. ACR exposure demonstrably reduced organismal viability, evidenced by decline in survival rate, offspring emergence and deficits in activity, sleep and locomotory behaviors. Using a high-resolution respirometry assay, the role of mitochondria respiratory system in ACR-mediated toxicity in the flies was investigated. Acrylamide caused dysregulation in mitochondrial bioenergetics and respiratory capacity leading to an impaired OXPHOS activity and electron transport, ultimately contributing to the pathological process of ACR-toxicity. Furthermore, ACR exacerbated apoptosis and induced oxidative stress in D. melanogaster. The up-regulation of mRNA transcription of Reaper, Debcl and Dark genes and down-regulation of DIAP1, an ubiquitylation catalyzing enzyme, suggests that ACR promotes apoptosis through disruption of caspase and pro-apoptotic protein ubiquitination and a mitochondria-dependent pathway in Drosophila melanogaster. Conclusively, this study provides valuable insights into the cellular mechanism underlying ACR-mediated toxicity. Additionally, our study reinforces the utility of D. melanogaster as a translational tool for elucidating the complex mechanisms of ACR toxicity.

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.

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.

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.

Metatranscriptomic analysis identifies different viral-like sequences in two neotropical Mansoniini mosquito species.

Mosquitoes interact with a wide range of viruses including both arboviruses and insect-specific viruses. This study aimed to characterize the RNA viruses that are interacting with Mansonia wilsoni and Coquillettidia hermanoi mosquito species. The total RNA extracted from mosquito pools were sequenced on a Ion torrent platform. Viral contigs were identified against viral databases and their evolutionary relationship were reconstructed. We identified a total of 107 viral sequences, 11 of which were assigned as endogenous viral elements, and at least six known viral families were identified. Phylogenetic reconstructions were performed for 4 viral families. All Mansoniini viruses investigated through phylogenetic analysis are closely related to insect-specific viruses found in other mosquito species although with considerable divergence at the amino acid level, suggesting that we have detected new viral lineages. This study enhanced our understanding about the virome of two sylvatic Mansoniini mosquitoes.

Drosophila parasitoid wasps bears a distinct DNA transposon profile.

BACKGROUND: The majority of Eukaryotic genomes are composed of a small portion of stable (non-mobile) genes and a large fraction of parasitic mobile elements such as transposable elements and endogenous viruses: the Mobilome. Such important component of many genomes are normally underscored in genomic analysis and detailed characterized mobilomes only exists for model species. In this study, we used a combination of de novo and homology approaches to characterize the Mobilome of two non-model parasitoid wasp species. RESULTS: The different methodologies employed for TE characterization recovered TEs with different features as TE consensus number and size. Moreover, some TEs were detected only by one or few methodologies. RepeatExplorer and dnaPipeTE estimated a low TE content of 5.86 and 4.57% for Braconidae wasp and 5.22% and 7.42% for L. boulardi species, respectively. Both mobilomes are composed by a miscellaneous of ancient and recent elements. Braconidae wasps presented a large diversity of Maverick/Polintons Class II TEs while other TE superfamilies were more equally diverse in both species. Phylogenetic analysis of reconstructed elements showed that vertical transfer is the main mode of transmission. CONCLUSION: Different methodologies should be used complementarity in order to achieve better mobilome characterization. Both wasps genomes have one of the lower mobilome estimates among all Hymenoptera genomes studied so far and presented a higher proportion of Class II than Class I TEs. The large majority of superfamilies analyzed phylogenetically showed that the elements are being inherited by vertical transfer. Overall, we achieved a deep characterization of the mobilome in two non-model parasitoid wasps improving our understanding of their evolution.

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.

Genetic exchange in eukaryotes through horizontal transfer: connected by the mobilome.

BACKGROUND: All living species contain genetic information that was once shared by their common ancestor. DNA is being inherited through generations by vertical transmission (VT) from parents to offspring and from ancestor to descendant species. This process was considered the sole pathway by which biological entities exchange inheritable information. However, Horizontal Transfer (HT), the exchange of genetic information by other means than parents to offspring, was discovered in prokaryotes along with strong evidence showing that it is a very important process by which prokaryotes acquire new genes. MAIN BODY: For some time now, it has been a scientific consensus that HT events were rare and non-relevant for evolution of eukaryotic species, but there is growing evidence supporting that HT is an important and frequent phenomenon in eukaryotes as well. CONCLUSION: Here, we will discuss the latest findings regarding HT among eukaryotes, mainly HT of transposons (HTT), establishing HTT once and for all as an important phenomenon that should be taken into consideration to fully understand eukaryotes genome evolution. In addition, we will discuss the latest development methods to detect such events in a broader scale and highlight the new approaches which should be pursued by researchers to fill the knowledge gaps regarding HTT among eukaryotes.

Effects of heat and UV radiation on the mobilization of transposon mariner-Mos1.

There are many complex interactions between transposable elements (TEs) and host genomes. Environmental changes that induce stressful conditions help to contribute for increasing complexity of these interactions. The transposon mariner-Mos1 increases its mobilization under mild heat stress. It has putative heat shock elements (HSEs), which are probably activated by heat shock factors (HSFs). Ultraviolet radiation (UVC) is a stressor that has been suggested as able to activate heat shock protein genes (Hsp). In this study, we test the hypothesis that if UVC induces Hsp expression, as heat does, it could also promote mariner-Mos1 transposition and mobilization. The Drosophila simulans white-peach is a mutant lineage that indicates the mariner-Mos1 transposition phenotypically through the formation of mosaic eyes. This lineage was exposed to UVC or mild heat stress (28 °C) in order to evaluate the induction of mariner-Mos1 expression by RT-qPCR, as well as the mariner-Mos1 mobilization activity based on the count number of red spots in the eyes. The effects of both treatments on the developmental time of flies and cell cycle progression were also investigated. Both the analysis of eyes and mariner-Mos1 gene expression indicate that UVC radiation has no effect in mariner-Mos1 transposition, although heat increases the expression and mobilization of this TE soon after the treatment. However, the expression of Hsp70 gene increased after 24 h of UVC exposure, suggesting different pathway of activation. These results showed that heat promotes mariner-Mos1 mobilization, although UVC does not induce the expression or mobilization of this TE.

An evaluation of the ecological relationship between Drosophila species and their parasitoid wasps as an opportunity for horizontal transposon transfer.

Evidences of horizontal transfer, the exchange of genetic material between reproductively isolated species, have accumulated over the last decades, including for multicellular eukaryotic organisms. However, the mechanisms and ecological relationships that promote such phenomenon is still poorly known. Host-parasite interaction is one type of relationship usually pointed in the literature that could potentially increase the probability of the horizontal transfer between species, because the species involved in such relationships are generally in close contact. Transposable elements, which are well-known genomic parasites, are DNA entities that tend to be involved in horizontal transfer due to their ability to mobilize between different genomic locations. Using Drosophila species and their parasitoid wasps as a host-parasite model, we evaluated the hypothesis that horizontal transposon transfers (HTTs) are more frequent in this set of species than in species that do not exhibit a close ecological and phylogenetic relationship. For this purpose, we sequenced two sets of species using a metagenomic and single-species genomic sampling approach through next-generation DNA sequencing. The first set was composed of five generalist Drosophila (D. maculifrons, D. bandeirantorum, D. polymorpha, D. mercatorum and D. willistoni) species and their associated parasitoid wasps, whereas the second set was composed of D. incompta, which is a flower specialist species, and its parasitoid wasp. We did not find strong evidence of HTT in the two sets of Drosophila and wasp parasites. However, at least five cases of HTT were observed between the generalist and specialist Drosophila species. Moreover, we detected an HT event involving a Wolbachia lineage between generalist and specialist species, indicating that these endosymbiotic bacteria could play a role as HTT vectors. In summary, our results do not support the hypothesis of prevalent HTT between species with a host-parasite relationship, at least for the studied wasp-Drosophila pairs. Moreover, it suggests that other mechanisms or parasites are involved in promoting HTT between Drosophila species as the Wolbachia endosymbiotic bacteria.

Horizontal transposon transfer in eukarya: detection, bias, and perspectives.

The genetic similarity observed among species is normally attributed to the existence of a common ancestor. However, a growing body of evidence suggests that the exchange of genetic material is not limited to the transfer from parent to offspring but can also occur through horizontal transfer (HT). Transposable elements (TEs) are DNA fragments with an innate propensity for HT; they are mobile and possess parasitic characteristics that allow them to exist and proliferate within host genomes. However, horizontal transposon transfer (HTT) is not easily detected, primarily because the complex TE life cycle can generate phylogenetic patterns similar to those expected for HTT events. The increasingly large number of new genome projects, in all branches of life, has provided an unprecedented opportunity to evaluate the TE content and HTT events in these species, although a standardized method of HTT detection is required before trends in the HTT rates can be evaluated in a wide range of eukaryotic taxa and predictions about these events can be made. Thus, we propose a straightforward hypothesis test that can be used by TE specialists and nonspecialists alike to discriminate between HTT events and natural TE life cycle patterns. We also discuss several plausible explanations and predictions for the distribution and frequency of HTT and for the inherent biases of HTT detection. Finally, we discuss some of the methodological concerns for HTT detection that may result in the underestimation and overestimation of HTT rates during eukaryotic genome evolution.

hAT transposable elements and their derivatives: an analysis in the 12 Drosophila genomes.

Transposable elements (TEs) comprise a significant fraction of the genome, and some models of the TE "life cycle" suggest that, in the last phases of the cycle, TEs should be represented, in the genomes, by inactive and degenerated copies. In this study, we analyzed, using a bioinformatics approach, the autonomous hAT elements and their derivatives (active non-autonomous, MITE relatives and degenerated copies) in 12 Drosophila genomes. We found 28 hAT elements that had derivatives. Most copies had features that suggested that they were active, while only a few degenerated copies were found. Because hAT elements comprise an evolutionarily old superfamily, one should expect to find many degenerated copies within the genome, although this was not observed in our study. These results suggest that primarily active copies of hAT elements are maintained in the euchromatic regions of the genome and that degenerated copies are removed from the genome by natural selection.

Evolution of Tom, 297, 17.6 and rover retrotransposons in Drosophilidae species.

LTR retrotransposons are the most abundant transposable elements in Drosophila and are believed to have contributed significantly to genome evolution. Different reports have shown that many LTR retrotransposon families in Drosophila melanogaster emerged from recent evolutionary episodes of transpositional activity. To contribute to the knowledge of the evolutionary history of Drosophila LTR retrotransposons and the mechanisms that control their abundance, distribution and diversity, we conducted analyses of four related families of LTR retrotransposons, 297, 17.6, rover and Tom. Our results show that these elements seem to be restricted to species from the D. melanogaster group, except for 17.6, which is also present in D. virilis and D. mojavensis. Genetic divergences and phylogenetic analyses of a 1-kb fragment region of the pol gene illustrate that the evolutionary dynamics of Tom, 297, 17.6 and rover retrotransposons are similar in several aspects, such as low codon bias, the action of purifying selection and phylogenies that are incongruent with those of the host species. We found an extremely complex association among the retrotransposon sequences, indicating that different processes shaped the evolutionary history of these elements, and we detected a very high number of possible horizontal transfer events, corroborating the importance of lateral transmission in the evolution and maintenance of LTR retrotransposons.

Isolation of high quality DNA: a protocol combining “rennet” and glass milk

High quality DNA is essential for many molecular biology techniques. However, the reagents used for that purpose usually are expensive and/or cause a high environmental impact. Here, we describe two alternative protocols that use inexpensive reagents and are not hazardous to the environment. The first protocol utilizes the enzyme chymosin, normally used as “rennet” in cheese production and which is easily obtained on the commercial market. The second protocol uses “rennet DNA extraction protocol” combined with the DNA binding capacity of glass powder (glass milk), which can easily be “home made”. The first protocol is used when a high yield of DNA is needed, whereas the second protocol is used for production of a higher quality DNA, being able to work with sparse samples.

Characterization of new hAT transposable elements in 12 Drosophila genomes.

In silico searches for sequences homologous to hAT elements in 12 Drosophila genomes have allowed us to identify 37 new hAT elements (8 in D. ananassae, 11 in D. mojavensis, 2 in D. sechellia, 1 in D. simulans, 2 in D. virilis, 3 in D. yakuba, 3 in D. persimilis, 1 in D. grimshawi, 5 in D. willistoni and 1 in D. pseudobscura). The size of these elements varies from 2,359 to 4,962 bp and the terminal inverted repeats (TIRs) show lengths ranging from 10 to 24 bp. Several elements show intact transposase ORFs, suggesting that they are active. Conserved amino acid motifs were identified that correspond to those important for transposase activity. These elements are highly variable and phylogenetic analysis showed that they can be clustered into four different families. Incongruencies were observed between the phylogenies of the transposable elements and those of their hosts, suggesting that horizontal transfer may have occurred between some of the species.

Genetic transformation of Drosophila willistoni using piggyBac transposon and GFP

Studies were carried out on the use of piggyBac transposable element as vector and the green fluorescent protein (EGFP) from the jellyfish, Aquorea victoria, as a genetic marker for the transformation of Drosophila willistoni. Preblastoderm embryos of D. willistoni white mutant were microinjected with a plasmid containing the EGFP marker and the piggyBac ITRs, together with a helper plasmid containing the piggyBac transposase placed under the control of the D. melanogaster hsp70 promoter. G0 adults transformants were recovered at a frequency of approximately 67 percent. Expression of EGFP in larvae, pupae and adults was observed up to the third generation, suggesting that this transposon was not stable in D. willistoni. Transformed individuals displayed high levels of EGFP expression during larvae and adult stages in the eye, abdomen, thorax and legs, suggesting a wide expression pattern in this species than reported to other species of Drosophilidae.

Descrevemos neste trabalho a transformação genética de Drosophila willistoni empregando o elemento transponível piggyBac como vetor e o gene EGFP (green fluorescent protein ) retirado da água-viva Aquorea victoria, como marcador de transformação. Embriões de D. willistoni em estágio pré-blastoderme, mutantes para o gene white, foram microinjetados com plasmídio contendo o marcador EGFP e as regiões ITRs do transposon piggyBac concomitantemente com um plasmídio auxiliar possuindo o gene da transposase de piggyBac sobre o controle do promotor do gene hsp70 de Drosophila melanogaster. Adultos transformantes Go foram gerados em uma taxa de 67 por cento. A expressão de GFP em larvas, pupas e adultos foi observada somente até a terceira geração, sugerindo que este transposon não é estável em D. willistoni. Os indivíduos transformados exigem um alto nível de expressão de EGFP durante os estágios de larva e, também em adultos o gene marcador é expresso nos olhos, abdome, tórax e patas, mostrando um padrão de expressão mais amplo nesta espécie do que o registrado para outros drosofilídeos.

Insertion sequences as variability generators in the Mycoplasma hyopneumoniae and M. synoviae genomes

We have analyzed the sequenced genomes of three strains of Mycoplasma hyopneumoniae and one strain of M. synoviae, and have found three and two different transposable element families, respectively in each species. In M. hyopneumoniae, the Insertion Sequences of the IS4 family is represented by ISMHp1, a putatively active element. The IS3 family is represented by several degenerated sequences. A third element called tMH was found, which shows some characteristics reminiscent of retrotransposons. In M. synoviae, three different possibly active IS4 elements are present (ISMHp1-like; ISMs1 and IS1634-like elements). The IS30 family is represented by the degenerated IS1630-like element. The IS1634-like element is shown to be involved in chromosomal rearrangements and horizontal gene transfer (HGT). The ISMHp1-like element is shown to relate to the HGT of a 25-kb region from M. gallisepticum to M. synoviae. The fractions of these genomes that correspond to mobile elements varied from 1.35 to 3.13 percent in M. hyopneumonia strains and was 2.08 percent in M. synoviae. Although these species possess reduced genomes, they maintain mobile elements, perhaps as a mechanism for genetic variability production.