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 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 TEs 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.

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.

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.

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.

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.

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.

Two new species of Mycodrosophila (Diptera, Drosophilidae) proposed by molecular and morphological approaches, with a key to American species

ABSTRACT There are approximately 130 species of MycodrosophilaOldenberg, 1914 worldwide, although only nine species were recorded in American countries so far, three of which are exclusively Nearctic, five exclusively Neotropical and one found in both biogeographic regions (Mycodrosophila projectans). Such a small number of American species is likely a consequence of collecting bias, which favors the capture of frugivorous drosophilids, and to the general absence of Neotropical Mycodrosophila studies in the last 50 years. Here, we describe two commonly sampled species of Mycodrosophila from the Amazonian and Pampa Brazilian biomes, which share morphological similarities with Mycodrosophila neoprojectans and M. projectans, respectively. We compared sequences of the mitochondrial gene cytochrome oxidase subunit I (COI), external morphology characteristics and male terminalia among these species. Based on a DNA barcoding approach coupled to morphological differences, we proposed the delimitation of two new species, Mycodrosophila hofmanni sp. nov. and Mycodrosophila valentae sp. nov. An updated key to identifying Neotropical and Nearctic Mycodrosophila species is also provided.

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.

Identification of influential events concerning the Antarctic ozone hole over southern Brazil and the biological effects induced by UVB and UVA radiation in an endemic treefrog species.

The increased incidence of solar ultraviolet radiation (UV) due to ozone depletion has been affecting both terrestrial and aquatic ecosystems and it may help to explain the enigmatic decline of amphibian populations in specific localities. In this work, influential events concerning the Antarctic ozone hole were identified in a dataset containing 35 years of ozone measurements over southern Brazil. The effects of environmental doses of UVB and UVA radiation were addressed on the morphology and development of Hypsiboas pulchellus tadpole (Anura: Hylidae), as well as on the induction of malformation after the conclusion of metamorphosis. These analyzes were complemented by the detection of micronucleus formation in blood cells. 72 ozone depletion events were identified from 1979 to 2013. Surprisingly, their yearly frequency increased three-fold during the last 17 years. The results clearly show that H. pulchellus tadpole are much more sensitive to UVB than UVA light, which reduces their survival and developmental rates. Additionally, the rates of micronucleus formation by UVB were considerably higher compared to UVA even after the activation of photolyases enzymes by a further photoreactivation treatment. Consequently, a higher occurrence of malformation was observed in UVB-irradiated individuals. These results demonstrate the severe genotoxic impact of UVB radiation on this treefrog species and its importance for further studies aimed to assess the impact of the increased levels of solar UVB radiation on declining species of the Hylidae family.

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.

Involvement of oxidative stress in 4-vinylcyclohexene-induced toxicity in Drosophila melanogaster.

4-Vinylcyclohexene (VCH) is a dimer of 1,3-butadiene produced as a by-product of pesticides, plastic, rubber, flame retardants, and tire production. Although, several studies have reported the ovotoxicity of VCH, information on a possible involvement of oxidative stress in the toxicity of this occupational chemical is scarce. Hence, this study was carried out to investigate further possible mechanisms of toxicity of VCH with a specific emphasis on oxidative stress using a Drosophila melanogaster model. D. melanogaster (both genders) of 1 to 3 days old were exposed to different concentrations of VCH (10 µM-1 mM) in the diet for 5 days. Subsequently, the survival and negative geotaxis assays and the quantification of reactive oxygen species (ROS) generation were determined. In addition, we evaluated RT-PCR expressions of selected oxidative stress and antioxidant mRNA genes (HSP27, 70, and 83, SOD, Nrf-2, MAPK2, and catalase). Furthermore, catalase, glutathione-S-transferase (GST), delta aminolevulinic acid dehydratase (δ-ALA-D), and acetylcholinesterase (AChE) activities were determined. VCH exposure impaired negative geotaxic behavior and induced the mRNA of SOD, Nrf-2, and MAPK2 genes expressions. There were increases in catalase and ROS production, as well as inhibitions of GST, δ-ALA-D, and AChE activities (P<0.05). Our results suggest that the VCH mechanism of toxicity is associated with oxidative damage, as evidenced by the alteration in the oxidative stress-antioxidant balance, and possible neurotoxic consequences due to decreased AChE activity, and impairments in negative geotaxic behavior. Thus, we conclude that D. melanogaster is a useful model for investigating the toxicity of VCH exposure, and here, we have provided further insights on the mechanism of VCH-induced toxicity.

Neuroprotective effect of diphenyl diselenide in a experimental stroke model: maintenance of redox system in mitochondria of brain regions.

Acute stroke is a major risk for morbidity and mortality in aging population. Mitochondrion has been the focus of a wide stroke-related research. This study investigated if treatment or pre-treatment with diphenyl diselenide (PhSe)2 can prevent mitochondrial damage in cerebral structures of rats induced by an ischemia and reperfusion (I/R) model. Adult male Wistar rats were assigned into five experimental groups: sham operation, ischemia/reperfusion, pre-treated + I/R, treated + I/R, and Sham + (PhSe)2. Neurological score showed the damage caused by I/R, which was partially prevented by (PhSe)2. Moreover, mitochondria of hippocampus and cortex were impaired by I/R through an increase of reactive oxygen species production, mitochondrial membrane potential (ΔΨm) and electrons flow alteration, activity of complex I deregulation as well as mitochondrial swelling. However, the ischemic damage did not induce an increase in pro-apoptotic proteins expression, but demonstrated an enhanced expression of Hsp70. The mitochondrial redox state was also altered (GSH/GSSG ratio, MnSOD, and GPx activities). Our results revealed that all treatments with (PhSe)2 significantly reduced the mitochondrial damage induced by I/R. These findings suggest that neuroprotective properties of (PhSe)2 may be attributed to the maintenance of mitochondrial redox balance.

The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

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.