Horizontal transfer and the widespread presence of Galileo transposons in Drosophilidae (Insecta: Diptera).

Galileo is a transposon notoriously involved with inversions in Drosophila buzzatii by ectopic recombination. Although widespread in Drosophila, little is known about this transposon in other lineages of Drosophilidae. Here, the abundance of the canonical Galileo and its evolutionary history in Drosophilidae genomes was estimated and reconstructed across genera within its two subfamilies. Sequences of this transposon were masked in these genomes and their transposase sequences were recovered using BLASTn. Phylogenetic analyses were employed to reconstruct their evolutionary history and compare it to that of host genomes. Galileo was found in nearly all 163 species, however, only 37 harbored nearly complete transposase sequences. In the remaining, Galileo was found highly fragmented. Copies from related species were clustered, however horizontal transfer events were detected between the melanogaster and montium groups of Drosophila, and between the latter and the Lordiphosa genus. The similarity of sequences found in the virilis and willistoni groups of Drosophila was found to be a consequence of lineage sorting. Therefore, the evolution of Galileo is primarily marked by vertical transmission and long-term inactivation, mainly through the deletion of open reading frames. The latter has the potential to lead copies of this transposon to become miniature inverted-repeat transposable elements.

Phylomitogenomics of two Neotropical species of long-legged crickets Endecous Saussure, 1878 (Orthoptera: Phalangopsidae).

Mitochondrial genomes have provided significant insights into the evolution of several insects. A typical mitogenome contains 37 genes, and variations in gene order can indicate evolutionary relationships between species. In this study, we have assembled the first complete mitogenomes of Endecous chape and E. onthophagus and analyzed the phylogenetic implications for the Gryllidea infraorder. We performed DNA extractions and genome sequencing for both Endecous species. Subsequently, we searched for raw data in the Sequence Read Archive (SRA) in NCBI. Using the SRA data, we assembled the partial mitogenome of Dianemobius nigrofasciatus and annotated the protein-coding genes (PCGs) for nine species. Phylogenomic relationships were reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI), utilizing the PCGs from 49 Gryllidea species. The mitogenome lengths of E. chape and E. onthophagus are 16,266 bp and 16,023 bp, respectively, while D. nigrofasciatus has a length of 15,359 bp. Our results indicate that species within the infraorder exhibit four types of gene order arrangements that align with their phylogenetic relationships. Both phylogenomic trees displayed strong support, and the ML corroborated with the literature. Gryllidea species have significantly contributed to various fields, and studying their mitogenomes can provide valuable insights into this infraorder evolution.

Evolution and diversification of the O-methyltransferase (OMT) gene family in Solanaceae.

O-methyltransferases (OMTs) are a group of enzymes involved in several fundamental biological processes in plants, including lignin biosynthesis, pigmentation, and aroma production. Despite the intensive investigation of the role of OMTs in plant secondary metabolism, the evolution and diversification of this gene family in Solanaceae remain poorly understood. Here, we conducted a genome-wide survey of OMT genes in six Solanaceae species, reconstructing gene phylogenetic trees, predicting the potential involvement in biological processes, and investigating the exon/intron structure and chromosomal location. We identified 57 caffeoyl-CoA OMTs (CCoAOMTs) and 196 caffeic acid OMTs (COMTs) in the studied species. We observed a conserved gene block on chromosome 2 that consisted of tandem duplicated copies of OMT genes. Our results suggest that the expansion of the OMT gene family in Solanaceae was driven by whole genome duplication, segmental duplication, and tandem duplication, with multiple genes being retained by neofunctionalization and subfunctionalization. This study represents an essential first step in unraveling the evolutionary history of OMTs in Solanaceae. Our findings deepen our understanding of the crucial role of OMTs in several biological processes and highlight their significance as potential biotechnological targets.

Preliminary population studies of the grassland swallowtail butterfly Euryades corethrus (Lepidoptera, Papilionidae).

Euryades corethrus is a Troidini butterfly (Papilionidae, Papilioninae), endemic to grasslands in southern Brazil, Uruguay, Argentina and Paraguay. Formerly abundant, nowadays it is in the Red list of endangered species for those areas. During its larval stage, it feeds on Aristolochia spp, commonly found in southern grasslands. These native grassland areas are diminishing, being converted to crops and pastures, causing habitat loss for Aristolochia and E. corethrus. This study aimed to assess the genetic diversity, population structure and demographic history of E. corethrus. We sampled eight populations from Rio Grande do Sul, Brazil and based on Cytochrome Oxidase subunit I (COI) molecular marker, our results suggest a low genetic variability between populations, presence of gene flow and, consequently, lack of population structure. A single maternally inherited-genetic marker is insufficient for population-level decisions, but barcoding is a useful tool during early stages of population investigation, bringing out genomic diversity patterns within the target species. Those populations likely faced a bottleneck followed by a rapid expansion during the last glaciation and subsequent stabilization in effective population size. Habitat loss is a threat, which might cause isolation, loss of genetic variability and, ultimately, extinction of E. corethrus if no habitat conservation policy is adopted.

Patterns of genome size evolution versus fraction of repetitive elements in statu nascendi species: the case of the willistoni subgroup of Drosophila (Diptera, Drosophilidae).

Genome size evolution is known to be related with transposable elements, yet such relation in incipient species remains poorly understood. For decades, the willistoni subgroup of Drosophila has been a model for evolutionary studies because of the different evolutionary stages and degrees of reproductive isolation its species present. Our main question here was how speciation influences genome size evolution and the fraction of repetitive elements, with a focus on transposable elements. We quantitatively compared the mobilome of four species and two subspecies belonging to this subgroup with their genome size, and performed comparative phylogenetic analyses. Our results showed that genome size and the fraction of repetitive elements evolved according to the evolutionary history of these species, but the content of transposable elements showed some discrepancies. Signals of recent transposition events were detected for different superfamilies. Their low genomic GC content suggests that in these species transposable element mobilization might be facilitated by relaxed natural selection. Additionally, a possible role of the superfamily DNA/TcMar-Tigger in the expansion of these genomes was also detected. We hypothesize that the undergoing process of speciation could be promoting the observed increase in the fraction of repetitive elements and, consequently, genome size.

Canavalia ensiformis lectin induced oxidative stress mediate both toxicity and genotoxicity in Drosophila melanogaster.

Mannose/glucose-binding lectin from Canavalia ensiformis seeds (Concanavalin A - ConA) has several biological applications, such as mitogenic and antitumor activity. However, most of the mechanisms involved in the in vivo toxicity of ConA are not well known. In this study, the Drosophila melanogaster model was used to assess the toxicity and genotoxicity of different concentrations of native ConA (4.4, 17.5 and 70 µg/mL) in inhibited and denatured forms of ConA. The data show that native ConA affected: the survival, in the order of 30.6 %, and the locomotor performance of the flies; reduced cell viability to levels below 50 % (4.4 and 17.5 µg/mL); reduced nitric oxide levels; caused lipid peroxidation and increased protein and non-protein thiol content. In the Comet assay, native ConA (17.5 e 70 µg/mL) caused DNA damage higher than 50 %. In contrast, treatments with inhibited and denatured ConA did not affect oxidative stress markers and did not cause DNA damage. We believe that protein-carbohydrate interactions between ConA and carbohydrates of the plasma membrane are probably the major events involved in these activities, suggesting that native ConA activates mechanisms that induce oxidative stress and consequently DNA damage.

Study of four Neotropical species of tree crickets Oecanthus Serville, 1831 (Orthoptera, Gryllidae) using cytogenetic and molecular markers.

Karyotypes in the worldwide subfamily Oecanthinae show variations in diploid number, chromosome morphology, and sex-chromosome system. This study described the chromosome set and phylogenetic relationships of four Neotropical species, Oecanthus lineolatus, O. valensis, O. pallidus, and O. pictus. We used classical cytogenetics and Bayesian Inference for phylogenetic reconstruction, using the mitochondrial genes COI, 12S rRNA, and 16S rRNA; and analyzed the phylogenetic patterns of changes in chromosome numbers, using ChromEvol. We observed differences in chromosome number among species and two different sex-chromosome systems. Oecanthus pictus showed 2n = 21, X0♂/22, XX♀; O. lineolatus, 2n = 20, XY♂/XX♀; and O. valensis and O. pallidus, 2n = 18, XY♂/XX♀. The karyotype of Oecanthus was asymmetric, one group with large chromosomes and variation in heterochromatin distribution, and another with small acrocentric chromosomes. The phylogenetic tree recovered two main groups: one with the Palearctic species and another with species from different bioregions, but with low posterior probability. The Neotropical species grouped separately, O. valensis and O. pictus with Nearctic and Ethiopian species, and O. pallidus and O. lineolatus in another, well-supported clade. Together, the phylogenic and chromosome data suggest descending dysploidy events during the evolution of the group.

Interpopulation variation of transposable elements of the hAT superfamily in Drosophila willistoni (Diptera: Drosophilidae): in-situ approach.

Transposable elements are abundant and dynamic part of the genome, influencing organisms in different ways through their presence or mobilization, or by acting directly on pre- and post-transcriptional regulatory regions. We compared and evaluated the presence, structure, and copy number of three hAT superfamily transposons (hobo, BuT2, and mar) in five strains of Drosophila willistoni species. These D. willistoni strains are of different geographical origins, sampled across the north-south occurrence of this species. We used sequenced clones of the hAT elements in fluorescence in-situ hybridizations in the polytene chromosomes of three strains of D. willistoni. We also analyzed the structural characteristics and number of copies of these hAT elements in the 10 currently available sequenced genomes of the willistoni group. We found that hobo, BuT2, and mar were widely distributed in D. willistoni polytene chromosomes and sequenced genomes of the willistoni group, except for mar, which is restricted to the subgroup willistoni. Furthermore, the elements hobo, BuT2, and mar have different evolutionary histories. The transposon differences among D. willistoni strains, such as variation in the number, structure, and chromosomal distribution of hAT transposons, could reflect the genomic and chromosomal plasticity of D. willistoni species in adapting to highly variable environments.

Impact of gamma radiation dose on sterility and quality parameters of Anastrepha fraterculus (Diptera: Tephritidae).

Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae) is a major fruit pest, which is basicaly controlled using insecticides, which represents a risk to beneficial arthropods, human health and food contamination. The sterile insect technique (SIT) is a potential alternative tool for the management of this pest, however, only conflicting data is found regarding the optimal dose to achieve sterility. Thus, this study evaluated the effect of gamma radiation doses (0, 40, 50, 60 and 70 Gy) on male and female reproductive sterility, gonads morphometry, emergence, flight ability, and longevity under nutritional stress of A. fraterculus. Full female sterility was achieved at 50 Gy, while full male sterility was achieved at 70 Gy. Both ovarian and testicular sizes were affected by irradiation, while no influence was observed on the quality parameters evaluated. Our results suggest that 70 Gy applied 48 h before adult emergence can be used to sterilize A. fraterculus in a SIT programme.

Barcoding a can of worms: testing cox1 performance as a DNA barcode of Nematoda.

Accurate taxonomic identifications and species delimitations are a fundamental problem in biology. The complex taxonomy of Nematoda is primarily based on morphology, which is often dubious. DNA barcoding emerged as a handy tool to identify specimens and assess diversity, but its applications in Nematoda are incipient. We evaluated cytochrome c oxidase subunit I (cox1) efficiency as a DNA barcode for nematodes scrutinising 5241 sequences retrieved from BOLD and GenBank. The samples included genera with medical, agricultural, or ecological relevance: Anguillicola, Caenorhabditis, Heterodera, Meloidogyne, Onchocerca, Strongyloides, and Trichinella. We assessed cox1 performance through barcode gap and Probability of Correct Identification (PCI) analyses, and estimated species richness through Automatic Barcode Gap Discovery (ABGD). Each genus presented distinct gap ranges, mirroring the evolutionary diversity within Nematoda. Thus, to survey the diversity of the phylum, a careful definition of thresholds for lower taxonomic levels should be considered. PCIs were around 70% for both databases, highlighting operational biases and challenges in nematode taxonomy. ABGD inferred higher richness than the taxonomic labels informed by databases. The prevalence of specimen misidentifications and dubious species delimitations emphasise the value of integrative approaches to nematode taxonomy and systematics. Overall, cox1 is a relevant tool for integrative taxonomy of nematodes.

The identity of some specimens previously (mis)identified as Rhinoleucophenga obesa (Loew) (Diptera: Drosophilidae) in Brazil, based on morphological and molecular data, with implications on distribution.

Rhinoleucophenga Hendel is an endemic genus of the New World with most species recorded in Brazil. Rhinoleucophenga obesa (Loew) seemed to be the most widespread species, being recorded in the United States of America, Mexico and Brazil. In the Neotropical region, identifications of R. obesa were commonly based on the description of non-type specimens determined by Costa Lima (1935) and Malogolowkin (1946), although their identities were doubtful. However, the recent redescription of R. obesa from the type-series from Texas, USA, confirmed a long period of misidentifications and a new species, R. cantareira Vilela Bächli, was proposed based on Brazilian specimens. Thus, review of morphological and molecular traits of specimens previously identified as R. obesa in Neotropical biomes are necessary to check the identity and distribution of a group of sibling species: R. obesa, R. cantareira, R. gigantea (Thomson), R. pallida Hendel and R. pampeana Poppe et al. In the present paper, specimens previously determined as R. obesa, sampled in different Brazilian localities, were compared by morphological and molecular traits. Plates of female terminalia of R. cantareira and R. gigantea are presented for the first time. The spermathecal capsules (as well as the male epandrium) revealed useful characteristics to differentiate those species; these morphological differences were corroborated by a sequence fragment of COI. The specimens on which were based the descriptions of Rhinoleucophenga obesa sensu Costa Lima (1935) and sensu Malogolowkin (1946) were reviewed and confirmed as R. cantareira. Other Brazilian specimens from different localities, previously misidentified as R. obesa, were determined as R. gigantea or R. cantareira, with new records for both species. Therefore, the present study corresponds to the advance of identity definition and distribution of sibling species of Rhinoleucophenga commonly sampled in Neotropical inventory studies.

Evolution of DNMT2 in drosophilids: Evidence for positive and purifying selection and insights into new protein (pathways) interactions.

The DNA methyltransferase 2 (DNMT2) protein is the most conserved member of the DNA methyltransferase family. Nevertheless, its substrate specificity is still controversial and elusive. The genomic role and determinants of DNA methylation are poorly understood in invertebrates, and several mechanisms and associations are suggested. In Drosophila, the only known DNMT gene is Dnmt2. Here we present our findings from a wide search for Dnmt2 homologs in 68 species of Drosophilidae. We investigated its molecular evolution, and in our phylogenetic analyses the main clades of Drosophilidae species were recovered. We tested whether the Dnmt2 has evolved neutrally or under positive selection along the subgenera Drosophila and Sophophora and investigated positive selection in relation to several physicochemical properties. Despite of a major selective constraint on Dnmt2, we detected six sites under positive selection. Regarding the DNMT2 protein, 12 sites under positive-destabilizing selection were found, which suggests a selection that favors structural and functional shifts in the protein. The search for new potential protein partners with DNMT2 revealed 15 proteins with high evolutionary rate covariation (ERC), indicating a plurality of DNMT2 functions in different pathways. These events might represent signs of molecular adaptation, with molecular peculiarities arising from the diversity of evolutionary histories experienced by drosophilids.

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.

Effects of Irradiation Dose on Sterility Induction and Quality Parameters of Drosophila suzukii (Diptera: Drosophilidae).

Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae) is a widely distributed pest of soft-skinned and stone fruits that is controlled mainly with pesticides. An alternative to the chemical control is the sterile insect technique (SIT), an ecologically friendly method of pest management that could be used against D. suzukii. The objective of the present study was to evaluate the effects of gamma radiation on reproductive sterility, ovarian morphometry, and quality parameters of D. suzukii. Full female sterility was achieved at 75 Gy, while an adequate level of male sterility (99.67%) was obtained at 200 Gy. The ovarian size showed an exponential decay in function of irradiation dose increase. There was no significant influence of irradiation dose on the quality parameters evaluated. Our data suggest that gamma radiation can be recommended to be used in an SIT program for D. suzukii.

Combining morphology and molecular data to improve Drosophila paulistorum (Diptera, Drosophilidae) taxonomic status.

The willistoni species subgroup has been the subject of several studies since the latter half of the past century and is considered a Neotropical model for evolutionary studies, given the many levels of reproductive isolation and different evolutionary stages occurring within them. Here we present for the first time a phylogenetic reconstruction combining morphological characters and molecular data obtained from 8 gene fragments (COI, COII, Cytb, Adh, Ddc, Hb, kl-3 and per). Some relationships were incongruent when comparing morphological and molecular data. Also, morphological data presented some unresolved polytomies, which could reflect the very recent divergence of the subgroup. The total evidence phylogenetic reconstruction presented well-supported relationships and summarized the results of all analyses. The diversification of the willistoni subgroup began about 7.3 Ma with the split of D. insularis while D.paulistorum complex has a much more recent diversification history, which began about 2.1 Ma and apparently has not completed the speciation process, since the average time to sister species separation is one million years, and some entities of the D. paulistorum complex diverge between 0.3 and 1 Ma. Based on the obtained data, we propose the categorization of the former "semispecies" of D. paulistorum as a subspecies and describe the subspecies D. paulistorum amazonian, D. paulistorum andeanbrazilian, D. paulistorum centroamerican, D. paulistorum interior, D. paulistorum orinocan and D. paulistorum transitional.

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

Abstract 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.

Evolution of DNMT2 in drosophilids: Evidence for positive and purifying selection and insights into new protein (pathways) interactions

Abstract The DNA methyltransferase 2 (DNMT2) protein is the most conserved member of the DNA methyltransferase family. Nevertheless, its substrate specificity is still controversial and elusive. The genomic role and determinants of DNA methylation are poorly understood in invertebrates, and several mechanisms and associations are suggested. In Drosophila, the only known DNMT gene is Dnmt2. Here we present our findings from a wide search for Dnmt2 homologs in 68 species of Drosophilidae. We investigated its molecular evolution, and in our phylogenetic analyses the main clades of Drosophilidae species were recovered. We tested whether the Dnmt2 has evolved neutrally or under positive selection along the subgenera Drosophila and Sophophora and investigated positive selection in relation to several physicochemical properties. Despite of a major selective constraint on Dnmt2, we detected six sites under positive selection. Regarding the DNMT2 protein, 12 sites under positive-destabilizing selection were found, which suggests a selection that favors structural and functional shifts in the protein. The search for new potential protein partners with DNMT2 revealed 15 proteins with high evolutionary rate covariation (ERC), indicating a plurality of DNMT2 functions in different pathways. These events might represent signs of molecular adaptation, with molecular peculiarities arising from the diversity of evolutionary histories experienced by drosophilids.

Deciphering the Routes of invasion of Drosophila suzukii by Means of ABC Random Forest.

Deciphering invasion routes from molecular data is crucial to understanding biological invasions, including identifying bottlenecks in population size and admixture among distinct populations. Here, we unravel the invasion routes of the invasive pest Drosophila suzukii using a multi-locus microsatellite dataset (25 loci on 23 worldwide sampling locations). To do this, we use approximate Bayesian computation (ABC), which has improved the reconstruction of invasion routes, but can be computationally expensive. We use our study to illustrate the use of a new, more efficient, ABC method, ABC random forest (ABC-RF) and compare it to a standard ABC method (ABC-LDA). We find that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well. All introduced populations passed through bottlenecks, and analyses reveal five distinct admixture events. These findings can inform hypotheses concerning how this species evolved between different and independent source and invasive populations. Methodological comparisons indicate that ABC-RF and ABC-LDA show concordant results if ABC-LDA is based on a large number of simulated datasets but that ABC-RF out-performs ABC-LDA when using a comparable and more manageable number of simulated datasets, especially when analyzing complex introduction scenarios.

Can sibling species of the Drosophila willistoni subgroup be recognized through combined microscopy techniques?

ABSTRACT In several arthropod groups, male genitalia is the most important feature for species identification, especially in cryptic species. Cryptic species are very common in the Drosophila genus, and the Neotropical Drosophila willistoni species group is a good example. This group currently includes 24 species divided into three subgroups: alagitans, bocainensis and willistoni. There are six sibling species in the willistoni subgroup – D. willistoni, D. insularis, D. tropicalis, D. equinoxialis, D. pavlovskiana and D. paulistorum, which is a species complex composed of six semispecies – Amazonian, Andean-Brazilian, Centroamerican, Interior, Orinocan and Transitional. The objective of this study was to characterize male genitalia of the willistoni subgroup, including the D. paulistorum species complex, using scanning electron microscopy and light microscopy. We also tried to contribute to the identification of these cryptic species and to add some comments about evolutionary history, based on male genitalia characters. Despite being cryptic species, some differences were found among the siblings, including the Drosophila paulistorum semispecies.

BuT2 is a member of the third major group of hAT transposons and is involved in horizontal transfer events in the genus Drosophila.

The hAT superfamily comprises a large and diverse array of DNA transposons found in all supergroups of eukaryotes. Here we characterized the Drosophila buzzatii BuT2 element and found that it harbors a five-exon gene encoding a 643-aa putatively functional transposase. A phylogeny built with 85 hAT transposases yielded, in addition to the two major groups already described, Ac and Buster, a third one comprising 20 sequences that includes BuT2, Tip100, hAT-4_BM, and RP-hAT1. This third group is here named Tip. In addition, we studied the phylogenetic distribution and evolution of BuT2 by in silico searches and molecular approaches. Our data revealed BuT2 was, most often, vertically transmitted during the evolution of genus Drosophila being lost independently in several species. Nevertheless, we propose the occurrence of three horizontal transfer events to explain its distribution and conservation among species. Another aspect of BuT2 evolution and life cycle is the presence of short related sequences, which contain similar 5' and 3' regions, including the terminal inverted repeats. These sequences that can be considered as miniature inverted repeat transposable elements probably originated by internal deletion of complete copies and show evidences of recent mobilization.