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.info:eu-repo/semantics/publishedVersio

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

Funding Information: This article is a chapter of the PhD thesis of the first author. We would like to thank Dr. Vera Lúcia da Silva Valente Gayeski for allowing us to use the Drosophila lab and all its equipment to perform the wetlab experiments, as well as supporting us in all stages of this work. Grant sponsor: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Brazil. Grant number: 163268/2015-0.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.publishersversionpublishe

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

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

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 benefi cial arthropods, human health and food contamination. The sterile insect technique (SIT) is a potential alternative tool for the management of this pest, however, only confl icting 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, fl ight 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 infl uence 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

Estudo da mobilização de transposons através de transformação genética

A mobilização dos elementos de transposição (TEs) está sujeita a um conjunto complexo de mecanismos regulatórios que envolvem não apenas proteínas codificadas pelos próprios transposons, mas também pela célula hospedeira. Entretanto, até o momento, se conhece pouco sobre os fatores que levam à mobilização de TEs nos genomas hospedeiros. De uma maneira geral, fatores que expõem um organismo a situações de estresse levam à mobilização de transposons. Em função disso, propomos a adaptação, a nossas condições de laboratório, de uma metodologia de transformação genética para ser utilizada como ferramenta, na tentativa de contribuir na elucidação dos mecanismos de mobilização do elemento transponível hobo. Para o estabelecimento desta metodologia utilizamos como ferramentas um microscópio óptico binocular e um micromanipulador adaptado LEITZ. Neste micromanipulador foi acoplado um sistema de compressão a ar, composto por uma seringa de vidro de 20 ml e um dispositivo para infusão endovenosa (butterfly), onde é encaixada uma ponteira de micropipeta. Na ponteira, foi colada, com adesivo instantâneo, uma agulha feita a partir de capilares de vidro. O processo de microinjeção ocorreu em uma lâmina de microscopia contendo uma fita adesiva dupla-face que serve de suporte para os ovos a serem injetados. Com o auxílio de uma agulha histológica, ovos do mutante white de Drosophila simulans, previamente desidratados, foram decorionados manualmente e colocados enfileirados sobre este suporte para evitar deslocamentos durante a injeção. Posteriormente, os embriões foram imersos em “óleo de halocarbono”, e, em seguida, foi realizada a microinjeção de DNA. Para esta etapa do trabalho foram testados vários vetores de transformação, sendo mais eficaz o elemento transponível piggyBac, com o vetor pBac[3xp3-GFPafm], o qual possui um promotor que conduz uma forte expressão da proteína GFP (Green Fluorescent Protein) em tecidos do olho de moscas adultas, o que, sob luz laser azul, fluoresce com uma intensa cor verde. Com este vetor conseguimos a detecção de transformantes, e a comprovação da eficiência da metodologia estabelecida. A segunda etapa do trabalho teve como objetivo estudar a mobilização do transposon hobo por meio de microinjeção de DNA. Para isso foram utilizados os mutantes white, cuja mutação é causada pela inserção do elemento hobo “relic” (hoboVA) neste gene, e dpp-like de D. simulans. Como vetor de transformação foram utilizados, independentemente, os plasmídeos pHFL1 (hobo canônico), e BlueScript como controle. Os adultos microinjetados sobreviventes (G0) foram individualmente cruzados com outro da mesma linhagem, e, a partir deste cruzamento, foram estabelecidas cinco isolinhagens de cada G0 acompanhadas até a 3ª geração. Todos os indivíduos foram analisados quanto ao fenótipo, para possíveis eventos de reversão de mutação, e alguns, analisados molecularmente por Southern Blot. Foram observadas diversas alterações morfológicas, essencialmente nas asas, tanto em G0 como em seus descendentes. Na linhagem dpp-like foram observados eventos de reversão ao fenótipo selvagem, porém, essa característica não foi transmitida à descendência, indicando ausência de mobilização do elemento hobo, pelo menos em células germinativas. Para o mutante white, nenhum processo de reversão foi observado, sendo o número de indivíduos com alteração de asas menos pronunciado. No entanto, foi possível isolar uma nova isolinhagem dpp-like, provavelmente fruto de uma inserção de novo no gene dpp. Na análise molecular foi detectada a mobilização do elemento hobo em duas isolinhagens de dpp-like de um total de 27 isolinhagens analisadas, sugerindo uma taxa de mobilização do hobo canônico de 7,4%. Entretanto, não foi possível observar a mobilização de hoboVA através da reversão das mutações white e dpp-like. Isso mostra que a taxa de mobilização desse elemento deve ser inferior à do hobo canônico, e sugere que deve haver um mecanismo de regulação de transposição em hobo.Transposable elements (TEs) mobilization depends on complex regulatory mechanisms that involve not only proteins codified by the transposons themselves, but also by the host cell. However, until the present, the factors that lead to the TEs mobilization in the host genomes are not well-known. Generally, factors that expose an organism to stress conditions lead to transposon mobilization. Therefore, we propose the adaptation of a transformation methodology to our laboratorial conditions, in order to investigate the mobilization mechanism of the hobo transposable element. To devise this methodology two tools were used: a binocular optic microscope and an adapted LEITZ micromanipulator. An air compression system comprising a 20 ml glass syringe and an intra-venous injection “butterfly” tube attached to a micro-pipette tip was coupled to the micromanipulator. A needle made of glass capillary tube was glued with instant adhesive to the micro-pipette tip. The micro-injection process was performed by using a microscope slide containing a double side adhesive band which served as support to the eggs to be injected. Aided by a histological needle, white mutant Drosophila simulans eggs, previously de-hydrated, were manually decorionied and lined up upon this support to avoid moving during the injection. After this step, the embryos were immersed in “halocarbon oil” and the DNA micro-injection was performed. In this part of the work several transformation vectors were tested, and the most efficient transposable element was the piggyBac with the pBac[3xp3-GFPafm] plasmid, which has a promoter that drives strong expression of the GFP (green fluorescent protein) in the eye-tissues of the adult flies; that fluoresce with a strong green color when exposed to blue laser light. With this vector we achieved the detection of transformants, as well as the proofing of the efficiency of the established methodology. The second step of the work was focused on the study of the hobo transposon mobilization by means of DNA microinjection. For this, white mutants, whose mutation is caused by the insertion of the hobo “relic” element (hoboVA) in this gene, and dpp-like of D. simulans, were used. The plasmids pHFL1 (canonical hobo) and BlueScript (control) were independently used as transformation vectors. The micro-injected adult survivors (Go) were individually crossed with other from the same line. From this crossing five isolines from each Go were obtained and observed until the 3rd generation. All individuals phenotype were analyzed in order to detect possible mutation reversion events, and some of them were molecularly analyzed by Southern Blot. Several morphological alterations were observed, essentially in the wings of the Go individuals as well as in their descendents. In the dpp-like line, events of reversion to wild phenotype were observed, however this characteristic was not transmitted to the descendents, indicating the absence of hobo element mobilization in the germ-line. No reversion process was observed for the white mutant, and the number of individuals with wings alterations was less expressive. However it was possible to insulate a new dpp-like isoline probably resulting of de novo insertion in the dpp gene. The molecular analysis showed the hobo element mobilization in two dpp-like isolines in a total of 27, suggesting a hobo canonic mobilization tax of 7.4%. Nevertheless, it was not possible to observe the hoboVA mobilization through white and dpp-like mutation reversion, showing that the mobilization tax of this element might be inferior to the canonic hobo and suggesting that may exist a regulatory mechanism of hobo transposition

Estudo da mobilização de transposons através de transformação genética

A mobilização dos elementos de transposição (TEs) está sujeita a um conjunto complexo de mecanismos regulatórios que envolvem não apenas proteínas codificadas pelos próprios transposons, mas também pela célula hospedeira. Entretanto, até o momento, se conhece pouco sobre os fatores que levam à mobilização de TEs nos genomas hospedeiros. De uma maneira geral, fatores que expõem um organismo a situações de estresse levam à mobilização de transposons. Em função disso, propomos a adaptação, a nossas condições de laboratório, de uma metodologia de transformação genética para ser utilizada como ferramenta, na tentativa de contribuir na elucidação dos mecanismos de mobilização do elemento transponível hobo. Para o estabelecimento desta metodologia utilizamos como ferramentas um microscópio óptico binocular e um micromanipulador adaptado LEITZ. Neste micromanipulador foi acoplado um sistema de compressão a ar, composto por uma seringa de vidro de 20 ml e um dispositivo para infusão endovenosa (butterfly), onde é encaixada uma ponteira de micropipeta. Na ponteira, foi colada, com adesivo instantâneo, uma agulha feita a partir de capilares de vidro. O processo de microinjeção ocorreu em uma lâmina de microscopia contendo uma fita adesiva dupla-face que serve de suporte para os ovos a serem injetados. Com o auxílio de uma agulha histológica, ovos do mutante white de Drosophila simulans, previamente desidratados, foram decorionados manualmente e colocados enfileirados sobre este suporte para evitar deslocamentos durante a injeção. Posteriormente, os embriões foram imersos em “óleo de halocarbono”, e, em seguida, foi realizada a microinjeção de DNA. Para esta etapa do trabalho foram testados vários vetores de transformação, sendo mais eficaz o elemento transponível piggyBac, com o vetor pBac[3xp3-GFPafm], o qual possui um promotor que conduz uma forte expressão da proteína GFP (Green Fluorescent Protein) em tecidos do olho de moscas adultas, o que, sob luz laser azul, fluoresce com uma intensa cor verde. Com este vetor conseguimos a detecção de transformantes, e a comprovação da eficiência da metodologia estabelecida. A segunda etapa do trabalho teve como objetivo estudar a mobilização do transposon hobo por meio de microinjeção de DNA. Para isso foram utilizados os mutantes white, cuja mutação é causada pela inserção do elemento hobo “relic” (hoboVA) neste gene, e dpp-like de D. simulans. Como vetor de transformação foram utilizados, independentemente, os plasmídeos pHFL1 (hobo canônico), e BlueScript como controle. Os adultos microinjetados sobreviventes (G0) foram individualmente cruzados com outro da mesma linhagem, e, a partir deste cruzamento, foram estabelecidas cinco isolinhagens de cada G0 acompanhadas até a 3ª geração. Todos os indivíduos foram analisados quanto ao fenótipo, para possíveis eventos de reversão de mutação, e alguns, analisados molecularmente por Southern Blot. Foram observadas diversas alterações morfológicas, essencialmente nas asas, tanto em G0 como em seus descendentes. Na linhagem dpp-like foram observados eventos de reversão ao fenótipo selvagem, porém, essa característica não foi transmitida à descendência, indicando ausência de mobilização do elemento hobo, pelo menos em células germinativas. Para o mutante white, nenhum processo de reversão foi observado, sendo o número de indivíduos com alteração de asas menos pronunciado. No entanto, foi possível isolar uma nova isolinhagem dpp-like, provavelmente fruto de uma inserção de novo no gene dpp. Na análise molecular foi detectada a mobilização do elemento hobo em duas isolinhagens de dpp-like de um total de 27 isolinhagens analisadas, sugerindo uma taxa de mobilização do hobo canônico de 7,4%. Entretanto, não foi possível observar a mobilização de hoboVA através da reversão das mutações white e dpp-like. Isso mostra que a taxa de mobilização desse elemento deve ser inferior à do hobo canônico, e sugere que deve haver um mecanismo de regulação de transposição em hobo.Transposable elements (TEs) mobilization depends on complex regulatory mechanisms that involve not only proteins codified by the transposons themselves, but also by the host cell. However, until the present, the factors that lead to the TEs mobilization in the host genomes are not well-known. Generally, factors that expose an organism to stress conditions lead to transposon mobilization. Therefore, we propose the adaptation of a transformation methodology to our laboratorial conditions, in order to investigate the mobilization mechanism of the hobo transposable element. To devise this methodology two tools were used: a binocular optic microscope and an adapted LEITZ micromanipulator. An air compression system comprising a 20 ml glass syringe and an intra-venous injection “butterfly” tube attached to a micro-pipette tip was coupled to the micromanipulator. A needle made of glass capillary tube was glued with instant adhesive to the micro-pipette tip. The micro-injection process was performed by using a microscope slide containing a double side adhesive band which served as support to the eggs to be injected. Aided by a histological needle, white mutant Drosophila simulans eggs, previously de-hydrated, were manually decorionied and lined up upon this support to avoid moving during the injection. After this step, the embryos were immersed in “halocarbon oil” and the DNA micro-injection was performed. In this part of the work several transformation vectors were tested, and the most efficient transposable element was the piggyBac with the pBac[3xp3-GFPafm] plasmid, which has a promoter that drives strong expression of the GFP (green fluorescent protein) in the eye-tissues of the adult flies; that fluoresce with a strong green color when exposed to blue laser light. With this vector we achieved the detection of transformants, as well as the proofing of the efficiency of the established methodology. The second step of the work was focused on the study of the hobo transposon mobilization by means of DNA microinjection. For this, white mutants, whose mutation is caused by the insertion of the hobo “relic” element (hoboVA) in this gene, and dpp-like of D. simulans, were used. The plasmids pHFL1 (canonical hobo) and BlueScript (control) were independently used as transformation vectors. The micro-injected adult survivors (Go) were individually crossed with other from the same line. From this crossing five isolines from each Go were obtained and observed until the 3rd generation. All individuals phenotype were analyzed in order to detect possible mutation reversion events, and some of them were molecularly analyzed by Southern Blot. Several morphological alterations were observed, essentially in the wings of the Go individuals as well as in their descendents. In the dpp-like line, events of reversion to wild phenotype were observed, however this characteristic was not transmitted to the descendents, indicating the absence of hobo element mobilization in the germ-line. No reversion process was observed for the white mutant, and the number of individuals with wings alterations was less expressive. However it was possible to insulate a new dpp-like isoline probably resulting of de novo insertion in the dpp gene. The molecular analysis showed the hobo element mobilization in two dpp-like isolines in a total of 27, suggesting a hobo canonic mobilization tax of 7.4%. Nevertheless, it was not possible to observe the hoboVA mobilization through white and dpp-like mutation reversion, showing that the mobilization tax of this element might be inferior to the canonic hobo and suggesting that may exist a regulatory mechanism of hobo transposition