Rosane Nunes Garcia

Sex-specific methylation in Drosophila: an investigation of the Sophophora subgenus.

Epigenetic phenomena have been widely characterized in the genomes of vertebrates and DNA methylation is a key mechanism of epigenetic regulation. The DNA methylation systems of invertebrates and vertebrates show several notable differences. However, the evolutionary implications of those differences only recently began to be revealed. Our study investigated the recurrence of sex-specific methylation, as previously described for the species Drosophila willistoni, in other species of the Sophophora subgenus that present close evolutionary relationship. The MSRE and Southern blot techniques were used to analyze rDNA of some species of the willistoni, melanogaster, saltans and obscura groups of Drosophila and the results suggested that differential DNA methylation between sexes only occurs in Drosophila tropicalis and D. insularis, two sibling species of the willistoni subgroup. However, only using the MSRE technique we could detect sex-specific patterns of DNA methylation in all species of willistoni subgroup. These results indicate that DNA methylation may present important differences, even between closely related species, shedding new light on this Neotropical species complex.

First evidence of methylation in the genome of Drosophila willistoni

DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogasterdDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.

Pupal Melanization in Heliconius erato phyllis (Lepidoptera; Nymphalidae): Genetic and Environmental Effects

This paper deals with estimates of heritability and the effect of two different kinds of environment on the melanization of pupae in the butterfly Heliconius erato phyllis. The results for heritability showed greater differences when the offspring were regressed on male or female values for pupal colour. Pupal colour followed a system of discrete scores, from 2 for the light pupae to 5 for the darkest one. Estimate of the heritability (h2) was 0.44 when the average score of the siblings were regressed on the mid-parental value. When regressed on the male parent, h2 was larger (0.54), being very low when regressed on the female parent (0.09). Estimate by the analysis of variance was also 0.44 when both male and female sibs were included in the analysis (for male sibs, h2=0.43 and for female sibs, 0.47). To test for the effect of the environment, each brood was divided in three, one being the control, the second subjected to a black environment when reaching the fifth instar and the third to a white environment. Those in the black environment originated dark pupae that scored 5 or 4; for those in the white environment, there was no difference with the controls. Caterpillars when entering the prepupal stage were also subjected to the black treatment (early and late prepupal stage, respectively, for treatments called black A and black B). A significant effect was observed only when early prepupal stage was subjected to the black environment.

New histochemical and morphological findings in the female genital tract of Boophilus microplus (Acari, Ixodidae): an attempt toward the elucidation of fertilization in ticks

At present not only is the site of fertilization in ticks still unknown but it is also unclear as to how this mystery can be solved. Signs of fertilization can be observed throughout the female genital tract and these can be clues for the elucidation of the unsolved questions relating to ticks fertilization. In Boophilus microplus (Canestrini, 1887) the most important signs are the following: the final eversion of the acrosomal canal in females ready for oviposition; the presence of small tubules, resembling the subplasmalemal process of the spermatozoon between the oviduct cells; budding nuclei throughout the female genital tract; and the two Feulgen and DAPI positive areas in the oocyte at vitelogenesis. These morphological characteristics suggest that fertilization takes place in the internal cylinder which extends from the uterus to the ovary itself.

Mitotic and meiotic chromosomes of a southern Brazilian population of Boophilus microplus (Acari, Ixodidae)

Using conventional staining with acetic orcein and C-banding techniques it was investigated constitutive heterochromatin chromosomal polymorphisms and the mitotic and the meiotic behavior of male and female chromosomes of Boophilus microplus (Canestrini, 1887). Some differences were detected in the population of southern Brazil as compared to the data of other authors for populations in other latitudes. The differences being mainly concerned with the distribution of constitutive centromeric heterochromatin and variation in the length of heterochromatic blocks in the pericentromeric regions of some chromosome pairs.

Changes in the oviducts after the fertilization of the cattle tick Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae)

Summary In the ticks the exact site and mechanism of fertilization is still largely unknown. This study on Boophilus microplus females describes the significant changes in the morphology and histology of the oviducts which occurs from feeding until oviposition. Our results show that in B. microplus the oviducts are formed by an internal and an external cylinder. The internal cylinder is divided into a main region, a ring-like region and a transitional region. The cells of the main region maintain close contact with spermatozoa, while in the ring-like region there are no spermatozoa, and only when this region disappears, spermatozoa are found in the ovary even at oviposition. The space between the cylinders contains several cell types, including contractile and granular cells. The ovary does not have granular cells. The cylinders are externally invested with a membrane. The internal cylinder extends throughout the length of the reproductive system from the uterus to the ovary itself, and this suggests that the internal cylinder is not merely a passageway for spermatozoa and ova but might be actively involved in the mechanism of fertilization. These observations suggest that the cells of the internal cylinder, including funicular cells, might be oocytes.

The male genital accessory gland complex of the cattle tick Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae)

A topographical and histological study of the male genital accessory gland complex of Boophilus microplus was undertaken. Ten lobes were found, the most prominent of which is the single dorso-median lobe, subdivided into antero-dorsal and postero-dorsal lobes. The other lobes are: a pair of postero-ventral lobes, a pair of lateral lobes (subdivided into dorso-lateral and postero-lateral lobes), a pair of antero-ventral lobes, a single medio-ventral lobe and a pair of latero-ventral lobes. These lobes were observed to present individual topographical and histological characteristics, with independent openings, hence the conclusion is that they form a gland complex. The secretory nature of the dorso-median lobe duct was also analysed.