Early spermatogenesis inRhynchosciara

Summary The development of differentiation of spermatogonia in the testes ofRhynchosciara is a relatively synchronous process. The testes are composed of three cell types. The first are small cells, possibly aberrant spermatogonia which have failed to differentiate, which actively synthesize RNA and DNA from the beginning of the first instar to the beginning of meiosis. The second cell type consists of large polytene cells whose cytoplasm anastomoses extensively and which may have a Sertoli cell-like function. The third cell type, the spermatogonia, synthesizes RNA from the first instar until the beginning of meiosis. However, DNA synthesis as well as mitotic division stops at mid-fourth instar. Groups of these cells are connected via cytoplasmic bridges, probably formed as a result of incomplete cytokinesis. These groups of cells develop synchronously. Shortly after the cessation of mitotic activity the mitochondria acquire unique inclusions. These consist of a mass of electron-lucid material which continues to accumulate throughout the last half of the fourth instar. Following the completion of meiosis, the mitochondria begin to fuse so that in the mature sperm there is a single mass of material in the matrix of what appears to be a single elongated mitochondrion. This research was sponsored by the United States Atomic Energy Commission under contract with the Union Carbide Corporation.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.     

Synthesis of satellite DNA in Rhynchosciara hollaenderi

During the latter part of the fourth instar development of Rhynchosciara larvae, DNA synthesis occurs in both germ cells and somatic cells, even though these cells do not undergo mitosis. CsCl density gradient analysis has revealed the synthesis of d(AT) satellite DNAs in the testis and in somatic tissues such as salivary gland, fat body, and gastric cecum. In these studies it has been shown that there is a tissue-specific variation in the relative proportions of synthesis of d(AT) satellite and main-band DNA in the testis during the fourth instar. The initiation of synthesis of the d(AT) satellite in the testis corresponds with the appearance and formation of the highly characteristic mitochondria which develop during the maturation of the spermatocytes. This satellite DNA has been shown to be circular and has a density of 1.681 gm/cm³ in CsCl. Synthesis of a similar circular DNA cannot be detected in somatic tissues, although these tissues do synthesize a d(AT) satellite of similar density.     

A molecular cytogenetic comparison between Rhynchosciara americana and Rhynchosciara hollaenderi (Diptera: Sciaridae).

The polytene chromosomes of Rhynchosciara americana and R. hollaenderi, a pair of sibling species in the americana-like group of Rhynchosciara, were compared using a number of techniques, including in situ hybridization. With classical cytological techniques, the only differences observed were in the morphology of centromeric and telomeric heterochromatin, in the size of a DNA and RNA puff, and in the presence of an inversion polymorphism in R. hollaenderi. However, after in situ hybridization with rDNA and poly-r(A) probes, differences between the two species appeared at a number of sites. Differences in poly-r(A) sites were especially informative in establishing phylogenetic relationships between these two species and a third species currently being examined from this group. Chromosomal evolution between these species appears to have occurred mainly through differential amplification and transposition of repetitive sequence DNA, of which dA:dT tracts are an important component. The R. hollaenderi karyotype is tentatively considered more ancestral than that of R. americana because it has features present in the third Rhynchosciara species. Explanations for the monomorphisms observed in Rhynchosciara species and mechanisms of speciation in the group are considered within the context of the species' complex behavior.     

Specialized features of Rhynchosciara americana embryogenesis

Insect embryo development is a complex process which requires nuclear and cellular division, cell shape alteration, and cell movement. This process needs to be orchestrated in a specific spatial and temporal fashion. Different insect species, despite similarities, present distinct morphogenetic pathways. We used the dipteran R. americana as a comparative model for embryo morphogenesis studies, following embryo development with different histochemical and immunohistochemical procedures. Despite the phylogenetic proximity with D. melanogaster, R. americana presents a peculiar morphogenesis. We show that at the initial phases of development, from egg fertilization to blastoderm formation, R. americana is similar to Drosophila. The first cleavages are nuclear and cellularization only begins after nuclei spread throughout the egg’s cortex. However after this stage a series of cell movements establishes a short compact germ band anlage, which gastrulates in a pattern quite different from Drosophila. After gastrulation the germ band elongates anterior–posteriorly and segmentation occurs simultaneously along the embryo. Embryo development from egg fertilization to larva hatching takes about 12 days. Our results show that R. americana presents a different morphogenetic pathway which does not fit in the current short, intermediate or long germ band classification.     

Action of hydroxyurea on chromosome physiology in Rhynchosciara angelae

Experiments have been performed to investigate the action of hydroxyurea (H.U.) on the polytene chromosomes of the salivary gland of Rhynchosciara angelae. After different times of H.U. treatment, larvae were injected with ³H-thymidine for a pulse of 10 min. DNA puffs were analysed especially in those regions where differential incorporation of thymidine occurs. H.U. progressively inhibited thymidine incorporation all over the chromosome. The maximum of inhibition occurs 9 hours after the treatment. However, after 227 hours the chromosome label was similar to that in the controls and puff 2B recovered its original size. The puff 3C showed a delay in its appearance. Our results show that H.U. inhibits temporarily the opening rate of the puff, as well as DNA synthesis. There is no reaction on RNA puffs.This work was supported by a grant from the National Institutes of Health (GM 17590-03), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) of which one of us (G.M.M.S.) was a fellow during this research, and the Conselho Nacional de Pesquisas (CNPq).     

Synthesis of mitochondrial DNA in spermatocytes of Rhynchosciara hollaenderi

During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm³; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 10⁶. Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 10⁵ daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.     

Developmental puffing patterns in salivary gland chromosomes of Rhynchosciara hollaenderi

An analysis of puff formation and regression has been carried out in 3 morphologically distinct regions of the Rhynchosciara hollaenderi salivary gland during mid-larval through pupal development. Puffing differences among these 3 regions have been found and analysed for both RNA and DNA puffs. The presence of such differences suggests that the gland regions may also be functionally differentiated. — Developmentally specific sequences of puffs have been distinguished and correlated with morphological and physiological events which occur during the development of Rhynchosciara. The DNA puffs as well as the RNA puffs enlarge and regress at predictably specific developmental stages. The presence of particular puffing sequences in the late larval to pupal period has been compared with the occurrence of known changes in the developmental ecdysone titre for Rhynchosciara. Certain aspects of this developmental picture appear to fit the ecdysone-stimulated puffing model for Drosophila, but other aspects indicate that the Drosophila-based model may not be completely applicable to Rhynchosciara.     

Characterisation of histones in the salivary glands of Rhynchosciara americana larvae

The histones from the salivary glands of Rhynchosciara americana larvae were identified. The electrophoretic patterns of the proteins studied resemble that of calf thymus histones, including the H₁ histone, which in Rhynchosciara has a lower electrophoretic mobility in urea/polyacrylamide gels but shows a molecular weight identical to the corresponding histone of calf thymus, as judged by SDS-polyacrylamide gel electrophoresis.     

A larval haemolymph protein in the eggs of Rhynchosciara americana

In Rhynchosciara americana larvae, a protein referred to as “protein 10” comprises one of the major plasma protein of the last instar. This protein was purified and an antiserum against it shows that an identical protein is present in the eggs from this fly. Protein 10 has an estimated molecular weight of 43,000 and an isoelectric pH of 6.6. Examination of protein 10 from eggs and from haemolymph by limited proteolysis indicates that the two are structurally identical. Protein 10 is synthesized in large amounts by the larval fat bodies up until the end of the feeding stage. Estimation by radial immunodiffusion shows that protein 10 is stored in the larval haemolymph, attaining a maximum level at the end of feeding stage and then decreasing until very little remains in the young adult. By the middle of the pupal stage the ovaries begin to sequester and acummulate the protein 10 which is deposited in the eggs.     

Embryonic expression of the engrailed homologue of Rhynchosciara americana

The segment polarity gene engrailed is involved in the determination of segment posterior identity in Drosophila. engrailed has been largely used for comparative developmental studies due to its evolutionary conservation from nematodes to humans. By in situ hybridization of an engrailed cDNA probe from Drosophila to polytene chromosomes of fourth instar larvae of Rhynchosciara americana we have shown that engrailed-like sequences must be localized in band 6 of chromosome A in this species. The pattern of engrailed protein expression during R. americana embryo development is diffuse at first evolving into a nuclear striped pattern after quite a length of time. In addition, our results suggest a possible developmentally regulated molecular modification of engrailed protein in R. americana embryos.