Ultrastructural aspects of histolytic processes in the salivary gland cells during metamorphic stages in Rhynchosciara hollaenderi (Diptera, Sciaridae).

The cytoplasm of Rhynchosciara hollaenderi late larval, prepupal and pupal salivary gland cells was studied at the ultrastructural level. In the second half of the 4th instar, evidence of an intensive secretory activity is visible in the form of numerous secretory granules in the apical area of the cells. At the same stage, the endoplasmic reticulum cisternae adjacent to Golgi groups are active in the transfer of vesicular elements. At later stages this activity rapidly diminishes. Before the appearance of the DNA puffs, i.e. at the end of the 4th instar, mitochondria begin to show a granular deposit and normal mitochondria decrease in number. These with the granular deposit form clusters and initiate formation of single autophagic vacuoles before the appearance of the DNA puffs. Later, at the time, when the 2B puff opens, the autophagic vacuoles appear in great number. Simultaneously with the formation of the autophagic vacuoles the presence of acid phosphatase in the Golgi vesicles and in autophagic vacuoles was shown. In the last stages investigated (late pupae) acid phosphatase is present free in the cytoplasm and at the same time disappearance of free ribosomes, pycnosis of polytene chromosomes and breakage of nuclear membranes occur. It is concluded that the histolysis of the salivary gland cells begins before the large DNA puffs appear, then it becomes very intensive and continues after these puffs undergo regression.     

The chromosomes of Rhynchosciara baschanti (Diptera: Sciaridae): molecular cytogenetic comparisons with taxa in the americana-like group.

Polytene chromosome analysis is presented for Rhynchosciara baschanti, a species belonging to the americana-like group of Rhynchosciara. R. baschanti chromosomes show morphological differences in centromeric and telomeric regions compared to two other members within the group, R. americana and R. hollaenderi. In addition, fixed band and autosomal inversion differences were noted. Physical mapping data showed synteny among the taxa under study for DNA puffs and single-copy or histone gene probes, whereas rDNA and poly-(r)A probes showed different diagnostic patterns. The activity of developmentally active genes and the pattern of thymidine incorporation into DNA puff sites of R. baschanti are consistent with those found in the two previously studied species, except for lower levels of expression at some of these sites. These results suggest that differential duplication of specific DNA sequences, in particular repetitive and homopolymeric DNA, has played a role in the chromosomal evolution of these Rhynchosciara species. Inversions and band dimorphisms have also occurred, but the processes leading to their maintenance and fixation appear to have been slow, since these three species are in general chromosomally monomorphic.     

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.     

Intrinsic Bent DNA Sites in the Developmentally Amplified C3-22 Gene Promoter of Rhynchosciara americana (Diptera: Sciaridae)

The Rhynchosciara americana C3-22 gene is located in an amplified domain and is developmentally expressed. The aim of the present work was to identify intrinsically bent DNA sites in a segment containing the gene promoter and downstream sequence. The results indicated that this gene is flanked by intrinsically bent DNA sites. Three bent DNA sites (b^?3, b^?2, and b^?1) were localized in the promoter, and one was localized downstream of the gene (b⁺¹). These sites had helical parameters that confirmed the curved structure, as well as segments with left-handed superhelical writhe. In silico analysis of the promoters of four other insect genes, which encode secreted polypeptides, showed that they all had curved structures and similar helical parameters. Correlation with other results indicates that the detected intrinsically bent DNA sites that flank the C3-22 gene might be a consensus feature of the gene structure in the amplified domains.     

Intrinsic bent DNA sites in the developmentally amplified C3-22 gene promoter of Rhynchosciara americana (Diptera: Sciaridae)

The Rhynchosciara americana C3-22 gene is located in an amplified domain and is developmentally expressed. The aim of the present work was to identify intrinsically bent DNA sites in a segment containing the gene promoter and downstream sequence. The results indicated that this gene is flanked by intrinsically bent DNA sites. Three bent DNA sites (b(-3), b(-2), and b(-1)) were localized in the promoter, and one was localized downstream of the gene (b(+1)). These sites had helical parameters that confirmed the curved structure, as well as segments with left-handed superhelical writhe. In silico analysis of the promoters of four other insect genes, which encode secreted polypeptides, showed that they all had curved structures and similar helical parameters. Correlation with other results indicates that the detected intrinsically bent DNA sites that flank the C3-22 gene might be a consensus feature of the gene structure in the amplified domains.     

X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (Diptera,Sciaridae)

Five new translocations recovered from irradiated sperm, each having a break-point in the proximal X heterochromatin, have been designated T23, T29, T32, T40, and T70. These translocations, together with Oak Ridge T1, make possible the precise localization of several genetic components including the X centromere, the controlling element, and the ribosomal cistrons. Only the data pertaining to centromere location are presented here. The ribosomal cistrons and controlling element will be dealt with separately. Full cytological details are given for each of the five translocations. The break-points on the X define three blocks of heterochromatin designated H₁, H₂, and H₃. Together they comprise the right arm of the X. H₃ is the smallest and forms the very end of the chromosome. H₁ lies immediately to the right of the centromere. In T23 and T70 the breakpoints are located between H₂ and H₃; in T29 and T32 between H₂ and H₁. In Oak Ridge T1 the break-point lies between H₁ and the centromere and in T40 between the centromere and the whole left arm of the chromosome. For the first time it has been possible to determine the exact breakpoints of the long paracentric inversion that is found on the X homologue.This series of papers is dedicated to Professor Sally Hughes-Schrader —cytologist, naturalist, scholar — who in her eighty-second year is still exulting in the wonders of chromosome behavior and still endowed with that understanding and grace which have heightened immeasurably the lives of those who have known her     

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.     

Genetic and cytogenetic analysis of the fruit fly Rhagoletis cerasi (Diptera: Tephritidae).

The European cherry fruit fly, Rhagoletis cerasi, is a major agricultural pest for which biological, genetic, and cytogenetic information is limited. We report here a cytogenetic analysis of 4 natural Greek populations of R. cerasi, all of them infected with the endosymbiotic bacterium Wolbachia pipientis. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of 6 pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement has shown a total of 5 long chromosomes (10 polytene arms) that correspond to the 5 autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The most prominent landmarks of each polytene chromosome, the "weak points", and the unusual asynapsis of homologous pairs of polytene chromosomes at certain regions of the polytene elements are also presented and discussed.     

Development of a nuclear polyhedrosis in cells of Rhynchosciara angelae (Diptera, Sciaridae) and patterns of DNA synthesis in the infected cells

An ultrastructural and autoradiographic study of the infection of cells of Rhynchosciara angelae by a nuclear polyhedrosis virus (RPV) is presented. RPV is a DNA virus and causes a dramatic increase in the volume of the infected cells and in the sizes of chromosomes and their DNA contents. The structure of the nucleoli changes with the infection and the changes are mainly related to an increase of DNA synthesis. The concentration of ribosomes increases in the cytoplasm of the infected cells. Autoradiographic study of the DNA synthesis showed that it varies with the infective process.Four patterns of DNA synthesis, in relation to the host chromosomes and the virus, were disclosed by means of tritiated thymidine incorporation in the infected nuclei. The patterns are: (1) incorporation mainly in the chromosomes, (2) incorporation in the chromosomes and in the nucleoplasm, (3) incorporation only in the nucleoplasm, and (4) incorporation mainly in the chromosomes in dissociation. There is indication of a succession 1→2 and 3→4. The succession of patterns indicates that the virus induces first the increase of synthesis of host cell DNA and RNA. The bulk of the synthesis of viral DNA is evident only after the host cell DNA and RNA machinery is amplified. The aspects of the formation of viral membrane indicate that it is a de novo process in which the membrane material is capable of self-assembly.     

Phylogeny of Prosciara Frey and related genera (Diptera: Sciaridae)

The phylogeny of Phytosciara Frey, Prosciara Frey, Dolichosciara Tuomikoski (together forming Phytosciara sensu Tuomikoski), Lobosciara Steffan and Bradysia Winnertz was studied by parsimony analysis using the computer programs Pee‐Wee and NONA. The analysis was based on sixty‐four morphological characters from adult males, coded for thirty‐nine ingroup and two outgroup terminals. Three cladograms of the maximum fit (Pee‐Wee) and one of the minimum length (NONA) were obtained, some of them with polytomies. Many characters showed much homoplasy in the cladograms. The solution by the fittest cladograms was chosen as the main hypothesis of the phylogeny. Of the groups studied, only Lobosciara appeared monophyletic. The relationships of the taxa in their now revised monophyletic sense are: Dolichosciara + (Lobosciara + ((‘Prosciara’ fuscina group + ‘P’. vulcanata) + (‘P’. mima group + ((‘P’. perfida + (Bradysia + Phytosciara)) + Prosciara)))). Phytosciara Frey, Prosciara, Dolichosciara and Lobosciara in their revised concepts are regarded as genera.     

Physical and kinetic properties of a plasma-membrane-bound beta-D-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva).

The midgut caecal cells from Rhynchosciara americana larvae possess a plasma-membrane-bound beta-D-glucosidase (cellobiase, EC 3.2.1.21), which is recovered (75-95%) in soluble form both after treatment with Triton X-100 and after treatment with papain. The Triton X-100-solubilized beta-D-glucosidase displays Mr106000 and pI 5.4, whereas the papain-released beta-D-glucosidase shows Mr65000 and pI 4.7. Thermal inactivations of the detergent-solubilized and the papain-released forms of beta-D-glucosidase both follow apparent first-order kinetics with similar half-lives. The papain-released beta-D-glucosidase, after being purified by density-gradient centrifugation, hydrolyses beta-D-glucosides, beta-D-galactosides and beta-D-fucosides at the same active site, as inferred from experiments of competition between substrates. The beta-D-glucosidase seems to operate in accordance with rapid-equilibrium kinetics, since the Km (0.61 mM) for the enzyme is constant over a wide range of pH. The hydrolysis of the beta-D-glucosidic bond catalysed by the beta-D-glucosidase occurs without inversion of configuration, delta-gluconolactone is a strong (Ki 0.5 microM) inhibitor of the enzyme and substituents in the substrate aglycone affect the catalytic constant of the reaction. These data support the assumption that the mechanism of the reaction catalysed by the beta-D-glucosidase involves the intermediary formation of a carbonium ion, rather than a glucosyl-enzyme intermediate.     

The genus Pnyxiopalpus gen.n. (Diptera: Sciaridae)

Pnyxiopalpus gen.n. (type-species P. raptor sp.n.), Oriental in distribution, includes the following species: P. acanthipes sp.n. (Malay Peninsula), P. aculeatus sp.n. (Borneo), P. adebratti sp.n. (Borneo), P. aphrodite sp.n. (Sumatra), P. dentaneus sp.n. (Sulawesi), P. fossor sp.n. (Borneo), P. fuscinellus sp.n. (Borneo), P. hamatus sp.n. (Borneo), P. latifalx sp.n. (Borneo), P. macrocellus sp.n. (Malay Peninsula), P. microdon sp.n. (Sumatra), P. nepenthophilus sp.n. (Malay Peninsula), P. noona sp.n. (Palawan), P. raptor sp.n. (Borneo), P. reticulatus sp.n. (Malay Peninsula) and P. simplex sp.n. (Borneo). Males and females are keyed and described. Pnyxiopalpus has a number of characters previously unknown in Sciaridae. In contrast to other Sciaridae, interspecific morphological variation is greater for the females compared to the males. Based on a parsimony analysis of sixty-four morphological characters, Pnyxiopalpus is monophyletic, and its sister group appears to be Spathobdella Frey + {[Peyerimhoffia Kieffer + (Faratsiho Paulian + Pnyxia Johannsen)] + [Hyperlasion Schmitz + (Hermapterosciara Mohrig & Mamaev + Parapnyxia Mohrig & Mamaev)]}. According to the most parsimonious solution, the monophyly of Hyperlasion, Lycoriella Frey and Plastosciara Berg in their current sense is questioned. Aptery and one-segmented maxillary palp, usually regarded as important in sciarid classification, show a lot of homoplasy.     

Relationships between newly synthesized proteins and DNA puff patterns in salivary glands of Rhynchosciara americana

An asymmetrical pericentric inversion in the onion fly, Hylemya antiqua was studied. Somatic pairing was studied in young eggs from test-and sibcrossed inversion heterozygous females which gave four and seven distinguishable karyotypes respectively. From these seven, three are balanced: the normal type, the inversion heterozygote and homozygote, and four are unbalanced recombinant karyotypes descending from crossovers in the loop. In all types at all mitotic stages the centromeres are paired. The telomeres only show association during prophase but this decreases from mid to late prophase. Quantitative analysis of the four different cross-over products as produced by inversion heterozygous females showed the presence of nonrandom disjunction. A significant disparity was observed, viz. the normal chromosome was taken up preferentially into the functional gamete compared to the inverted chromosome. Dragging of long chromatids in the asymmetric dyad during M I-A I is a possible explanation of this feature.