Project description:The success of neopteran insects, with 1 million species described, is associated with developmental innovations like the holometaboly and the evolution from short to long germ-band embryogenesis. To unveil the mechanisms underlining these innovations, we compared gene expression during the ontogeny of two extreme neopterans, the cockroach Blattella germanica (polyneopteran, hemimetabolan and short germ-band species), and the fly Drosophila melanogaster (endopterygote, holometabolan and long germ-band species). Results revealed that genes associated with metamorphosis are predominantly expressed in late nymphal stages in B. germanica and in early-mid embryo in D. melanogaster. In B. germanica the maternal to zygotic transition (MZT) concentrates early in embryogenesis, when juvenile hormone factors are significantly expressed. In D. melanogaster, the MZT extends throughout embryogenesis, during which juvenile hormone factors appear unimportant. These differences possibly reflect broad trends in the evolution of development within neopterans, related to the germ-band type and the metamorphosis mode.
Project description:microRNAs (miRNAs) have been reported as key regulators in the post-transcriptional process in eukaryotic cells. In insects most of the studies have been reported in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphonpisum) have had their miRNAs identified. Therefore, the study on miRNAs of the evolutionarily basal hemimetabolan Blattella germanica, may provide valuable insights on the structural and functional evolution of miRNAs. Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 411 miRNA candidates conserved in other insects and 1017 candidates specific of B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that reads can be used as quantitative method. Novel miRNA candidates were validated by decreasing levels of expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates. Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and Dicer-1 RNAi assay is a reliable method to validate novel miRNAs.
Project description:microRNAs (miRNAs) have been reported as key regulators in the post-transcriptional process in eukaryotic cells. In insects most of the studies have been reported in holometabolans while only recently two hemimetabolansM-BM- (Locusta migratoria and Acyrthosiphonpisum) have had their miRNAs identified. Therefore, the study on miRNAs of the evolutionarily basal hemimetabolanM-BM- Blattella germanica, may provide valuable insights on the structural and functional evolution of miRNAs. Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body andM-BM- ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 411 miRNA candidates conserved in other insects and 1017 candidates specific of B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that reads can be used as quantitative method. Novel miRNA candidates were validated by decreasing levels of expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates. Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and Dicer-1 RNAi assay is a reliable method to validate novel miRNAs. Small RNAs were sequenced whole body of the last instar nymph and adult ovaries of the cockroach Blattella germanica.
Project description:Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0-6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared - presumably by Mir-309 and associated scavenger miRNAs. miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny - patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.
Project description:Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes.