A Soluble Pyrophosphatase Is Essential to Oogenesis and Is Required for Polyphosphate Metabolism in the Red Flour Beetle (Tribolium castaneum)

Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been mainly investigated in mammalian cells with few studies on insects. Some studies have demonstrated that a pyrophosphatase regulates polyphosphate metabolism, and most of them were performed on trypanosomatids. Here, we investigated the effects of sPPase gene knocked down in oogenesis and polyphosphate metabolism in the red flour beetle (Tribolium castaneum). A single sPPase gene was identified in insect genome and is maternally provided at the mRNA level and not restricted to any embryonic or extraembryonic region during embryogenesis. After injection of Tc-sPPase dsRNA, female survival was reduced to 15% of the control (dsNeo RNA), and egg laying was completely impaired. The morphological analysis by nuclear DAPI staining of the ovarioles in Tc-sPPase dsRNA-injected females showed that the ovariole number is diminished, degenerated oocytes can be observed, and germarium is reduced. The polyphosphate level was increased in cytoplasmic and nuclear fractions in Tc-sPPase RNAi; Concomitantly, the exopolyphosphatase activity decreased in both fractions. Altogether, these data suggest a role for sPPase in the regulation on polyphosphate metabolism in insects and provide evidence that Tc-sPPase is essential to oogenesis

Biologia evolutiva do desenvolvimento de artropodes: uma visão brasileira

Os artrópodes possuem o maior número de espécies dentro do reino animal e possuem também uma grande variabilidade morfológica. No Brasil, nosso grupo tem focado em estudar o desenvolvimento embrionário de organismos pertencentes ao filo Arthropoda, particularmente de organismos vetores de doenças ou causadores de grandes prejuízos econômicos, como as pragas de estocagem. Estes estudos têm revelado similaridades e diferenças na biologia do desenvolvimento dos artrópodes. Entre os estágios embrionários conservados encontra-se a banda germinal, que é bastante similar para todo o grupo (estágio filotípico), enquanto que os estágios iniciais, sob influência maternal e os estágios finais da embriogênese, são mais distintos. Estes resultados, junto com demais resultados da literatura, evidenciam uma história evolutiva comum e a constituição de um grupo monofilético dentre os artrópodes. Com o desenvolvimento de novas tecnologias e o surgimento da Biologia Evolutiva do Desenvolvimento (EVO-DEVO), tem sido possível investigar o controle morfogenético do desenvolvimento embrionário e pós-embrionário, comparando grupos taxonômicos distintos. No presente trabalho discutiremos a importância de ter outros organismos modelo além da mosca-da-fruta (Drosophila melanogaster) para estudos de biologia do desenvolvimento de artrópodes. Estes estudos incluem espécies de interesse médico como o hemiptera Rhodnius prolixus e o carrapato Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) bem como pragas de estocagem, o besouro Tribolium castaneum (Coleoptera)

Bacterial community dominance in a sewage-driven eutrophic coastal lagoon by next generation sequencing: initial findings

Abstract: This study investigates the presence of bacterial dominance in one of the most studied sewage-driven eutrophic coastal lagoons, the Imboassica Lagoon in Macaé (RJ), Brazil, utilizing high-throughput sequencing of 16S rDNA. Water samples were collected from three sites within the lagoon. Total microbial DNA was extracted, and the V3-V4 region of the 16S rRNA gene was amplified and sequenced on the Illumina MiSeq platform. A total of 744,879 partial 16S rRNA sequences were clustered, revealing the absence of a single bacterial dominance in the sewage-driven eutrophic coastal lagoon. The prominent phyla detected in the lagoon were Cyanobacteria (27.8%), Proteobacteria (23.7%), and Actinobacteria (14.6%). Proteobacteria emerged as the most abundant phylum in the sewage-impacted lagoon site, whereas Cyanobacteria dominated the other two sampling sites. Among families, Synechococcaceae predominated with genus Synechococcus exhibited the highest prevalence. Families of potentially toxic Cyanobacteria represented less than 1% of the total families. The sewage-impacted lagoon section displayed greater bacterial diversity and richness. The dominance of bacterial communities associated with raw sewage, such as members of the Enterobacteriaceae family, was not confirmed, constituting only 0.75% of the families in the most affected site. This study presents the initial analysis of the bacterial community in the Imboassica Lagoon and suggests that dominance in the lagoon responds to the eutrophication and sewage discharge

Evolution and multiple roles of the Pancrustacea specific transcription factor <i>zelda</i> in insects

<div><p>Gene regulatory networks (GRNs) evolve as a result of the coevolutionary processes acting on transcription factors (TFs) and the cis-regulatory modules they bind. The zinc-finger TF <i>zelda (zld)</i> is essential for the maternal-to-zygotic transition (MZT) in <i>Drosophila melanogaster</i>, where it directly binds over thousand cis-regulatory modules to regulate chromatin accessibility. <i>D</i>. <i>melanogaster</i> displays a long germ type of embryonic development, where all segments are simultaneously generated along the whole egg. However, it remains unclear if <i>zld</i> is also involved in the MZT of short-germ insects (including those from basal lineages) or in other biological processes. Here we show that <i>zld</i> is an innovation of the Pancrustacea lineage, being absent in more distant arthropods (e.g. chelicerates) and other organisms. To better understand <i>zld´s</i> ancestral function, we thoroughly investigated its roles in a short-germ beetle, <i>Tribolium castaneum</i>, using molecular biology and computational approaches. Our results demonstrate roles for <i>zld</i> not only during the MZT, but also in posterior segmentation and patterning of imaginal disc derived structures. Further, we also demonstrate that <i>zld</i> is critical for posterior segmentation in the hemipteran <i>Rhodnius prolixus</i>, indicating this function predates the origin of holometabolous insects and was subsequently lost in long-germ insects. Our results unveil new roles of <i>zld</i> in different biological contexts and suggest that changes in expression of <i>zld</i> (and probably other major TFs) are critical in the evolution of insect GRNs.</p></div

<i>Tc-zelda</i> larval RNAi affects elytra enclosure and molting.

<p><i>Tribolium castaneum</i> larvae were injected during early (3rd) and late (6th) stages as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref050" target="_blank">50</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref080" target="_blank">80</a>]. (A) Relative expression of <i>Tc-zld</i> during pupal stages after <i>Tc-zld</i> dsRNA or <i>neo</i> dsRNA injection. qRT-PCR was normalized using <i>Tc-rps3</i> gene, as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref078" target="_blank">78</a>]. (B,C) Morphology of late pupae obtained from early larvae injected with <i>zld</i> or <i>neo</i> dsRNA. Differential pigmentation in the head (arrow) and reduced wings (arrowhead) were observed in <i>zld</i> dsRNA and not in <i>dsneo</i> dsRNA pupae. (D) Number of emerging adult beetles eleven days after larval injection (DALI) at late (6th) stage. (E,F). Adults obtained by late larval injections of <i>neo</i> (E) or <i>zld</i> (F) dsRNA. Hindwings are usually not properly folded underneath the forewing (elytra).</p

<i>Tc-zld</i> knockdown in larval stages affects elytra and wings in adult stage.

<p>(A) Control elytron extracted from a <i>dsneo</i> adult shows a parallel venation pattern and rigid chitinous structure. (B) <i>Tc-zld</i> RNAi elytron displays a disrupted vein pattern and a less resistant structure. (C,D) Hindwings were dissected and photographed. Although the overall morphological pattern is not affected in both wings, <i>zld</i> dsRNA wings show severe dehydration after ethanol fixation. (E) PD indexes comparison between <i>dsneo</i> and <i>dszld</i> RNAi hindwings and forewings, a PD index reflects the shape of the wing based on its dimensions ratio [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref052" target="_blank">52</a>]. The statistical analysis was carried out by unpaired t-test assuming unequal variances (asterisks refer to P<0.0001 while ns stands for “no significance”), indicating that <i>Tc-zelda</i> RNAi affects hindwing shape. (F) <i>Tc-zld</i> RNAi elytra and hindwing do not show statistically significant differences in length when compared to their respective <i>dsneo</i> RNAi controls.</p

<i>zld</i> is required for the generation of the posterior region in the hemiptera <i>Rhodnius prolixus</i>.

<p>(A,C,E) <i>R</i>. <i>prolixus</i> control (<i>dsneo</i> embryos). (B,D,F,F’) A representative embryo collected from <i>R</i>. <i>prolixus zld</i> pRNAi. (F,F’) Embryo inside the chorion, ventral and dorsal views. (C,E) Embryo removed from the egg shell. (G,H) Schematic drawings of control and <i>zld</i> pRNAi embryos. (A) DAPI stainings of control (A,E) and <i>zld</i> RNAi (B) embryos. (D) Asterisk denotes an eye which can be observed at the ventral side (F). (D) After dissection, the eye can be identified due to its characteristic red pigmentation and shape. Scale bar corresponds to 500μM.</p

Tc-Zld proteins from insects and crustaceans.

<p>(A) Multiple sequence alignment of Zelda proteins, representing major groups of arthropods. (B) Conserved protein architecture features of Zelda proteins. Asterisks and x marks represent presence and absence, respectively. Question marks denote that the feature is either partially preserved or could be flagged as absent due to sequencing or assembly errors (e.g. wrong start codons). The cladogram was organized according to a previously reported phylogenetic study [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref079" target="_blank">79</a>]. Two outgroups without <i>zld</i> orthologs were also included.</p

Computational identification of <i>Tc-zelda</i> target genes.

<p>(A) TOMTOM comparison of <i>D</i>. <i>melanogaster</i> motif similar to the TAGteam [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref007" target="_blank">7</a>] obtained by DREME and putative <i>T</i>. <i>castaneum zld</i> DREME motif. (B) Venn diagram of the <i>D</i>. <i>melanogaster</i> ChIP-Seq MZT regulated genes (green) from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref008" target="_blank">8</a>] and <i>D</i>. <i>melanogaster</i> genes predicted by FIMO analysis with the putative DREME motif (yellow). (C) Venn diagram of the <i>D</i>. <i>melanogaster</i> Dm-Zld MZT targets (green) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref008" target="_blank">8</a>] and <i>D</i>. <i>melanogaster</i> one-to-one orthologs of putative <i>Tc-zld</i> targets.</p