Piezo regulates epithelial topology and promotes precision in organ size control.

Mechanosensitive Piezo channels regulate cell division, cell extrusion, and cell death. However, systems-level functions of Piezo in regulating organogenesis remain poorly understood. Here, we demonstrate that Piezo controls epithelial cell topology to ensure precise organ growth by integrating live-imaging experiments with pharmacological and genetic perturbations and computational modeling. Notably, the knockout or knockdown of Piezo increases bilateral asymmetry in wing size. Piezo's multifaceted functions can be deconstructed as either autonomous or non-autonomous based on a comparison between tissue-compartment-level perturbations or between genetic perturbation populations at the whole-tissue level. A computational model that posits cell proliferation and apoptosis regulation through modulation of the cutoff tension required for Piezo channel activation explains key cell and tissue phenotypes arising from perturbations of Piezo expression levels. Our findings demonstrate that Piezo promotes robustness in regulating epithelial topology and is necessary for precise organ size control.

The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc.

Background: G proteins mediate cell responses to various ligands and play key roles in organ development. Dysregulation of G-proteins or Ca 2+ signaling impacts many human diseases and results in birth defects. However, the downstream effectors of specific G proteins in developmental regulatory networks are still poorly understood. Methods: We employed the Gal4/UAS binary system to inhibit or overexpress Gαq in the wing disc, followed by phenotypic analysis. Immunohistochemistry and next-gen RNA sequencing identified the downstream effectors and the signaling cascades affected by the disruption of Gαq homeostasis. Results: Here, we characterized how the G protein subunit Gαq tunes the size and shape of the wing in the larval and adult stages of development. Downregulation of Gαq in the wing disc reduced wing growth and delayed larval development. Gαq overexpression is sufficient to promote global Ca 2+ waves in the wing disc with a concomitant reduction in the Drosophila final wing size and a delay in pupariation. The reduced wing size phenotype is further enhanced when downregulating downstream components of the core Ca 2+ signaling toolkit, suggesting that downstream Ca 2+ signaling partially ameliorates the reduction in wing size. In contrast, Gαq -mediated pupariation delay is rescued by inhibition of IP 3 R, a key regulator of Ca 2+ signaling. This suggests that Gαq regulates developmental phenotypes through both Ca 2+ -dependent and Ca 2+ -independent mechanisms. RNA seq analysis shows that disruption of Gαq homeostasis affects nuclear hormone receptors, JAK/STAT pathway, and immune response genes. Notably, disruption of Gαq homeostasis increases expression levels of Dilp8, a key regulator of growth and pupariation timing. Conclusion: Gαq activity contributes to cell size regulation and wing metamorphosis. Disruption to Gαq homeostasis in the peripheral wing disc organ delays larval development through ecdysone signaling inhibition. Overall, Gαq signaling mediates key modules of organ size regulation and epithelial homeostasis through the dual action of Ca 2+ -dependent and independent mechanisms.

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies.

BACKGROUND: New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from 'finished'. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies. RESULTS: We evaluated and employed 3 gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies, we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: 6 with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and 3 with new assemblies based on re-scaffolding or long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: 7 for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further 7 with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi. CONCLUSIONS: Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our evaluations show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.

A standard cytogenetic map of Culex quinquefasciatus polytene chromosomes in application for fine-scale physical mapping.

BACKGROUND: Southern house mosquito Culex quinquefasciatus belongs to the C. pipiens cryptic species complex, with global distribution and unclear taxonomy. Mosquitoes of the complex can transmit human and animal pathogens, such as filarial worm, West Nile virus and avian malarial Plasmodium. Physical gene mapping is crucial to understanding genome organization, function, and systematic relationships of cryptic species, and is a basis for developing new vector control strategies. However, physical mapping was not established previously for Culex due to the lack of well-structured polytene chromosomes. METHODS: Inbreeding was used to diminish inversion polymorphism and asynapsis of chromosomal homologs. Identification of larvae of the same developmental stage using the shape of imaginal discs allowed achievement of uniformity in chromosomal banding pattern. This together with high-resolution phase-contrast photography enabled the development of a cytogenetic map. Fluorescent in situ hybridization was used for gene mapping. RESULTS: A detailed cytogenetic map of C. quinquefasciatus polytene chromosomes was produced. Landmarks for chromosome recognition and cytological boundaries for two inversions were identified. Locations of 23 genes belonging to 16 genomic supercontigs, and 2 cDNA were established. Six supercontigs were oriented and one was found putatively misassembled. The cytogenetic map was linked to the previously developed genetic linkage groups by corresponding positions of 2 genetic markers and 10 supercontigs carrying genetic markers. Polytene chromosomes were numbered according to the genetic linkage groups. CONCLUSIONS: This study developed a new standard cytogenetic photomap of the polytene chromosomes for C. quinquefasciatus and was applied for the fine-scale physical mapping. It allowed us to infer chromosomal position of 1333 of annotated genes belonging to 16 genomic supercontigs and find orientation of 6 of these supercontigs; the new cytogenetic and previously developed genetic linkage maps were integrated based on 12 matches. The map will further assist in finding chromosomal position of the medically important and other genes, contributing into improvement of the genome assembly. Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships. This, in turn, will contribute into future development of vector and disease control strategies.

Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi.

BACKGROUND: Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range. RESULTS: Here, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism. CONCLUSIONS: The genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.

An automated homology-based approach for identifying transposable elements.

BACKGROUND: Transposable elements (TEs) are mobile sequences found in nearly all eukaryotic genomes. They have the ability to move and replicate within a genome, often influencing genome evolution and gene expression. The identification of TEs is an important part of every genome project. The number of sequenced genomes is rapidly rising, and the need to identify TEs within them is also growing. The ability to do this automatically and effectively in a manner similar to the methods used for genes is of increasing importance. There exist many difficulties in identifying TEs, including their tendency to degrade over time and that many do not adhere to a conserved structure. In this work, we describe a homology-based approach for the automatic identification of high-quality consensus TEs, aimed for use in the analysis of newly sequenced genomes. RESULTS: We describe a homology-based approach for the automatic identification of TEs in genomes. Our modular approach is dependent on a thorough and high-quality library of representative TEs. The implementation of the approach, named TESeeker, is BLAST-based, but also makes use of the CAP3 assembly program and the ClustalW2 multiple sequence alignment tool, as well as numerous BioPerl scripts. We apply our approach to newly sequenced genomes and successfully identify consensus TEs that are up to 99% identical to manually annotated TEs. CONCLUSIONS: While TEs are known to be a major force in the evolution of genomes, the automatic identification of TEs in genomes is far from mature. In particular, there is a lack of automated homology-based approaches that produce high-quality TEs. Our approach is able to generate high-quality consensus TE sequences automatically, requiring the user to only provide a few basic parameters. This approach is intentionally modular, allowing researchers to use components separately or iteratively. Our approach is most effective for TEs with intact reading frames. The implementation, TESeeker, is available for download as a virtual appliance, while the library of representative TEs is available as a separate download.

Evolutionary dynamics of the Ty3/gypsy LTR retrotransposons in the genome of Anopheles gambiae.

Ty3/gypsy elements represent one of the most abundant and diverse LTR-retrotransposon (LTRr) groups in the Anopheles gambiae genome, but their evolutionary dynamics have not been explored in detail. Here, we conduct an in silico analysis of the distribution and abundance of the full complement of 1045 copies in the updated AgamP3 assembly. Chromosomal distribution of Ty3/gypsy elements is inversely related to arm length, with densities being greatest on the X, and greater on the short versus long arms of both autosomes. Taking into account the different heterochromatic and euchromatic compartments of the genome, our data suggest that the relative abundance of Ty3/gypsy LTRrs along each chromosome arm is determined mainly by the different proportions of heterochromatin, particularly pericentric heterochromatin, relative to total arm length. Additionally, the breakpoint regions of chromosomal inversion 2La appears to be a haven for LTRrs. These elements are underrepresented more than 7-fold in euchromatin, where 33% of the Ty3/gypsy copies are associated with genes. The euchromatin on chromosome 3R shows a faster turnover rate of Ty3/gypsy elements, characterized by a deficit of proviral sequences and the lowest average sequence divergence of any autosomal region analyzed in this study. This probably reflects a principal role of purifying selection against insertion for the preservation of longer conserved syntenyc blocks with adaptive importance located in 3R. Although some Ty3/gypsy LTRrs show evidence of recent activity, an important fraction are inactive remnants of relatively ancient insertions apparently subject to genetic drift. Consistent with these computational predictions, an analysis of the occupancy rate of putatively older insertions in natural populations suggested that the degenerate copies have been fixed across the species range in this mosquito, and also are shared with the sibling species Anopheles arabiensis.

A physical map for an Asian malaria mosquito, Anopheles stephensi.

Physical mapping is a useful approach for studying genome organization and evolution as well as for genome sequence assembly. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to develop high-resolution physical maps. We report a 0.6-Mb-resolution physical map consisting of 422 DNA markers hybridized to 379 chromosomal sites of the Anopheles stephensi polytene chromosomes. This makes An. stephensi second only to Anopheles gambiae in density of a physical map among malaria mosquitoes. Three hundred sixty-three (363) probes hybridized to single chromosomal sites, whereas 59 clones yielded multiple signals. This physical map provided a suitable basis for comparative genomics, which was used for determining inversion breakpoints, duplications, and origin of novel genes across species.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle.

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

Compound-specific bromine isotope analysis of brominated diphenyl ethers using gas chromatography multiple collector/inductively coupled plasma mass spectrometry.

The bromine isotope composition is potentially diagnostic in both degradation monitoring and source apportionment of organobromines in the environment. A method for compound-specific bromine isotope analysis (delta(81)Br) based on gas chromatography multiple collector inductively coupled plasma mass spectrometry (GC/ICPMS) was developed for common brominated diaromatic compounds. Brominated diphenyl ethers (BDEs) in Bromkal 70-5DE, a technical flame-retardant mixture containing mainly BDEs #47, #99 and #100, were used as test substances, with standard bracketing for the samples achieved through co-injected monobromobenzene (MBB) with a known delta(81)Br of -0.39 per thousand vs. Standard Mean Ocean Bromine (SMOBr). Three different heated transfer lines were constructed and tested to achieve efficient conduction of the BDEs from the gas chromatograph to the ICPMS instrument. The MBB was analyzed with a precision of 0.4 per thousand (1 s, n = 18). The precision for BDEs was 1.4-1.8 per thousand (1 s, n = 10-12 depending on the congener). The lower precision for the BDEs than for MBB may reflect the heat required to prevent condensation of the analytes in ICP torch assembly. The use of an internal standard of similar chemical structure to the analytes alleviates this problem, as illustrated by a difference of 0.3 +/- 0.7 per thousand (1 s, n = 6) between the delta(81)Br values of co-injected methoxy BDE-47 and BDE-47 extracted from whale blubber. Improvements in precision and accuracy may be achieved by the use of a more efficient heating of the torch assembly in conjunction with a set of internal standards that match the target compounds.

Characterization of an abundant and novel methyl- and methoxy-substituted brominated diphenyl ether isolated from whale blubber.

A previously unidentified yet abundant substituted polybrominated diphenyl ether (PBDE) was isolated from a northern bottlenose whale (Hyperoodon ampullatus) found dead in the Skagerrak, North Sea. A combination of gas chromatography, high and low resolution mass spectrometry and nuclear magnetic resonance spectroscopy (NMR) ((1)H, (1)H-(1)H and (1)H-(13)C) after isolation with preparative capillary gas chromatography (PCGC) lead to the identification of the unknown substance as 6-MeO-5-Me-2,2',3,4'-tetrabromo diphenyl ether (6-MeO-5-Me-BDE42). To our knowledge this is only the second time PCGC has been used to isolate individual organohalogen compounds present in trace amounts for identification with NMR. The concentration of this novel bioaccumulated compound was estimated to be about 100 ng g(-1) lipid, which was 2.5 times higher compared with the most abundant MeO-PBDE congeners.

Polybrominated and mixed brominated/chlorinated dibenzo-p-dioxins in sponge (Ephydatia fluviatilis) from the Baltic Sea.

Polybrominated dibenzo-p-dioxins (PBDDs) have recently been found in the Baltic Sea at concentrations 1000 times above that of the chlorinated analogs (PCDDs), yet their sources are undefined. Marine production of organobrominated compounds by sponges is well documented. The objective of the current study was to investigate the potential for an aquatic sponge (Ephydatia fluviatilis), common to the Baltic Sea, to produce PBDDs and other organobromine compounds in the field. Mono- to pentaBDDs as well as several mixed brominated/chlorinated dibenzo-p-dioxins (Br/Cl-DDs), PCDDs and methoxylated polybrominated diphenyl ethers (MeO-PBDEs) were quantified in sponge from the SW Baltic. Concentrations of individual PBDDs in the range 1-80 ng per g extractable organic matter were similar as in blue mussels from the Baltic Sea and about 25 000 times higher than 2,3,7,8-tetraCDD. To the best of our knowledge, this is the first time Br/Cl-DDs are reported in biota from a background environment. While this study does not point out sponges as a dominant source, the concentrations of PBDDs in sponge relative to related anthropogenic compounds such as PBDEs and PCDDs as well as the relative abundance of brominated dioxins and furans strengthens the idea of natural production.

Update of the Anopheles gambiae PEST genome assembly.

BACKGROUND: The genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA. RESULTS: Polytene chromosome in situ hybridization with cDNA clones was used to place 15 unmapped scaffolds (sizes totaling 5.34 Mbp) in the pericentromeric regions of the chromosomes and oriented a further 9 scaffolds. Additional analysis by in situ hybridization of bacterial artificial chromosome (BAC) clones placed 1.32 Mbp (5 scaffolds) in the physical gaps between scaffolds on euchromatic parts of the chromosomes. The Y chromosome sequence information (0.18 Mbp) remains highly incomplete and fragmented among 55 short scaffolds. Analysis of BAC end sequences showed that 22 inter-scaffold gaps were spanned by BAC clones. Unmapped scaffolds were also aligned to the chromosome assemblies in silico, identifying regions totaling 8.18 Mbp (144 scaffolds) that are probably represented in the genome project by two alternative assemblies. An additional 3.53 Mbp of alternative assembly was identified within mapped scaffolds. Scaffolds comprising 1.97 Mbp (679 small scaffolds) were identified as probably derived from contaminating bacterial DNA. In total, about 33% of previously unmapped sequences were placed on the chromosomes. CONCLUSION: This study has used new approaches to improve the physical map and assembly of the A. gambiae genome.

Spectral characterization of two bioaccumulated methoxylated polybrominated diphenyl ethers.

Two methoxylated polybrominated diphenyl ethers (MeO-PBDEs) were isolated from a True's beaked whale (Mesoplodon mirus) and identified by NMR (1H, 1H-1H and 1H-13C) and high resolution mass spectrometry as 2-(2',4'-dibromophenoxy)-3,5-dibromoanisole (6-MeO-BDE47) and 2-(2',4'-dibromophenoxy)-4,6-dibromoanisole (2'-MeO-BDE68). Previously the structures of these bioaccumulated compounds have been determined by comparison of their mass spectra and gas chromatographic (GC) retention times with those of authentic standards. While this method is accepted and generally successful, NMR of the isolated compounds allows us to definitively identify the congeners. Our characterizations are consistent with those made for MeO-PBDEs in other organisms, identified by chromatographic methods.

Evolution of supergene families associated with insecticide resistance.

The emergence of insecticide resistance in the mosquito poses a serious threat to the efficacy of many malaria control programs. We have searched the Anopheles gambiae genome for members of the three major enzyme families- the carboxylesterases, glutathione transferases, and cytochrome P450s-that are primarily responsible for metabolic resistance to insecticides. A comparative genomic analysis with Drosophila melanogaster reveals that a considerable expansion of these supergene families has occurred in the mosquito. Low gene orthology and little chromosomal synteny paradoxically contrast the easily identified orthologous groups of genes presumably seeded by common ancestors. In A. gambiae, the independent expansion of paralogous genes is mainly a consequence of the formation of clusters among locally duplicated genes. These expansions may reflect the functional diversification of supergene families consistent with major differences in the life history and ecology of these organisms. These data provide a basis for identifying the resistance-associated enzymes within these families. This will enable the resistance status of mosquitoes, flies, and possibly other holometabolous insects to be monitored. The analyses also provide the means for identifying previously unknown molecules involved in fundamental biological processes such as development.