In-vivo Optical Tomography of Small Scattering Specimens: time-lapse 3D imaging of the head eversion process in Drosophila melanogaster

5 fig.Even though in vivo imaging approaches have witnessed several new and important developments, specimens that exhibit high light scattering properties such as Drosophila melanogaster pupae are still not easily accessible with current optical imaging techniques, obtaining images only from subsurface features. This means that in order to obtain 3D volumetric information these specimens need to be studied either after fixation and a chemical clearing process, through an imaging window - thus perturbing physiological development -, or during early stages of development when the scattering contribution is negligible. In this paper we showcase how Optical Projection Tomography may be used to obtain volumetric images of the head eversion process in vivo in Drosophila melanogaster pupae, both in control and headless mutant specimens. Additionally, we demonstrate the use of Helical Optical Projection Tomography (hOPT) as a tool for high throughput 4D-imaging of several specimens simultaneously.This work was supported in part by Project ‘‘THALES – BSRC ‘Alexander Fleming’ – Development and employment of Minos-based genetic and functional genomic technologies in model organisms (MINOS)’’ – MIS: 376898, the Fellowship for Young International Scientist of the Chinese Academy of Sciences Grant No. 2010Y2GA03 and the NSFC-NIH Biomedical collaborative research program 81261120414. A. Arranz acknowledges support from Marie Curie Intra-European Fellowship Program FP7-PEOPLE-2010-IEF. J. Ripoll acknowledges support from EC FP7 CIG grant HIGH-THROUGHPUT TOMO, and Spanish MINECO grant MESO-IMAGING FIS2013-41802-R. The authors would like to thank Dr. S. Oehler for the help with the GFP-expressing flies, and G. Livadaras and G. Zacharakis for help with the Drosophila stocks

Use of Mutagenesis, Genetic Mapping and Next Generation Transcriptomics to Investigate Insecticide Resistance Mechanisms

Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w−]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over- and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ∼1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results

Wolbachia Prophage DNA Adenine Methyltransferase Genes in Different Drosophila-Wolbachia Associations

Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studing of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility

Toxicology and cytogenetic analysis of a Drosophila melanogaster mutant resistant to Imidacloprid and DDT

Resistance to all major insecticide classes has developed in numerous and diverse insect field populations. Imidacloprid, the worldwide most used neonicotinoid, has been extensively applied during the last decade for the control of different insect pests. Lately, cases of sporadic resistance also to neonicotinoids, including Imidacloprid, have been reported. Drosophila melanogaster is one of the most popular model organisms in biology and, although not a pest species, a promising model system for insecticide resistance research. In this study, we present a toxicological and karyotypic analysis of a Drosophila mutant (MiT[w-]3R2) resistant to Imidacloprid and cross-resistant to DDT. Karyotype analysis of polytene chromosome of MiT[w-]3R2 flies did not identify any apparent structural change of the polytene chromosome linked with the resistance phenotype. [Projekat Ministarstva nauke Republike Srbije, br. 173012

Minos as a Genetic and Genomic Tool in Drosophila melanogaster

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5′-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient “jump-starting” scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and ∼55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila

Gene Organization of the dnaA Region of Wolbachia

The dnaA region of Wolbachia, an intracellular bacterial parasite of insects, is unique. A glnA cognate was found upstream of the dnaA gene, while neither of the two open reading frames detected downstream of dnaA has any homologue in the database. This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies