Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera

<p>Abstract</p> <p>Background</p> <p>Moths have evolved highly successful mating systems, relying on species-specific mixtures of sex pheromone components for long-distance mate communication. Acyl-CoA desaturases are key enzymes in the biosynthesis of these compounds and to a large extent they account for the great diversity of pheromone structures in Lepidoptera. A novel desaturase gene subfamily that displays Δ11 catalytic activities has been highlighted to account for most of the unique pheromone signatures of the taxonomically advanced ditrysian species. To assess the mechanisms driving pheromone evolution, information is needed about the signalling machinery of primitive moths. The currant shoot borer, <it>Lampronia capitella</it>, is the sole reported primitive non-ditrysian moth known to use unsaturated fatty-acid derivatives as sex-pheromone. By combining biochemical and molecular approaches we elucidated the biosynthesis paths of its main pheromone component, the (<it>Z,Z</it>)-9,11-tetradecadien-1-ol and bring new insights into the time point of the recruitment of the key Δ11-desaturase gene subfamily in moth pheromone biosynthesis.</p> <p>Results</p> <p>The reconstructed evolutionary tree of desaturases evidenced two ditrysian-specific lineages (the Δ11 and Δ9 (18C>16C)) to have orthologs in the primitive moth <it>L. capitella </it>despite being absent in Diptera and other insect genomes. Four acyl-CoA desaturase cDNAs were isolated from the pheromone gland, three of which are related to Δ9-desaturases whereas the fourth cDNA clusters with Δ11-desaturases. We demonstrated that this transcript (<it>Lca</it>-KPVQ) exclusively accounts for both steps of desaturation involved in pheromone biosynthesis. This enzyme possesses a Z11-desaturase activity that allows transforming the palmitate precursor (C16:0) into (<it>Z</it>)-11-hexadecenoic acid and the (<it>Z</it>)-9-tetradecenoic acid into the conjugated intermediate (<it>Z,Z</it>)-9,11-tetradecadienoic acid.</p> <p>Conclusion</p> <p>The involvement of a single Z11-desaturase in pheromone biosynthesis of a non-ditrysian moth species, supports that the duplication event leading to the origin of the Lepidoptera-specific Δ11-desaturase gene subfamily took place before radiation of ditrysian moths and their divergence from other heteroneuran lineages. Our findings uncover that this novel class of enzymes affords complex combinations of unique unsaturated fatty acyl-moieties of variable chain-lengths, regio- and stereo-specificities since early in moth history and contributes a notable innovation in the early evolution of moth-pheromones.</p

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

<p>Abstract</p> <p>Background</p> <p>Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture.</p> <p>Methods</p> <p>Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale <it>in silico </it>image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM).</p> <p>Results</p> <p>We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates.</p> <p>Conclusion</p> <p>The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes.</p

Global transcriptional analysis of pheromone biosynthesis-related genes in the female turnip moth, Agrotis segetum (Noctuidae) using a custom-made cDNA microarray.

Using a custom-made cDNA microarray, global transcriptional analyses were conducted to identify genes differentially regulated in the pheromone gland as compared to the remaining insect tissue of the moth Agrotis segetum (Noctuidae). A two-fold or larger difference in relative expression levels was found for 227 of 864 genes investigated comparing the two tissues. Unexpectedly, an antennal binding protein homologue, containing a pheromone-binding/general odorant-binding protein PFAM domain, was expressed at a 56-fold higher level in the pheromone gland. Relatively higher expression levels in the pheromone gland were also found for other gene representatives putatively encoding odorant-binding proteins and chemosensory proteins, as well as a number of gene representatives putatively encoding proteins involved in juvenile hormone interactions. The largest relative up-regulation (84-fold) in the pheromone gland was found for a gene encoding a Delta11-desaturase homologue implicated in desaturation of pheromone precursors. For three gene representatives, the expression patterns were independently verified by quantitative real-time PCR (qPCR). Additionally the expression pattern in the pheromone gland for the Delta11-desaturase homologue was shown by qPCR to follow the previously known pattern of pheromone production in female A. segetum, both with respect to age and circadian rhythm, whereas the expression of a Delta9-desaturase and a chemosensory protein homologue did not share this pattern

Global transcriptional analysis of pheromone biosynthesis-related genes in the female turnip moth, Agrotis segetum (Noctuidae) using a custom-made cDNA microarray.

Using a custom-made cDNA microarray, global transcriptional analyses were conducted to identify genes differentially regulated in the pheromone gland as compared to the remaining insect tissue of the moth Agrotis segetum (Noctuidae). A two-fold or larger difference in relative expression levels was found for 227 of 864 genes investigated comparing the two tissues. Unexpectedly, an antennal binding protein homologue, containing a pheromone-binding/general odorant-binding protein PFAM domain, was expressed at a 56-fold higher level in the pheromone gland. Relatively higher expression levels in the pheromone gland were also found for other gene representatives putatively encoding odorant-binding proteins and chemosensory proteins, as well as a number of gene representatives putatively encoding proteins involved in juvenile hormone interactions. The largest relative up-regulation (84-fold) in the pheromone gland was found for a gene encoding a Delta11-desaturase homologue implicated in desaturation of pheromone precursors. For three gene representatives, the expression patterns were independently verified by quantitative real-time PCR (qPCR). Additionally the expression pattern in the pheromone gland for the Delta11-desaturase homologue was shown by qPCR to follow the previously known pattern of pheromone production in female A. segetum, both with respect to age and circadian rhythm, whereas the expression of a Delta9-desaturase and a chemosensory protein homologue did not share this pattern

Transcriptional analysis of the pheromone gland of the turnip moth, Agrotis segetum (Noctuidae), reveals candidate genes involved in pheromone production.

Moths generally rely on pheromone communication for mate finding. The pheromone components of most moths are produced by a common pathway of fatty-acid biosynthesis coupled with species-specific modifications of the final products. Some genes involved in moth pheromone production have previously been described, whereas others remain to be characterized and thus the molecular mechanisms accounting for the production of species-specific blends are far from understood. The turnip moth, Agrotis segetum, has a multicomponent pheromone, consisting of at least four components derived from palmitic and stearic acid. Different populations produce and respond to different pheromone blends, which makes this species an excellent model for research on genes and molecular mechanisms involved in moth pheromone production. For this purpose, we performed an expressed sequence tag (EST) analysis of two cDNA libraries, one representing the female pheromone gland and the other representing the remainder of the insect body. Among 2285 ESTs analysed altogether, we identified a unigene set of 707 putative gene representatives. The comparative distribution of those in the two libraries showed the transcriptomes of the tissues to be clearly different. One third of the gene representatives were exclusively found in the pheromone gland. From sequence homology to public database information we assigned putative functional roles for a majority of the unigenes and then compared functional profiles of the two tissues. In the set of ESTs more abundant in the pheromone gland library, we found homologues of an acyl-CoA Delta11-desaturase, a G-protein subunit, a chemosensory protein as well as a juvenile hormone binding protein

To screen the effect of 30 different anti-cancer drugs, preprinted on a 384-well plate and incubated for three days with primary tumor cells from one patient

Using live/dead fluorescence cell discrimination to determine a drug sensitivity profile for primary human tumor cells with the aim of assisting individualized assay guided cancer therapy.<p><b>Copyright information:</b></p><p>Taken from "Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with virtual microscopy"</p><p>http://www.biomedcentral.com/1471-2342/8/13</p><p>BMC Medical Imaging 2008;8():13-13.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2515298.</p><p></p

To visualize thousands of cells growing in monolayer – for automatic and objective quantitation of epigenetic effects of the transformation associated viral nuclear antigen HHV8 LANA fused to green fluorescence protein in a transient transfection assay

The blue color is Hoecsht 33342 fluorescence showing DNA. The mosaic is built up from 100 images (10 columns × 10 rows) captured at 16X/NA O.5. Individual images covers an area of 522 μm × 398 μm. Images are captured at binning 1 (1344 × 1024 pixels) with 100 pixels overlap resulting in a final area of the seamless panorama, after image processing in the ImageLab/Virtual Microscope, of 4,82 mm × 3,58 mm.<p><b>Copyright information:</b></p><p>Taken from "Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with virtual microscopy"</p><p>http://www.biomedcentral.com/1471-2342/8/13</p><p>BMC Medical Imaging 2008;8():13-13.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2515298.</p><p></p

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with virtual microscopy-7

programming environment OpenLab Automator. A: Main program. B: Subroutine 1, Find Four Corners. C: Subroutine 2, Z Capture Loops. D: Slider control interface: Settings for excitation wavelengths, exposure times and field of view of the objective (XY-dimensions). E: Pop-up message, to guide the user through the settings of parameters. F: Pop-up Welcome message. G: Head interface of QuantCapture4, shows program status and control panel for start (Run), pause and ending the capture (Stop).<p><b>Copyright information:</b></p><p>Taken from "Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with virtual microscopy"</p><p>http://www.biomedcentral.com/1471-2342/8/13</p><p>BMC Medical Imaging 2008;8():13-13.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2515298.</p><p></p

This schematic image illustrates the virtual microscopy features of a montage presented in the ImageLab/Virtual Microscope software

Allow real-time browsing by building a representation of images from the aligned stack "on the fly".<p><b>Copyright information:</b></p><p>Taken from "Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with virtual microscopy"</p><p>http://www.biomedcentral.com/1471-2342/8/13</p><p>BMC Medical Imaging 2008;8():13-13.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2515298.</p><p></p