The case report by Nautiyal et al.  is an inActual measurement with exophthalmometry clearly demonstrates the lack of enophthalmos. As stated by Loewenfeld


The first of these can be attributed to a melting phase transition arising from the rotation of two trifluoromethyl groups. In the crystal structure, both trifluoromethyl groups are disordered over two sites with occupancy factors of 0.660 (17) and 0.340 (17) for the major and minor orientations, respectively. The introduction of trifluoromethyl groups inhibits π-stacking between the diazafluorene  units. Three short F⋯O contacts between 2.80 (3) and 2.95 (1) Å are observed in the crystal structure.The title compound, C Å                           b = 12.357 (3) Å                           c = 26.941 (6) Å                           V = 3090.4 (12) Å3                                                   Z = 4                           Kα radiationMo −1                        μ = 0.11 mmT = 173 (1) K                           0.38 × 0.30 × 0.20 mm                                 Rigaku/MSC Mercury CCD diffractometerAbsorption correction: none20599 measured reflections3171 independent reflectionsI > 2σ(I)2849 reflections with R                           int = 0.040                                                            R[F                           2 > 2σ(F                           2)] = 0.056                           wR(F                           2) = 0.146                           S = 1.05                           3171 reflections498 parameters24 restraintsH-atom parameters constrainedmax = 0.28 e Å−3                        Δρmin = −0.25 e Å−3                        Δρ                                 CrystalClear  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008PLATON (Spek, 2003Mercury (Macrae et al., 2006WinGX (Farrugia, 1999Data collection: 10.1107/S160053680803420X/hb2809sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S160053680803420X/hb2809Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Abstracttask clusters) that are common to efficient and effective practices in the digitization of biological specimen data and media. Examples of these clusters come from the observation of diverse digitization processes. The staff of iDigBio  visited active biological and paleontological collections digitization programs for the purpose of documenting and assessing current digitization practices and tools. These observations identified five task clusters that comprise the digitization process leading up to data publication: (1) pre-digitization curation and staging, (2) specimen image capture, (3) specimen image processing, (4) electronic data capture, and (5) georeferencing locality descriptions. While not all institutions are completing each of these task clusters for each specimen, these clusters describe a composite picture of digitization of biological and paleontological specimens across the programs that were observed. We describe these clusters, three workflow patterns that dominate the implemention of these clusters, and offer a set of workflow recommendations for digitization programs.This paper describes and illustrates five major clusters of related tasks (herein referred to as  These tasks typically include photography of specimens and labels, image processing, capture of label information as text, and locality georeferencing. The presentation of workflow characteristics in this paper provides the framework for analyzing the effectiveness and efficiency of workflows and for the development of new effective workflows. It should be noted that the workflows we observed represent a major departure from a historical practice of pulling a single specimen, creating a comprehensive database record, including researching localities, georeferences, collectors, taxon names, nomenclature, and other related details, then moving on to the next specimen  and the specimen itself (e.g. scientific name and identifying number). Digitization of label information includes capturing the text as characters, dividing the text into specific properties, and storing this information in a database. Digitization may also include capturing digital images and other media. References to media objects are added to the database records.http://www.gbif.org ), iDigBio (http://www.idigbio.org ) and the Thematic Collections Networks funded by the National Science Foundation’s Advancing Digitization of Biological Collections (ADBC) program (http://www.nsf.gov/pubs/2011/nsf11567/nsf11567.htm), Atlas of Living Australia , http://vbrant.eu/ ), and VertNet (http://www.vertnet.org ). However, little has been published that characterizes modern existing and effective digitization workflows for a broad range of collections . We believe such characterizations are an early step in the process of building a common framework for sharing efficiencies across biological and paleontological research collections.The collections community has recognized that digitization processes need to be made more efficient to meet pressing scientific and societal needs (a topic broadly reviewed by (URLs provided for first mention only. Please see Appendix 2 for URLs of software and websites.)PageBreakgrounded theory research methodology . We asked that PageBreakIn the digitization workflows we observed, protocols for the digitization of biological and paleontological specimens were typically divided into clusters of related tasks. The order in which these task clusters were accomplished was based on a combination of staff availability, equipment, space, facilities, institutional goals, and the type of collection being digitized. Hence, though there was a general pattern to the components included within a particular task cluster, the order of accomplishment of the clusters and the tasks within each cluster varied by institution.These five task clusters were important components of digitization, but not all were essential to meeting the digitization goals of every organization or of every specimen for every organization. These clusters are presented here in a common order of operation:• pre-digitization curation and staging,• specimen image capture,• specimen image processing,• electronic data capture, and• georeferencing specimen data.It should be noted that quality control and data cleaning tasks were integral to each of these task clusters  to a specimen, container, or cabinet,• discover important but previously unknown, lost, or dislocated holdings ,• update nomenclature and taxonomic interpretation,• reorganize the contents of cabinets, cases, trays, and containers, especially when these are the units of digitization,• vet type specimens, and• select exemplars for digitization, when that approach is appropriate.The last five activities in this list may require the greatest knowledge of the organismal group of any during digitization. Many institutions use students, interns, dependable volunteers, or other full- or part-time technicians to accomplish the other pre-digitization curatorial tasks on this list, including the selection of exemplars for digitizing. However, some institutions also reported success with allowing technicians to take on more responsibility for at least some of the last 5 tasks in the above list .In addition, as collections data become more generally available online, updating nomenclature and taxonomic interpretations and vetting type specimens can occur after the publication of data and images on the internet, providing an opportunity for off-site experts to comment on the specimens. The latter approach will avoid what can become a bottleneck in the digitization workflow caused by the limited availability of in-house taxonomic experts or well-trained technicians.Although the application of specimen barcodes is treated here as part of pre-digitization curation, this placement in the digitization workflow is not universal. Some institutions applied barcodes at or just prior to the time of image or data capture, depending on the customized order of operations. In all cases where barcodes were used, they were applied prior to image capture to allow for the barcode value to be seen in the image, and prior to data capture to ensure that the physical specimen identifier is accurately included in the electronic data record.PageBreak of specimens from a single collecting event, barcodes were sometimes affixed to or inserted into the enclosing container. In most instances, when a container was barcoded, the number of specimens within the container was recorded, but individual specimens within a common container and not segregated by separate vials were neither barcoded nor otherwise individually identified. When individual vials containing single specimens were aggregated into larger jars, a replica of the label for the containing jar was sometimes inserted into each vial. In a few cases, the container was barcoded as were the individual specimens within that container (e.g. with Lepidoptera). In this latter case, the specimens were digitized individually, with both the individual specimen and container barcodes recorded in the database.Barcodes were used for two primary purposes. For individual specimens, barcodes were affixed or pinned to the single specimen or inserted into a wet container that held a single specimen. For specimen groups, such as taxon trays, wet containers, or a collectionLinear, one-dimensional barcodes are relatively large and are used in cases where sufficient space is available, for example on vascular plant specimens, bryophyte and lichen packets, and other dry, flat specimens. A smaller version of this type of barcode, printed the size of a standard insect label, was also used in entomology collections. Space is an important constraint in barcode selection.One-dimensional barcodes used for insect collections had two advantages. They mimicked the other labels in size, thus conserving space between specimens, and, if positioned near the bottom of the pin, were easily viewed and hand scanned without removal.Two-dimensional barcodes were also used, especially for small specimens. They were preferred by some entomology collections because they could be included on an insect pin with the coded end clearly visible and easily scanned.Determining what to image varied by institution and collection type. Most herbaria imaged entire specimen sheets. Close-up images of particular morphological features  were also sometimes captured. Certain entomological , paleontological, and ornithological collections captured several images of the same specimen with various views .http://www.jpeg.org/committee.html ) (jpeg or jpg) file format for distribution on the internet. Some institutions preferred camera raw formats for archiving images as these formats retain all data originally recorded when the image was made. Others preferred the well-documented and widely used http://partners.adobe.com/public/developer/tiff/index.html ) (tiff or tif), which retains all of the original image data and most of the Exchangeable Image File Format (EXIF) data (a topic reviewed by http://www.adobe.com/products/photoshop.html ) and Lightroom (http://www.adobe.com/products/photoshop-lightroom.html ))PageBreak. It should be noted that capturing and preserving high quality specimen label images offers opportunities to take advantages of future improvements in image analysis . A few camera manufacturers  have adopted the digital negative format as the native output for some of their cameras.Manufacturer-controlled raw formats are not openly documented and are subject to change without public notice. Hence, in 2004, Adobe, Inc. developed the publicly documented digital negative format (dng) as well as a freely accessible software application that converts many proprietary raw formats to digital negatives with little or no data loss  or a custom-designed replica), SatScan ,http://www.canon.com ), Nikon Capture NX2 (http://www.nikonusa.com ), Photoshop, and Lightroom), image stacking equipment and software, for example Helicon Focus (http://www.heliconsoft.com/heliconfocus.html ) or Auto-Montage (http://www.syncroscopy.com/syncroscopy/automontage.asp ) ,• copy stand and/or specimen holder,http://www.mkdigitaldirect.com/products/lighting-systems/mk-photo-ebox-plus-1419.html )),• studio lighting, flash units, or light/diffuser box .The most common brand of camera in use across collections was a Canon DSLR equipped with a medium-length macro lens, although Nikon DSLR cameras were also sometimes used. Megapixel ratings generally ranged from about 17 to 21.5, but were sometimes lower or higher, depending upon the expected use of the images.PageBreakstudy were usually captured at megapixel ratings of 17 and above. Macro lenses in the range of 50–60 mm were common, but a few institutions used macro lenses in the range of 100–105 mm, which allowed for close focusing and performed well for smaller objects, such as small birds and mammals. Collections requiring macro images of very small specimens usually used a Leica microscope equipped with a Canon, Nikon, or Leica camera.It is instructive to note that generally, the larger the megapixel rating, the better the quality of the resulting images. Hence, images to be used for morphological To control for image quality, some institutions located the imaging station in a darkened or minimally lit windowless room. This prevented strong extraneous light, like that from a window, from contaminating or overpowering studio lighting or producing visible shadows on the resulting images. Light control was also sometimes accomplished by draping diffuser material across studio lights. A more elegant solution utilized a diffuser box with internal lighting that can be closed prior to image capture. Preferred for this was the MK Photo-eBox Plus Digital Lighting System, originally designed for photographing jewelry, coins, and collectibles. The box is slightly larger than a standard herbarium sheet, rests on a copy stand, includes halogen, fluorescent, and LED lighting, and is equipped with an oval port on the upper surface that allows an unobstructed camera view of the specimen. Herbaria using this system usually place the color bar and scale at the top of the sheet to preserve the aspect ratio of the resulting image, thus obviating the need for image cropping and reducing the number of steps required for image processing. Although the requirement to open and close the doors of the light box seemingly slowed the imaging rate, time lost was likely recaptured from a reduction in time spent on post-imaging batch cropping and light level adjustments.http://gpi.myspecies.info/ ). GPI specifications require that specimens be scanned at 600 ppi resolution, beyond the capacity of most DSLR cameras when used for whole sheet images of herbarium specimens. HerbScan uses a flatbed scanner  and a platform that raises the specimen sheet to the face of the inverted scanner. Scanning requires 4-6 minutes per scan for a maximum effective rate of about ten images per hour. Because the specimen sheet is pressed against the rigid glass face of the scanner, the acceptable depth of the specimen sheet is limited to about 1.5 cm, hence some specimens are too bulky for this equipment.HerbScan is the imaging system used for scanning type specimens for the Global Plants Initiative (GPI) project ,• adjusting camera height,• changing or attaching lenses, and• loading ancillary image management/processing software.In some institutions, especially those where all specimens are similarly sized (e.g. herbaria), camera settings and equipment mountings were usually not changed from session to session and required only a spot check prior to commencing a new imaging session. With collections of variously sized organisms , camera distance to subject was frequently adjusted, lighting re-arranged, camera settings altered, and custom or specialized specimen holders repositioned. In some instances, grouping like-size specimens alleviated the need for continuous camera adjustment and increased workflow efficiency. In these situations, the potential increase in imaging error due to increased demands for technician judgment were effectively offset by a higher level of detail in written protocols, elevated attention to specialized training, and diligent monitoring during the early phases of a new technician’s tenure. Institutions that imaged only labels that required only moderate resolution sometimes dispensed with much of the equipment listed above in favor of a small digital camera and less elaborate copy stand that afforded more mobility .PageBreakProcuring and organizing the next batch of specimens for imaging was sometimes facilitated by ensuring proximity of the specimens to the imaging station. Institutions used mobile carts or cabinets to transport specimens from the pre-digitization curation or data entry areas to a location in close proximity to the imaging station. Moving specimens from station to station rather than returning them to storage cabinets and re-retrieving them reduced the amount of time devoted to travel and handling. From our observations, workflows that began with image capture, imaged every specimen, and extracted data directly from the image rather than the physical specimen effectively eliminated the need to handle or move specimens beyond the imaging stage, facilitating re-storage immediately following imaging . To ensuImage acquisition focuses on the process of camera operation for image capture. For collections with standard sized specimens (e.g. herbaria), the process involved repeating a rote procedure for each new specimen. Even for such collections, however, the technician was required to pay close attention to quality by periodically examining images to ensure that:• lighting, exposure, and focus remained constant,• file naming progressed according to plan,• exposure was correct,• focus remained sharp,• images lacked imperfections such as blemishes or streaking,• files were not corrupted, and• barcodes or identifiers were in place and readable.For wet collections, exemplar specimens were usually removed from the container before imaging. One successful technique we observed for imaging fish, reptiles, amphibians, and other organisms with a reflective epidermis submerged them in a shallow, ethanol-filled container, allowed the ripples to settle, and acquired the image through the ethanol. This method increased detail by reducing reflectance and increasing contrast. Coating fossil specimens with a thin layer of alcohol also increases contrast and provides for a sharper image .PageBreak clearly visible in the resulting image. One institution (Museum of Comparative Zoology) designed and constructed a custom specimen holder (http://www.imagemagick.org/ ).Protocols and workflows for efficiently imaging insects—with the possible exceptions of bees, ants, and butterflies—are under development and continue to pose special challenges. In nearly every case where we observed butterflies being imaged, specimens were removed from the pinning substrate, labels were carefully removed and placed on a custom-designed holder with the labels and barcodes (or other identifier)n holder  with sufn holder . Other in holder . Some inImaging productivity varied by collection. For herbaria, rates per imaging station ranged from as few as 10 sheets per hour using a single HerbScan, to 75–120 sheets per hour using a camera (average rate slightly less than 100 sheets per hour). Imaging rates for insects are not well documented and their derivation is sometimes confounded by the inclusion of data entry and image acquisition in a single, linear workflow that makes it difficult to segregate strictly imaging tasks from data entry. For example, the imaging step might include removing the label from the pin, taking the photo, and putting the label(s) back on the specimen pin.Image processing involves all tasks performed on an image or group of images following image capture. Nine tasks are addressed here, reflecting common practices:• quality control,• barcode capture,• file conversion,• image cropping,• color balance or light level adjustments,• image stacking,• redaction,• file transfer, and• optical character recognition (OCR).Some institutions include one or more of these nine tasks  at other stages of the digitization process, as noted in the discussion below.http://www.snsb.info/SNSBInfoOpenWiki/attach/Attachments/JSTOR-Plants-Handbook.pdf ).Quality control was usually effected by selecting and examining sample images at regular intervals. In some institutions, all images were visually scanned for obvious deficiencies before individual images were selected for more thorough review. Selected images were evaluated for correct focus and exposure, blemishes, scan lines, mismatches between file names and barcode values (in situations where these are expected to match), and other obvious signs of imperfections or errors. Imperfections in camera images usually related to incorrect focus or exposure. Institutions using HerbScan, especially as part of the GPI, followed a more elaborate and rigorous process (not detailed here) that included converting images to high contrast in Photoshop and running scripts that track pixilation and banding, and that expose scanner-produced flaws such as minute streaks and lines caused by wear and tear on scanner parts. The standard for GPI images, coupled with mechanical parameters of the scanners, demanded these enhanced quality control procedures  software to scan the image file for a barcode value and rename the file to the barcode value detected. The benefits of the latter approach included reduction of potential naming errors and greater efficiency due to reduced camera manipulation.Based on our observations, collections that chose to use barcode values as filenames generally used one of several options. Most high-end DSLR cameras allow for cuhttp://finereader.abbyy.com/corporate/) at the herbarium of Valdosta State University, where barcodes were carefully affixed in precise horizontal or vertical orientation. A fourth approach used custom-designed software to intercept the filename generated by the camera, simultaneously creating an associated record in the database for later data entry from PageBreakthe image. Image filenames were unique for the collection, and the image files were usually stored in a repository and linked to database records through a software interface.However, OCR software sometimes failed at detecting barcodes within images due to image quality or other issues, resulting in files not being appropriately renamed. According to our observations, barcode extraction failure rates on bryophyte packets ranged from 0.2–3%, based on tests with ABBYY Finereader Corporate edition (http://code.google.com/p/zxing/ ), compared the value to the image’s EXIF data, and created a database record containing the image filename and barcode value. This allowed database records to be created by software without regard to the image’s filename. The key point of this process is that camera-generated filenames can be stored verbatim in a database if software is responsible for associating image files with specimen records .Cropping is used to trim excess image data in order to achieve an acceptable aspect ratio or to reduce unnecessary borders surrounding the specimen. Where cropping was utilized, it was accomplished in large batches that did not require monitoring once set into motion. However, cropping was not universal.In general practice, it is considered unwise to use photo manipulation software to alter color balance, saturation, sharpness, or other image features . Doing shttp://en.wikipedia.org/wiki/Focus_stacking ) involved recording several images of a stationary specimen at varying depths of field, processing them through a stacking algorithm that essentially merged the several layers into a single image while preserving properly focused pixels in each layer. The result was a sharply focused image throughout the specimen’s depth. Software packages in common use included proprietary Auto-Montage . Stacking worked best with cameras that supported a live view of the specimen in conjunction with camera control software that allowed precise focus control targeted to small percentage regions of the specimen.Specimens with significant depth, such as fossils, some insects, birds, mammals, and even some herbarium sheets, make it difficult to achieve sharp focus throughout the depth of field. Institutions used one of several stacking software packages to rectify this problem. Focus stacking ), Microsoft Voice Recognition (a standard component of the Microsoft Windows® operating system), and Dragon Naturally Speaking  are three software packages being used or tested. We note that programmers at the Botanical Research Institute of Texas (BRIT) are testing the Application Programming Interface that is packaged with the Microsoft Windows® operating system.We believe that voice recognition shows great potential for data capture and that the comparatively small cost for appropriate commercial products will be offset by greater workflow efficiencies. Most modern operating systems include built-in voice recognition capabilities of various qualities that should be tested using a high quality microphone. From our experience, the potential drawback to this technology is that substantial training to particular voices is often required for the software to perform adequately, which may limit its use where several data entry technicians are involved or when the rate of technician turnover is high. In addition, PageBreakwe noted from our interviews that simultaneous data entry by several technicians in close proximity might lead to distortion and interference, or be distracting to workers.Advances in voice recognition technology are evident in computer, tablet, and smart phone applications. Nevertheless, we saw only a single use of this technology, and this only for capturing a limited set of data, but we note that some institutions are experimenting with this technology. IBM ViaVoice  at BRIT and the Symbiota Software Project (http://symbiota.org/tiki/tiki-index.php ) at Arizona State University. Each of these interfaces simultaneously displays a specimen image, an OCR-rendered version of label data extracted from the image, and a collection of database fields into which data can be transferred. Apiary allows users to demarcate OCR regions of interest within the image and highlight OCR-generated text that can be transferred to associated data fields by mouse click. Symbiota provides for moving data to fields manually, but additionally includes functionality for searching the databases of the Consortium of North American Byrophyte Herbaria (http://symbiota.org/bryophytes/ ) and Consortium of North American Lichen Herbaria  for previously digitized duplicates from which data can be imported.Optical character recognition (OCR) was also being used or considered by several institutions. Two of the most effective uses we observed included the Apiary Project (http://code.google.com/p/tesseract-ocr/ ), OCRopus (http://code.google.com/p/ocropus/ ), and JOCR (GOCR) (http://jocr.sourceforge.net/ ), all of which are open source, and the proprietary ABBYY Finereader corporate version (http://www.abbyy.com/ ) and Adobe Acrobat Professional version (http://www.adobe.com/products/acrobatpro.html ), both of which can batch process large numbers of images. There is significant interest in natural language processing (NLP), which is designed to parse OCR text into fields, as well as intelligent character recognition (ICR) or handwriting analysis, but effective systems for using these technologies to extract data from biological specimens were not observed.Other institutions routinely process all images through OCR and store the OCR-generated output in text files, or import it into a field within the database for subsequent editing, data cleaning, and searching. Popular OCR software packages included Tesseract (http://specifysoftware.org/ ) (via Workbench), Brahms (http://herbaria.plants.ox.ac.uk/bol/ ) (via Rapid DataPageBreak Entry http://herbaria.plants.ox.ac.uk/bol/BRAHMS/Documentation ), and KE EMu (http://www.kesoftware.com/ ) provide this capability. Issues to resolve when importing legacy or external data include data quality, mapping imported data fields to those in the preferred database, dealing with imported fields that do not have database correlates, and time required for post-import data cleanup. In many cases, importing and transforming legacy data can be efficiently managed, resulting in large dataset acquisitions for relatively small investment in time, especially when compared to keystroking.In some instances data entry is accomplished by electronic import from spreadsheets or other delimited lists. Some software interfaces, e.g. Specify  and Biogeomancer  are software applications designed to assist in assigning latitude/longitude coordinates to textually described localities. Both of these applications convert locality descriptions into coordinate pairs based on statements of state, county, orthogonal direction, distance, and place names of geographical features. Both also provide protocols for uploading datasets for processing and bulk georeferencing similar localities. Each returns a map of the estimated location of each described locality, including a point-radius estimate of precision. Map interfaces allow technicians to manipulate and refine the georeferenced locations of these points before recording a final determination of the point’s coordinates. Technician manipulation was required for points to be reliable. Both Geolocate and Biogeomancer are free to use. The third method we observed was based on the use of standard and customized map layers in conjunction with GIS software (such as ArcMap http://webhelp.esri.com/arcgisdesktop/9.2/index.cfm?TopicName=An_overview_of_ArcMap ) and paper maps to pinpoint locations. For best results, all of these systems rely on a technician’s knowledge of the region in which a collection is made, facility with desktop GIS or online mapping software, general understanding of maps and mapping, and ability to recognize habitat signatures on aerial photographs.We observed three georeferencing methodologies in use where coordinate values were not present on the specimen. Geolocate , which requires specimen handling only during image aquisition.The image to data to distribution pattern fits institutions that image all specimens (e.g. most herbaria) and captures data from these images. It reduces specimen handling and with it the likelihood of specimen damage, increases efficiency by eliminating the need for return trips to storage locations, and offers the capacity to incorporate Optical Character Recognition and similar technologies within the data capture workflow.The Based on our observations, interviews, discussions, and readings, we offer the following recommendations for establishing and improving biological and paleontogical collections digitization programs.1. With planning, the pre-digitization curation step is an opportunity for the goals of specimen digitization and collection curation to be merged into an efficient workflow. Curation tasks that cannot be efficiently addressed in the workflow can be identified so that adequate resources can be assigned to them in the future .PageBreakmore efficient (2. Biodiversity informatics managers and other digitization personnel should look for bottlenecks in digitization workflows and seek ways to make them fficient . We reco3. There should be clear institutional policies guiding which specimens to expose to public access, including policies governing whether to redact or not redact locality data for sensitive species  and ensu4. Barcodes should be used only as identifiers; encoded barcode data should not incorporate taxonomic or related information that might change with time.5. Where possible, the aspect ratio of specimen to camera should be synchronized to eliminate the need for image cropping.6. Image processing should not include color balancing or other adjustments that result in images inaccurately reflecting actual specimens .7. A color bar and scale should be visible in all images .8. Protocols for periodic quality control should be established for all stages in the digitization workflow to ensure data accuracy and the production of high quality digital images .9. For institutions in which imaging is paramount, acquiring images of labels prior to data entry reduces specimen handling by allowing for data extraction from images rather than from specimens.10. Attention to the digitization of gray and published literature related to specimen data is an important consideration and should be accomplished whenever possible cf. .11. Georeferencing should be treated as an essential part of digitization protocols .12. Quality control should be integral to all steps in the digitization workflow, including post-digitization review and targeted testing should be designed to expose data inconsistencies or suspected anomalies .13. Detailed written protocols should guide every step of the digitization workflow, be uniquely designed for a given institution, and be amended regularly to reflect emerging technologies and improved efficiencies. These protocols should be electronically stored in a common folder that allows technicians to insert comments and suggestions to be reviewed and potentially adopted by biodiversity informatics managers.14. Selection of data entry and imaging technicians should be guided by employability skill sets strongly associated with success in digitization tasks, with particular attention to potential technicians’ attention to detail, orientation to increased efficiency, and commitment to high productivity.PageBreak15. Institution-wide digitization tasks should be periodically evaluated for overall progress, organizational collaboration and cooperation, and compatibility with new and emerging technology, with plans to use results of the evaluation to implement improvements .16. Digitization workflows should be coordinated by a designated biodiversity informatics manager with IT experience, preferably from a biological sciences and collections background, to bridge the potential knowledge gap between collections managers and information technology professionals .17. Biodiversity informatics managers should construct a frequently asked questions document that outlines common problems and offers instructions about how to address these problems, whom to contact with questions about specific categories of problems, and guidelines for which types of problems should be elevated to a higher administrative level.18. Institutions should utilize a digitization workflow strategy that captures problems, remedies, lessons learned, and technician input for use in improving digitization protocols, and remain open to investigating possible changes in current practice .19. Determining an appropriate storage format for archived images is an important decision that should precede image capture. Here we recommend capturing images in native camera raw and converting them from camera raw to dng or tif (a topic addressed by


Nothing is known, however, about; thedevelopmental mechanisms governing butterfly oogenesis, how polarity in theoocyte is established, or which particular maternal effect genes regulateearly embryogenesis. To gain insights into these developmental mechanismsand to identify the conserved and divergent aspects of butterfly oogenesis,we analysed a Pararge aegeria females expressed74.5% of the genes that are known to be essential for D.melanogaster oogenesis. We discuss the genes involved in allaspects of oogenesis, including vitellogenesis and choriogenesis, plus thoseimplicated in hormonal control of oogenesis and transgenerational hormonaleffects in great detail. Compared to other insects, a number of significantdifferences were observed in; the genes involved in stem cell maintenanceand differentiation in the germarium, establishment of oocyte polarity, andin several aspects of maternal regulation of zygotic development.A total of 17306 contigs were annotated, with 30% possibly novel or highlydivergent sequences observed. This study provides valuable resources to investigate a number of divergentaspects of butterfly oogenesis requiring further research. In order to fullyunscramble butterfly oogenesis, we also now also have the resources toinvestigate expression patterns of oogenesis genes under a range ofenvironmental conditions, and to establish their function. Drosophila melanogaster, areinvolved in setting up; 1) the location of the germ plasm and subsequent germ cellline development in the offspring . Th. ThBomby by mex-3. Pararge-3 Table . Furthernslation . All of anogaster,65, presDrosophila melanogaster includes maternal hunchback(hb) transcripts into the egg, the protein of which will form an APgradient during early embryogenesis and cooperate with Bcd to specify theanterior of the embryo, whilst being repressed at the posterior by Nos [hb RNA or protein is transferred to the egg, as well as in thesignificance of the maternal contribution to the Hb gradient for AP patterning,the transcription of hb during oogenesis appears conserved [Schistocerca americana embryo, maternal hbtranscripts appear to be involved in distinguishing embryonic fromextra-embryonic cells along the AP axis, whilst in D. melanogastermaternal and zygotic Hb are redundant for AP patterning of the embryo [B. mori, the hb transcripts detected appear tobe transcribed by the zygote, not the mother [Pararge aegeria also did not express hb duringoogenesis ),which indeed appear to have divided up these functions [B. mori does not have agerm plasm, the location of maternal B. mori nos-O transcripts in theembryo seems to correspond with where the PGCs will form [nos paralogs, with the exception of nos-P,are expressed during oogenesis in both B. mori and P. aegeria,with maternal transcripts detectable in P. aegeria eggs genome [nos sequences showsnos-P to be quite different from the other paralogs  genome  and phylD. melanogaster nos RNA is accomplishedduring oogenesis by proteins encoded by glorund (glo) and in the earlyembryo by smaug (smg) [D. melanogaster oocytes [P. aegeria ortholog of smg was found, which waspresent as RNA in the oocyte, but not of glo  that preventsrapid deadenylation at the posterior pole, establishing nos as aposterior defining gene [osk ortholog [nosparalogs may not being involved in AP axis formation during oogenesis. Indeed,P. aegeria also does not possess an ortholog of osk(Table P. aegeriagenome).Translational repression of ug (smg) . Transcr oocytes . A P. aelo Table . Furtheranogaster. Rapid d nos RNA . AlthougR4 Table . In D.ming gene . DitrysiPectinophora gossypiella[osk, which has been argued to have been co-opted as theessential gene in germ plasm formation in holometabolous insects [Pararge aegeria may not possess an osk ortholog,but it does express two genes, which in D. melanogaster silenceosk translationally during oogenesis; bruno[cup[D.melanogaster , vasa (vas) and valois(vls) [valois (vls) could not be found in the P.aegeria transcriptome  . Only vae Tables .tud, vas and vls , Brother ofYb (BoYb) and Sister of Yb (SoYb) [P.aegeria transcriptome , TDRD7 andspindle E/homeless (TDRD9) [P. aegeria  . Indeed, (TDRD9) ,110 and  with osk. It is l with osk.piwi,aubergine (aub) and argonaute 3 (AGO3) [P. aegeria pathway genes often disrupt the axes of the developingoocyte, through their effects on the microtubule cytoskeleton; for examplemaelstrom (mael), zucchini (zuc) andsquash (squ) affect DV polarity [aub in D.melanogaster in silencing osk translation during oogenesis [armitage (armi)affects axis formation and is involved in osk translational silencingin D. melanogaster[zuc nor squ was found in the P.aegeria transcriptome, but mael and armi were . All thraFigure  and 10. em cells . Furtherpolarity ,115. Theanogaster. NeithereTables  and 10.D. melanogaster these can befound in nurse cells, but also appear to be involved in compartmentalisation ofmRNA decay and translation repression, for example of osk[EDC4/Ge-1 and pacman(pcm), genes that encode the essential components of P-bodies wereexpressed in P. aegeria  are aggregates of translationallyinactive ribonucleoproteins (RNPs). In le of osk,117. Witogenesis . This waD.melanogaster, a number of (late-acting) maternal-effect genes areessential in germline formation during early embryogenesis  and imp[germ cell-less (gcl) and polar granulecomponent (pgc)  is involved in germ cell migration inD. melanogaster embryos  and imp. However) Tables .These gte Table .Drosophila melanogaster uses an elaborate network of genes to patternthe DV axis during embryogenesis on the basis of the oocyte polarity establishedduring oogenesis , nudel (ndl), gastrulationdefective (gd), snake (snk), easter(ea), spn27A, spz, tube (tub)and pelle (pll) (Tables weckle (wek) have yet been found outsideDrosophila, and wek was also not found in P.aegeria , rather than toll (tl). A siminespace . This pria Table . In D.m)Tables  and 13. igration , and DV igration . While Ppathways . As discster toll. It thusPararge aegeria did express cactus (cact) anddorsal (dl)  is essential for subsequent DV patterning in the D. melanogasterembryo. Dorsal protein activates some genes, whilst repressing others along theDV axis [dpp ventrally and the proteinencoded by grainyhead (NTF-1/grh) acts asco-repressor [grh is deposited maternally into the oocyte to betranslated and used ventrally during embryogenesis [dpp by a Dorsal gradient does not, however,occur in T. casteneum[D. melanogaster embryo and its concentration isfurther restricted ventro-laterally by Short gastrulation (Sog), which in D.melanogaster may also be maternally provided [vrille (vri) gene encodes a Bzip transcriptionfactor that interacts in D. melanogaster with Dpp signalling, acting asdominant maternal enhancers of embryonic DV patterning defects caused byea and dpp mutations [eclair (eca) andbaiser (bai) are essential for the activity of maternalTkv, a type I Dpp receptor [Pararge aegeria females did transfer maternal transcriptsof grh, dpp, tkv, eca, bai and vri into the oocyte,but did not express sog maternally  Table . DorsalDV axis ,124. Whiprovided . Rather provided . The vriutations . Two P24receptor . ParargeDrosophila melanogaster females express a group of genes called theyema genes  during oogenesis,with most of them displaying strict maternal expression. This may be ofimportance in the development of the central nervous system of the embryo [yema genes arenot known and there are no orthologs outside Drosophila[P. aegeriatranscriptome ,elav, brainiac (brn), Fmr1, braintumor (brat), mnb, and terribly reduced opticlobes (trol)  biogenesis [torso (tor) encodes areceptor whose ligand is most probably encoded for by trunk(trk). Furthermore, the protein encoded by torsolike(tsl) plays a role upstream of trk in activating the Torreceptor in a localised manner, and is thought to be essential for terminalspecification [tor and tsl are involved in terminalspecification in T. castaneum, different tissues are patterned andTorso signalling plays a role in defining the posterior growth zone duringembryogenesis in this short germband insect [Apis mellifera) has the genetsl, but not tor and trk in its genome [Pararge aegeria does not express clear orthologs of eithertor or trk during oogenesis, but does express tsl(Table Bombyx mori does have a RTKin its genome (BGIBMGA003976), which shows similarity to torso, as wellas to tie-like and Cad96Ca. Pararge aegeria did notexpress tie-like , although it hadgreater similarity to the receptor tyrosine kinase Fps85D than totor. This transcript is present in both P. aegeria oocytesand ovarioles, but its role in oogenesis has not been described in theliterature. It is clear that P. aegeria uses RTK signalling duringoogenesis and that the sequences of its ligands and receptors have diverged fromthose of other insects. However, at present it is unclear in which functionalcontext RTK signalling takes place.The Torso receptor tyrosine kinase (RTK) pathway has been implicated in a numberof different processes during ogenesis  and in pogenesis . The matfication . Althougd insect . Torso ss genome . The hons genome . ParargeslTable . Bombyx D. melanogaster , sex combs onmidleg (scm), and Arginine methyltransferase 1 and8  andsimilar to D. melanogaster polyhomeotic (ph-p) gene. Both playversatile and important roles in D. melanogaster oogenesis,particularly in ovarian follicle formation [Pararge aegeria females did express and transfer orthologsof other PcG genes into the oocyte. These include the polycomb repressivecomplex 1 (PRC1) genes sex combs extra (sce),polycomb (ph), posterior sex combs(psc), the PRC2 genes extra sex combs (esc),Enhancer of zeste (E(z)) and the polycomb related genesEnhancer of polycomb (E(ph)) and additional sexcombs (asx)  important in oocyte production ,matrimony (mtrm), microcephalin (MCPH1)and chiffon (chif) .A large number of genes that regulate mitosis have been studied in a reproductivecontext in ri Table . Among talin MCPHand chifD. melanogaster germarium [bruno is not onlyessential in regulating the translation of a number of genes during oocytedifferentiation, but it also appears to be involved in regulating the silencingof Cdk1 activity in order to achieve primary meiotic arrest [bruno was expressed by P.aegeria females (Table The cell cycle becomes arrested in meiotic prophase I in the majority ofMetazoans oocytes. This is initiated during the first stages of oogenesis inregion 2 of the ermarium . The intc arrest . It shouc arrest . As indies Table .D. melanogaster germarium [D. melanogaster for SCformation and thus possibly chiasmata formation: crossover suppressor on 2of Manheim (c(2)M); crossover suppressor on 3 ofGowen (c(3)G); corona (cona) andnipped-B , corona (cona),orientation disrupter (ord) and mei-S332, for example, is bothessential in D. melanogaster for maternal provisioning of the eggduring vitellogenesis and to ensure secondary meiotic arrest by stage 14 ofoogenesis in metaphase I [P. aegeriafemales did express this gene during oogenesis  interacts with polo kinase(polo) in mitotic regulation particularly during earlyembryogenesis, and is maternally provided  and LpR [yl/VgR RNA into previtellogenicoocytes, thus preparing the oocyte for Vtg uptake [Pararge aegeria females expressed not only Vtg/Vg,apoLp-III, apoLp, their receptors yl/VgRand LpR, but also the genes described in D. melanogastervitellogenic endocytosis , sec5, sec6,garnet (G) and jagunal (jagn), such as Vitellogenin (Vtg/Vg) and Apolipophorins (ApoLPs) ,9. Predoanogaster. Vitelloanogaster. LLTPs a and LpR . Nurse cg uptake . Pararge)Figure .B. mori and released as ecdysteroids during yolk uptake in theembryo as a result of dephosphorylation by ecdysteroid-phosphate phosphatase (EPPase)[Pararge aegeria did express EPPase and Samia cynthia (ESP) had not been found [G. mellonella yolk protein 2 have been discovered inD. plexippus and Plodia interpunctella, whilstESP does show significant sequence similarity with genes encodingthe KK-42 binding proteins in Antheraea moth species [D. melanogaster, in particular lipase-1 and 3(lip-1 and 3) [. Rather interestingly, although not functioningas a yolk protein, lip-1, but not lip-3, is expressed invitellogenic follicles in D. melanogaster[lip-1, and possibly lip-3 , was expressed by P. aegeria, whilst no clearortholog of a minor yolk protein was found . ParargeseTable . Further protein . The genen found . More re species  (Additio1 and 3) . Lepidopanogaster. An orthnd Table .P. aegeria ovarioles is anortholog of the slime mold Physarum polycephalum genespherulin-2A. No transcripts were found for this gene in eggs  Chico precludes vitellogenesis, whilst a sharp increasein 20-hydroxy-ecdysone (20E) relative to juvenile hormone (JH) results inapoptosis of the egg chamber before vitellogenesis is initiated or completed [D. melanogaster[bbx genes are expressed predominantly in the brain, butsome may also be expressed in ovaries [B. mori, possess a large number ofbbx-like genes in their genome [D. plexippus appears tohave only three such genes [bbxA1-like andbbxA3-like) were transcribed in P. aegeria ovarioles,whilst a third partial IRS transcript showed more sequence similarity tochico than to any bbx-like gene (Table insulin-like receptor (InR) was also expressed by P.aegeria during oogenesis  and the lipid storagedroplet proteins 1 and 2 (Lsd1 and Lsd2). Please refer to Table Reproductive output depends on female nutritional status which not only affectsthe rate and duration of oogenesis significantly, but also whetherprevitellogenic egg chambers will enter the vitellogenic stage or apoptose . Two sigompleted ,155. Altanogaster. The bbx ovaries . Moths, r genome , but thech genes . OrtholoApart from nutritional status, environmental factors such as temperature canaffect hormone concentrations, providing a possibility for environmental controlof reproductive output ,26. The methoprene-tolerant (met) [taiman (tai) in D.melanogaster[tai gene was originally discovered as a gene that wasexpressed in follicle cells in the functional context of border cell migrationand was described as an ecdysone co-receptor  can also been found in the genome of D. plexippus[There has been extensive investigation of JH signalling ,26, but nt (met) -160. It anogaster,160. Theplexippus.kruppel-homolog 1 (krh1) has beendescribed as a JH response gene, inhibiting 20E induced broad(br) expression in D. melanogaster, but not in thespecific context of oogenesis [khr1 and br were expressed by P.aegeria females , which regulates polyamine biosynthesisand appears to be essential for vitellogenesis [odc and its antagonist gutfeeling(oda), also a mitotic cell-cycle regulator, were expressed inP. aegeria. Maternal transcripts of odc and odawere found in eggs  and the JH esterases (JHEs) . JHEs fuB. mori and in aJHbp CDSs were identified in P.aegeria ovaries; JHbp, cytosolic JHbp(cJHbp), hemolymph JHbp (hJHbp) and a sequenceshowing strong orthology to takeout (to) identified in D.melanogaster as involved in JH binding  to enableimmobilisation, regulate degradation or enable transport . Four cong Table . Transcr, like P. aegeria, have been argued tobest match the criteria for group 4 [B.mori and D. melanogaster[There is a significant amount of life-history variation among insects andconsequently in the relative importance of 20E and JH on oogenesis , even wi group 4  where JHanogaster,146.Bombyx mori appears to be capable of producing ecdysteroidsin the ovaries [D. melanogaster[Drosophila melanogaster expresses start1 duringoogenesis in significant amounts in nurse cells, most likely in response toecdysone signalling. The cholesterol transporter Start1 may in turn facilitateecdysteroid production from cholesterol-based precursors [D.melanogaster cholesterol conversion in the ovaries is defective inthe avoidance of repellents (dare), which encodes an Adrenodoxinreductase [D. melanogaster the SGT1 protein homologecdysoneless (ecd) and disembodied (dib)have been described as essential for ecdysone, both for functionality and itsproduction in the ovaries [D. melanogaster start1 arehypothesised to be deposited into the egg to facilitate ecdysteroid signallingin the developing embryo [P. aegeria females did not expressdib, but did express ecd, start1, and dare. Weobserved the transfer of transcripts of all three genes into the oocytes and its partner ultraspiracle  in B. mori) in theovaries , thetranscription factor gene E74 and the Broad-Complex gene Br-C[B. mori andtheir expression increases during vitellogenesis [E74, all of these genes wereexpressed in P. aegeria  during choriogenesis [P. aegeria females did not expressESP, but did express the related gene lip-3 is expressed in all follicle cells and is essential for DVpatterning of the embryo in D. melanogaster[D. melanogaster embryo [Pararge aegeria females expressed ndl and as inD. melanogaster, no transcripts were found in the oocyte is formedhalfway through vitellogenesis , for whie oocyte . This al [B. mori, althouganogaster. Ndl is anogaster. As suchr embryo . ParargeteTable . It remaD. melanogaster VMPgenes outside the genus Drosophila. The best-characterised VMPgene in Lepidoptera is VMP30[P. aegeria ovarioles. Once again, notranscripts were found in the oocyte (Table Insect vitelline membrane protein (VMP) genes show tremendous sequence diversity.For example, no clear orthologs can be found for  is VMP30, for white Table .D. melanogaster and clusters of choriongenes are selectively amplified or expressed at very high levels [P. aegeria did not express anortholog of G1/S specific cycE, which in D. melanogaster isessential for chorion gene amplification and endocycling in general  in D. melanogaster[P. aegeria (Table B. morihave multiple cis-regulatory binding sites for CCAAT/enhancer bindingprotein (C/EBP) transcription factors and their expression levels are C/EBPconcentration dependent [D. melanogaster ortholog of C/EBP is slbo,which is also expressed in follicle cells though predominantly involved inborder cell migration , its negative regulator tribbles (trbl) andHMGa  [B. mori[D. plexippus[cf1 and CbZ were transcribed by P.aegeria, with transcripts of the latter rather intriguingly found to bepresent in the oocyte  . Further [B. mori and orthplexippus. HoweverD. melanogaster cp geneswith those identified in Lepidoptera, including P. aegeria, is very lowindeed , which forms part of the eggshell and is produced by the follicle cells [P. aegeriaoogenesis (Tables Chorion protein (cp) genes evolve possibly even faster than vitelline membraneprotein genes  and sequed Table . The infstrength . Furtherfication . Expressfication ,179. As le cells . BmEP80 D.melanogaster and B. mori, with nurse and follicle cellsundergoing apoptosis as oogenesis progresses, while complete egg chambers mayapoptose in response to environmentally induced hormonal signals such asstarvation [D. melanogasterovaries, where the effector caspase Dcp-1 and the inhibitor of apoptosis proteinBIR-superfamily domain protein Bruce regulate both autophagy and starvation-induced cell death [B. mori, and the results of the study by Zhang and co-workersshowed that most of these genes are highly conserved [D. melanogaster , which is required for posterior follicle cell maturation [D. melanogaster oogenesis  and mob as tumor suppressor   . It is essential for regulating tissue size and growth . Hippo sturation . Ortholo1) Table . Merlin/M1 Table , which iM2Table . In D. mlisation , but clei) Table . The latD. melanogaster, for example, Hsp60C is essential inorganising and maintaining cytoskeletal and cell adhesion components and thusfor establishing AP and DV oocyte polarity [D. melanogaster oogenesis(Tables Heat shock proteins (Hsps) provide a possible mechanism for environmental controlof development in ovaries and as maternal effects. The transcription of genesencoding Hsps, or molecular chaperones in general, is not only regulated inresponse to various environmental factors (e.g. temperature), but is alsoessential during many developmental processes, including oogenesis. It isthought that Hsps are important for both developmental buffering anddifferentiation ,186(furtpolarity . A large)Tables .D. melanogaster oogenesisand early embryogenesis  are among the most highly transcribed genes duringMetazoan oogenesis, as large amounts of the translation machinery are neededboth during oogenesis and by the developing embryo . Just li are amonis Table .P. aegeria and present as maternal transcripts in the oocytes, has been implicated in DV axisformation [Orthologs of the majority of the genes identified from the literature as beinginvolved in immune response during oogenesis were also found to be expressed byesTable  and 2. AesTable , these iesTable .Drosophormation .Wolbachia sp. is anendocytosymbiont in many arthropod species affecting oogenesis in a multitude ofways and the Bacterium is maternally transmitted [D. mauritiana, Wolbachia increases egg production byaffecting the maintenance and division of germ-line stem cells [Asobara tabida, Wolbachiaconfers a reproductive advantage to the females by properly regulating apoptosisduring oogenesis via its regulation of iron metabolism and ferritinexpression [D. melanogaster highly infected females sufferfrom a range of oogenesis defects mediated via grk signalling [Pararge aegeria females were also found to be infected withWolbachia, but how this affects oogenesis in this species is atpresent not known. However, we did observe that the gene encoding an ortholog ofthe Ferritin 2 light chain protein (FER2-LCH) was amongst the most highlytranscribed genes during P. aegeria oogenesis M19/wisp)tail elongation of bcd, toll, and tor transcriptsupon egg activation. It is thus important for proper patterning of the embryo [Given that P. aegeria females did not expressbcd and tor, it remains to be investigated whether wisp isof any importance in patterning of the embryo.As discussed elsewhere in this paper, after vitellogenesis both the aphase I ,194. Unlaphase I , egg actme Table ,which mp)Table . In D. mansition . The D. e embryo , but is e embryo . Given tD. melanogaster oogenesis)were transcribed by P. aegeria and transcripts were transferred to theoocytes. As this was an ovarian transcriptome study, the precise functional contextin which these genes were transcribed has not been identified. Differences in thefunctional context in which particular genes are expressed are to be expectedcompared to model organisms such as D. melanogaster and even B.mori. What is perhaps more revealing, however, is the absence of certaintranscripts in the database, in particular where these transcripts concern paradigmsof maternal regulation for various aspects of early insect embryogenesis [Pararge aegeria differed most significantly from D.melanogaster (and quite a number of other insect species), both in terms ofstem cell maintenance or differentiation in the germarium and in establishing (andmaintaining) polarity along AP, DV and at the termini of the oocyte. In particular,although Pararge aegeria females expressed an ortholog of aspi/krn-like EGF ligand and possibly its receptor, manycomponents of the EGF pathway involved in patterning of the axes in D.melanogaster embryos, as well as pipe andmirror, were not expressed. This may either suggest that there is not muchevidence for a significant role of EGF signalling in establishing P.aegeria oocyte polarity, or that its functional role and genes involved isdivergent from other insects. This requires further study, as well as the functionalrole and significance of Dpp and Notch signalling in this context.A large proportion of the genes currently described in the literature as beingessential during insect oogenesis . Although progress has beenmade in investigating B. mori embryonic patterning [P.aegeria may prove an ideal model these future studies.Although the more derived species such as and-like , it is mand-like ,54, as teristics . It is vtterning ,54, how Unfortunately, maternal effect gene expression and regulation have receivedsignificantly less research attention in Lepidoptera compared to vitellogenesis,choriogenesis and reproductive physiology . This isP. aegeria as an eco-evo-devo modelsystem for the study of butterfly oogenesis. In order to fully unscramble butterflyoogenesis, an investigation of the spatio-temporal expression patterns of the genesdiscussed in this study, as well as establishment of their function, is required.Further studies are also required to establish the function and expression patternsof the uncharacterised contigs identified in this study, which make up 30% of thetotal contigs found, and are undoubtedly composed of genes that are of highimportance in butterfly oogenesis.This study has taken a much-needed first step in determining the conserved anddivergent elements of the butterfly oogenesis GRN  and establishes P. aegeria. This population originatedfrom a woodland population from the south of Belgium  and by the time of the experiment, the butterflies had been rearedin the laboratory for 10 generations. Newly hatched larvae were placed on pottedhost plants (4 larvae per plant) of Poa trivialis L. with access toad libitum food and were reared until eclosion in a climate roomunder a regime  that promotes direct development(i.e. no diapause). On the day of eclosion  females from this laboratory stock placed individually in netted cages (0.5m3) along with a potted P. trivialis plant foroviposition and an artificial flower containing a 10% honey solution [As butterflies were used in this study, no ethical approval was required. Eggswere collected from a large outbred laboratory population of solution . Later tEggs from 50 mated 4-day old females were collected within 20 minutes of beinglaid, which is well before the onset of cleavage and thus early embryogenesis inbutterflies . The eggThe homogenate (both of eggs and ovarioles/ovary) was first centrifuged at 13000gfor 10min primarily to remove the yolk, after which the supernatant was vortexedwith 200μl of chloroform. Phases were separated at 13000g for 15min at roomtemperature. The aqueous phase was removed and precipitated in 0.5ml isopropanol . The RNAPararge aegeria egg and ovary RNA was sequenced by Source BioScience using Illumina short read RNA-Seq technology. Both total RNAsamples went through polyA selection, fragmentation and double stranded cDNAconversion to produce two separate libraries (300bp insert size) in accordancewith the Illumina mRNA-seq library preparation protocol . Sequencing was performed on the Illumina Genome Analyzer IIx platform withone flowcell lane allocated to each library. A total of 61,400,070 single-readsof 38 base pairs (bp) in length were obtained from the ovary and egg flowcelllanes which were pooled to produce a de novo assembly in CLC GenomicsWorkbench v4.0  using the default settings for shortread data (automatic word and bubble size) [le size) . The assde novo assembly usingTopHat on the Galaxy server [The RNA-seq data was analysed by FASTQC on the Galaxy platform ,199. Aday server . FPKM vay server  with quade novo assembly  was discarded from each rank, using the next best hit (which may bein a lower rank or group)  used for annotation, includingthose genes annotated manually, were used as input in the BLAST2GO software  and assi°C for 30 min after an initial 5 min denaturation step at70°C. Negative reverse transcription (NRT) controls were run inparallel without both Verso RT enzyme mix and primers. A final heat deactivationat 95°C for 2 min was also implemented to deactivate the RTenhancer. The resulting cDNA was stored at −20°C.For of a subset of 19 genes the expression in the ovarioles and the presence oftranscripts in the oocyte were confirmed further by means of RT-qPCR . Only those primers exhibiting the best stability were selected. Eachprimer pair was tested on a 3-step 5-fold dilution series of the ovary cDNA intriplicate, which enabled the primer pair efficiencies to be determined usingthe CFX Manager software . Primers withadequate efficiency (>65%) were then used for investigating the transcriptabundance in the egg and ovary cDNA  on white 96-well plates in ABsolute Blue qPCR SYBR Green Mastermix with the recommended amount of ROX reference dye was used to reveal whetherany individual transcript was used as a maternal effect gene transcript or wasmerely necessary for oocyte production. Relative transcript abundance in theovaries and eggs were obtained using the relative expression software tool RESTv2.0.13.0 software package , which uP. aegeria transcriptome investigatedby means of qPCR, transcript abundance calculated on the basis of Cq values bymeans of the methods described in [41 = 2.37, P = 0.011;Additional file The number of reads mapping to a transcript of a particular gene in RNA-seq datawas argued to be correlated linearly with the number of transcripts of that gene . Rather ribed in  showed sThe sequence read data reported in this manuscript have been deposited in theNCBI Sequence Read Archive and are available under the accession numbersSRR771147 (ovarian reads) and SRR772253 (oocyte reads). Additional file GRN: Gene Regulatory Network; eco-evo-devo: Ecological evolutionary development; AP:Anterior-posterior; DV: Dorso-ventral; RNA-seq: RNA-sequencing; RNP:Ribonucleoprotein; RTK: Receptor Tyrosine Kinase; CDK: Cyclin-dependent kinase; SC:Synaptonemal Complex; RN: Recombination Nodules; IRS: Insulin Receptor Substrate;20E: 20-hydroxy-ecdysone; JH: Juvenile Hormone; FPKM: Fragments Per Kilobase of exonper Million of fragments mapped; ORF: Open Reading Frame; SS: Similarity Score; GO:Gene Ontology; RT-qPCR: Real-time reverse transcription quantitative polymerasechain reaction; NRT: Negative reverse transcription; NTC: No template controlThe authors declare that they have no competing interests.nanos. JT assisted in manualannotation of the transcriptome. MG and CJB designed and supervised the study,performed the manual annotation of the transcriptome, and co-wrote the manuscript.All authors have provided comments on earlier drafts of the manuscript and approvedthe final version of the manuscript for publication.JMC collected and analysed RT-qPCR data, designed the automatic annotation pipeline,performed bioinformatic analyses, and co-wrote the manuscript. SCB assisted inRT-qPCR study design and data collection. RP and DRFC prepared RNA samples forRNA-seq. AC performed phylogenetic analyses of Oogenesis genes. Contains a tabulated and fully referenced list ofgenes identified from the literature, which have been studied in the contextof insect oogenesis and maternal regulation of early embryogenesis. The vastmajority of papers concern the fruitfly Drosophila melanogaster andthe silkmoth Bombyx mori. Many genes have multiple functions duringoogenesis, but to avoid repetition, and keep the size of the Tablemanageable, each gene has been listed only once in the functional contextfor which it is probably best known. Referencing has been kept to a minimum,highlighting key papers and databases. Hyperlinks have been provided foralmost all of the genes listed, which will provide full database informationon their myriad functions and further references. Presence (Y) or absence(N) of orthologs in the Pararge aegeria combined oocyte andovariole transcriptome are indicated.Click here for fileAnnotation summary of the combined transcriptome of the Pararge aegeria ovarioles and oocytes. Details the results of both automaticand manual annotation of 25266 contigs. Egg and ovary FPKM values aregiven for each contig. Each column contains a pop-up comment box with anexplanation of the column contents.Click here for fileOverview of the primer pair properties and performance in qPCRconditions. Gives an overview of the forward and reverse primersdesigned for qPCR of a set of 19 oogenesis and 3 housekeeping genes.Efficiency and R2 values are provided for each of theprimers.Click here for fileData generated by the CFX96 qPCR experiments. Details themeasurements from a total of 8 96-well white plates. Cq are given foreach gene of interest or reference gene.Click here for fileRelative Abundance Data generated by REST. Gives the results fromusing REST v2.0.13.0 to process Cq measurements and efficiencies inorder to estimate relative transcript abundance, and thus comparerelative transcript abundance between ovaries and eggs.Click here for fileTranscript abundance: Cq - FPKM correlation. Provides the resultsof the correlation analyses between two measures of transcriptabundance: Cq and FPKM-values.Click here for fileMapping of raw RNA-seq reads against egfr and wingless coding sequences as predicted from the draft Pararge aegeria genome. Provides the complete egfr andwingless (wg) CDS fasta information from ourunpublished P. aegeria genome. Furthermore, raw RNA-seq readswere mapped against these sequences and coverage determined.Click here for filePhylogenetic analysis of Nanos. Provides a phylogenetic analysisof insect Nanos protein sequences.Click here for filePhylogenetic analyses of both chorion and minor yolk proteins inLepidoptera. Provides the phylogenetic analyses of both chorionand minor yolk proteins in Lepidoptera.Click here for fileOocyte and ovarian RNA quality. Provides the Agilent BioAnalyzerElectropherograms detailing oocyte and ovarian RNA quality prior to cDNAsynthesis.Click here for fileFiltering of BLAST hits in the automated annotation. Provides avisualisation of the similarity score distribution and thresholdsapplied in the automated annotation of the P. aegeriatranscriptome.Click here for fileAutomated annotation based on different BLAST Strategies.Provides a summary of the automated annotation method, detailing thedifferent queries.Click here for fileDistribution of similarity classes across BLAST sources. Providesdetails regarding the number of Pararge aegeria contigs in eachof the similarity classes, according to the BLAST strategy used in theautomated annotation.Click here for fileThermocycler and qPCR reaction setup. Provides details regardingthe reaction conditions and thermocycler programming parameters forsuccessful qPCR amplification for each qPCR measurement reported in thisstudy.Click here for fileCombined annotated ovarian and oocyte transcriptome of Pararge aegeria. Provides the fasta format sequences of the contigs, which inAdditional file 2 had a YES in the SubmitFlag column (i.e. to besubmitted to NCBI TSA). Suggested annotated names are given on the basisof the BLAST results listed in Additional file 2, and as described inthe main text. The start and end of the open reading frames can be foundin the final two columns of Additional file 2.Click here for file


N = 138) were divided into three groups: patients with a history of suicide attempts (N = 23); patients with current suicidal ideation (N = 59); and patients without current suicidal ideation (N = 56). After controlling for covariates, no significant differences were found among the three groups on any measure of temperament or character except self-directedness and self-transcendence. The self-transcendence scores of the lifetime suicide-attempt group were significantly higher compared with those of the suicidal-ideation group; post hoc analysis revealed that self-directedness was significantly lower in the suicide-attempt group compared with the non-suicidal group. The results from the present study suggest that remitted depression patients with a history of suicide attempts do not differ from non-attempters in temperament, but do differ in certain character traits.The aim of this study was to evaluate the Temperament and Character Inventory (TCI) scores of a sample of Korean patients with remitted depression who had attempted suicide and reported suicidal ideation and to compare their scores with those of remitted depressed patients without suicidal ideation. Adult depression patients who had completed 12 weeks of follow-up ( Suicide is a major public health problem worldwide, especially in patients with depression. The World Health Organization (WHO) estimates that 873,000 people commit suicide each year worldwide. This represents 1.4% of the total global burden of disease Although a variety of risk factors for suicidal behavior have been identified among persons with depression, including prior suicide attempts, hopelessness, co-morbid alcohol/substance abuse, further depressive episodes, and earlier age of onset of a major depressive disorder (MDD) In several previous studies, personality, as represented in Cloninger's temperament and character model and measured by the Temperament and Character Inventory (TCI), has been investigated in suicidal patients. TCI is a widely used inventory that evaluates four major dimensions of temperament: harm avoidance (HA), novelty seeking (NS), reward dependence (RD), and persistence (PE), together with three major character dimensions: self-directedness (SD), cooperativeness (CO), and self-transcendence (ST) Two temperament traits have been repeatedly associated with suicidal behavior: higher NS To date, no study has specifically compared remitted depression patients who have attempted suicide to patients with and without suicidal ideation in the context of Cloninger's temperament and character model as measured by the TCI. The aim of the present study was to compare the personality traits, indexed by TCI scores, of Korean patients with remitted major depressive disorder across three groups: patients with a history of suicide attempts (suicide-attempt group); patients with current suicidal ideation but without a history of suicide attempts ; and patients with neither current suicidal ideation nor a history of suicide attempts .The present study used data from the ongoing Clinical Research Center for Depression study (CRESCEND), whose design has been detailed elsewhere Between January 2006 and August 2008, CRESCEND enrolled 1,183 patients with depressive disorders  from 18 South Korean hospitals . Enrolment occurred in a naturalistic clinical environment from both outpatient and inpatient settings irrespective of depression subtype or physical co-morbidities. The study was approved by all of the relevant university and/or hospital institutional review boards and was conducted according to the Declaration of Helsinki and good clinical practices. All participants reviewed the consent form, and written informed consent was obtained by research staff prior to their participation.All patients with depressive disorders reviewed at the study hospitals were approached regarding participation. The clinicians assessing and diagnosing the patients applied the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria The CRESCEND research protocol recommended assessments at baseline, 1, 2, 4, 8, 12, 24, and 52 weeks and annually thereafter. The analysis described herein was restricted to data obtained at baseline and week 12 because the TCI was only completed at week 12. Socio-demographic and clinical characteristics were evaluated at baseline. Baseline and week 12 scores were used in this analysis.We categorized patients into three groups: patients without current suicidal ideation and without a history of suicide attempts ; patients with current suicidal ideation but without a history of suicide attempts ; and patients with a history of suicide attempts with or without current suicidal ideation (suicide-attempt group). To determine suicidality, we investigated patients' suicide attempt histories, and we administered the Beck Scale for Suicide Ideation (SSI-B) p<0.1), we excluded variables with low variance and/or significance. Finally, a post hoc analysis was performed to compare the three groups. Statistical significance was set at p<0.05 for all tests. All statistical analyses were conducted using the Statistical Analysis System software package .Descriptive statistics are provided for socio-demographic data and psychometric tests. The chi-square test, Fisher's exact test, and analysis of variance (ANOVA) were conducted to compare the groups across categorical and continuous variables. The three groups' TCI scores were compared via analysis of covariance (ANCOVA), with sociodemographic variables that were significant at baseline and clinical variables significant at week 12 used as covariates. If two or more significant variables exhibited strong correlations , and 23 patients (16.7%) had a history of suicide attempts. Included patients were more likely to be employed and had lower baseline scores on the CGI-S, HAMD, BDI, and SSI-B and higher scores on the SOFAS and WHOQOL compared with the excluded patients (data not shown).Of 1,183 total participants, 253 (21.4%) patients who had attempted suicide and 407 (34.4%) other patients completed follow-up to the week 12 visit. Information from the TCI was available for 325 patients (79.9%); 138 patients (42.5%) met the inclusion and exclusion criteria for the analysis described herein. The majority of the patients were female , age of onset , marital status , and religious status . Among the baseline psychiatric symptom scales, the HAMA , BDI , WHOQOL-BREF , and SSI-B  differed significantly across the three groups.The baseline socio-demographic and clinical characteristics of the non-suicidal, suicidal-ideation, and suicide-attempt groups are summarized in Post hoc analysis indicated that the suicide-attempt group was significantly younger compared with the suicidal-ideation (p = 0.004) and non-suicidal (p = 0.001) groups. Age of onset was higher in the suicidal-ideation group compared with the suicide-attempt group (p = 0.042). Patients in the suicide-attempt group were more likely to be unmarried/divorced/widowed (p = 0.005) and to have no religious affiliation (p = 0.026) compared with the other groups. Compared with the non-suicidal group, the baseline HAMA total score was higher in the suicidal-ideation (p = 0.015) and suicide-attempt (p = 0.019) groups. Baseline total BDI scores were higher in the suicidal-ideation group compared with the non-suicidal group (p = 0.004), and baseline WHOQOL scores were significantly lower in the suicide-attempt group compared with the non-suicidal group (p = 0.005).p = 0.009).At week 12, the total HAMD, BDI, CGI-S, WHOQOL-BREF, and SOFAS scores were not significantly different across groups. However, total HAMA scores were higher in the suicide-attempt group compared with the non-suicidal group , and the SD scores of the suicide-attempt group were significantly lower compared with the non-suicidal group (p = 0.033).In the second phase of the analysis, ANCOVA was performed to compare group TCI scores at week 12, with age, gender, and week 12 HAMA total scores used as covariates. Age of onset, marital status and religion were excluded because they were correlated with age (data not shown), although they were significantly different across groups at baseline . There wT and SD . ST scorThe aim of present study was to evaluate patients who were currently in remission from MDD, with or without suicidal ideation and prior suicide attempts, to identify a distinct personality profile for suicide risk based on the TCI. We compared TCI scores in remitted MDD patients with and without suicidal ideation and in patients who had attempted suicide. The suicide-attempt group had higher ST scores compared with the suicidal-ideation group and had lower SD scores compared with the non-suicidal group. Interestingly, there were no significant group differences in HA, NS, RD, CO, and PE scores among groups.Several previous studies have reported that high HA, NS, and ST and low SD scores were associated with the risk for suicide In the present study, the suicide-attempt group had higher ST scores compared with the suicidal-ideation group and lower SD scores compared with the non-suicidal group. In a study with schizophrenic patients, high ST was associated with previous suicide attempts n = 59) in remitted depressive patients in the present study. However, the widely accepted relationship between impulsivity and suicidality It is unclear why suicidal ideation was observed so frequently (42.8%, The present study had a number of strengths. First, the study used data from the CRESCEND study, which is the largest-scale depression cohort study ever conducted in Asia and includes a wide range of data pertaining to the socio-demographic and clinical characteristics of depressed Korean patients. Second, we excluded depressed patients with psychiatric comorbidities to control for the confounding effects of personality disorders or other Axis I disorders, such as anxiety disorders, eating disorders, and substance misuse-related disorders. Third, we included only patients who were in remission from depressive episodes, and assessed TCI at week 12 to control for the possible effects of depressive symptom severity.The present study also has several limitations. First, we included only Korean patients because cultural issues may lead to variation when assessing dimensions of temperament or character In conclusion, we did not observe differences among patients who attempted suicide and those with and without suicidal ideation in any of the dimensions of the TCI, with the exception of ST and SD. Longitudinal studies that include younger age groups are required to disentangle the personality profile of individuals with high suicidality from underlying psychopathology. The findings of the present study may further the delineation of this complex phenotype.


The use of sulfur mustard (SM) as a chemical weapon for warfare has once again assumed center stage, endangering civilian and the military safety. SM causes rapid local skin vesication and late-onset systemic toxicity. Most studies on SM rely on obtaining tissue and blood for characterizing burn pathogenesis and assessment of systemic pathology, respectively. However the present study focuses on developing a non-invasive method to predict mortality from high dose skin SM exposure. We demonstrate that exposure to SM leads to a dose dependent increase in wound area size on the dorsal surface of mice that is accompanied by a progressive loss in body weight loss, blood cytopenia, bone marrow destruction, and death. Thus our model utilizes local skin destruction and systemic outcome measures as variables to predict mortality in a novel skin-based model of tissue injury. Based on our recent work using vitamin D (25(OH)D) as an intervention to treat toxicity from SM-related compounds, we explored the use of 25 (OH)D in mitigating the toxic effects of SM. Here we show that 25(OH)D offers protection against SM and is the first known demonstration of an intervention that prevents SM-induced mortality. Furthermore, 25 (OH)D represents a safe, novel, and readily translatable potential countermeasure following mass toxic exposure. Sulfur mustard (SM) has been used as a chemical weapon in World War I and during inter-war periods causing life-threatening injury for which there is currently no specific treatment. Indeed the recent past has once again witnessed the horrors of SM re-emerging as an arsenal for combat so the pressure is on, more than ever, to intensify the search for effective intervention. In this study we use SM as the inducer of chemical injury which possesses a range of toxicities with exposure to varying intensities. Low dose cutaneous exposure to SM results in erythema and burning , while hPathogen-free, six week old female C57/BL6J mice were purchased from Jackson Laboratories . Animals were quarantined according to Battelle standard operating procedure (SOP). All animals receive standard laboratory diet. Anesthetic calculations were based on weights obtained during quarantine. An anesthesia cocktail of a 10 ml/kg mixture of ketamine hydrochloride  and xylazine hydrochloride  was administered by intraperitoneal (IP) injection. Hair on the dorsal back of mice was removed by clippers and depilation as previously described . Mice weSM was supplied by U.S. Army Edgewood Chemical Biological Center . The SM purity value was >96 percent as determined by gas chromatography. As part of the dose finding study, SM dissolved in DMSO , was applied at 8 different concentrations  over an area approximately 8 mm in diameter on the dorsal surface of each mouse. To determine a stable LD70 model, doses of 50, 55 and 60 mg/kg were applied on mice. Control mice were applied DMSO only.25(OH)D (Sigma-Aldrich) was reconstituted in ethanol and further diluted in mineral oil Sigma-Aldrich Chemical Company, Inc. , for use. 5 ng of 25(OH)D was injected intraperitoneally using a 27 gauge needle  1 h following SM exposure. Untreated mice receiving SM only were injected with mineral oil.Wounds were measured with a digital caliper daily for 5 days following SM exposure. All mice in the study were weighed daily till day 5 or till drop in body weight met euthanasia criteria.A drop of blood was collected from SM exposed and control mice and transferred onto a glass slide by tail snip, smeared using a microscope slide (Fisher Scientific), fixed in 100% methanol (Fisher Scientific), and stained with Wright-Giemsa to observe cell types and enumerate red blood cells per high power field (HPF).Blood was collected from mice undergoing sacrifice on day 5 and analyzed for complete blood count using a hematology analyzer at Battelle R&D facility Sternums and skin of euthanized mice were removed and fixed overnight in 10% formalin diluted in PBS (Fisher Scientific). Sternums were embedded in paraffin, sectioned (8 µm thickness), stained with H&E and observed under the microscope to evaluate SM-induced cellular depletion.Freshly harvested skin and sternum designated for immunofluorescence studies were OCT-embedded and analyzed as previously described . PrimaryTibiae and femurs of mice were isolated to harvest bone marrow cells as described before . Viable A 2 sided unpaired t-test was used to compare mean values. The Kaplan-Meier method was used to plot the survival distributions of all groups (SM and SM + 25(OH)D) which were then compared using the log-rank test. A Cox regression analysis was used to assess mortality hazard as a function of lesion size and body weight loss. Data are presented as mean ± s.e.m, and p values ≤0.05 were considered significant.2 circular area (8 mm diameter). The challenge dose range (4.8–116 mg/kg SM) was selected in order to reduce confidence limits around LD90 and obtain a statistically significant probit slope. Skin lesions were evident in all SM challenged groups with representative images shown in We developed a SM skin exposure mouse model in which dorsally shaved mice received 8 different concentrations of SM topically applied (in DMSO) onto a 50 mm2  and 68 mg/kg  (data not shown). Interestingly, we observe no mortality with exposure to SM at doses ≤40 mg/kg, although doses ≥ 45 mg/kg appeared to be a critical threshold for death resulting in a stable LD70 at 50 mg/kg D in rescuing mice from NM-induced pathology (70 model in mice that is attenuated by 25(OH)D.The data presented defines 50 mg/kg SM in DMSO as the minimum dose that elicits a sufficient local (skin lesion) and systemic  response to generate an LDathology , 25(OH)Dathology . These dOur findings suggest that skin exposure to SM precipitates in an acute phenotype in mice characterized by skin ulceration, body weight loss and death, all of which progress in a dose dependent manner. We demonstrate that the early effects following challenge with SM include skin destruction that penetrates deep into the dermis through massive infiltration of activated macrophages producing elevated levels of iNOS. In most experimental animal models studied cutaneous wounds from SM burn progress with an inflammatory burst which critically influences the kinetics of wound healing . While mVitamin D is widely available and has been shown to be beneficial in a NM skin vesication model and thus could have a broad public health impact in the event of chemical weapon exposure. A prospective study on patients with leg ulcers are associated with acute vitamin D deficiency that upon treatment with vitamin D shows a trend towards accelerated healing  suggestiIn conclusion this study establishes that SM exposure to skin sustains skin vesication including formation of inflammatory foci at the site of exposure. Local skin destruction is accompanied by progressive weight loss that contributes to mortality. Our results provide the first evidence that early intervention with 25(OH)D offers protection from SM induced injury. Utilizing progressive increase in wound area and loss in body weight as the two key variables, our model presents a stable platform for advancing research to test the efficacy of potential countermeasures like 25(OH)D that would offer treatment options to exposed victims.