Data from Moulting and development in a freshwater prawn from the Late Cretaceous of Morocco

Description

Abstract

Arthropods are the most diverse phylum on Earth, inhabiting virtually all ecosystems and playing important roles in the natural environment as well as in agriculture and human health. Their segmented body plans allow modularity and evolvability, with a spectacularly large variety of life histories. Their exoskeleton imposes periodic moults, but also provides opportunities for specialisation or plasticity. Thus moulting is both a key step in each arthropod’s life history, and a key to understanding arthropod diversity. This project brings together researchers with complementary expertise to address three main goals unified through the theme of arthropod moulting as a key life history trait. The first is to establish broad evolutionary trends across groups of extant and extinct arthropods by building and analysing a comprehensive compendium of arthropod moulting characteristics. The second main goal is to determine how moulting modes observed today evolved from ancient diversity. The comparisons aim to define which aspects –in terms of genes and pathways or networks and the biological processes they control– of moulting are ancestral versus derived, to then relate these to ecological changes and species diversifications and trace the evolutionary paths of moulting. This leads to the final goal focusing specifically on the evolutionary flexibility of moulting and its role in arthropod terrestrialisation events.

This project compiles currently dispersed knowledge from all relevant fields (paleontology to genomics) into an integrated database, MoultDB, covering a large number and variety of species, from which broad trends in the characteristics and evolution of moulting in arthropods can be established. This answers the first goal, and serves as a foundation for the other project goals. The project has generated genomes and moulting transcriptomes in diverse arthropods. Comparative analysis of these data, guided by re-analysis of paleontological and phylogenetic trends, allows us to answer the second goal. Building on outcomes from the first two goals, moulting characteristics (extant and extinct), as well as genetic and molecular processes that direct moulting are identified, for example in terrestrial versus aquatic taxa to learn how moulting plasticity could have facilitated or constrained these transitions.

This research allows us to obtain a much improved understanding of the evolutionary and functional mechanisms which underpin arthropod diversity and adaptation, both in the broad picture and in specific examples. The integration of different data and expertise sheds new light on existing fossils, and allowing for a better interpretation of genomic and functional data. MoultDB provides important resources for the community and future research, and set an example of integrating paleontological, morphological and genomic data together.

A major result of this project is MoultDB, a knowledge-base that integrates paleontological, morphological, and genomic information and data. The back-end, the API and a front-end of MoutlDB have been programmed, and database entries describe moulting characteristics of extant and extinct taxa. A citizen science approach to help populate MoultDB has been employed, based on images of arthropod moulting uploaded and linked to a project on the iNaturalist website. To support these goals, the R package Taxonbridge has been published, and machine learning approaches are being integrated.

Several descriptive projects on arthropod fossils that provide information on moulting and development are results of this project. While the molecular pathways and developmental biology of moulting are well documented in insects (especially holometabolous insects), information is sparse for other arthropods, as outlined in an indepth review produced during this project. Morphological observations of modern arthropod moulting sequences and development make up further results of this project. Genome sequencing, assembly and annotation, as well as gene expression data, for several arthropod taxa also result from this project.

Technical info

Programs used:
Online Supplementary Material 1: Microsoft Excel (Microsoft 365, Version 2505, Build 16.0.18827.20102)
Online Supplementary Material 2: R (version 4.2.1); ggplot2 package (version 3.4.4)
Online Supplementary Material 4: Microsoft Excel (Microsoft 365, Version 2505, Build 16.0.18827.20102)
Online Supplementary Material 5: R (version 4.2.1)

File Formats and Compatible Software:
Online Supplementary Material 1: .xlsx (excel; version used for R-script) and .csv (plain text format that can be opened with free spreadsheet software or any text editor)
Online Supplementary Material 2: .R (R-studio or other text editor)
Online Supplementary Material 3: PNG, JPG and TIFF (any standard photo viewer or image editing software); multispectral TIFF (ImageJ)
Online Supplementary Material 4: .xls (excel; version used for R-script) and .csv (plain text format that can be opened with free spreadsheet software or any text editor)
Online Supplementary Material 5: .R (R-studio or other text editor)

Filenames and organisation in Online Supplementary Material 3:
Each figure in the manuscript has a corresponding folder including the figure and its raw images.

Figure 3: - Files are organised into folders based on the type of imaging used (Multispectral, UV, Visible). Within each of these folders, subfolders are named according to the corresponding figure panel letter and the specimen's accession number.
      - These contain raw PNG or JPG. For visible light (Filename = Accession number_Imaging type). For UV light (Filename = Accession number_Imaging Type_Number Of Views_Camera Setting). 
      - The multispectral contains a PNG of the false-colour RGB overlays and the TIFF file containing the raw grayscale images used for this false-colour RGB overlays (Filename = "R"_RedCombinationxIlluminationWavelengthFFilterNumber-exposuretime_ "G"_GreenCombinationxIlluminationWavelengthFFilterNumber-exposuretime_"B"_BlueCombinationxIlluminationWavelengthFFilterNumber-exposuretime.TIFF)

Figure 5: - These folders are named according to the corresponding figure panel letter and the specimen's accession number.
      - These all contain BSE raw TIFF, the same image is either with the legend included (Filename = Accession number_"BSE"_"legend"), without (Filename = Accession number_"BSE"), or both.

Figure 7 and 8: - These folders are named according to the corresponding figure panel letter and the specimen's accession number; If an image shows a morphological detail captured than this is mentioned in the title (Filename = Panel Letter, accession number, and body part); If the image was taken using an imaging method other than standard visible light (e.g., SEM, Keyence, UV), the technique is indicated in the filename (Filename = Panel Letter, Accession number, body part, and imaging technique when necessary)
          - These are contains raw PNG, JPG or raw TIFF (Filename = Accession number_Imaging type)
      - In BES raw TIFF, the image is either with legend included (Filename = Accession number_"BSE"_"legend"), without (Filename = Accession number_"BSE"), or both. One is a calcium-eds map, which is specified in the filename and folder with ("BSE-EDS calcium map").

Figure production: 
- Adobe Photoshop (22.5.1); Adobe Illustrator (25.4.1); Affinity Designer (2.6.3); Affinity photo (2.5.3); ImageJ (1.53t)

Data collection: 
Online Supplementary 1: The measurements were made on scaled digital images of specimens using Adobe Photoshop 22.5.1. 
Online Supplementary 4: UV−vis−NIR emission spectroscopy was measured on the cuticle of moults and the cuticle and muscles of carcasses, using a Specbos 1211UV spectroradiometer (JETI). UV illumination was provided by a U1c 6W 365 nm UV LED flashlight (JAXMAN), and a long-pass filter (cutoff wavelength: 410 nm) was placed in front of the detector to remove diffuse reflection of the excitation by the sample. Spectra (up to 1000 nm) were collected from ~1 mm2 area pinpointed using the spectroradiometer's internal target spot laser. 

Imaging details:
- UV and Visible: Specimens were photographed under visible and UV light using a Canon camera (EOS-800D) equipped with a macro lens (EFS 60 mm 1:2.8) and the EOS Utility photo software. UV light was applied with a 365 nm UV LED flashlight (JAXMAN U1c, 6W). 
- Keyence: Digital microscopy images were taken with a Keyence microscope (VHX-7000) fitted with a dual objective zoom lens (VH-ZST), using the ZS-20 macro lens (20-200x). 
- Backscattered electron micrographs (BSE): Images of the cuticle macrostructure were captured using a Zeiss Gemini 500 scanning electron microscope (SEM) in variable pressure (environmental) mode on uncoated specimens. The SEM was operated with the backscattered electron detector (BSD), at an electron high tension voltage (EHT) set to 15 kV, an aperture of 60 µm and the chamber pressure maintained at 40 Pa during imaging.
- The BSE-EDS calcium map: was generated using the same settings as described above, with the Oxford AZtec Microanalysis System (Oxford X-max 150 detector, software version 4.2 SP1; Oxford Instruments, High Wycombe, UK)
- Multispectral: Using a broad spectrum of light wavelengths from UV-A to the near-infrared (NIR) to enhance contrast within the fossil remains. Each selected carcass (MHNM-KK-OT 75a, 60a, 78a, UC 102-1) and moult (MNHN.F.A88748, MHNM-KK-OT 82b, 64b, UC 102-2, UC 135-2) was imaged under the same conditions (i.e. same distance from the camera, same LED intensity, same exposure time  for three illumination-emission wavelength combinations : 385 nm / 472±15 nm (luminescence), 660 nm / 719±30 nm (reflectance + luminescence) and 460 nm / 472±15 nm (reflectance), which each produce a grayscale image. For each specimen, the three resulting greyscale images were combined into false-colour RGB overlays (red: illumination 385 nm / detection 472±15 nm, green: 660 nm / 719±30 nm (green) and blue, 460 nm / 472±15 nm)