Eos 60d

To test the effect of feeding treatments on oocyte metrics, gonads were dissected from the nine females and ovaries were rinsed in 0.2-μm filtered seawater (FSW) to remove loose oocytes. Ovary lobes were treated in 10⁻⁵ M 1-methyladenine to induce ovulation. Released oocytes were transferred into containers with filtered seawater and wet mounted on glass slides for microscopic examination. Oocytes were photographed with a camera (Canon EOS 60D) mounted on a microscope (Leica DM300) with a calibrated ocular micrometer. Ayukai et al. [54 ] developed a criterion to estimate oocyte quality in A. planci based on morphometric characteristics. Oocytes that were relatively large (> 0.15 mm in diameter), spherical (round), and uniform in size and shape often achieved successful fertilization, embryogenesis, and gastrulation [54 ]. Diameters (d_oocyte) of the long and short axes of 100 randomly selected mature oocytes (have undergone germinal vesicle breakdown) from each treatment were measured using Image J [53 (link)]. Oocyte volume (v_oocyte) was calculated using the formula for an oblate spheroid: 4/3 × π × (long axis radius)² × short axis radius. Oocyte sphericity is the ratio of the long and short axis diameter measurements.

To measure the length of the third and fourth legs, legs of adult spiders were cut off at the proximal end of the trochanter and placed flat on a coverslip. A photo of the leg was taken with a Canon EOS 60D digital camera attached to a Leica MZ16 stereomicroscope. The leg length was determined by measuring the length of each individual segment. The length of the claw tuft was measured in side view as the distance between the tips of the most proximal and the most distal setae.
Images of the pretarsi of five P. lanigera and nine S. pubescens were recorded using scanning electron microscopy (SEM). Spiders were anaesthetized, and their third and fourth legs mounted on SEM stubs, and freeze-dried at  – 20 °C over silica crystals for at least 2 weeks. Before imaging, spiders were transferred into a desiccator to warm up to room temperature and then sputter-coated with gold for 3 min (40 mA current) using an Emitech K550X sputter coater. Images were recorded with a Zeiss EVO LS10 SEM at 15 kV and with a FEI XL 30-FEG SEM at 10 kV.

The primary localities for the Afon Gam Biota are stream sections at Ceunant-y-garreg-ddu (national grid reference for base of section: SH82103600) and Amnodd Bwll (GR: SH80703683). Additional exceptionally preserved fossils have been recovered from Cefn Glas Quarry (GR: SH80643767) and an overgrown exposure at Amnodd Wen (GR: SH81653747). Searches for similar preservation were conducted without success in the Dol-cyn-Afon Formation at Afon Gam Quarry (GR: SH751422) and Aran Fawddwy (including Pared yr Ychain, centred at GR: SH844227, and outcrops around SH840223), and in the contemporaneous Shineton Shales of Shropshire (including Coundmoor Brook, GR: SJ55530150; Maddock’s Hill Quarry, SJ64500885 and exposures at Nipstone Rock, SO35679648). The material was photographed using a Nikon D80 with Sigma 105 mm macro lens and extension tubes, and photomicrographs were taken using a Canon Eos60D attached to a Leica M125 stereomicroscope. Specimens are deposited in the Nanjing Institute of Geology and Palaeontology (NIGP), the Natural History Museum, London (NHM) and the National Museum of Wales, Cardiff (NMW).