M165c stereomicroscope

Zebrafish (Danio rerio) strains used in this study were albino lines. Husbandry, breeding and microinjection of approx. 50 CFU of bacteria into the yolk sac of 2 dpf embryos was performed following the procedure described in the study by Fehr et al. (2015)28 .
A set of uninjected embryos, incubated in E3 maintenance medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) was included in order to determine the quality of the embryos; embryos injected with DPBS served as controls. Injected embryos were transferred into 24-well plates (1 embryo per well) in 1 ml E3 medium per well, incubated at 28 °C and observed for signs of disease and survival under a Leica M165 C stereomicroscope twice a day. In order to follow the course of infection embryos or larvae were collected at several time points, namely at 0, 24, 48 and 72 h post infection (hpi) and individually treated for bacterial enumeration.
Research was conducted with approval (NO 216/2012) from the Veterinary Office, Public Health Department, Canton of Zurich (Switzerland). The applied methods were carried out following the approved guidelines.

Fungal isolates were cultured on four media types; PDA, oatmeal agar (OA), malt extract agar (MEA) (Boerema et al. 2004 (link); Chen et al. 2015a (link)), and carnation leaf agar (CLA). The colonies were measured at 7 d, and morphology examined after 12–14 d incubation in the same light and temperature conditions described above. Images of the colonies were captured by an Epson Perfection V700 scanner at a 300 dpi resolution. Colony colour was determined on surface and reverse using the colour charts of Rayner (1970) . Isolates were characterised microscopically from the PDA plates. Lactic acid (100 % v/v) was used as the mounting fluid. Specimens were examined using a Leica DM5500B compound microscope with a Leica DFC 500 camera fitted to capture images under Nomarski differential interference contrast illumination. Micromorphological measurements and descriptions of pycnidia, pycnidial wall cells and conidia were taken from up to 20 samples, and septation and colour recorded. Images of pycnidia were taken from CLA plates using a Leica M165C stereo microscope and Lecia DFC 500 camera. The NaOH spot test on MEA culture plates helped distinguish taxa (Boerema et al. 2004 (link)).

All focal individuals, males and females, were assayed for body size on day 3 of adulthood, the first day of peak reproduction. Pictures were taken individually on plates in a microscope with a mounted camera (Leica M165C stereomicroscope, Lumenera Infinity 2‐5C digital microscope camera). We also photographed females on day 6 of adulthood, because previous studies have found that female body size after peak reproduction (days 3 and 4 of adulthood) may change, by continued growth or shrinkage, and this could potentially be different between the ancestral population and the treatment populations (Lind et al., 2016; Shi & Murphy, 2014). The microscope pictures were analysed using Fiji (ImageJ http://imagej.nih.gov/ij/) plugin called Wormsizer (Moore et al., 2013), where total body volume (in cubic picometre, pm³) was used as a body size measurement.

Selected parapodia of the holotype (XMU-Pol-2021-105), paratype 10 (XMU-Pol-2021-209) and paratype 12 (XMU-Pol-2021-214) were dissected with iridectomy scissors and permanently mounted on slides for observation of their gross morphology and chaetae, as well as for photography. To observe the minute teeth inside the pharynx, the anterior region of the paratype 6 (XMU-Pol-2021-204), paratype 11 (XMU-Pol-2021-213), and paratype 13 (XMU-Pol-2021-215) were hyalinized with graded series of glycerol (30%, 60%, 100%), mounted on slides and compressed slowly with a cover glass. Photographs of the whole specimen and parapodia (with chaetae) were taken using a camera DMC5400 mounted on a Leica M165C stereomicroscope. Photographs were taken at different focuses and stacked into fully focused images using Helicon Focus v. 7 as described by Wang et al. (2018) . The anterior region of the paratype 5 (XMU-Pol-2021-203) and paratype 12 (XMU-Pol-2021-214) were treated by critical point drying and fixed on a conductive adhesive for gold plating. Photographs of the anterior end were taken using Phenom ProX scanning electron microscope (SEM). The classification of the type of parapodia following Jarvis (2011) : biramous with many emergent notochaetae; sub-biramous with few emergent notochaetae; sesquiramous with acicula inside cirrophore and without emergent chaetae.

Three-day-old larvae were utilized for the tail fin amputation experiments. In each experiment, three different treatment groups (20 larvae, unless otherwise indicated) were used: a vehicle (DMSO) treatment group (negative control), a group treated with beclomethasone (25 μM, positive control) [32 (link)], and a group treated with Rg1 (120 μM). All groups were pretreated with vehicle/beclomethasone/Rg1 for 2 h before tail fin amputations, and received the same treatment for 4 h after the amputation. Next, larvae were anesthetized in egg water containing 0.02% buffered amino benzoic acid ethyl ester (tricaine; Sigma-Aldrich, St. Louis, MO, USA). Larvae were placed on petri dishes coated with 2% agarose under a Leica M165C stereomicroscope, and the tail fins were partly amputated using a 1 mm sapphire blade (World Precision Instruments). For quantification of leukocyte migration, larvae were fixed overnight in 4% paraformaldehyde (PFA) at 4 °C. For gene expression analysis by qPCR, an additional nonamputated, vehicle-treated group was used, and all samples contained 15 larvae, which were collected in TRIzol reagent (Invitrogen, Waltham, MA, USA) and stored at −20 °C. For all experiments, three independent replicates were performed.

Three-day-old zebrafish larvae were utilized for the tail fin amputation experiments. In each experiment, 20 larvae were used in each treatment group. The administration included a vehicle (Veh) group treated with 0.01% DMSO as the negative control, a group treated with Beclo of 25 μM as the positive control, and groups subjected to the following treatments: RPN powder at 50 μg/ml; RPN extract (RPNE) at 50 μg/ml; SPN powder at 50 μg/ml; SPN extract (SPNE) at 30 μg/ml; and three concentrations (at 30, 60, and 90 μM) each for Rh₁, Rk₃, Rh₄, 20(R)-Rg₃, and 20(S)-Rg₃. In a pilot experiment, no toxicity on the survival of zebrafish larvae was observed for any group (data not shown). All groups were pretreated with Veh/Beclo/experimental treatment for 2 h before tail fin amputation, and received the same treatment for 4 h after the amputation. Next, larvae were anesthetized in egg water containing 0.02% buffered amino benzoic acid ethyl ester (Sigma-Aldrich Chemie N.V., Zwijndrecht, Netherlands). Larvae were placed on petri dishes coated with 2% agarose under a Leica M165C stereomicroscope, and the tail fins were partly amputated using a 1 mm sapphire blade. For quantification of leukocyte migration, larvae were fixed overnight in 4% paraformaldehyde at 4°C.

External morphology (wing pattern) has been examined in 72 dried, pinned and mostly set specimens (62 out of 72 specimens were DNA-barcoded) (S1 Table). In addition, we studied male genitalia of 56 moths (46 of these moths were DNA-barcoded) (S1 Table). Morphometric analysis was carried out on genital preparations of 33 males from the presumable invaded area (Bulgaria, Finland, Hungary, Italy, Poland, Western Russia and Siberia) and of 21 males from the presumable native region (Russian Far East, Japan, South Korea). Genitalia dissections and slide mounts followed by the standard technique [50 ]. Length of the four genital parameters: phallus, left valva, and spines of left and right basal processes (hereinafter–left spine and right spines) was measured on a Leica M 165C stereomicroscope and expressed in μm (Fig 2). Male genitalia of P. issikii is asymmetric, with a big right valva (Fig 2), which was impossible to measure precisely due to its deformation when arranging slides with genital structures. Thus it was not included into analysis.