Anisoptera

The presented sleep scoring routine enables fast and easy semi-automated definitions of the vigilance/sleep states wakefulness (WAKE), non-REM sleep (NREMS) and REM sleep (REMS), especially for beginners in this field. The underlying algorithms applied in the present program are based on the vigilance/sleep state classification algorithm introduced by Louis et al. for rats, modified for mice by Fenzl and co-workers [6] (link), [3] (link).
The program was written in LabVIEW 8.5 (National Instruments, Austin, TX, USA). Starting the sleep scoring software a graphical user interface GUI will be opened and the user can choose between three executable programs: A manual ARTIFACT DETECTION routine, the SLEEP SCORING routine for semi-automated sleep scoring and a RESCORING routine to enable manual re-evaluation of distinct automatically scored EEG/EMG sequences.
In total, 14 mice were used for this study. All mice where kept in their home cages (26 cm × 26 cm with wood chips and nesting material) during the whole experiment under constant light/dark cycles (12/12 h; 220 lx in the light period) and constant temperature (24 °C ± 1 °C) within noise reduced recording chambers. Water and food was supplied ad libitum. All mice were allowed to recover from surgery for 14 days. Within that period all mice were also adapted to the laboratory light/dark cycles. After recovery, EEG and EMG recordings started 23 h a day for at least 3 consecutive days. The one hour gap (at the end of the dark period) after 23 h of recording and the beginning of the next 23 h recording was used for animal maintenance, if needed.
The animals were surgically prepared under isoflurane anesthesia (Univentor 410 anesthesia unit, agnthós, Sweden) within a stereotactic frame (Angle Two Leica Biosystems, USA). After surgical tolerance was reached, the animals’ eyes were covered with eye ointment to prevent desiccating and each animal received Meloxicam (0.5 mg/kg) to reduce postoperative pain. The analgesic was additionally given into the water bottle (0.5 mg/kg) for 8 days following surgery. EEG and EMG electrodes were composed of gold wire with ball shaped endings of approximately 100 μm diameter and were surgically implanted in all animals. The electrodes were soldered on socked boards (Typ 861-87-008, preci-dip, Switzerland) that were used to connect the recording cable [7] , [4] (link). The recording cable consisted of a headstage amplifier (1x amp., npi electronics, Germany), connected to a commutator (SL-10, slip-ring commutator, Dragonfly, USA). The commutator was balanced on a swivel equipped with a counter weight (custom made, Streicher, Austria). This design facilitated weight-neutral, free movements of the animals in all three dimensions within the home cage. Signals of two EEG electrodes, a ground electrode and a differential electrode together with two EMG signals were fed into individual amplifiers (type DPA-2FL, npi electronics, Germany). The amplification factor of the EEG and EMG recording was set to 1000. Before analog to digital conversion, EEG signals and EMG signals were band pass filtered to the 0.1–100 Hz range. Digital sampling rate was 250 Hz (Power 1401-3 AD-board and SPIKE2, Cambridge Electronic Design, UK). The EMG signals were additionally band pass-filtered online (40–90 Hz). All signals were stored for offline analysis.
All experiments were conducted under the guidelines of national and international animal welfare protocols and were approved by the Bundesministerium für Wissenschaft, Forschung und Wirtschaft, Austria (BMWF-66.008/0011-II/3b/2013 and BMWFW-66.008/0011-WF/V/3b/2014).

The Swift Normalase Amplicon Panels (SNAP) kit (PN: SN-5X296 (core) COVG1V2-96 (amplicon primers), Integrated DNA Technologies) was used on RNA from wastewater samples that were positive for SARS-CoV-2 RNA to prepare the multiplex next-generation sequencing (NGS) amplicon libraries and indexed using the SN91384 series of dual indexing oligos, yielding up to 1,536 index pairs per pool. A miniaturized version of the protocol was used with the following modifications: the Superscript IV VILO (Thermo Fisher) cDNA synthesis reaction was scaled down to approximately one-twelfth the normal reaction volume with 0.333 µl of enzyme mix and 1.333 µl of RNA being used. The multiplex amplicon amplification and Ampure XP bead purification steps were scaled down approximately one-sixth the normal reaction volume. The index adapter PCR and Ampure XP bead purification steps were scaled down to approximately two-thirteenths the normal reaction volume. The final library resuspension volume was 29 µl. Of each library, 1 µl was pooled for an initial shallow NGS run on a MiSeq (Illumina) using a Nano flow cell. This equal volume pool was used to estimate the differential volumes required for similar read depths across samples using a NovaSeq SP or S4 flow cell (Illumina). Between 5 µl and 0.2 µl of library material, depending on the data provided from the MiSeq Nano run, was pipetted into a single pool for the NovaSeq run. Transfer volumes were capped at 5 µl to reduce pipetting time and because these types of ‘high-volume’ samples typically contained a higher proportion of likely adapter dimers that inhibit flow cell performance for all samples. A Dragonfly Discovery (SPT Labtech) was used to dispense reaction master mixes or water depending on the step. A BlueWasher (BlueCatBio) was used for high-throughput centrifugal 384-well plate washing during the AmpureXP bead reaction cleanup steps. An IKA MS3 Control linear plate mixer (IKA Works Inc.) set to 2,600 r.p.m. for 5 min was used to resuspend the AmpureXP beads during the rehydration steps. A Mosquito Genomics HV 16 channel robotic liquid handler (SPT Labtech) was used to dispense the RNA, the reaction master mixes and prepare the equal volume pools for the initial MiSeq Nano (Illumina) balancing runs. A Mosquito X1 single-channel ‘hit picker’ robotic liquid handler (SPT Labtech) was used for the final library balancing for the NovaSeq (Illumina) NGS lanes.
Sequencing data were analysed using the C-VIEW (COVID-19 Viral Epidemiology Workflow) platform for initial quality control and SARS-CoV-2 lineage assignment and phylogenetics. In brief, sequencing reads were aligned with minimap2 (ref. ^{30 (link)}), and primer sequence trimming and quality filtering were applied using the iVar trim method^{20 (link)}. Sequencing depth and SNV calls were obtained using samtools mpileup^{31 (link)} and the iVar variants method^{20 (link)}.
Controls were included at all stages of sample processing (viral concentration, extraction, qPCR and sequencing) to assess potential inhibition and cross-contamination. Most of the sample processing steps were performed by liquid handling robots for consistency and to minimize human error. Replicates were included for all wastewater samples. If any of the controls failed or indicated cross-contamination, the entire batch was rerun. The clinical samples and wastewater samples were processed separately for sequencing due to significant differences in viral load between the two sample types.

Two experiments were performed (Fig 1). In experiment I, the influence of biomass on sequence abundance and the reproducibility of the method were tested using 31 stonefly specimens of the same species (Dinocras cephalotes), i.e., standardizing for a single species. In experiment II, species detection rates were tested using the standard barcoding primers LCO1490 and HCO2198 [41 (link)] and controlling for tissue biomass.
Ethics statement: No protected species and areas were sampled for this study with the exception of the dragonfly larvae Cordulegaster sampled from the Deilbach (N51.3282, E7.1619). Here, special permissions were obtained beforehand from the Kreisverwaltung Ennepe-Ruhr and Mettmann. No further permissions were required for sampling all other non-protected species from the Felderbach (N51.3450, E7.1703) and Ruhr University Bochum pond (N51.4457, E7.2656).

Electroretinograms and fundal imaging was performed as described in Findlay et al., 2018 (link). PCM1-SNAP retinal labeling was carried out under inhaled anesthesia. 1.5 μl of 0.6 μM SNAP-Cell 647-SiR (New England Biolabs) was injected into the mouse vitreous under direct visualization using a Zeiss operating microscope. After 2 hr, mice were sacrificed by cervical dislocation and eyes enucleated. Keratectomy, sclerectomy and lensectomy were performed and whole retinas isolated. Flat mount petaloid retinal explants were made and mounted, photoreceptor side up, on Menzel_Glaser Superfrost Plus Gold slides (Thermo Fisher Scientific; K5800AMNZ72). Nuclei were stained with DAPI and mounted in Prolong Gold under coverslip. Slices were imaged on an Andor Dragonfly spinning disc confocal.

Brightfield images in Figure 2 and Figure 2—figure supplement 1 were imaged on a Hamumatsu Nanozoomer XR with ×20 and ×40 objectives. Macroscope images in Figure 1 and Figure 2 were imaged on a Nikon AZ100 Macroscope. Figure 1—figure supplement 3 was imaged on Leica Stellaris DMI8 equiped with 4 (HyD X/HyD S) GaSP detectors with ×40 or ×60 oil objectives. Fluorescent images in Figure 2, Figure 3A, Figure 3—figure supplement 1A, B, Figure 3—figure supplement 2A, Figure 3—figure supplement 3, and Figure 7—figure supplement 1 were taken on a Nikon A1+Confocal with Oil 60 or ×100 objectives with 405, Argon 561 and 640 lasers and GaSP detectors. Fluorescent images in Figure 1, Figure 2—figure supplement 1D, Figure 4D, E, K, Figure 4—figure supplement 1, and Figure 6—figure supplement 1 were taken with Andor Dragonfly and Mosaic Spinning Disc confocal. Images in Figure 3B, O, Figure 3—figure supplement 1C, H, I, Figure 3—figure supplement 2C–E, and Figure 6E, F were taken with Nikon SORA with 405 nm 120 mW, 488 nm 200 mW, and 561 nm 150 mW lasers, ×100 1.35 NA Si Apochromat objective and a Photometrics Prime 95B 11 mm pixel camera. High-speed video microscopy was performed on a Nikon Ti microscope with a ×60 Nikon Plan Apo VC ×60/1.20 water immersion objective, and Prime BSI, A19B204007 camera, imaged at 250 fps. 3D-SIM imaging in Figure 4B, C, H, Figure 5, Figure 5—figure supplement 1, Figure 6A, B, Figure 7, and Figure 8 was performed using the GE Healthcare DeltaVision OMX-SR microscope equipped with the ×60/1.42 NA oil-immersion objective and three cMOS cameras. Immersion oil with refractive index of 1.518 was used for most experiments, and z stacks of 5–6 µm were collected every 0.125 µm. Images were reconstructed using GE Healthcare SoftWorx 6.5.2 using default parameters. Images for quantifications were collected at the widefield setting using the same microscope. Figure 8—figure supplement 1 was imaged using a DeltaVision Elite high-resolution imaging system equipped with a sCMOS 2048x2048 pixel camera (GE Healthcare). Z-stacks (0.2 μm step) were collected using a ×60 1.42 NA plan apochromat oil-immersion objective (Olympus) and deconvolved using softWoRx (v6.0, GE Healthcare).