2.3. Protocol 1. Complete information of live-imaged specimens with preserved voucher exuviae
2.3.1 Culturing and morphological data. Plankton-collected y-larvae were sorted into petri dishes and reared further in mini-cultures of up to five morphologically distinct types or forms as outlined in Olesen et al., (2022). These were then followed alive daily in culture by video under a light microscope (LM). Live specimens were transferred between petri dishes and the light microscope in a glass Pasteur pipette. The final naupliar exuviae of the developing specimens were mounted in glycerin jelly on glass slides (GL; Grygier et al., 2019), or (although only attempted for a few specimens) on SEM-stubs for high-resolution morphological investigations (Grygier et al., 2019).
2.3.2 DNA extraction. Specimens were extracted using both the GeneReleaser and DNeasy kits. For GeneReleaser, we follow a slightly modified protocol from that outlined in Schizas et al., (1997). 1 µL of 10xPCR buffer and 9 µL ddH2O were mixed in a 250 µL Eppenorph tube, to which a single y-larva was added. Protein denaturization was initiated by incubating the tubes at 94-95°C for 2 mins, and the specimens were subsequently transferred to ice. We then added 1 µL protein kinase K (QIAGEN, Chatsworth, CA, USA) and vortexed the samples thoroughly before incubating them at 55°C for 15 min and 70°C for 10 min. Subsequently, we added 10 µL of GeneReleaser (BioVentures, Inc, Murfreesboro TN, USA) and finally incubated the samples under the following thermal program: 65°C for 15 sec, 8°C for 15 sec, 65°C for 45 sec, 97°C for 90 sec, 8°C for 30 sec, 65°C for 90 sec, 97°C for 30 sec, 65°C for 30 sec, 80°C for 3 min, and lastly 4°C for 10 min to halt the reaction. The tubes were then centrifuged for 1 min, after which 15 µL supernatant and 10 µL AE-buffer (QIAGEN, Chatsworth, CA, USA) were transferred to fresh Eppendorph tubes. To locate and retrieve voucher “exuviae”, the samples were spun down and inspected under a dissection microscope. Any exuviae were recovered and mounted as described below.
For the simplified DNeasy method, we transferred individual y-larval specimens from their vials into small, individual petri dishes. After gently vortexing the dish by hand, the specimens were often easy locatable at the center of the dish, after which they were transferred to a 250 µL Eppendorf tube in a 0.5-1.0 µL droplet of ethanol by using a 2.5 µL pipette fitted with a sterile filter tip. The vial(s), each containing a single y-larva specimen, were then incubated for 2 min at 36°C to evaporate the remaining ethanol. 40 µL AE buffer and 4uL Protease K (Qiagen, DNeasy kit) were subsequently added to each vial, which were then incubated for 1h at 56°C followed by enzyme inactivation at 72°C for 12 min. The vials were then spun down and placed in a rack. The “exuviae”, now located at the bottom of their vial, were removed in a 1µL droplet by using a 2.5µL pipette fitted with a filter tip and either placed directly in pre-heated glycerin jelly on a glass slide or in a droplet in a petri dish with a small note written on the lid above the droplet indicating the specimen’s ID code for later mounting. We usually performed PCR of “legacy” markers (12S, 16S, 18S, and 28S rDNA, COX1, and H3) before mounting the exuviae to avoid potential contamination prior to PCR. Using the pipette tip or an eyelash taped to a stick, y-naupliar exuviae (those of the last y-nauplius or an earlier stage) were rotated so the dorsal side faced upward, and y-cyprids were placed on their lateral side. We found intact exuviae in vials that had been stored at -18°C for up to 3 years, suggesting that continued freezing does not impact the quality of prospective vouchers.
We also tested the non-destructive protocol provided by Cornils (2015), which uses Protease K and ATL-buffer instead of AE buffer for DNA extraction. Their protocol consistently worked for a range of copepod species, but for y-larvae yielded faint bands, fragmented exuviae, and noisy chromatograms (i.e., low quality DNA template amplicons).
2.3.3 PCR. PCR was done as described in the Methods and Materials section. The vials were sequenced with their PCR-identifier, complete specimen-designation, and gene listed sequentially (e.g., 1-JA-2019-001-Hansenocaris-demodex_COX1, 2-JA-2019-100-Hansenocaris-demodex_COX1, etc).