Methods

1) Collection of material

New material will be collected primarily using a WP3 net (mesh size ≥300 μm) with a non-filtering cod-end and low (0.3 ms-1) towing speed, or other nets adapted for collection of gelatinous zooplankton. The large opening, slow towing speed and non-filtering cod-end combined increase the chances for the collecting pelagic ctenophores in good condition for further processing.

Modified WP3 plankton net. Photo: Aino Hosia.

In addition, underwater photography will be used by scuba divers and ROVs to be able to combine taxonomic scrutiny together with photographic vouchers of fresh, live specimens linked to molecular IDs for accurate species identification and to develop a modern interactive version of the traditional dichotomous taxonomic identification key.

2) Sorting and identification

Samples collected will be processed immediately after collection. Unfortunately, ctenophores fixed in ethanol can no longer be used as morphological vouchers due to shrinkage and distortion caused by the fixative. The aim is therefore to carefully document the general appearance and morphological details of live specimens prior to fixation for DNA barcoding. This documentation will be done photographically using small aquaria with side lighting and an SLR camera and/or a stereomicroscope mounted camera, as well as by drawing. Specimens will subsequently be selected for both sequencing (fixed in absolute ethanol; see section “Molecular species identification”) and as voucher specimens to be included in BOLD with the barcode and in the museum collections (Photographic ID).

Sorting gelatinous zooplankton from live sample, with a help of light table. Photos: Sanna Majaneva & Aino Hosia

3) Molecular species identification

DNA isolation

The first step of identification of taxa is the extraction of DNA. For ctenophores, most suitable method for DNA extraction has been following the modified Chelex rapid boiling protocol according to Granhag et al. 2012.

DNA amplification and sequencing

The next step is to amplify the targeted gene sequences of the samples by polymerase chain reaction technique (PCR) that allows unlimited amplification of the targeted gene region. The 18S rDNA is known to be highly conserved among ctenophore species, with a maximum divergence between two species of 87 bp, i.e. less than 5 % but provides currently the most sufficient reference library. The more variable ITS region has often been suggested for use in detailed species identification whereas the common barcode CO1 mtDNA reference library is limited down to few species. Thus, the GooseAlien project will use combination of all three gene regions.

Ctenophore 18S rDNA sequences. Photo: Sanna Majaneva

4) Curating:

A reference collection of Norwegian ctenophores based on new samples will be established at Tromsø University Museum. Due to the fragility of ctenophores, traditional reference collection with voucher specimens preserved either in formalin or ethanol is not possible with ctenophores; the reference collection will therefore be established with photographic IDs.

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