TITLE: 11. Taxon Group: Polychaeta
AUTHORS: Patrick Adkins, Chris Fletcher, Inez Januszczak

[DESCRIPTION]
This is part of the collection "DToL Taxon-specific Standard Operating Procedures (SOPs) for Marine Metazoa", lead by the Other Metazoa Working Group. The SOP collection contains guidance on how to process the various marine Metazoa species within the scope of the Darwin Tree of Life project. The guidance specifically refers to the tissue samples needed for DNA barcoding (which takes place at the Natural History Museum (NHM) and at the Marine Biological Association (MBA)) and outlines the dissected tissues required for whole genome sequencing, which takes place at the Wellcome Sanger Institute. Every specimen is submitted for DNA barcoding first before potentially being sent to the Wellcome Sanger Institute.

Definition: Polychaetes are a paraphyletic class of generally marine annelid worms. Each body segment has a pair of protrusions called parapodia that bear many bristles made of chitin (called chaetae). More than 10,000 species are described in this class.

Including: All species/specimens larger than 5mm.

Excluding: Specimens smaller than 5mm.

See the Guidelines for important details and checklists.

Acknowledgements
Thank you to Emma Sherlock and Lenka Nealova at the Natural History Museum for reviewing this SOP. 

[GUIDELINES]
Field sampling:

1. Environment to be sampled: Marine and brackish.

2. Trap/method of sampling: Specimens caught for genome sequencing will be live caught; single target or bulk capture (up to 10 specimens of the same species collected if possible).

This may be done on shore by hand/net; sub tidally by divers/snorkelers by hand/net or from a vessel using appropriate capture methods e.g. dredge/trawl/grab.

Light traps may also be appropriate for some taxa.

Bulk habitat/substrate may also be collected where appropriate and then tray sorted.

Often, species can be identified live when anaesthetised (use appropriate relaxants for specific taxa). Live specimens can also be put temporarily in the fridge to help slow them down during identification.

 

For genome sequencing:

3. Specimens can be sampled and frozen whilst still alive. Some taxa can be very difficult to dissect when frozen. Fast and accurate dissection on ice and flash freezing of tissue as soon as possible may be most appropriate for some species. 

Photography:

4. Photograph the whole worm dorsally and ventrally. Close ups should include the head and other taxonomically important features for example the parapodia, elytra and operculum. Every species will have its own identifying features so these should be captured appropriately 

For species in the subclass Sedentaria photograph the tube they are dwelling in if appropriate.

5. The image should be taken in the highest quality resolution - a macro lens is recommended.  The photos should be of high enough resolution to be diagnostic, when possible.  

Photograph to include a unique identifier (e.g. QR code, specimen barcode) where possible; when no voucher specimen parts are retained the photograph will serve as voucher and should include identifying features. 

Dissection for DNA barcoding:

6. The mid body without the gut should be used for barcoding. Rinse in clean water and dab on clean tissue to remove gut contaminants and any excess mucus.

Once the tissue for barcoding is removed, that tissue should be placed in 100% ethanol. The rest of the frozen/live organism can then be dissected.  

Dissection for whole genome sequencing:

7. The mid body without the gut can also be used for whole genome sequencing. Rinse in clean water and dab on clean tissue to remove gut contaminants and any excess mucus.

 

Dissect tissue up to ten, lentil-sized pieces in separate tubes if possible.

Tissue should be frozen at at least -80°, for example in dry ice, a liquid nitrogen charged dry shipper or in a -80° freezer.

Storage of frozen tissue:

8. If barcoded tissue passes the DNA barcoding stage, subsequent frozen tissue of specimen to be sent to Wellcome Sanger Institute. 

 

9. Leftover tissue from specimens must be sent to NHM for vouchering and long term storage.

Storage of voucher:

10. Vouchers to be sent to and kept at NHM.

11. Vouchered tissue to be preserved in 80% ethanol.

 

Photo guide below:

  


[STEPS]