TITLE:  Taxon group: Non-larval Arthropods (TSS1)
AUTHORS: Lyndall Pereira da Conceicoa, Olga Sivell, Laura Sivess, Chris Fletcher, Gavin R. Broad, Liam Crowley, Inez Januszczak

[DESCRIPTION]
This is part of the collection "DToL Taxon-specific Standard Operating Procedure (SOP) for the Terrestrial and Freshwater Arthropods Working Group". The SOP collection contains guidance on how to process the various terrestrial and freshwater arthropod taxa 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 outlines the dissected tissues required for whole genome sequencing (WGS), 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.

TSS1 = Taxon Specific SOP for Non-larval Arthropods

Definition: Adult terrestrial and freshwater arthropods and immature life stages of hemimetabolous taxa; over 5 mm body length.

Including: Coleoptera, Hymenoptera, Diptera, Lepidoptera, other insect orders (Blattodea, Dermaptera, Ephemeroptera, Hemiptera, Mecoptera, Megaloptera, Neuroptera, Odonata, Orthoptera, Plecoptera, Raphidioptera, Trichoptera), Amphipoda, Isopoda, Diplopoda, Chilopoda, Araneae, Opiliones, Scorpiones. 

Excluding: Specimens smaller than 5mm
          
Pictorial dissection guides available. 

See the Guidelines for important details and checklist.

[GUIDELINES]

 

Including: Arachnidae (especially Acari, Pseudoscorpiones), Amphipoda (under 10mm), Collembola, Diplura, Protura, Symphyla, Pauropoda and terrestrial insects (incl. terrestrial and aquatic life stages of freshwater taxa) smaller than 5mm: Archaeognatha, Phthiraptera, Psocoptera, Siphonaptera, Strepsiptera, Thysanoptera, Zygentoma, also small species from remaining orders, e.g. Blattodea (‘Isoptera’), Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mecoptera (Boreidae), Hemiptera.


Field sampling:

1. Environment to be sampled: Terrestrial and freshwater.

2. Trap/method of sampling: Bulk capture and single specimen targeted (up to five specimens of the same species collected if possible).If aquatic, sorted in a tray of water with specimens still alive.
 

For genome sequencing:

Tissue preparation:

3. Specimens must must be sampled and frozen while still alive.
 

Photography:

4. Photography appropriate for taxon as detailed here:

  

 

Dissection: 

5. Ensure all tube barcodes are linked with the original specimen.

5.1 Specimen must be larger than a lentil (~5 mm).

5.2 Leg(s) are to be dissected for DNA barcoding; the number will depend on size of the specimen, number of legs present and volume of tissue.

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

5.3 Dissection for whole genome: 

One dissected section per tube is required with a unique barcode/ relevant unique identifier (e.g. QR code, NHMUK number) associated with the tube number.

Please use the following recommendations for taxon-specific dissection:

Insecta: In separate tubes: head, thorax, abdomen if they take up 5mm chunks; depending on body part size can be fragmented further. For specimens under 5mm see section for TSS3. 

Amphipoda: Remove pleopods for DNA barcoding and whole genome. The rest of the whole body would act as a voucher. This is recommended for specimens over 10mm. For specimens under 10mm, please refer to section for TSS3. 

Araneae: In separate tubes: cephalothorax, abdomen, legs (if any left after taking samples for barcoding); depending on size to be further fragmented. 

Pedipalps of male specimens and abdomen of female specimens should be preserved in ethanol and retained as vouchers. 

Opiliones: In separate tubes: cephalothorax, abdomen, legs (if any left after taking samples for barcoding). 

Scorpiones: In separate tubes: prosoma (cephalothorax), mesosoma, metasoma, pedipalps (pincers); can be fragmented further if needed.

Diplopoda:Mid section of the body (trunk) should be dissected into approximately 5 mm chunks.
Head and seven anterior body segments, also last leg-bearing segment and terminal segments including telson should be preserved in ethanol and retained as vouchers.

Chilopoda: Mid body should be dissected into 5mm chunks.
Head and two anterior leg-bearing segments, also two to three last leg-bearing segments including last pair of legs should be preserved in ethanol and retained as vouchers.

Isopoda: Body dissected into 5 mm chunks (anterior & posterior).
Pleon and hind legs should be preserved in ethanol and retained as vouchers. 

Immature life stages of hemimetabolous taxa: To be dissected as their adult stage. Pictorial dissection guides TSS1 available below for more specific dissection information.

5.4 Up to 10 sections per specimen to be dissected for genome sequencing (into separate FluidX tubes); could be more depending on arthropod size. Should be dissected into 5 mm chunks.


 


Storage of frozen tissue:

6. If prepared tube with frozen dissected tissue passes the DNA barcoding stage, it is to be sent to Wellcome Sanger Institute. 

Please refer to DNA barcoding SOP v2.1.

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


Storage of voucher:

8. Vouchers to be sent to/kept at NHM, to act as a voucher. 

9. Vouchered tissue preserved in 70-90% ethanol. 

Pictorial dissection guides TSS1 available below for more taxon specific vouchering information.

Voucher methods

For certain families genitalia may need to be dissected out (Diplopoda, Chilopoda, Araneae, Calyptrate flies) or dissected/slide mounted (various insect groups) to allow for positive identification. 

The dissected genitalia or slides should be retained as a voucher, in a vial with 70-90% ethanol. Genitalia are removed by cutting the end of the abdomen off the freezing specimen (ideally before it is completely frozen and then liable to ping off).

Wings in cellophane envelope: Lepidoptera, Mecoptera, Odonata, Raphidioptera

For some species of specialist interest, eg. in Lepidoptera, the abdomen can also be stored as a voucher for diagnostic purposes, alongside the winds.


SOPs checked by experts. 




TSS1: Pictorial dissection guides 
  
  
  
  
  
  

  

 

   
  

  

  

  

  

  

  

  

  

  

  

  

  

  


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