TITLE: Global Malaise Trap Project and LIFEPLAN Malaise sampling
AUTHORS: Jayme E Sones, Gaia Giedre Banelyte, Jeremy R. deWaard, Arielle M Farrell, Hanna M.K. Rogers, Bess Hardwick, Deirdre Kerdraon, Kate HJ Perez

[DESCRIPTION]
Lifeplan is a global biodiversity monitoring project with the aim of assessing the current state of biodiversity worldwide, and using this knowledge to generate predictions of how biodiversity might look in the future. 

In this protocol we describe the materials and method used to sample flying insects with a Malaise trap for the Global Malaise Trap Project and the LIFEPLAN project on a global scale and in a wide variety of environmental conditions and habitats. The aim is to identify species in further analysis (e.g. image recognition, DNA sequencing) and create species lists for different locations across the globe. This protocol contains a detailed description from setup of the Malaise traps in the field to weekly data collection, as well as steps to reduce ethanol in the samples and collect necessary information to help in species identification.

We identify the equipment used in Lifeplan, but also give technical specifications of that equipment so that other users of this protocol can find equivalent alternative equipment. We also specify what metadata should be collected with the Malaise trap data. The technical solution we use to collect metadata in the Lifeplan project is described in detail in the full Lifeplan protocol.

It is critical that we employ standardized operating procedures for the Malaise trapping. Our coordinated efforts will ensure specimen preservation for sequence analysis and high data quality, permitting the comparison of sites at a global scale. For global standardization with the BIOSCAN initiative, of which LIFEPLAN is a part. LIFEPLAN is based on bulk processing (metabarcoding) of samples and automatic image recognition which are outside of the scope of this protocol and will be described elsewhere.

[BEFORE_START]
Ensure that all proper specimen permissions are obtained (i.e. from local authorities, property owners, etc.). Consider possibilities of wildlife disturbance and/or human vandalism; the trap may be relocated if consistent issues persist after deployment.

[GUIDELINES]
This protocol is used by the BIOSCAN initiative for the Global Malaise Program and the LIFEPLAN project

The aim of this protocol is to provide a standardised Malaise trap sampling method to collect invertebrates using a Malaise trap across the world and include the same metadata for all samples.

This protocol will produce samples of weekly invertebrate catches conserved in pure ethanol linked to the following information:
Unique sample ID
GPS coordinate of the Malaise trap
Country in which the sample is collected
Name of the person that collected the sample
Date at which the sample was placed
Date at which the sample was collected

 For the LIFEPLAN project, the metadata is automatically collected with the use of the LIFEPLAN app. A QR code linked to a unique 6 character code for each sample is scanned when the sample is placed, then scanned again when the sample is collected. For the Lifeplan project, the Malaise trap is placed close to the centre point or middle of the LIFEPLAN sampling site.


[STEPS]
SECTION: Malaise trap sampling
4. Label your bottle clearly with the sample location, placement date and other relevant information. Be aware that ethanol erases most markers; to avoid that you can use a pencil.

Note: For the LIFEPLAN project, this information is collected by scanning a QR code in the LIFEPLAN App.
SECTION: Placement and deployment of the Malaise trap
1. The trap design depends on insects moving towards the highest and lightest part of the trap. 
When mounting, make sure that the collecting part of the trap (i.e., the trap head) is directed towards the equator (north in the Southern hemisphere and south in the Northern hemisphere). 
Also, check that the collecting part is the highest part of the trap, and avoid placing the trap on a downhill slope with the collecting part lowest. When possible, position the trap in areas with low undergrowth; forest edges or clearings and elevated sites are recommended.


SECTION: Placement and deployment of the Malaise trap
2. At your field site, assemble the trap securely, according to the Malaise trap instruction sheet. When possible, tie the front and/or back ropes to nearby trees for added support. If your plot does not have trees or there is a risk of high winds, attach the trap poles to a 1.5 to 2 meter stake or post at its highest points to protect the trap against falling over from high winds.
   

2. 
SECTION: Malaise trap sampling
5. Fill approximately 2/3 of a sampling bottle with 95% ethanol in the laboratory, close the lid tightly.
SECTION: Malaise trap sampling
6. Tightly affix the prepared collection bottle to the trap head; cover the bottle with foil to prevent the
UV light from damaging the DNA, tie the white ropes on the trap around the bottle to secure it.
Do not cover the top bottle with foil, light needs to shine into the trap for it to work.

  

SECTION: Malaise trap sampling
7. To collect a sample, untie the bottle and remove the foil. Note the sample code and collection date. You can also write on a blank unlined paper using a pencil and put that paper inside the bottle. Close the bottle tightly with the lid. If possible, store the collected sample in a cooling container in the field.
 


SECTION: Malaise trap sampling
8. Before installing a new bottle, wipe the trap head with a clean paper wipe while wearing gloves to avoid dead insects remaining between weeks.
SECTION: Malaise trap sampling
9. Repeat from step 4 to place the next sampling bottle.
SECTION: Decanting ethanol in the laboratory for transferring samples to smaller containers or shipping
11. Put on single use gloves. Clean the bench surface with sterilizing liquid and sterile wipes, then wipe once again with 70% ethanol and leave to dry.
SECTION: Decanting ethanol in the laboratory for transferring samples to smaller containers or shipping
12. Clean the nitex filter and the connecting ring with ethanol. Use one filter per Malaise trap or site.
SECTION: Decanting ethanol in the laboratory for transferring samples to smaller containers or shipping
13. Place a nitex filter on the bottle, leave a gap for air at the top. Secure with a connecting ring or a rubber band.

  

SECTION: Decanting ethanol in the laboratory for transferring samples to smaller containers or shipping
14. Decant off most of the ethanol from each bottle into the container for waste ethanol.

  

SECTION: Malaise trap sampling
10. Once in the lab, ensure that entire insect mass is fully submerged in ethanol before storage; add fresh ethanol to the sample bottle as needed. Store in a -20 °C freezer as soon as possible.
SECTION: Malaise trap sampling
3. Sampling frequency:
Collect the sample once a week during the insect activity period (i.e., during weeks with an average temperature above freezing; occasional frosts are not an issue). Remove the sample on the same day each week. 
SECTION: Decanting ethanol in the laboratory for transferring samples to smaller containers or shipping
15. Carefully remove the connecting ring. Turn the nitex filter around and wash the sample contents using a squeeze bottle containing 95% ethanol back into the sample bottle. Make sure that the amount of ethanol in the sample does not exceed the desired amount.
SECTION: Decanting ethanol in the laboratory for transferring samples to smaller containers or shipping
16. Immediately store the sample in a -20°C freezer until ready for analysis or shipment.