TITLE: CARD COUNT AND VIAL DEPLOYMENT/RETRIEVAL PROTOCOL
AUTHORS: Grace Sandel, Laura Barragan, Erica Garibay, Kiran Bengard, Ellis Gelt, Sage Moloney

[DESCRIPTION]
Procedure to conduct "Crazy Yellow Ant Sampling on Tetiaroa"
This protocol provides instructions for performing card counts and deploying/retrieving vials in order to quantify the prevalence of yellow crazy ants, Anoplolepis gracilipes, in given transects around Onetahi, Tetiaroa using two kinds of ant baits--sugar water and peanut butter. In addition, the experiment tested yellow crazy ant preference for sugar water bait versus the peanut butter bait. 

[BEFORE_START]
- Recommend wearing long sleeves, pants, a sun hat, and closed toed shoes
- Recommend putting on sunscreen and bug spray

[GUIDELINES]
- Do not trespass
- Analyze site safety before conducting a sample
- Avoid trampling flora 
- If a point is inside a building, just go to the next point or as close to the point as possible
- If it starts raining, stop deploying vials and card counts

[STEPS]
SECTION: Card Count Protocol
1. If needed, scrape the ground (at least a 10x10cm area) at the site so the card sits flat on the ground (i.e. if there are branches or leaf litter, scrape them away).
SECTION: Card Count Protocol
3. Place the card down on the ground.
SECTION: Card Count Protocol
4. For 30 seconds, count the number of yellow crazy ants that cross the card.
SECTION: Card Count Protocol
5. After 30 seconds have passed, pick up the card and remove all ants from it. 
SECTION: Card Count Protocol
2. If you scraped the ground, wait 30 seconds before placing the card down. If you did not scrape the ground, place the card down without waiting.
SECTION: Card Count Protocol
6. Enter the number of ants counted in the card count into the appropriate field of the EpiCollect5 form.
SECTION: Vial Protocol
7. Preparation: Divide the atoll into a series of transects labeled A-Z. Then on each transect create points 1-X. The points should create a grid on the island. 
SECTION: Vial Protocol
7.1. Prepare 25 sugar plastic vials by soaking a napkin in sugar water and placing at the bottom of the vial                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

SECTION: Vial Protocol
7.2. Prepare 12 peanut butter (PB) plastic vials by placing 1 TBS of peanut butter at the bottom of each vial, making sure not to get any PB on the sides of the vial.

SECTION: Vial Protocol
8. Deployment: Navigate to the site using Gaia GPS
SECTION: Vial Protocol
8.1. At each site, place one sugar vial. Place a peanut butter vial down directly next to the sugar water vial only if ants are spotted in the nearby surrounding area. 
 

SECTION: Vial Protocol
8.2. Fill out Epicollect form detailing: deployed/retrieved, site number, and time collected (automatic)
SECTION: Vial Protocol
8.3. Repeat steps 3-5 at each site. Go in reverse order in the following section for retrieval. 
SECTION: Vial Protocol
9. Retrieval: After 90 minutes, return to placed vials. 

SECTION: Vial Protocol
9.1. Pick up and quickly cap vials, not letting any ants escape in the process. 

SECTION: Vial Protocol
9.2. Fill out form on EpiCollect5. Count crazy ants on each vial. If there are too many ants, head back to lab. Freeze vials and count ants afterward. 
SECTION: Vial Protocol
10. Cleanup: Clean out test tubes and throw away samples.