TITLE: Golden monkey fecal sampling protocol
AUTHORS: Amanda Johnston

[DESCRIPTION]
This project aims to use data collected from fecal samples to asses the effects of habitat loss on two populations of endangered monkey. The protocol outlines the steps for collecting and processing fecal samples from groups of habituated and un-habituated monkey groups suitable for multiple habitat types. The sub-samples processed from the feces will be used for host (monkey) DNA extraction, helminth DNA extraction, and morphological parasite ID using mini-FLOTAC and sedimentation. 

[BEFORE_START]
Before going into the Field:
Check the field materials list. Make sure to have at least 15 pairs of gloves and 15 tubes filled with 4 ml RNAlater, enough to be able to collect samples from an entire group. Before going into the field, use labeling tape to label the  caps of the tubes filled with RNAlater with 1 through 15. Then, weigh the mass of each each tube, including the RNAlater and the cap. Record the mass  for each vial in a notebook, using a new page each day. Each page should contain only the date, the list of numbers 1-15, and the corresponding mass for each numbered vial. It is ok to re-use vials between days, just be sure that no numbers are repeated within a day. 

Boots and hands need to be sanitized with alcohol before entering the forest and a mask must be worn at all times in the forest except when you are eating or drinking.

Each sample you collect will be assigned a unique ID code which you will record on the data sheet and on each collection container before filling them with fecal matter. These instructions therefore begin with how to fill out the data sheet and assign the ID code, and then explain how to collect the samples. 

[GUIDELINES]
Overview:
This protocol describes procedures for collecting and processing 30-50 (target: 48) fecal samples from golden monkeys in each of two populations, VNP and GMNP. Samples will be collected opportunistically during golden monkey group follows when team members observe defecation by a monkey from whom no samples have yet been collected.

In VNP, only one sample will be taken per identifiable individual (8-15 per group).

In GMNP we also aim to collect one sample per unique individual (4-12 per group)  but because these monkeys are not individually recognized we will use a combination of age-sex class and any uniquely identifying characteristics such as scars or bent digits to distinguish individuals. 

[STEPS]
SECTION: Field sample collection and sub-sample preparation
4. After locating the fecal bolus, use a wooden spatula to remove leaves, sticks, vegetative debris, and soil as much as possible from the outside of the bolus.
 

SECTION: Field sample collection and sub-sample preparation
7. Fill in the field data sheet, recording the time, GPS location, and age/sex class of the individual, and in VNP the identity of the individual if possible. Also note any identifying characteristics of the monkey (ex. bent digits or tail, bald spots) and irregularities of the fecal sample such as  presence of blood, mucus, or worms. Any information that does not fit on the field data sheet can be added to the note data sheet.

Age/sex class abbreviations:
AM = adult male
AF = adult female
SM = subadult male
J = juvenile
SECTION: Field sample collection and sub-sample preparation
8. Seal the remaining feces in the glove for transport to the field station and use a sharpie marker to label the glove with the ID code.





 

SECTION: Station lab sub-sample preparation
13. Tare the 8 ml collection tube labeled with OH on the scale and use a flame sterilized spatula to measure 1 g of feces into it, removing or adding fecal matter as necessary to reach 1 g  within ± 0.1 g. Record the final mass of the sub-sample on the lab data sheet under Mass OH. Then, fill the collection tube with 96% ethanol so that the tube is filled. Note any major sample loss during  measurement or sample abnormalities in the notes section of the lab data sheet.
 

SECTION: Station lab sub-sample preparation
14. In a similar way, measure two 3 g  sub-samples  into the appropriately labeled collection tubes (F1 and F2) and record each sample mass on the lab data sheet under Mass F1 and Mass F2 respectively. Fill each tube with 10% formalin so that there is no air.
 

SECTION: Station lab sub-sample preparation
14.1. If you cannot measure out 2 full 3 g  samples, measure one full 3 g  sample into the tube labeled F1. Then, measure the remaining feces into the tube labeled F2 and record the mass (which will be less than 3 g) under Mass F2 on the lab data sheet. 


SECTION: Data recording
2. Below is the heading of the field data sheet. Site refers to the specific park, VNP or GMNP, or pine forest outside GMNP.  Group ID refers to the identity of the group if known and should include all groups observed that day, separated by a comma. If the group does not have a name (GMNP) note the approximate location of the group's home range (ex. in GMNP: core forest south-eastern edge) 

Make note of any abnormalities in collected feces such as presence of blood, mucus, or worms. Unique marks used to identify individuals (ex. bent digits or tail, bald spots) should also be recorded. If you need more space, you can record additional notes on the notes sheet. Be sure that you match the sample ID across sheets.  

 

SECTION: Sample/data storage
15. Seal sample tube caps with a strip of parafilm (as shown below) before storing. Store samples in sample boxes labeled with the project name (Johnston GM fecal samples), location, and reagent code (D, OH, F1, F2). Boxes should be kept at Room temperature  and in a dark location. Make sure that all samples are stored upright so that any air pockets are at the top of the tube, minimizing the likelihood fecal matter will be exposed to air.

  

SECTION: Station lab sub-sample preparation
12. Use labeling tape to label 3 collection tubes for parasite sub-samples with the ID code followed by the reagent code. For samples in formalin, add a 1 or 2 after the reagent code (F) for the first and second 3 g sub-samples. 

Reagent codes:
OH = 96% ethanol
F = 10% formalin
 

 

SECTION: Field sample collection and sub-sample preparation
6. Use labeling tape to label the sub-sample tube with the ID code followed by a D for DNA. Also record the tube number (from when you measured the mass of the sample tubes with RNAlater) on the field data sheet.
 

SECTION: Data recording
3. The unique ID code consists of three parts with a hyphen separating each:
 The first letter of the site
 The date (month and day) written as 2 digits and a 3 letter month abbreviation (ex. 8 May would be 08-MAY)
 The sample number where 1 is the first sample collected that day and each following sample is assigned the next consecutive number (2,3,4, and so on)

Site and team codes:
V = VNP
G = GMNP
P = Pine Forest
 

For each team, sample numbers increase consecutively on each day, no matter how many groups you follow. They reset to 1 only on a new day.
SECTION: Sample/data storage
16. After a set of data sheets are complete (all information about all samples listed is filled out), take a picture or scan them. Once a week, email all new digital copies to project leads. Physical data sheets should be stored together in chronological order in a dry location and duplicated by photocopying when complete. 

SECTION: Data recording
1. You should use one set of data sheets (field, lab, and note) per group. If you fill up a data sheet set, start a new set for that group if necessary and mark 1/2 on the first set and 2/2 on the new datasheets. Make all notations in pen. If you make a mistake, cross it out it and then begin again on the next line. Do not write over a previous incorrect entry to try and fix it. Relevant abbreviations are defined in later steps. When preparing samples, double check that the sample's ID code on your containers matches the ID code across all data sheets.
SECTION: Field sample collection and sub-sample preparation
5. Look for the pointy end of the fecal bolus (this is the last part of the feces to leave the monkey). From the outside layer of the pointy end, use a wooden spatula (tongue dispenser) to scrape off  2 mL of feces (this is half the volume of the liquid in the tube). Place this fecal matter in 4 mL  of RNAlater in an 8 ml tube.

 
 

SECTION: Station lab sub-sample preparation
10. Weigh the tube containing the DNA fecal sample, and record the DNA-tube w/ sample mass (tube, cap, RNAlater and feces) on the lab data sheet. Then subtract the tube + RNAlater mass from the DNA-tube w/ sample mass.
SECTION: Station lab sub-sample preparation
9. Prepare the lab data sheet (example below) by filling in the ID codes for all samples collected on that day, the dates,  and the tube # and the tube + RNAlater mass that corresponds with each sample. 


 

 

SECTION: Station lab sub-sample preparation
11. Place a sheet of weigh paper on the scale and tare it. Then carefully invert the fecal bolus in the glove onto the weigh paper to measure the total mass of the fecal bolus. Record that number on the lab data sheet under bolus mass.