eDNA - Midterm Flashcards
Advantages of eDNA (state 5)
- Non-invasive
- More efficient (for aquatics) - highly accurate for species detection
- More cost effective
- Able to detect presence of pathogens and diseases
- Minimizes risk of pathogen transfer between sites
What are the 7 major steps in eDNA analyses in order of their process?
- Survey design
- Sample collection
- Sample filtration
- Filter preservation
- eDNA extraction
- Analysis (qPCR)
- Interpretation
What are 4 major limitations of eDNA?
- Can not accurately quantify species abundance (only presence not detected)
- Contamination can occur and cause a false positive
- Interpretation requires the person to have knowledge of the 3 processes that influence detection
- External sources of DNA can lead to false detection (like equipment)
What are the 3 factors to consider with eDNA?
Production
Transport
Degradation
In terms of PRODUCTION, what must be considered?
- The rate of eDNA production release into the habitat varies across species, their life stage, and individuals
- eDNA methods can not distinguish between different life stages
- eDNA methods can not indicate abundance
In terms of TRANSPORT, what must be considered?
- The rate of transport (spread/diffusion) of eDNA through water systems vary. This rate of transport is unknown for most species
In terms of DEGRADATION, what must be considered?
- The rate of degradation strongly influences the amount of eDNA present in a sample
- Life span of DNA in the environment depends several factors like water temp, pH levels, type of flow, etc.
How many days can eDNA persist in water source (general rule of thumb)?
7 - 21 Days after the removal of the organism from the system
Why must one carefully consider their study objective before using eDNA?
- eDNA cannot determine a link to concentration of eDNA in a habitat to species abundance
When should you survey using eDNA?
- When the timing is right (coincide with breeding season or biological timing windows)
What is an advantage of increasing survey effort?
- yields high confidence in results
What is a disadvantage of increasing survey effort?
- increases project labour and cost
What guides the level of sampling effort needed for both eDNA or traditional sampling methods?
- OBJECTIVES!
What 4 factors influence the number of samples collected at a site?
- Objectives
- Budget
- System (lotic or lentic)
- Target species
What is the recommended number of samples for:
a) well studied high confidence species detection probabilities
b) Unknown detection probabilities
c) New species or practitioner
a) 1 sample sufficient
b) 3 samples recommended
c) 1 negative control sample per processing session is recommended
What is the recommended spacing for samples?
- up to 50 m apart collected at locations where target species is likely to occur
What is the recommended sample volume to be taken?
- 1 L per sample, with 3 samples per site
- Should be consistent within study design for a single study when detection probabilities will be compared
Where should sample be taken?
- Surface water
- Lotic sample collected from surface water in the thalweg
- Multiple samples should be collected from same feature, in up stream manner to avoid contamination
When sampling in multiple time periods, what pattern of sampling produces:
a) strong results
b) stronger results
c) strongest results
a) sampling across seasons within one time year
b) sampling in one season across multiple years
c) sampling across multiple seasons over multiple years
What are the 2 methods for storage of field samples?
- filters can be preserved and stored in molecular grade ethanol in the dark and remain viable for at least 6 months
- samples can be folded inward and placed in coin envelope in a bag with self-indicating silica beads until desiccated
- Once extracted, can be stored indefinitely
When should you NOT collect samples in a lake/stream?
- After or during high rainfall event or high flow events
Provide 4 steps to avoid cross contamination
- Do not leave sample gear unprotected/exposed
- Do not enter water upstream of sample site, or within 3 m in lentic environment
- Ensure sampling occurs before other field studies happen where personnel enter the water
- Adhere to “hygiene protocols for amphibian field staff” to prevent spread of infectious diseases
Describe the process of collecting water samples
- LABEL lid and bottle with time, date, site, staff
- CLEAN gloves, and refrain from touching anything other than bottle
- Fill, shake, rinse bottle and empty away from site
- RINSE x3 total
- FILL sample bottles from surface water (typically 3 bottles, about 10 m apart)
- Put samples in COOLER with ice
- Collect water QUALITY with water quality meter
- DECONTAMINATE clothing
When should filtering occur?
- Within 24 hours of sample collection
What order should samples be filtered?
- same order they were collected
Where should filtering occur?
- workspace dedicated to filtration
How should samples be preserved?
- cellulose membrane is filtered and the membrane is preserved in a vial with molecular grade ETHANOL or a coin-envelope stored in a bag with self indicating silica beads
How should samples be labeled?
- Ethanol resistant marker
- Project ID, site name, collection date, staff
How should filter paper be handled once filtration is complete?
- Using decontaminated forceps
- Never touched by hand!
What are some cautions needed when interpreting positive results?
- May represent true positive or false positive (Type 1 error)
- For larger lentic or lotic water bodies, it is possible to have uneven distribution of target taxa eDNA
What are some factors that may affect the reliability of eDNA results? (7 total)
- eDNA may have been introduced through other means (feces)
- May have been introduced by contamination
- Environmental conditions
- Low densities of target organisms in the habitat
- Large distance from source organism
- Suspended materials in samples causing reduced sensitivity
- Timing of sample collection
