prey dna detectability half -lives: turning pcr positives...

Prey DNA Detectability Half-Lives: Turning PCR Positives into Quantitative Predation Rates

Bastian Egeter

https://bastianegeter.wordpress.com/https://cibio.up.pt/people/details/egeterbas/projects/505

http://inbio-envmetagen.pt/

Grant agreement 668981

https://bastianegeter.wordpress.com/

Outline

Case study of diet analysis to quantify predation

Why aren’t there more studies like this?

Grant agreement 668981

Pre-human

X 7

Background

Polynesian settlers (500-1300 AD)

Pre-human

Pacific rat

X 7

Background

Polynesian settlers (500-1300 AD)

European settlers (1840)

Pre-human

Pacific ratHedgehog

Ferret Pig

Cat

Possum

Norway rat and black rat

Weasel

Stoat

Mouse

X 4

Background

New Zealand Frogs

All in top 60 EDGE list: Evolutionarily Distinct and Globally EndangeredArchey’s frog Hochstetter’s frog

Maud Island frog Hamilton’s frog

New Zealand Frogs

Frost DR, Grant T, Faivovich J, Bain RH, Haas A, Haddad CFB, De Sa RO, Channing A, Wilkinson M, Donnellan SC, Raxworthy CJ, Campbell JA, Blotto BL, Moler P, DrewesRC, Nussbaum RA, Lynch JD, Green DM and Wheeler WC (2006) The amphibian tree of life. Bull Am Mus Nat Hist 297: 1—291

Essner Jr RL, Suffian DJ, Bishop PJ, Reilly SM 2010 Landing in basal frogs: evidence of saltational patterns in the evolution of anuran locomotion. Naturwissenschaften 97: 935

Watch video at:https://www.eurekalert.org/pub_releases/2010-07/s-pfd072110.php

https://www.eurekalert.org/pub_releases/2010-07/s-pfd072110.php

Case study central question: How many frogs are being eaten by introduced mammals?

Bell BD, Carver S, Mitchell NJ and Pledger S (2004) The recent decline of a New Zealand endemic: how and why did populations of Archey’s frog Leiopelma archeyicrash over 1996–2001? BiolConserv 120: 189—199

• 1080: €13m/yr• €50m/yr pest control

in general• half a million hectares• kills game species• kills some birds• a form of habitat

restoration


Egeter B, Robertson BC and Bishop PJ (2015) A synthesis of direct evidence of predation on amphibians in New Zealand, in the context of global invasion biology. Herpetological Review 46: 512-519

Who is eating frogs?

n = 217 n = 20

Frog carcasses following rat predation

© Tertia Thurley& Dr. Ben Bell, 1994 © Dr. Chris Smal, 2009

© Bastian Egeter, 2010Egeter B, Bishop PJ and Robertson BC (2011) DNA detects frog predation. FrogLog99: 36-37

Frog carcasses following rat predation

Krull C, Egeter B (2016) Feral pig (Sus scrofa) predation of a green and golden bell frog (Litoria aurea). New Zealand Journal of Ecology 40(1): 191-195.


Hedgehog consuming southern bell frog in New ZealandPhoto: Dr. Stephen Chadwick

How many prey individuals are being taken by a particular

predator?

How many frogs are being eaten by introduced mammals?

𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑

= 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒑𝒑𝒐𝒐 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒄𝒄𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒄𝒄 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 × 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑

×𝒑𝒑𝒑𝒑𝒕𝒕𝒑𝒑 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒑𝒑𝒂𝒂𝒑𝒑𝒑𝒑𝒂𝒂𝒑𝒑𝒂𝒂𝒂𝒂𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒄𝒄𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑

𝒑𝒑 = 𝒑𝒑𝒑𝒑 ×𝒑𝒑𝑷𝑷𝑷𝑷𝒑𝒑𝑫𝑫𝑷𝑷

Modified from: Dempster JP (1967) The control of Pieris rapae with DDT. I. The natural mortality of the young stages of Pieris. Journal of Applied Ecology 4(2):485-500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 2 4 6 8 10 12 14 16 18 20

Prob

abili

ty o

f det

ectin

g pr

ey D

NA

Hours since prey ingestion

Half-life

Prey detectability half-lives

n = 10+

What have detection half-lives been used for so far?

• Proposed by Chen et al. 2000 to rank predator importance

Chen Y, Giles KL, Payton ME and Greenstone MH (2000) Identifying key cereal aphid predators by molecular gut analysis. Mol Ecol 9: 1887-1898

aphid (prey) lacewing ladybird

4h8.8h

2.2X more likely to detect aphid DNA from ladybirds

Greenstone MH, Szendrei Z, Payton ME, Rowley DL, Coudron TC and Weber DC (2010) Choosing natural enemies for conservation biological control: use of the prey detectability half-life to rank key predators of Colorado potato beetle. Entomol Exp Appl 136: 97-107


Colorado potato beetle (prey) Ladybird (adults)

Lebia grandis (adults)

spined soldier bug (adults)

Prey occurrence from 11 to 95% spined soldier bug (nymphs)

two-spotted stink bug (adults)

two-spotted stink bug (nymphs)

1

2

3

4

5

6

Greenstone MH, Szendrei Z, Payton ME, Rowley DL, Coudron TC and Weber DC (2010) Choosing natural enemies for conservation biological control: use of the prey detectability half-life to rank key predators of Colorado potato beetle. Entomol Exp Appl 136: 97-107


Colorado potato beetle (prey) Ladybird (adults)

Lebia grandis (adults)

spined soldier bug (adults)

Prey occurrence from 11 to 95% spined soldier bug (nymphs)

two-spotted stink bug (adults)

two-spotted stink bug (nymphs)

1

2

3

4

5

6

1

2

3

4

5

6

Review of 37 studies (2003-2014) none quantified impact of predation on prey

ApproachesLab feeding trials Diet analysis

Trapping of wild mammals

tPA

p

d Searching for frogs

tDP


Feeding trials (tDP)Stage 1: Faeces

Stage 2: Stomachs


Results: Morphological analysis

1 hr 3 hr

3 hr1 hr

Hedgehog Hedgehog

Norway rat Ship rat

Results: Prey DNA amplification

Norway RatStomach contents

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30 35 40

Prob

abili

ty o

f det

ectin

g fr

ogs a

s pre

y

Hours since prey ingestion

ship rat

mouse

Norway rat FF

Norway rat EF

hedgehog

Half-life

0.5h 10.6hGIT

6h* 12-16hGIT

Detection period (tDP)

Egeter B, Bishop PJ and Robertson BC (2015) Detecting frogs as prey in the diets of introduced mammals: a comparison between morphological and DNA-based diet analyses. Molecular Ecology Resources 15: 306—316

single line: stomachsdouble lines: faeces arrows: known GIT transit times

• Using faeces lengthens half-life > 3-fold

• Prey detection rate: 2% to 70% stomachs

• and 0% to 53% in faeces• Ship rats: 0% to 80% stomachs; 7.3h

(95%CIs 4.4-11.29h)

Detection period (tDP)

Egeter B, Bishop PJ and Robertson BC (2015) Detecting frogs as prey in the diets of introduced mammals: a comparison between morphological and DNA-based diet analyses. Molecular Ecology Resources 15: 306—316

Study sites


Nights prey available

1 3 5 7 9 11 13 15 17 19 21 23 250.000.050.100.150.200.250.300.350.400.450.50

1 3 5

Arch

ey’s

frog

enc

ount

er ra

te (f

rogs

-m)

Site 1 Site 2

Day

trapping period

tPA


Num

ber o

f shi

p ra

ts c

augh

t eac

h ni

ght

Cumulative number of ship rats caught

Rough average across sites

6.4 ship rats / ha

d


Predator density

Samples testing positive

5/60 ship rat stomach samples (8.3%)

p𝒑𝒑 = 𝒑𝒑𝒑𝒑 ×

𝒑𝒑𝑷𝑷𝑷𝑷𝒑𝒑𝑫𝑫𝑷𝑷

Detection methodsuccess

Study Area Site Rats Prey Morphological DNA

Whareorino Forest 1 16 Archey´s frog No 1

Whareorino Forest 2 44 Archey´s frog No 4

Positive8%

Negative92%

if we assumed that we were detecting all predation events here then p=r(also assumes constant sampling effort and uniform rate of predation events)

Predation rate of ship rats on frogs

WF Site 1 WF Site 2 WF Site 1&2

WF – WhareorinoForest

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Frog

s/ra

t/ni

ght P (uncorrected)

P (corrected for Tdp)

P (stomach emptying time alone)

Predation rate of ship rats on frogs

WF Site 1 WF Site 2 WF Site 1&2

WF – WhareorinoForest

Corrected predation rate 1.65X (1.06X to 2.72X) greater than uncorrected P

0.00

0.50

1.00

1.50

2.00

2.50

Frog

s/ha

/nig

ht

P (uncorrected)

P (corrected for Tdp)

P (stomach emptying time alone)

% of frog population eaten• 26 – 110 frogs / rat annually• 0.4 – 2 frogs / ha / night• 146 – 730 frogs / ha annually• 2600 – 9600 frogs / ha• 1.5 % to 28.1 % of population

Summary• Survey for frogs as prey using DNA• Measure detection half-lives in mammalian stomachs• Compare detection half-lives in faeces vs stomachs • Estimate the impact of rats on frogs• Estimate predation rate using DNA

Why is this type of study not more common?

Predation

Distribution

Abundance

Spatiotemporal population

trends

Niche use and

resource partitioning

Circadian rhythms

Evolution

Global conservation/ Resource management

Problem species

Impact of introduced faunaPest predator: e.g. preying on pollinators, crop damage →

biomanipulation

High Value Species

Commercially valued prey: e.g. game

Commercially valued predator: e.g. fish

Conservation-valued prey

Conservation-valued predator

Top down effect on environmental microbiome, nutrient resources →

agricultural productivity


• %F versus bulk– PCR vs HTS

Baker R, Buckland A, Sheaves M (2014) Fish gut content analysis: robust measures of diet composition. Fish and Fisheries 15: 170–177

Sample sizes > 100

Ontogenetic dietary models for

Platycephalus fuscus(n = 357)

Baker R, Sheaves M (2005) Redefining the piscivore assemblage of shallow estuarine nursery habitats. Marine Ecology Progress Series 291, 197–213.



• Lots of work• People and their study

species• Not many predation models

Dempster model


Predicted number of prey attacked

Obs

erve

d nu

mbe

r of p

rey

atta

cked

Naranjo SE and Hagler JR (2001) Toward the quantification of predation with predator gut immunoassays: a new approach integrating functional response behavior. Biological Control 20: 175-189

Bayesian models

• Average time since feeding• Reaches same conclusions as other models,

but better measures of uncertainty

Welch KD, Schofield MR, Chapman EG and Harwood JD (2014) Comparing rates of springtail predation by web‐building spiders using Bayesian inference. Molecular ecology 23: 3814-3825



• Lots of work• People and their study

species• Not many predation models• One (or more) prey

One (or more) prey

Carreon‐Martinez LB, Wellband KW, Johnson TB, Ludsin SA and Heath DD (2014) Novel molecular approach demonstrates that turbid river plumes reduce predation mortality on larval fish. Molecular ecology 23: 5366-5377

Microsat variation in stomach contents vs microsat variation in prey population. Minimum number prey necessary to explain the observed number of microsats (n=5)



• Lots of work• People and their study species• Not many predation models• One (or more) prey• Lab diversity (collaboration)• Factors affecting half-lives

• Number of individuals in a sample• Multiple prey approach, HTS• A few good mesocosm experiments• Same questions as metabarcoding• Approximating half-life based on known GIT• Answer questions about biocontrol, biomanipulation,

invasive species, resource management, agri-environment, pollinators, pure ecology

The future

Acknowledgements

SRARNZ

EcogeckoConsultants

Phil Bishop, Bruce Robertson, Cailín Roe, Luke Easton, Kim Garrett, Murray McKenzie, Ken Miller, Nicky McHugh, Tania King, Karen Judge, Deb Wilson, Otago Frog Team, Lisa Daglish, Kate McKenzie, Oliver

Overdyck, Sarah Cosio, Alison Davis, Duncan Emerson, Dave Smith, Tim Darviole, Allison Ernst, Debra Kriger, Jess, Pia Goldsmith, James Barnaville, Lucy Ferris, Rhodri Hardy, Michaela Blomquist, Beth Haines, Rob

Wood, Justyna Miklas, Alan Mackern, Edgrado Moreno, Christy Reynolds, Roc Taull, Kate Morozova, Devin, Stephanie Hicks and Chris Smal

prey dna detectability half -lives: turning pcr positives...

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