Advances in compound-specific stable isotope analysis of amino acids�
Yoshito Chikaraishi and Yuko Takizawa

Applications to invisible food webs

Trophic adequacy & habitability(1-1) Ant’s fungus garden(2-1) Trophic shift in Hawaiian petrel

We revealed that this symbiosis is a discrete four level food chain,
wherein bacteria function as the apex carnivores, animals and fungi aremeso-consumers, and the sole herbivores are fungi.

We revealed millennial-scale trophic shift of AAs in bone collagen in a wideranging oceanic seabird, the Hawaiian petrel (Pterodroma sandwichensis), a
phenomenon potentially related to the conflict within industrial fishing.

Perspectives on the isotope analysis(3-1) Energetic vs. functional position in food webs
Isotopic discrimination in N mirrors “How much metabolic energy is produced” inorganisms, implying that δ-values tell us “energetic” hierarchy among organisms
in food web, but “energetic” does not always equal to “functional”.	
Functional TP pyramid
TP = 3.x

Top predators	
Energetical TP pyramid
4

TP = 4Multiple carnivores
Carnivores

TP = 33

TP = 22TP = 2.x
Omnivores

TP = 2.9 ± 0.2

Ants28.31±0.90

25

4.51±1.24

30

0

23.80±1.06

51020

25

Primary Producers

Takizawa et al., ECE, 2017

TP = 1.0 ± 0.130

23.32±1.13

4.50±1.43

0

510

20

25

TP = 4.1 ± 0.1(300-400y BP)

A large ε (e.g., 16‰)

F (residual Glu)

Bacteria

Fungi

Modern (N=8)

TP = 4.0 ± 0.2

26.32±1.12

TP = 2.0 ± 0.1

20.79±1.09

5.52±1.08

25
25

30
30

0

δ15NGlu (‰)510

20

25TP = 3.8 ± 0.2(Modern)28

F=0.53(2-2) Isoscape in a terraced field
We can illustrate spatial�(and temporal) gradient in δ15NPhe of environments,
which is useful for identifying the habitat preference among organisms.

df2nDry(N poor)

1st2F

r
loo

df

3F
floorrloo

Goto et al., in prep

Deamination preferentially eliminates 14N as ammonia, leaving behind theenriched 15N in the residual pool of amino acids.
Decarboxylation preferentially eliminates 12C as CO2, leaving behind the enriched
13C on intermediates (e.g., pyruvic acid and α-ketoglutaric acid), which can bepropagated into amino acids via re-biosynthesis (or metabolic routing).

1F

Wet(N rich)

【Δδ15N-Δδ13C of Sea slug feeds on Sponge】

δ15NPhe of orange leaves

3

30.98 ± 0.09

21

δ15N (‰)10

100-10

#1

#2

#3

#4

#1

#2

#3

#1

#2

#3

#4

#1

#2

#3Venus flytrap
Tropical pitcher plant(Dionaea muscipula) (Nepenthes alata)

-10

32
120

100Pitcher plant
Flypaper trap
(Sarracenia purpurea) (Drosera anglica)

3.21 ± 0.09

2120

#1

#2#3-10#1#2

TPGlu/Phe = ~3.0

ε− NH3
3PG+ NH3

PEPε− NH3Crickets @ 2F

Pyruvate

TPGlu/Phe = ~2.8
L. doenitzi(3.4 ± 1.8 ‰)CO2

ε+ NH3

Ala(Val, Leu)

εCO2ε

TCAcycle

H. Axyridis type-a C. septempunctata(3.7 ± 1.4‰)(6.2 ± 1.6 ‰)H. Axyridis type-b(5.9 ± 2.4 ‰)M. sexmaculatus(4.9 ± 3.1‰)
H. axyridis type-c(5.0 ± 2.7 ‰)10− NH388Ala6Ile44LeuRe-synthesis(incorporation of 13C into AAs)

2AlaIleLeuPheGly0PheT P G+ NH3Deamination(elimination of 14N from AAs)

Val

6Val
Gly-2Glu (Ile)

Glu

Glu0

TPGlu/Phe = ~3.1

#3Re-synthesis(metabolic routing)

12102

Acetyl-CoA

T. emma(5.9 ± 1.4 ‰)V. Micado(6.1 ± 2.4 ‰)1412Gly+ NH3

Ladybugs @ 2F
Caterpillars
on S. purpurea

14Glucose

F. japonica

0Venus flytrap
without insect

Nest 1F(12.2 ± 0.8‰)Δδ15N (‰)

2.28 ± 0.04

Frequency

1.91 ± 0.30

Nest 2F(6.5 ± 0.9‰)(Phe)

Frequency

2.00 ± 0.19

Nest 3F(4.5 ± 0.8‰)

Deamination

2.27 ± 0.09

TPGlu/Phe(2) Without insect (3) Their consumersFrequency

Ants

2047

Energy(3-2) Metabolic flux via CSIA N & C

δ15NPhe (‰)

3r

453

Biomass

Ostrom et al., 2017, Proceedings of the Royal Society B

We revealed that Insectivores are really carnivores. Like animals, they
assimilate and digest diets (i.e., insect-derived amino acids) that is frequently
accounts to >50% of proteins in the plant biomass.

472A small ε (e.g., 4‰)

F=0.08F=0.28ε (= Δδ15N, ‰)(1-2) Insectivores

4928A standard ε (e.g., 8‰)δ15NGlu − δ15NPhe (‰)

δ15NPhe (‰)

Steffan et al., 2015, PNAS

(1) Four wild insectivores

1Rayleigh model for the enzymatic deamination of Glu	

27.91±1.59

25

TP = 1

TP = 4.2 ± 0.1(2000-2500y BP)

Late Expansion (N=8)

Plant leaves

Herbivores

Pre-human (N=5)

-2-10123G
G: Glycolysis
D: Decarboxylationof pyruvate
T: TCA cycle

456456GlyVal

PT

Leu

AlaIleGluPhe-10123Δδ13C (‰)

Unpublished data

δ15NPhe (‰)

Unpublished data

Unpublished data