Animal NitrogenOverview of N cycling

farm animalsa few unfortunate songbirds

road-kill down underNitrogen Isotopes in Mammalian Herbivores: Hair 15N Values

from a Controlled Feeding Study(Sponheimer et al., 2003)

Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an

omnivorous songbird(Pearson et al., 2003)

Kangaroo metabolism does not cause the relationship between bone collagen δ15N and water availability

(Murphy et al., 2006)

N Cycle(human)

amino acid pool•throughout body•significant mixing

protein turnover•Some proteins turnover faster than others•some tagged (oxidized or other means)

•Highconversion to fat/glucoseAmmonia/urea excretion

N Cycle(human)

Dietary protein•Low

•deficiency of essential aa’s

First transfer amine group to carrier

Ketoglutarate → Glutamate

Deamination

Then deaminate Glutamateto produce ammonia

in liver or kidney

First transfer amine group to carrier

Ketoglutarate → Glutamate

Synthesis

Then to amino acid

in liver or kidney

*Direction of these reactions controlled by [ ] ofGlutamateKetoglutarateAmmoniaratio of oxidized to reduced enzymes

Synthesis

Deamination

Urea cycle

Urea cycle controlled by acetyl CoA and glutamateincrease in [ ] after protein rich meal

kidney liver

Nitrogen excretion animals

Ammonia NH3

•Simplest form, but toxic•fully aquatic animals

Urea (NH2)2CO•Still toxic more complex than ammonia•mammals some herps (frogs), cartilaginous fish

Uric Acid C5H4N4O3

•Least toxic•egg layers (bird, reptiles, insects)•precipitates from egg

A few things

1. animals assimilate dietary components with varying efficiencies

2. animal tissues fractionate the isotopes in their diet3. animals allocate nutrients in their diet differentially

to specific tissues ‘isotopic routing’4. animals retain δ15N, excreting δ14N (~6‰)5. protein balance is a key to fractionation

low dietary protein = “protein sparing” reserve dietary protein for tissue maintenance rather than catabolizing it for energy (Castellini and Rea 1992).

high dietary protein = use diet protein for tissue synthesis and catabolize excess

Nitrogen Isotopes in Mammalian Herbivores: Hair 15N Values from a

Controlled Feeding Study(Sponheimer et al., 2003)

Goals:

Determine the importance of

1. hindgut vs foregut fermentors

2. dietary protein levels

on herbivore δ15N values.

Nitrogen uptake herbivores

Hindgut• Horses, rabbits, birds, iguanas, green turtle• Limited cycling of urea nitrogen

fermentation, N cycling, protein balance

ForegutRuminants (can synthesize proteins from inorganic

nitrogen compounds)• multi compartmental stomachs• cows, llamasRuminant-like• kangaroos, wallabies, hoatzin

cycle/mix N from diet and selfdeamination and de novo protein syntheses

Diet-Hair Fractionation

Same diet, fair bit of variationrabbits and alpaca vary 3.6‰, > 1 trophic level!

Foregut fermenters are enriched vs hindgut fermenting rabbitsBut not to horses…

↑ dietary protein (9-19%)causes enrichment δ15N (1.5-2.8‰)

Not what they expected!

This refutes N cycling hypo(states that low protein group ↑δ15N)

feces explanation poorfeces is δ15N enriched (0.5-3.0‰), low protein = less urine loss and greater relative (not absolute) %N loss via feces, ↑δ15N loss

High Protein vs Low Protein Herbivores

Effects of elemental composition on the incorporation of dietary nitrogen and carbon

isotopic signatures in an omnivorous songbird.(Pearson et al., 2003)

Goals•Determine turnover rates of δ15N and

δ13C in whole blood and plasma.

•δ15N and δ13C diet-tissue fractionation factors for plasma, whole blood, and feathers.

•Influence of high protein (%N) and low protein (%C) concentrations on fractionation factors.

yellow-rumped warbler

•32 captive wild-caught migratory birds•‘controlled’ for age & sex

•Acclimation diet 32% insect•Experimental diet

20%,49%,73%, 97% insects•Sampled

•21 days, mass, blood (plasma, wb), feathers (entire)•Determined

•C&N δ values of different diets•turnover rates•Discrimination•Isotopic signatures of diet on different tissues

Materials and methods

Diets: %Insect, Isotopes, & Concentrations

Attempted to created diets along a linear continuum of increasinga) isotopic signature (didn’t quite work for 15N)b) elemental concentration

by increasing the % insect protein in diet

Only 0.12‰ difference in δ15N values among diets.

Diet containing most insects did not have highest δ15N value(diet with lowest proportion of insects did not have the lowest

δ15N value)

Banana Effect (δ15N 0.5 - 5.3‰)

Diets: %Insect, Isotopes, & Concentrations

Turnover Rates

Isotope incorporation kinetics model(O’Brien et. Al 2000)

Δdt = discrimination factorr = fractional turnover rate

Half-life =

Turnover Rates: Half-life Plasma & Blood

Half-life estimates plasma: δ13C 0.4-0.7 days δ15N: 0.5-1.7 days

Half-life whole blood: δ13C ~4-6 days (diet 1=33 days!) δ15N 7.45-27.7 daysWhole blood is variable!

Discrimination: Plasma, Feather, and Blood

15N values plasma & whole blood enriched 1.7 to 3.0‰

“Apparent” fractionation factor for feathers

15N enriched (3.2-3.6‰)

Fractionation factors increased linearly

with elemental concentration in diet

for N

↑ %N

↑ urine w/ ↑ 14N

in

out

↑ tissueδ15N

Importance of Elemental Concentrations

Both isotopic signature of diet and fractionation factors influence the ultimate isotopic signature of tissues

(at least plasma).

Supports the importance of using concentration-dependent mixing models when reconstructing diet.

Results•Discrimination factors depend on diet and tissue

•Fractionation factors to reconstruct diet requires an estimate of elemental concentrations in the diet.

•Turnover ratesPlasma 1 day (short) Whole blood 1 wk (longer)

• Carbon and nitrogen fractionation factors increase linearly with elemental concentration in the diet.

• Relationship between the isotopic signature of the diet and the sum of a given tissue’s (at least plasma) isotopic signature + fractionation factor was also positive & linear.

USE CONCENTRATION-DEPENDENT MIXING MODELS WHEN ATTEMPTING TO ESTIMATE THE RELATIVE

CONTRIBUTION OF DIFFERENT FOOD SOURCES TO AN ANIMAL’S DIET!!!

Kangaroo metabolism does not cause the relationship between bone collagen δ15N

and water availability (Murphy & Bowman, 2006)

Goals

• Evaluate importance of water availability and dietary δ15N in determining δ15N values in herbivore bone collagen

• Indirectly determine if ↑ δ15N linked to animal metabolism

• Assessed if δ15N in grass and Kangaroo bone collagen are constant with respect to a Water Availability Index

• Examine other factors influencing δ15N in herbivore bone collagen

Does ↓ Water availability↑ δ15N in Animal Tissue?

Plants enriched in arid environs• ‘openness’ N cycle theory (Austin & Vitousek 1998)

– ↑ water in system = ↓ in ratio of N loss to intrasystem N turnover

• Cryptobiotic crusts

Why ↑ animal δ15N when in water limited systems?• Metabolic enrichment ‘theories’

– ↑ Urea osmolarity, urine excreted is more nitrogen (δ15N) concentrated (Ambrose & Deniro 1986, Sealy 1987)

• excrete more δ15N deplete urea when arid (Sponheimer 2003)• not experimentally shown for rats (Ambrose 2000)

– not tested rigorously…

BUT… can ↑ δ15N be explained by herbivore diet alone?

Methods

• 173 grass collections (3-4 primary spp/collection)

• 779 road killed roos– macropus sp, grazers…

• Water Availability Index estimated from mean annual actual and potential evapotranspiration

• Akaike’s Information Criterion (AIC)

Big study!

→

+

data

=

+

Results

Found relationship of δ15N and WAI similar between grass and kangaroo bone collagen

4.74‰ to 4.79 ‰ enrichment

~0.05‰ variation over entire range of data

When plotted against annual rainfall Murphy & Bowman’s δ15N relationship fits with

•Previous Kangaroo work

•Eutherian herbivoresNorth America & AfricamatchesSealey et al 1987 follows similar pattern

What about C3 vs C4 grasses?

Model gave little support for other variables:•slope•chenopod

•δ13C of bone collagen as proxy•negative and weak relationship

•Found lower δ15N in C4 plants (1.1‰)•C4 diet (high δ13C, low protein) = lower consumer δ15N

C4C3

C4C3

• Strong negative relationship of herbivore δ15N bone collagen and water availability.

• Near identical negative pattern of δ15N in grass and kangaroo bone collagen with water availability (near constant offset in slopes)

• Suggest dietary δ15N is main cause of negative relationship between δ15N of kangaroo bone collagen, with water availability and metabolic factors having little discernible effect.

Summary

Importance…

• Ties water availability directly to plant δ15N to animal δ15N values, with little ‘animal’ affect

• Huge support for historic trophic ecology and past climate change data that rely on direct relationship between herbivores and plants which not confounded by animal metabolism

Marine food webs are enriched in δ15N

(Kelly 2000)

Trophic Systems

(Hobson & Welch 1992)

Marine systems 3-4‰/trophic levelHerbivores ~3.2‰Carnivores 5‰

• Marine food chains tend to have longer food webs

• Diet affects, as ascend trophic chain, ↑ %N in dietexpect more catabolism = discrimination @ high

trophic level

• Trophic enrichment commonly produces 3:1 slope for δ15N and δ13C ratios

Trophic Systems

• N15 to get at rainfall abundance

Diet-Hair d15N Equilibration

Dietary 15N values changed from 2.5‰ to 7.8‰.Dietary equilibration took ~8-10 weeks

Hair

Kelly

δ15N in top consumers in C3, C4 and Marine food chains

(Kelly 2000)

Discrimination

• Tissue effectsFeathers more enriched than plasma or

wb

Diet Tissue Relationship

• C & N signatures linearly related with tissue signatures + discrimination factors

• Correlated linearly with metabolic rate of tissue

• Different species have different turnover rates for same tissues

Likely related to size, mass specific metabolic rates, life history factorshalf-life for wb C in bear > crow > quail > warbler

Turnover Rates

• Plasma (1-5 days)• Whole Blood (5-35 days) • Feces (• Feathers, Hair, Nails, Hoof (time when

grown, maybe a lag here)• Bone• Teeth

Turnover Rates

Importance of Elemental Concentrations

Phillips & Koch 2002

Implications

CO2 WUE N demandNPP

δ15N in plants

‘openness’ of N cycle

δ15N in herbivores

• Pearson• Funk/questions

- variability in initial mass and mass change following dietary switch among treatment groups (shows they like carbs

- Diets did not have ↑ δ15N values w/↑ % insects

- Fractionation vs. discrimination

CO2 effects on δ15N

(Coltrain et. Al. 2004)