metabolic substrate use of trimerotropis pallidipennis; a stable isotope approach

Metabolic substrate use of Trimerotropis pallidipennis; a stable isotope approach

Alyssa CorbettAugust 9, 2007

Study Species: Trimerotropis pallidipennis

Dorsal View

Sophia Engel

Population Distribution Map

Pfadt 1994

T. pallidipennis

• Commonly referred to as the Pallid-winged grasshopper• Reproduction

– Eggs hatch in spring– 5 instars– Adult stage reached in late spring

• Relatively large grasshopper – Males smaller than females

• Two traverse bands across

body width– Attracts mates– Camoflauge

Pfadt 1994

T. pallidipennis

-Consume a mixture of grasses and forbes

• Annual grasses in early spring• Perennial grasses and green

forbes in late spring• Research is still in progress

-Depends on quality and availability of plants

Food Selection

How do we know?

• Observation

• Stable carbon isotopes– “You are what you eat”– Animals are isotopically coupled to their diets– Stable isotopes allow us to trace carbon

fluxes through food webs

Stable Carbon Isotopes

• Abundance in Nature:

12C 98.89%13C 1.11%

• 12C is a lighter isotope, having one less neutron

Encarta

6P and 6N

6P and 7N

Carbon Isotope Ratio Equation

δ13C = Rsample – Rstandard * 1000 ,

Rstandard

Where R = 12C

13C

• Carbon standard set by marine belemnite sample found in the Pee Dee Formation (VPDB) = zero

• δ13C is negative because more 12C exists

Plant Physiology

• Different functional groups of plants have unique photosynthetic pathways

• C4 plants use an additional carbon molecule during a step of photosynthesis

• Thus, C4 plants have a heavier δ13C value

• C4 plants are well adapted to high daytime temperatures and intense sunlight

δ13C value -12 -14 -16 -18 -20 -22 -24 -26 -28 -30

C3C4

What will stable isotopes tell us?

δ13C = ?Feeding on mixed grasses and forbes Fasting period

δ13C = -23.0 ‰*Uncertain of substrate material *Uncertain of extent of substrate metabolized

Research Questions

Does increased duration of fasting period (after being on a known diet) affect:

• δ13C breath values?

• Body fat percentage?

• C:N ratios and δ13C values of body, muscle, and fat?

Methods

Captured 20 T. pallidipennis for each fasting period treatment:1. Fed2. Fasted 2 hrs3. Fasted 6 hrs4. Fasted 1 day5. Fasted 3 days

Methods•Animals kept in steel mesh cages

•Separated by treatment and date of collection

•Known diet of C3 plant material:

-Organic romaine lettuce -Organic rolled oats

Experimental Design

• Real-time breath test analysis (breath δ13C) All 100 samples

• Fat extractions (% body fat lost) 50 samples

• Isotopic analysis (C:N ratios and δ13C values)– Body– Muscle– Fat

50 samples each

Experimental Design

• Real-time breath test analysis (δ13C)

• Fat extractions (% body fat lost)

• Isotopic analysis (C:N ratios)– Body– Muscle– Fat

Real-time Breath-test Analysis

• Campbell Scientific TGA 100 trace gas analyzer

– Tunable diode laser absorption spectroscopy (TDL)

– Flow through system (100 ml)

– Measures [12CO2] and [13CO2]

– Calculates δ13C in real-time

– Can be used in the field

• Provided by Los Alamos National Laboratory

Real-time Breath-test Analysis

• Campbell Scientific TGA 100 trace gas analyzer

– Tunable diode laser absorption spectroscopy (TDL)

– Flow through system (100 ml)

– Measures [12CO2] and [13CO2]

– Calculates δ13C in real-time

– Can be used in the field

• Provided by Los Alamos National Laboratory

Chamber = 30-60 ml

ResultsBREATH-TEST ANALYSIS

-29

-28

-27

-26

-25

-24

-23

-22

-21

-20

No Treatment Fed 2 hr 6 hr 1 day 3 day

Fasting period

Del

ta 1

3C (p

er m

il)

C4 Plants (-14 to -19 ‰)

C3 Plants (-24 to -30 ‰)

DiscussionBREATH-TEST ANALYSIS

Consumeplant material

Metabolize plant material

Metabolize endogenousfat reserves

Breath δ13C ≈ -25 ‰

Increasin

g tim

e spen

t fastin

g

DiscussionBREATH-TEST ANALYSIS

Consumeplant material

Metabolize plant material

Metabolize endogenousfat reserves

Breath δ13C ≈ -25 ‰

Breath δ13C represents recently digested plants

δ13C ≈ -25 ‰

Increasin

g tim

e spen

t fastin

g

DiscussionBREATH-TEST ANALYSIS

Consumeplant material

Metabolize plant material

Metabolize endogenousfat reserves

Breath δ13C ≈ -25 ‰

Breath δ13C represents recently digested plants

δ13C ≈ -25 ‰

Breath δ13C represents content of endogenous

lipidsδ13C ≈ -27 ‰

Increasin

g tim

e spen

t fastin

g

After 6 hrs

DiscussionBREATH-TEST ANALYSIS

Consumeplant material

Metabolize plant material

Metabolize endogenousfat reserves

Increasin

g tim

e spen

t fastin

g

After 6 hrs

Based on a a study by Michael J. DeNiro in 1977:

“There are differences in the carbon isotopic composition of the major biochemical components of an organism. The lipid fraction has a lower δ13C value than the other major components (protein and carbohydrate fractions) and the total organism.”

Experimental Design

• Real-time breath test analysis (δ13C)

• Fat extractions (% body fat lost)

• Isotopic analysis (C:N ratios)– Whole body– Muscle– Fat

Fat Extractions• Objective

1. Separate fat reserves from other body tissue2. Calculate average percent body fat of each treatment

• MethodsSolvent

• Petroleum Ether

Soak • Allow lipids to separate

Extractions• Pipette off fat • Top off jars with solvent• Repeat 3x

Fat Extractions• Adding petroleum ether (solvent) • Soaking allows separation of lipids

ResultsFAT EXTRACTIONS

05

10152025

303540

4550

None Fed Fast 2h Fast 6h Fast 1 day Fast 3 day

Treatment

Per

cen

t B

od

y F

at

?

DiscussionFAT EXTRACTIONS

• Endogenous fat reserves are used for energy during periods of fasting

• Therefore, percent body fat will decrease as time spent fasting increases

Experimental Design

• Real-time breath test analysis (δ13C)

• Fat extractions (% body fat lost)

• Isotopic analysis (C:N ratios and δ13C)– Whole body– Muscle– Fat

Isotopic Analysis• Objectives:

– To determine C:N ratio and δ13C and of body, fat, and muscle tissue

– C:N ratios are representative of fat and protein composition of tissue

• Methods:– Grind dried animal tissue

– Mass spectrometry isotopic analysis(UNM Geology Dept.)

ResultsBODY δ13C ANALYSIS

• Hypothesis: Body δ13C = Breath δ13C

-28.00

-27.00

-26.00

-25.00

-24.00

-23.00

-22.00

-21.00

-20.00

None Fed 2 hr 6 hr 1 day 3 day

Treatment

δ13

C ‰

Breath δ13C

Body δ13C

ResultsMUSCLE δ13C ANALYSIS

• Hypothesis: Muscle δ13C > Body δ13CPfadt 1994

-27.00

-26.00

-25.00

-24.00

-23.00

-22.00

-21.00

None Fed 2 hr 6 hr 1 day 3 day

Treatment

δ13

C ‰ Muscle δ13C

Body δ13C

ResultsC:N RATIO ANALYSIS

• C:N ratio is representative of fat:protein content

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

None Fed 2 hr 6 hr 1 day 3 day

Treatment

C:N

Rat

io

Muscle C:N Ratio

Body C:N Ratio

ResultsFAT ANALYSIS

• Results not back from lab…

• Hypothesis – Fat δ13C < Breath δ13C – Fat C:N ratio < Body C:N ratio as duration of

time spent fasting increases

ResultsISOTOPIC ANALYSIS

• Review:

– Body δ13C = Breath δ13C– Muscle δ13C > Body δ13C – Fat δ13C < Body δ13C ?

– C:N ratios decrease as fat reserves are used during periods of fasting

Overview: Research Questions

Does increased duration of fasting periods affect:

• δ13C breath values? YES

• Body fat percentage? YES

• C:N ratios and δ13C body values? YES

Impact of ResultsWHY DOES THIS MATTER?

• Effect of fasting (periods of starvation, drought, poor nutrition) on T. pallidipennis

• Error rates associated with a temporal gap between collection and isotopic analysis of T. pallidipennis

• Breath-test analysis as an innovative tool for diet analysis of animals

Acknowledgements

Special Thanks to:• Sophia Engel• Jennifer Johnson• Robin Warne• Blair Wolf• Hilary Lease• John Craig• Jarrod Blue• John Dewitt

Funding Sources:• Sevilleta LTER• University of New Mexico• US Fish and Wildlife