metabolic substrate use of trimerotropis pallidipennis; a stable isotope approach
DESCRIPTION
Metabolic substrate use of Trimerotropis pallidipennis; a stable isotope approach. Alyssa Corbett August 9, 2007. Study Species: Trimerotropis pallidipennis. Dorsal View. Population Distribution Map. Sophia Engel. Pfadt 1994. T. pallidipennis. - PowerPoint PPT PresentationTRANSCRIPT
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