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Metabolic substrate use of Trimerotropis pallidipennis; a stable isotope approach

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.

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Metabolic substrate use of Trimerotropis pallidipennis; a stable isotope approach

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  1. Metabolic substrate use of Trimerotropis pallidipennis; a stable isotope approach Alyssa Corbett August 9, 2007

  2. Study Species:Trimerotropis pallidipennis Dorsal View Population Distribution Map Sophia Engel Pfadt 1994

  3. 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

  4. T. pallidipennis Food Selection -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

  5. 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

  6. Stable Carbon Isotopes • Abundance in Nature: 12C 98.89% 13C 1.11% • 12C is a lighter isotope, having one less neutron 6Pand 6N 6P and7N Encarta

  7. 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

  8. 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 C3 C4 δ13C value -12 -14 -16 -18 -20 -22 -24 -26 -28 -30

  9. What will stable isotopes tell us? Feeding on mixed grasses and forbes Fasting period δ13C = ? δ13C = -23.0 ‰ *Uncertain of substrate material *Uncertain of extent of substrate metabolized

  10. 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?

  11. Methods Captured 20 T. pallidipennis for each fasting period treatment: 1. Fed 2. Fasted 2 hrs 3. Fasted 6 hrs 4. Fasted 1 day 5. Fasted 3 days

  12. 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

  13. 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

  14. Experimental Design • Real-time breath test analysis (δ13C) • Fat extractions (% body fat lost) • Isotopic analysis (C:N ratios) • Body • Muscle • Fat

  15. 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

  16. 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

  17. Chamber = 30-60 ml

  18. ResultsBREATH-TEST ANALYSIS C4 Plants (-14 to -19 ‰) C3 Plants (-24 to -30 ‰)

  19. DiscussionBREATH-TEST ANALYSIS Increasing time spent fasting Breath δ13C ≈-25 ‰

  20. DiscussionBREATH-TEST ANALYSIS Increasing time spent fasting Breath δ13C ≈-25 ‰ Breath δ13C represents recently digested plants δ13C ≈-25 ‰

  21. DiscussionBREATH-TEST ANALYSIS Increasing time spent fasting Breath δ13C ≈-25 ‰ Breath δ13C represents recently digested plants δ13C ≈-25 ‰ After 6 hrs Breath δ13C represents content of endogenous lipids δ13C ≈-27 ‰

  22. DiscussionBREATH-TEST ANALYSIS Increasing time spent fasting 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.” After 6 hrs

  23. Experimental Design • Real-time breath test analysis (δ13C) • Fat extractions (% body fat lost) • Isotopic analysis (C:N ratios) • Whole body • Muscle • Fat

  24. Fat Extractions • Objective 1. Separate fat reserves from other body tissue 2. Calculate average percent body fat of each treatment • Methods Solvent • Petroleum Ether Soak • Allow lipids to separate Extractions • Pipette off fat • Top off jars with solvent • Repeat 3x

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

  26. ResultsFAT EXTRACTIONS ?

  27. DiscussionFAT EXTRACTIONS • Endogenous fat reserves are used for energy during periods of fasting • Therefore, percent body fat will decrease as time spent fasting increases

  28. Experimental Design • Real-time breath test analysis (δ13C) • Fat extractions (% body fat lost) • Isotopic analysis (C:N ratios and δ13C) • Whole body • Muscle • Fat

  29. 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.)

  30. ResultsBODY δ13C ANALYSIS • Hypothesis: Body δ13C = Breath δ13C

  31. ResultsMUSCLE δ13C ANALYSIS Pfadt 1994 • Hypothesis: Muscle δ13C > Body δ13C

  32. ResultsC:N RATIO ANALYSIS • C:N ratio is representative of fat:protein content

  33. 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

  34. 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

  35. 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

  36. 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

  37. 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 Acknowledgements

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