Objectives Research Problem 1: Animal growth and metabolism Bio 31 - Spring, 2015 1. Conduct a hypothesis-driven investigation of patterns and/or processes in the growth of insect herbivores. 2. Write a research paper, in the form of a contribution to the primary literature, describing the study. 3. Learn the methods and analyses that are fundamental to studies of animal growth, consumption, assimilation, and metabolism. Specifically, the:.! Principles, measurement, calculation, and interpretation of relative growth rates (Appendix 1);! Principles, calculation and interpretation of consumption rate, assimilation rate, apparent digestibility, and conversion efficiencies (Appendix 1);! Principles, measurement, calculation, and interpretation of respiration rate using an infrared gas analyzer (IRGA) and respiration accessory (RACC) (Appendices 2-4); Materials! Larvae of four species of insect herbivore (Table 1).! Potted plants of numerous plant species grown under an assortment of environmental conditions (Table 2).! Artificial diet appropriate for all the insects in Table 1.! Vials suitable for insect growth trials with detached leaves.! An electrobalance (± 0.1/ 0.01 mg)! An infrared gas analyzer that measures CO 2 concentrations in parts per million (ppm)! A respiration accessory (RACC) and gas-tight syringes that allow measurement of CO 2 concentrations in respiration flasks.! Respiration flasks of different sizes.! Mesh bags suitable for limiting the locomotion of animals within respiration flasks.! Environmental chambers set at constant temperatures of 12, 18, 24, and 30 C (all with photoperiod of 12:12 light:dark.) Table 1. Insect herbivores. 44444444444444444444444444444444444444444444444444444444444444444444444 Species ))))))))))))))) Common name )))))))))))))) Family )))))) Reported host plant )))))))))))))))))))))))))) ) Helicoverpa zea corn bollworm Noctuidae corn, cotton*, soybeans*, sunflowers*, >13 other crops Heliothis virescens tobacco budworm Noctuidae tobacco, cotton*, soybeans*, deergrass, toadflax; not corn or tomato Anticarsa gemmatalis velvetbean caterpillar Noctuidae soybeans and other legumes; also cotton and kudzu Chrysodeixis includens soybean looper Noctuidae soybean, sweet potato, peanut; also cotton, tomato, tobacco, others. * indicates good larval growth in previous Bio 31 laboratories
Table 2. Experimental conditions under which six species of plants have been grown for Physiological Ecology (Biology 31) laboratory experiments. Each species was grown in a factorial combination of normal and low nutrients (N, P, and K) crossed with cool or warm temperatures. Plants in all treatments have been watered sufficiently to avoid moisture stress. Entries indicate number of replicates. Cool (20 day, 15 night C) Warm (25-30 day, 20 night C) Plant species Normal N,P,K Low N,P,K Normal N,P,K Low N,P,K Lycopersicon esculentum (tomato) 6 6 12 12 Nicotiana alata (tobacco) 6 6 12 12 Glycine max (soybean) 7 7 7 7 Helianthus annuis (sunflower) 6 6 12 12 Zea mays (corn) 6 6 12 12 Hordeum vulgare (barley) 8 8 8 8 day: night photoperiod, 12:12 > 14: 10 hrs transition over 4 weeks cool in incubator / warm in greenhouse
APPENDIX 1. FLOW OF ENERGY AND MATTER THROUGH ORGANISMS RGR Production Environment RCR Gut RAR Animal ADMR Respiration Excretion Feces RCR = Relative consumption rate = g # g -1 # d -1 or kj # g -1 # d -1 RAR = Relative assimilation rate = g # g -1 # d -1 or kj # g -1 # d -1 RGR = Relative growth rate = g # g -1 # d -1 or kj # g -1 # d -1 days M f RGR t = Mi e, where: M f = final mass at time t, M i = initial mass, e = base of the natural logarithm, t = elapsed time in ln( M f ) ln( Mi) RGR = t DoublingTime = ln( 2) RGR ADMR = Average daily metabolic rate = g # g -1 # d -1 or kj # g -1 # d -1 AD = Apparent digestibility = g / g or kj / kj = RAR / RCR ECD = Efficiency of conversion of digested matter = g / g or kj / kj = RGR / RAR ECI = Efficiency of conversion of ingested matter = g / g or kj / kj = RGR / RCR Note that: RAR = RCR # AD RGR = RAR # ECD RGR = RCR # AD # ECD RGR = RCR # ECI RGR = RAR - ADMR etc.
Appendix 2 Calculating respiration rates using IRGA, RACC, and respiration flasks ppm := 85 ppm = IRGA reading from gas sample SysVol := 93 InjVol := 5 JarVol := 180 ppm ( SysVol + InjVol) A := 1000 A = 8.33 4 oz jar = 180 ml 8 oz jar = 360 ml 16 oz jar = 720 ml CO2 volume in injected sample (µl) B := A 1000 InjVol CO2 concentration in injected sample (ppm) B = 1.666 10 3 C := A JarVol InjVol Total CO2 in jar (µl) C = 299.88 C := ppm ( SysVol + InjVol) JarVol 1000 InjVol Total CO2 in jar (µl) C = 299.88 pco2 := C JarVol 1000 Partial pressure of CO2 in jar (assuming 1 atmosphere pressure) pco2 = 1.666 10 3 CO2ppm := pco2 10 6 CO2 concentration in jar (ppm) CO2ppm = 1.666 10 3 Respiration rates can be expressed as µl CO2 g fresh mass of animal -1 hour -1 Rate := C final C initial time mass So, with a 100 mg animal in a 4 oz flask, if the IRGA reading increases from 28 to 85 in two hours, the respiration rate would be 1004 µl g -1 h -1
Appendix 3. Sample respiration calculations in a spreadsheet Respiration calculations from IRGA and RACC SysVol 93 4 oz = 180 ml (Follows appendix 2) InjVol 5 8 oz = 360 ml Highlighted areas indicate variables to be entered; other fields are calculated. JarVol 180 16 oz = 720 ml For initial reading For final reading ID Temp (ºC) Mass (mg) Time(init) Time(final) Time (h) ppm(init) ppm(final) A(i) B(i) C(i) A(f) B(f) C(f) Respiration Rate (µl/g/h) TEST 25 100.0 14:00 16:00 2.0 28 85 2.74 549 99 8.33 1666 300 1005 Appendix 4. Sample data sheet. ID Species Temp (ºC) Mass (mg) Time(init) Time(final)ppm(init) ppm(final) 901 H. zea 25 100.0 14:00 16:00 28 85
Respiration data sheet for use with IRGA and RACC ID Species Temp (ºC) Mass (mg) Time(init) Time(final) ppm(init) ppm(final) 901 H. zea 25 100.0 14:00 16:00 28 85