Ecological Archives E086-140-A1

Phyllis D. Coley, John Lokvam, Kathleen Rudolph, Keryn Bromberg, Tara E. Sackett, Leslie Wright, Tania Brenes-Arguedas, Dan Dvorett, Seth Ring, Alex Ponge, Caroline Baptiste, R. Toby Pennington, and Thomas A. Kursar. 2005. Divergent defensive strategies of young leaves in two species of Inga. Ecology 86:2633–2643.

Appendix A. Methodological details of chemical extractions and bioassays.

Preparation of crude assay material and separation into component metabolite classes : To prepare crude extracts, a portion of the field-processed EtOH leaf suspension was filtered and the marc was extracted two to three times with each of the following solvents in order: 80% aqueous EtOH, 70% aqueous acetone, dichloromethane (DCM) and water at 24ºC and then at 70ºC. In turn, each fraction was concentrated under reduced pressure and all of the extracts were combined.

A separate portion of the EtOH leaf suspension was fractionated into marc and five classes of extractables which were given general descriptive names based on their chemical properties: non-protein amino acids, flavanoids, organic acids, lipids, and proteins (see Fig. A1). The EtOH leaf suspensions were filtered and the marc was re-extracted two to three additional times with 80% EtOH. EtOH was removed and the aqueous phase was extracted repeatedly with DCM, yielding an aqueous fraction, marc and a DCM extract. The young leaves of I. umbellifera and I. goldmanii do not contain saponins, so that a specific extraction step is not required for this class of compounds.

The aqueous fraction was submitted to solid-phase extraction on octadecylsilane using a flash chromatography column packed with Bakerbond Octadecyl (C 18) Prep LC Packing (J. T. Baker). The solid phase was equilibrated with 100% water, the aqueous fraction to be separated was applied to the column and was eluted with 100% water followed by and 100% MeOH. MeOH was removed from the organic fraction and the sample was lyophilized to yield Flavanoids I. The aqueous fraction was separated by ion exchange chromatography using a cation ion exchange resin that had been equilibrated with 2N HCl (Dowex 50, H+ mode). Water eluted polar neutral molecules and anions; a 2N NH 4OH elution removed cations. When lyophilized, these yielded the Organic acid (O-Acids) and Non-protein amino acid (NPAA’s) fractions, respectively.

The marc was extracted repeatedly with each the following solvents in order: DCM, 70% aqueous acetone, 22°C water and 75°C water. Bioassays and further analysis of the marc (called “marc plus phenolics”) indicated that phenolic compounds were still bound. Thus, the marc was further extracted with BuOH-12N HCl (19:1) in 95°C water to remove phenolics (called “marc minus phenolics”). The DCM extracts from the marc and from the ethanolic leaf extractions (described above) were combined and lyophilized to yield the Lipids fraction. The 70% aqueous acetone fraction was dried and lyophilized to yield the Flavanoids II fraction. Flavanoids I and II were combined into a single flavanoid fraction for bioassays. Similarly, lyophilizations of the low and high temperature water extracts yielded Proteins I and II which were combined into a single water-soluble protein fraction. All fractions and the marc were maintained at 1 mTorr until dry, usually three to four days, and stored at -20°C for later use in bioassays and chemical characterizations.

To compare investments in different fractions, the final masses of each fraction and the marc were determined after lyophilization. Losses of plant material due to irreversible binding to solid phases were determined for the solid-phase extraction and ion exchange steps. In both instances, all volumes were measured before and after chromatography, a sub-sample was lyophilized and weighed, allowing losses to be quantified.

Bioassays: In Utah, a Heliothis virescens colony was maintained at 25°C with 12:12 light:dark cycles and 60–75% relative humidity. Eggs were collected daily and treated for five minutes in 10% formaldehyde solution, rinsed for fifteen minutes with deionized water and dried for 1–2 h in a fume hood, placed in a culturing room until the eggs hatch 3 to 5 days later. For colony maintenance, larvae were reared, one per plastic cup (29.6 mL portion cup, Sysco, Houston, Texas), on a diet of pinto beans (140g dry mass, soak overnight in 500 mL of water, drain, add 500 mL fresh water and cook in pressure cooker for 10–15 min at pressure) homogenized with 0.144g Fe 2(SO 4) 3 ( Sigma; Odell et al. 1997), 1.0 g potassium sorbate (Sigma), 2.4 g methyl paraben (BioServ), 3.6 g ascorbic acid ( Fisher), 8.0 g cellulose (Alphacel, ICN Biomedical, Aurora, Ohio ), 22g Vitamin Diet Fortification Mixture (ICN), 60g brewer's yeast (ICN) and mixed with 2.48g of agar ( Sigma) that was brought to boil with 200 mL of water. Pupae were placed on vermiculite. Adults were provided with a sucrose solution (88 g/L) and with paper towels for laying eggs. For bioassays, recently hatched larvae of about 1 mg were reared under the same environmental conditions, allowed to feed for 8 days (reaching a weight of about 100 mg in the control) and then weighed.

In Panama, wild-caught Phoebis philea adults were kept in an insectary with food (1 vol honey: 9 vol boiled water) and Inga saplings and other substrates upon which they could lay eggs. Eggs were collected and hatched in the lab. Larvae were fed Cassia reticulata until they reached about 90 mg, after which they will consume artificial diet. Larvae were then placed on the experimental diet prepared similarly to the method described below, allowed to feed on the diet for 3 days and then weighed. For bioassays, extracts were incorporated into artificial agar diets.

The artificial diet (modified from Chan et al. 1978) is 85% water by weight, with a total diet dry mass of 4.50g. Stock I (13.8 mL) and 2.15g of Stock II (2.15 g) were mixed by homogenizing in a Polytron, a cellulose/plant metabolite/Vitamin II mixture was added and the medium incubated at 40° C. Stock I, prepared on the day of the bioassay, contained 1.0 mL of a choline chloride solution (30.0 g made up to 100 mL; BioServ), 1.76g ascorbic acid, 0.5 mL of Vitamin I solution and 20 mg chlortetracycline (Sigma) with pH adjusted to 6.5 with KOH and made up to 100 mL. Vitamin I solution contained 0.30 g nicotinamide, 0.30 g Ca pantothenate, 75 mg thiamine-HCl, 75 mg pyridoxine-HCl, 0.6 mg vitamin B12 and 1.50 g myo-inositol dissolved in 100 mL of water (all reagents from Sigma). Stock II contained 0.70 g potassium sorbate, 0.75 g methyl paraben, 58 mg Fe 2(SO 4) 3, 6.08 g sucrose (BioServ), 5.0 g Wesson Salt Mix (BioServ), 15 g wheat germ (BioServ) and 17.5 g casein (BioServ).

The cellulose/plant metabolite/Vitamin II mixture was composed of Inga extract in either solid form or dissolved in solvent, mixed with 1.56 g of cellulose plus 10 mL of Vitamin II solution. The mixture was dried overnight in a fume hood and then at high vacuum (<10 mTorr) for 12 hours to remove all traces of solvent, and stored at –20°C. Vitamin II solution contained riboflavin (1.5 mg), folic acid (7.5 mg) and biotin (6.0 mg) dissolved in 50 mL of ethanol and made up to 100 mL with water (all reagents from Sigma). In the marc assays, cellulose (1.56 g or 34.6% of dry mass) was left out and 1.56 g of marc was substituted. The marc was first ground finely for 30 seconds at 30 Hz/sec with a Retsch MM 200 Mixer Mill (Retsch GmbH and Co., Haan, Germany).  

The cellulose/plant metabolite/Vitamin II mixture was combined with agar (0.50 g), dissolved in 12.5 mL of water in a pressure cooker and poured into a plastic cup. Once cool, this was divided into 16 sections, one section and one larva were placed in each cup, with a hole poked in the lid with a 27G needle. One cup, without a larva, was weighed at the beginning and end of the experiment in order to determine water loss.

In bioassays, the final weights of the larvae were divided by the mean control weight (GRC = growth relative to control) and the GRC values were fitted to a dose-response function using the NLIN procedure in SAS.

GRC = a0 -  (a0 / { 1 - [(log C)/b2]b1} )

where C is the concentration in percent, b2 is the concentration that inhibits growth by 50% (GI50), b1 is the slope and a0 is the response at a high concentration. Control values were entered as a concentration of 10-12.

   FIG. A1. Flow diagram of extraction protocol.



Chan, B. G., A. C. J. Waiss, W. L. Stanley, and A. E. Goodban. 1978. A rapid diet preparation method for antibiotic phytochemical bioassay. Journal of Ecological Entomology 71:366–368.

Odell, T. M., M. A. Keena, and R. B. Willlis. 1997. Dietary influence of iron formulation on the development of gypsy moth (Lepidoptera: Lymantriidae) in laboratory colonies. Annals of the Entomological Society of America 90:149–154.

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