1Department of Biology, 119 Schmucker Science Center I, West Chester University, West Chester, PA 19383, USA.
2Department of Entomology, 501 ASI Building, The Pennsylvania State University, University Park, PA 16802, USA.
A two-hour agar cylinder artificial nodule bioassay was developed which exploited the burrowing behavior common to S. hispidulus neonate feeding. Response to this bioassay resulted in a dose-dependent increase in neonate larval recruitment to alfalfa nodule homogenate, up to one nodule equivalence, with potential feeding deterrency elicited at higher doses. It subsequently provided an appropriate avenue to isolate and identify key nodule finding and acceptance components in M. sativa for S. hispidulus. Through bioassay-driven fractionation of M. sativa root nodules including chromatographic analyses (TLC and HPLC), and independent testing of neonate responses to the pure standard compound, the amino acid asparagine was discovered to be phagostimulatory to S. hispidulus neonates at physiologically relevant concentrations. The response it elicits in the nodule may be potentiated by other amino acids, such as L-alanine and L-glutamate, and perhaps organic acid levels as well. Lack of interest by S. hispidulus neonates, as exhibited in aggregation studies and preference tests, for non-nodulated roots and pseudonodules or mutant nodules which possess much lower levels of the above amino acids, as well as poor survival and development on these aberrant plant tissues suggests a strong relationship between nodules amino acid content and insect feeding.