Progress in the isolation and characterization of a host hemolymph ovipositional kairomone for the endoparasitoid Microplitis croceipes.

The purpose of this study was to (1) define further the bioassay parameters of an ovipositional stimulating kairomone (OSK) for Microplitis croceipes found in the hemolymph of the corn earworm, Heliothis zea; (2) purify and isolate the OSK; and (3) determine the viability of the eggs oviposited into an artificial ovipositional substrate (AOS). Phenylthiourea (PTU), added to hemolymph to prevent melanization, and host feces which contains a host-seeking stimulant (Jones et al.: Sciences 173:842, 1971) [1], were eliminated as possible factors influencing egg laying in the bioassay. Extraction of hemolymph with ether, hexane, and to a lesser degree with methylene chloride removed lipids without loss of OSK activity. In contrast, extraction with polar solvents such as methanol tetrahydrofuran, and acetonitrile resulted in a loss of OSK activity. After ether extraction, the sample could be concentrated by rotoevaporation (90°C) or lyophilization without loss of OSK activity. Fractionation of the sample by gel permeation chromatography indicated a molecular weight of between 100–300 daltons. The OSK was extracted to a specific activity higher than crude hemolymph on two disposable solid-phase adsorbants, a normal-phase diol, and a reverse-phase phenyl material. Subsequent fractionation of hemolymph on a phenyl adsorbent column by HPLC indicated that the OSK contained at least two components. Ovipositional activity was obtained only when two fractions with different retention volumes were combined. Preparation of the AOS's from agar plus Goodwin's tissue culture medium maintained viability of the oviposited eggs. Thirty-seven percent of the eggs that were removed from the AOS's and held in culture media eventually hatched.