© 2004 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 110: 165 – 172, 2004 165 Blackwell Publishing, Ltd. Influence of handling and conditioning protocol on learning and memory of Microplitis croceipes M. Tertuliano 1 , D.M. Olson 2 , G.C. Rains 1 & W.J. Lewis 2, * 1 Department of Biological and Agricultural Engineering, University of Georgia, Tifton, GA 31793, USA; 2 USDA-ARS, PO Box 748, Tifton, GA 31793, USA Accepted: 4 November 2003 Key words: Microplitis croceipes, Hymenoptera, Braconidae, 3-octanone, training, reinforcement, hunger state, memory Abstract Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) learns odors in association with both hosts and food. The food-associated ‘seeking’ behavior of M. croceipes was investigated under various training protocols utilizing the conditioning odor, 3-octanone. We investigated the effects of odor training, or its lack, training duration, training frequency, time elapsed after training, wasp hunger state, and training reinforcement, on the food-seeking responses of M. croceipes females. We found that odor-trained females show strong food seeking responses, whereas non-odor-trained females do not respond to the odor, and that a single 10 s association with the odor whilst feeding on sugar water subsequently conditioned the wasps to exhibiting significant responses to it. Increases in training time to more than 10 s did not improve their responses. Repetition of the food–odor associations increased a wasp’s recall, as well as its response over time, compared to a single exposure. Repeated exposure to the learned odor in the absence of a food reward decreased the responses of less hungry individuals. However, the level of response increased significantly following a single reinforcement with the food–odor association. Understanding the factors that influence learning in parasitoids can enhance our ability to predict their foraging behavior, and opens up avenues for the development of effective biological detectors. Introduction Parasitic wasps detect and learn numerous chemical cues associated with their host and food resources, and use them in order to forage more effectively (Lewis & Martin, 1990; Lewis et al., 1991; Zanen & Cardé, 1991). Recent studies of the potential of wasps as biological detectors has also indicated that Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), a specialist parasitoid of three highly poly- phagous hosts species, can also detect and learn ‘foreign’ chemicals that are unlikely to have played a role as foraging cues during their evolution (Wäckers et al., 2002; Olson et al., 2003). This learning ability elicits specific behavioral responses to cues associated with both long- (e.g., flight) and short-range (e.g., area restricted search) foraging behaviors (Olson et al., 2003). This species can also be trained to simultaneously associate two different odors with food and host rewards (Lewis & Takasu, 1990), or successively to two different odors with the same reward (Takasu & Lewis, 1996), suggesting that they are able to store this informa- tion for future use according to their specific resource needs. Although the ability of these species to detect and learn a wide variety of chemicals has been determined, we still have very little information on the factors that affect their acquisition and memory of learned experiences. The optimal training protocol and age of females used for flight responses of this species after food associated conditioning were determined by Takasu & Lewis (1996); responses peak 2–5 days after emergence and after five, 30 s exposures to food and odor experience, which they remem- ber for at least 2 days. Takasu & Lewis (1996) also showed that female M. croceipes will cease to respond to learned odors after several experiences with the odor without a food reward, and that they will switch to a different odor they had recently experienced as rewarding. However, the effect of a state of hunger on responses over time, and the factors which affect learning and memory of this species to cues associated with the more short-range behavioral res- ponses are not known. We chose a food-associated seeking behavior of M. croceipes females, which is a modification of ‘area restricted search’ and other food-specific behaviors Correspondence: W. Joe Lewis, USDA/ARS, PO Box 748, Tifton, GA 31793, USA. E-mail: wjl@tifton.uga.edu