Pre- and post-imaginal associative learning for host-borne cues from different trophic contexts in the parasitic wasp Psyttalia concolor (Hymenoptera: Braconidae)

Insects rely extensively on learning for the purpose of all major life activities, including reproductive behavior. Among Hymenoptera, it is widely recognized that parasitic wasps can learn several visual and olfactory cues from the host-microhabitat, and the associative learning may occur both at the immature stage as well as at the adult stage. Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae) is able to attack fourteen tephritid species on different wild and cultivated plants, including pests of great economic importance, such as the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), and the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae). The present research investigates the olfactory stimuli able to affect the P. concolor host-seeking behavior, both during pre-imaginal and adult stage. First, we evaluate the influence of larval and early adult experience on subsequent host seeking in P. concolor females. In two choice ovipositional tests, P. concolor females showed to preferentially oviposit and have higher oviposition success rates on their natal host, but, when P. concolor females were excised from their host puparia, their natal host preference vanished, solidifying the evidence that early adult learning takes place for host selection. Then, we investigate if P. concolor females are able to negatively and positively associate olfactory stimuli. First, we examine how parasitic wasps rely to associative learning for danger to modify innate positive chemotaxis for C. capitata-induced plant volatiles. Trained wasps, nullified (lowest HIPV dosage tested) or reversed (highest HIPV dosage tested) their innate positive chemotaxis for HIPVs. Furthermore, to investigate the exact nature of the olfactory cues affecting the tri-trophic interaction P. concolor-B. oleae-O. europaea, the chemical stimuli arising from the olive fruits were identified. We investigated the presence of olfactory cues leading host location of P. concolor females toward B. oleae larvae infesting different olive cultivars through behavioral assays and gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC-MS) analyses. Volatile organic compound (VOC) emissions were peculiar for each cultivar analyzed, but two putative HIPVs were detected in infested fruits, regardless the cultivar, the monoterpene (E)-β-ocimene and the sesquiterpene (E-E)-α-farnesene. Furthermore, we analyzed also the VOC emissions of olives infested in laboratory under controlled conditions. Over seventy volatile compounds were identified on infested and healthy Ascolana fruits, and two were found to increase during B. oleae infestation: (E)-β-ocimene and 2-methyl-6-methylene-1,7-octadien-3-one, and four volatiles decreased: α-pinene, β-pinene, limonene and β-elemene. The attractiveness of the chemicals identified were evaluated in Y-tube assays, toward P. concolor mated females and virgin males. Female wasps showed attraction just for (E)-β-ocimene, while males were attracted by (E)-β-ocimene, α-pinene and limonene, highlighting the presence that the HIPV identified could be used by parasitic wasps as short-range attractant. Indeed, we hypothesized that P. concolor females could detect these volatiles in association with food (i.e. sweet exudates from tephritid-infested decaying fruits) and learn to respond favorably to these cues in subsequent experiences. Trained female P. concolor showed positive chemotaxis to the learned cues in the majority of tested odor combinations, except to both dosages of limonene. Our results add knowledge about the basic ecology of the tephritid parasitoid P. concolor and may improve its performances under field conditions, using HIPVs long lasting dispenser to maintain parasitoid population on a given crop or to monitor its presence. Indeed, in biological control programs P. concolor females could be conditioned before field release, both by training and/or using different mass-rearing hosts in order to prime their response and improve their ability to search for a given tephritid host