Hans M. Smid

GC-EAG-analysis of volatiles from Brussels sprouts plants damaged by two species of Pieris caterpillars: olfactory receptive range of a specialist and a generalist parasitoid wasp species

Summary. Feeding by Pieris brassicae or P. rapae caterpillars on Brussels sprouts plants induces the emission of synomones that attract natural enemies of the caterpillars, Cotesia glomerata, a generalist parasitoid, and C. rubecula, a specialist on P. rapae. Previous research on this tritrophic system has identified a large number of volatiles in the headspace of herbivore-damaged Brussels sprouts plants, and this paper addresses the question which of these volatiles are perceived by the two parasitoid species. Headspace odors from both P. brassicae- and P. rapae-damaged Brussels sprouts plants were analyzed by coupled gas chromatography electro- antennogram (GC-EAG) detection. Twenty volatiles evoked consistent EAG reactions in the antennae of both species and nineteen of these volatiles could be identified with GC-MS. One component that could not be identified due to its low concentration, evoked EAG responses in antennae of C. rubecula only. Possible consequences for searching behavior of the two parasitoid species are discussed.

Natural variation in learning rate and memory dynamics in parasitoid wasps: opportunities for converging ecology and neuroscience.

Although the neural and genetic pathways underlying learning and memory formation seem strikingly similar among species of distant animal phyla, several more subtle inter- and intraspecific differences become evident from studies on model organisms. The true significance of such variation can only be understood when integrating this with information on the ecological relevance. Here, we argue that parasitoid wasps provide an excellent opportunity for multi-disciplinary studies that integrate ultimate and proximate approaches. These insects display interspecific variation in learning rate and memory dynamics that reflects natural variation in a daunting foraging task that largely determines their fitness: finding the inconspicuous hosts to which they will assign their offspring to develop. We review bioassays used for oviposition learning, the ecological factors that are considered to underlie the observed differences in learning rate and memory dynamics, and the opportunities for convergence of ecology and neuroscience that are offered by using parasitoid wasps as model species. We advocate that variation in learning and memory traits has evolved to suit an insects lifestyle within its ecological niche.

Species-specific acquisition and consolidation of long-term memory in parasitic wasps

Long-term memory (LTM) formation usually requires repeated, spaced learning events and is achieved by the synthesis of specific proteins. Other memory forms require a single learning experience and are independent of protein synthesis. We investigated in two closely related parasitic wasp species, Cotesia glomerata and Cotesia rubecula, whether natural differences in foraging behaviour are correlated with differences in LTM acquisition and formation. These parasitic wasp species lay their eggs in young caterpillars of pierid butterflies and can learn to associate plant odours with a successful egg laying experience on caterpillars on the odour-producing plant. We used a classical conditioning set-up, while interfering with LTM formation through translation or transcription inhibitors. We show here that C. rubecula formed LTM after three spaced learning trials, whereas C. glomerata required only a single trial for LTM formation. After three spaced learning trials, LTM formation was complete within 4 h in C. glomerata, whereas in C. rubecula, LTM formation took 3 days. Linking neurobiology with ecology, we argue that this species-specific difference in LTM acquisition and formation is adaptive given the extreme differences in both the number of foraging decisions of the two wasp species and in the spatial distributions of their respective hosts in nature.

Herbivore-Induced Plant Volatiles Mediate In-Flight Host Discrimination by Parasitoids

Herbivore feeding induces plants to emit volatiles that are detectable and reliable cues for foraging parasitoids, which allows them to perform oriented host searching. We investigated whether these plant volatiles play a role in avoiding parasitoid competition by discriminating parasitized from unparasitized hosts in flight. In a wind tunnel set-up, we used mechanically damaged plants treated with regurgitant containing elicitors to simulate and standardize herbivore feeding. The solitary parasitoid Cotesia rubecula discriminated among volatile blends from Brussels sprouts plants treated with regurgitant of unparasitized Pieris rapae or P. brassicae caterpillars over blends emitted by plants treated with regurgitant of parasitized caterpillars. The gregarious Cotesia glomerata discriminated between volatiles induced by regurgitant from parasitized and unparasitized caterpillars of its major host species, P. brassicae. Gas chromatography-mass spectrometry analysis of headspace odors revealed that cabbage plants treated with regurgitant of parasitized P. brassicae caterpillars emitted lower amounts of volatiles than plants treated with unparasitized caterpillars. We demonstrate (1) that parasitoids can detect, in flight, whether their hosts contain competitors, and (2) that plants reduce the production of specific herbivore-induced volatiles after a successful recruitment of their bodyguards. As the induced volatiles bear biosynthetic and ecological costs to plants, downregulation of their production has adaptive value. These findings add a new level of intricacy to plant–parasitoid interactions.

Three-dimensional organization of the glomeruli in the antennal lobe of the parasitoid wasps Cotesia glomerata and C. rubecula

Abstract. Two closely related parasitoid wasp species, Cotesia glomerata and C. rubecula, differ in their use of associative learning. To investigate the neural basis underlying these differences, it is necessary to describe the olfactory pathway of both wasp species. This paper focuses on the organization of the glomeruli in the antennal lobe. Glomeruli were stained by retrograde axon tracing of all axons in the antennal nerve and observed by confocal laser scanning microscopy. Stacks of optical sections were processed with AMIRA software, and 3D digital models of the glomeruli were produced. The combined use of 2D images and 3D surface models of the antennal lobes enabled the identification of a set of corresponding glomeruli in both wasp species. This offers unique opportunities for the study of subtle differences involved in synaptic plasticity that may occur at the glomerular level and factors regulating this plasticity.

Differences in memory dynamics between two closely related parasitoid wasp species

The two closely related parasitoids Cotesia glomerata and C. rubecula (Hymenoptera: Braconidae) coexist in The Netherlands where they occupy slightly different niches. When searching for their caterpillar hosts, they use host plant odours that are released upon feeding by the caterpillars. The species differ in their preference for plant odours during host searching after an associative learning experience. Cotesia glomerata changes its preference for the odour of a particular plant species after an oviposition experience on that plant, whereas C. rubecula does not alter its naive preference. Using no-choice wind tunnel bioassays we tested, for both species, to what extent oviposition induces memory formation and whether this results from associative learning. In experiment 1 we characterized the temporal dynamics of the memory trace. In both species, oviposition experience induced increased response levels compared to those of naive wasps. Memory dynamics differed between the species. A single associative learning experience induced a stable long-lasting memory trace that persisted for at least 5 days in C. glomerata. In C. rubecula a memory trace for the odour was present during the first day after the oviposition experience but waned over the following days. From a second experiment we concluded that the increased response could be attributed to a combination of nonassociative and associative learning. We furthermore formulate the learning paradigm for the parasitoids and hypothesize that adaptation to different spatial distributions of the preferred host species has led to the observed differences in memory dynamics.

Hitch-hiking parasitic wasp learns to exploit butterfly antiaphrodisiac

Many insects possess a sexual communication system that is vulnerable to chemical espionage by parasitic wasps. We recently discovered that a hitch-hiking (H) egg parasitoid exploits the antiaphrodisiac pheromone benzyl cyanide (BC) of the Large Cabbage White butterfly Pieris brassicae. This pheromone is passed from male butterflies to females during mating to render them less attractive to conspecific males. When the tiny parasitic wasp Trichogramma brassicae detects the antiaphrodisiac, it rides on a mated female butterfly to a host plant and then parasitizes her freshly laid eggs. The present study demonstrates that a closely related generalist wasp, Trichogramma evanescens, exploits BC in a similar way, but only after learning. Interestingly, the wasp learns to associate an H response to the odors of a mated female P. brassicae butterfly with reinforcement by parasitizing freshly laid butterfly eggs. Behavioral assays, before which we specifically inhibited long-term memory (LTM) formation with a translation inhibitor, reveal that the wasp has formed protein synthesis-dependent LTM at 24 h after learning. To our knowledge, the combination of associatively learning to exploit the sexual communication system of a host and the formation of protein synthesis-dependent LTM after a single learning event has not been documented before. We expect it to be widespread in nature, because it is highly adaptive in many species of egg parasitoids. Our finding of the exploitation of an antiaphrodisiac by multiple species of parasitic wasps suggests its use by Pieris butterflies to be under strong selective pressure.

Natural variation in learning and memory dynamics studied by artificial selection on learning rate in parasitic wasps.

Animals form memory types that differ in duration and stability. The initial anaesthesia-sensitive memory (ASM) can be replaced by anaesthesia-resistant memory (ARM), and/or by protein synthesis-dependent, long-term memory (LTM). We previously showed that two closely related parasitic wasp species differ in learning rate and memory consolidation. In Cotesia glomerata, LTM lasting at least 24 h was formed after single-trial conditioning, whereas single-trial conditioning led to ARM that waned before 24 h in Cotesia rubecula. This species formed LTM only after repeated conditioning trials spaced in time. Here, we used artificial selection on learning rate to investigate whether selection for a low learning rate in C. glomerata would result in C. rubecula-like memory dynamics. Memory consolidation was tested by using cold-shock anaesthesia and protein synthesis inhibitors. After single-trial conditioning, ARM was consolidated within hours in unselected C. rubecula, but directly, without an intermediate ARM phase, into LTM in unselected C. glomerata. We obtained low learning rate selection lines of C. glomerata wasps that, like C. rubecula, did not form LTM after single-trial conditioning, to see whether such wasps would then consolidate ARM instead of LTM. We showed that this was not the case. The selected wasps formed LTM after repeated, spaced conditioning trials, but formed only ASM without consolidation of ARM or LTM after single-trial learning. Ecological consequences of this type of memory formation are discussed.

Reward Value Determines Memory Consolidation in Parasitic Wasps

Animals can store learned information in their brains through a series of distinct memory forms. Short-lasting memory forms can be followed by longer-lasting, consolidated memory forms. However, the factors determining variation in memory consolidation encountered in nature have thus far not been fully elucidated. Here, we show that two parasitic wasp species belonging to different families, Cotesia glomerata (Hymenoptera: Braconidae) and Trichogramma evanescens (Hymenoptera; Trichogrammatidae), similarly adjust the memory form they consolidate to a fitness-determining reward: egg-laying into a host-insect that serves as food for their offspring. Protein synthesis-dependent long-term memory (LTM) was consolidated after single-trial conditioning with a high-value host. However, single-trial conditioning with a low-value host induced consolidation of a shorter-lasting memory form. For Cotesia glomerata, we subsequently identified this shorter-lasting memory form as anesthesia-resistant memory (ARM) because it was not sensitive to protein synthesis inhibitors or anesthesia. Associative conditioning using a single reward of different value thus induced a physiologically different mechanism of memory formation in this species. We conclude that the memory form that is consolidated does not only change in response to relatively large differences in conditioning, such as the number and type of conditioning trials, but is also sensitive to more subtle differences, such as reward value. Reward-dependent consolidation of exclusive ARM or LTM provides excellent opportunities for within-species comparison of mechanisms underlying memory consolidation.

Variation in the specificity of plant volatiles and their use by a specialist and a generalist parasitoid.

Herbivore-induced plant volatiles (HIPV) provide important information that influences host location behaviour for insect natural enemies, such as parasitoid wasps, that develop in the bodies of herbivorous insects. The dietary breadth of both the parasitoid and its host may affect the extent to which a searching parasitoid relies on HIPV. Specialist species are expected to rely on specific volatile cues to which they respond innately, whereas generalists are expected to show a higher degree of phenotypic plasticity that depends on foraging experience in the parasitoid. We compared the response to HIPV emitted by different plant species damaged by host and nonhost caterpillars for two congeneric parasitoid species, the specialist Diadegma semiclausum and the generalist Diadegma fenestrale, attacking caterpillars of the diamondback moth, Plutella xylostella. For the three tested plant species, Brassica oleracea, a feral Brassica population and Sinapis alba, both parasitoid species preferred volatiles from host-infested plants over those produced by undamaged plants. However, both parasitoid species only distinguished between volatiles induced by host and nonhosts when the caterpillars had been feeding on B. oleracea, the plant on which they had been reared. Chemical analysis of the volatile blends could not explain volatile preferences of the parasitoids. Despite the difference in their dietary breadth, the two parasitoids responded similarly to HIPV and experience treatments. A flexible response to a wide array of volatile blends by parasitoids is probably important in nature, given that different generations of the host and the parasitoid probably develop on different food plants.