Comparison of detection ability of learned odors between males and females in the larval parasitoid Microplitis croceipes

Although female parasitic wasps are known to learn to associate odors with hosts and food, the ability of males to learn and detect odors has been neglected. We conducted laboratory experiments to compare the detection ability of learned odors between males and females in the larval parasitoid Microplitis croceipes (Cresson) (Hymenoptera: Braconidae). We first conditioned males and females to associate sucrose water with methyl benzoate, 3‐octanone, or cyclohexanone, and then observed their behavior toward various concentrations (40 ng l^?1?4 mg l^?1) of the trained odors. Conditioned male wasps responded as well as female wasps to various concentrations of the three odors. Response times by wasps to these three odors were not significantly different between sexes. For the three odors, response times of both sexes were longer at the intermediate concentrations (40–400 µg l^?1) than the higher or lower concentrations. The present study suggests that M. croceipes males can learn and respond to the three chemicals tested as well as the females, and conditioned males are as sensitive to learned odors as conditioned females. By using their sensitive learning and odor‐detection capabilities, M. croceipes males could search for food sources as efficiently as females under natural conditions.     

Influence of handling and conditioning protocol on learning and memory of Microplitis croceipes

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.     

棉铃虫成虫对两种关键花香气味的联系性学习

为了明确棉铃虫对苯乙醛和乙酸苯甲酯两种关键花香气味的联系性学习行为,在室内分别测定了不同学习训练的棉铃虫雌雄成虫对两种关键花香气味的选择偏好性反应和雄成虫的触角电位反应。偏好性测定结果表明,无花香气味接触经历的棉铃虫对苯乙醛和乙酸苯甲酯的选择频次均无显著差异,且单纯的花香气味接触经历也不能诱导棉铃虫气味偏好性的显著改变,唯有花香气味接触经历伴随蔗糖溶液进行强化训练时,才能诱导气味偏好性的显著性改变。从对两种花香气味的联系性学习看,棉铃虫更容易将苯乙醛与蔗糖溶液进行联系性学习,而不容易将乙酸苯甲酯与蔗糖溶液进行联系性学习。从性别差异看,雌虫比雄虫具有更强的将花香气味与蔗糖溶液进行联系性学习的能力。触角电位测定结果表明,不同学习训练的棉铃虫雄成虫对两种花香气味和绿叶气味顺-3-己烯-1-醇的EAG反应值之间不存在显著性差异。总之,花香气味伴随食物资源的联系性学习经历,能够明显提高棉铃虫成虫对花香气味的选择偏好,可以此为基础进一步优化花香引诱剂的配方设计和应用技术。     

Conditioning of appetitive behavior in the Hymenopteran parasitoid Microplitis croceipes

Work on insect learning has made ample use of conditioned behaviors in single learning tasks. Parasitic wasps are particularly suited to study more complex learning processes, as they alternate between host searching and foraging for sugar sources. We here describe a set of behaviors that unambiguously represents sugar feeding in the Hymenopteran parasitoid Microplitis croceipes (Cresson) (Hymenoptera: Braconidae). Furthermore, we demonstrate that this parasitoid can be conditioned to exhibit these behaviors in response to odor stimuli previously associated with feeding. In conjunction with a previously described ovipositor probing response in this species, our findings constitute the first instance in which multiple conditioned behaviors representing feeding and reproduction have been identified for one insect species. This provides a valuable tool for the study of multiple task learning.     

A comparison of color-, shape- and pattern-learning by the hymenopteran parasitoid Microplitis croceipes

Visual learning by the larval parasitoid, Microplitis croceipes Cresson (Hymenoptera: Braconidae) was investigated in flight chamber studies. During conditioning, free-ranging parasitoids were given a choice between two visual alternatives, only one of which offered a host larva. By using alternatives that differed in either color, shape or pattern, parasitoids could be conditioned to distinguish host sites on the basis of each of these visual cues. Tests during which no reward was offered were conducted following six rewards (ovipositions) at one of the two alternative stimuli. The test results reveal that M. croceipes learned to distinguish between shapes more readily than between colors or patterns. This high rate of shape learning in this parasitoid is in strong contrast to the learning capacity of honey bees, which have been shown to learn color better than pattern and pattern better than shapes. It is argued that the difference in learning capacities between M. croceipes and the honey bee may reflect the different selection pressures imposed on these two species by their natural ecological needs. The high rate of shape learning in  M. croceipes may be adaptive in dealing with the homochromatic but multishaped environment in which parasitoids have to locate their herbivorous hosts. Accepted: 30 January 1999     

Computational modeling and experimental validation of odor detection behaviors of classically conditioned parasitic wasp,Microplitis croceipes

A prototype chemical sensor named Wasp hound® that utilizes five classically conditioned parasitoid wasps, Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), to detect volatile odors was successfully implemented in a previous study. To improve the odor‐detecting ability of Wasp Hound®, searching behaviors of an individual wasp in a confined area are studied and modeled through stochastic differential equations in this paper. The wasps are conditioned to 20 mg of coffee when associated with food and subsequently, tested to 5, 10, 20, and 40 mg of coffee. A stochastic model is developed and validated based on three positive behavioral responses (walking, rotation around odor source, and self‐rotation) from conditioned wasps at four different test dosages. The model is capable to reproducing the behaviors of conditioned wasps, and can be used to improve the ability of Wasp Hound® to assess changes in odor concentration. The model simulation results show the behaviors of conditioned wasps are significantly different when tested at different coffee dosages. We conjecture that the searching behaviors of conditioned wasps are based on the temporal and spatial neuron activity of olfactory receptor neurons and glomeruli, which are strongly correlated to the training dosages. The overall results demonstrate the utility of mathematical models for interpreting experimental observations, gaining novel insights into the dynamic behavior of classically conditioned wasps, as well as broadening the practical uses of Wasp Hound. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:596–606, 2015     

Selection for associative learning of color stimuli reveals correlated evolution of this learning ability across multiple stimuli and rewards

We are only starting to understand how variation in cognitive ability can result from local adaptations to environmental conditions. A major question in this regard is to what extent selection on cognitive ability in a specific context affects that ability in general through correlated evolution. To address this question, we performed artificial selection on visual associative learning in female Nasonia vitripennis wasps. Using appetitive conditioning in which a visual stimulus was offered in association with a host reward, the ability to learn visual associations was enhanced within 10 generations of selection. To test for correlated evolution affecting this form of learning, the ability to readily form learned associations in females was also tested using an olfactory instead of a visual stimulus in the appetitive conditioning. Additionally, we assessed whether the improved associative learning ability was expressed across sexes by color‐conditioning males with a mating reward. Both females and males from the selected lines consistently demonstrated an increased associative learning ability compared to the control lines, independent of learning context or conditioned stimulus. No difference in relative volume of brain neuropils was detected between the selected and control lines.     

棉铃虫低龄幼虫对花香挥发物的联系性学习

鳞翅目昆虫的寄主选择主要是成虫的任务,但幼虫也可以精细调节取食部位。低龄幼虫记忆一些与食物伴随的化学信息能够提高适合度。当幼虫被迫离开寄主植物后,最好是搜索与此前取食过的寄主气味相似的植物,以便节省寄主转移的生理代谢成本。棉铃虫幼虫嗜食作物花器,因此推测花香挥发物可以代表幼虫食物的典型化学信息。采用人工饲料为无条件刺激,在条件化训练开始时,使初孵幼虫取食时暴露在7种花香挥发物下,随后7 d逐日测试低龄幼虫学习表现。结果发现,在7次测试中,苯乙醛选择频次有3次显著多于对照,芳樟醇条件化组在2次测定中处理被选频次显著或极显著多于对照,苯乙醇条件化组仅1次对处理选择频次显著多于对照,其他4种挥发物无论训练时间长短均不能造成嗅觉偏好性的改变,说明棉铃虫在取食过程中是选择性采集食物关键化学信息。然而,无经历组无论训练时间如何均不对苯乙醛表现出嗅觉偏好性,证实气味偏好性的改变并非生理性成熟所致。总之,和同种的成虫以及若虫期比较活泼的其他种类相比,棉铃虫低龄幼虫嗅觉联系性学习表现较差,并且嗅觉偏好性变化与条件化训练时长没有明确关系,结合棉铃虫的生态学习性讨论了可能的原因。     

Flight Toward a Learned Odor and Factors Inducing Landing of Female Brachymeria intermedia (Hymenoptera: Chalcididae), a Parasitoid of the Gypsy Moth, Lymantria dispar (Lepidoptera: Lymantriidae)

Through a single oviposition experience in the presence of vanilla odor, female Brachymeria intermedia were conditioned to fly toward a source of vanilla odor in a wind tunnel. Conditioning was an associative process, and wasps were able to fly toward odor sources at much lower doses than the dose used in conditioning. Furthermore, upon approaching vanilla-scented paper rolls hung on a vertical cylinder, conditioned wasps landed on them readily, whereas few wasps landed on real pupae. However, more wasps reached pupae or pupal cases than white paper rolls. Visual and olfactory cues appeared to mediate the foraging behavior of wasps in conflicting ways. Therefore, the extent to which olfactory learning mediates foraging success in this species remains unresolved.     

Three viruses found in the braconid parasitoid Microplitis croceipes and their implications in biological control programs

Productivity and longevity decreased in a laboratory colony of the parasitoid wasp Microplitis croceipes (Cresson) (Hymenoptera: Braconidae). Using light microscopy, it was determined that the colony was free of microsporidia. However, samples of the colony examined for pathogens by electron microscopy revealed three types of viruses: a nonpathogenic polydnavirus which is produced by all female wasps; a nonoccluded baculovirus which is pathogenic to late-stage pupae and adults; and a picorna-like virus which is present in larvae, pupae, and adults. The nonoccluded baculovirus was eliminated from the laboratory colony of M. croceipes by selection of progeny from wasps which had oviposited within 2 to 3 days after emergence from the cocoons and which had lived for at least 14 days post-emergence. Upon death, the wasps were examined by negative stain electron microscopy and only progeny from baculovirus-free wasps were retained. Parasitoid colonies should be systematically examined for pathogenic viruses that may reduce their productivity and efficacy as biological control agents. In addition, exotic parasitoids and predators should be evaluated for viruses and other pathogens while in quarantine.     

Insecticide odour interference with food-searching behaviour of Microplitis croceipes (Hymenoptera: Braconidae) in a laboratory arena

Response to odours plays an important role in resource location by natural enemies, particularly by parasitoid wasps. While a considerable research effort has been dedicated to studying the effects of insecticide intoxication on natural enemy search behaviour, it is yet unknown if the odours themselves interfere with distant chemoreception. We investigated this issue using the food-searching behaviour of Microplitis croceipes (Cresson)(Hymenoptera: Braconidae) in laboratory arenas as a model system. Odours of imidacloprid (Genesis®), spinosad (Entrust®), esfenvalerate (Asana®), methamidophos (Monitor®), and vanilla were tested for their ability to interfere with wasp response to the odour of honey. The wasps did not contact the chemicals. Honey odour was generally effective in triggering food-searching behaviour in both honey-fed (and thus conditioned to associate honey odour with food) and unfed, naïve wasps. Mixing honey with imidacloprid and spinosad did not affect wasp responses. The remaining compounds (esfenvalerate, methamidophos, and vanilla) significantly reduced the proportion of positively responding fed wasps, but only methamidophos had such an effect on the unfed wasps. Negative methamidophos effects became completely reversed when wasps were forced to feed on honey in the presence of methamidophos odour. Our results suggest that odours and provision of food may potentially be used to keep beneficial natural enemies away from insecticide-treated areas.     

Decrease of memory retention in a parasitic wasp: an effect of host manipulation by Wolbachia?

Several factors, such as cold exposure, aging, the number of experiences and viral infection, have been shown to affect learning ability in different organisms. Wolbachia has been found worldwide as an arthropod parasite/mutualist symbiont in a wide range of species, including insects. Differing effects have been identified on physiology and behavior by Wolbachia. However, the effect of Wolbachia infection on the learning ability of their host had never previously been studied. The current study carried out to compare learning ability and memory duration in 2 strains of the parasitoid Trichogramma brassicae: 1 uninfected and 1 infected by Wolbachia. Both strains were able to associate the novel odors with the reward of an oviposition into a host egg. However, the percentage of females that responded to the experimental design and displayed an ability to learn in these conditions was higher in the uninfected strain. Memory duration was longer in uninfected wasps (23.8 and 21.4 h after conditioning with peppermint and lemon, respectively) than in infected wasps (18.9 and 16.2 h after conditioning with peppermint and lemon, respectively). Memory retention increased in response to the number of conditioning sessions in both strains, but memory retention was always shorter in the infected wasps than in the uninfected ones. Wolbachia infection may select for reduced memory retention because shorter memory induces infected wasps to disperse in new environments and avoid competition with uninfected wasps by forgetting cues related to previously visited environments, thus increasing transmission of Wolbachia in new environments.     

Selection on olfactory response to semiochemicals from a plant-host complex in a parasitic wasp

Parasitic wasps (parasitoids) use volatiles from the plants infested by phytophagous insects to locate host herbivores, but their behavioural response to such semiochemicals is highly variable. Bi-directional selection on Cotesia glomerata (Hymenoptera: Braconidae) was conducted to investigate the importance of genetic variation in the olfactory response of parasitoids. Female wasps were assessed for flight orientation and landing success in response to the hexane extract from a plant-host complex in a wind tunnel. After the first generation of selection, two strains significantly differentiated in both flight orientation and landing success, and their divergence continued with further selection. The two selected strains genetically differentiated in olfactory perception rather than upwind flight ability. The realized heritability was estimated as 0.248 for flight orientation and 0.216 for landing success. The selection experiment further demonstrated that a prior exposure to the semiochemicals significantly enhanced the subsequent response of female wasps, independent of genetic differences. These results suggest that both genetic component and environmental conditioning have played an important role in the evolution of host selection and utilization by the parasitoid in a tritrophic system.     

Learning of host searching cues by the larval parasitoid Microplitis croceipes

We conducted laboratory experiments using a wind tunnel to determine the effects of prior experience on the learning and retention of learned responses in the larval parasitoid Microplitis croceipes (Cresson) (Hymenoptera: Braconidae). Although most females that had either antennated host frass or oviposited in a host in the presence of vanilla odor, made oriented flight to the odor from downwind in the wind tunnel at 30 min after experience, only those that had oviposited in a host with the odor responded to the odor at 24 h after experience. Females that had oviposited in the non‐natural host beet armyworm (BAW) larva with or without the odor did not respond to the odor at 30 min after experience. These results indicate that an oviposition in the host in the presence of odors strongly affects associative learning and the persistence of learned response to the odors. When females were allowed to antennate host frass in the presence of vanilla and to subsequently oviposit in a host within an interval of 5 min or less, their learned response to vanilla also persisted for 24 h. Similarly, when females were conditioned to link vanilla with host frass and then allowed to make an ovipositor contact with host hemolymph, their learned response persisted for 24 h. However, antennal contact with hemolymph after such conditioning of vanilla with host frass did not improve the persistence of learned response to the odor. These results indicate that ovipositor contact with host hemolymph during oviposition is partially responsible for an increased retention of learned response. Females responded to vanilla 48% of the time at 30 min after antennating host frass without the odor, but their response to the odor significantly decreased after oviposition in a BAW larva subsequent to the antennation of host frass. This result indicates that oviposition in a BAW larva decreases subsequent response to general odors. Based on the results, we discuss the foraging behavior of M. croceipes dependent on learning and subsequent experiences.     

Potential fitness consequences of associative learning in a parasitoid wasp

Several studies have documented associative learning in insects,but the adaptive value of such learning is not yet well understood.To evaluate this issue, we quantified long-term fitness consequencesof associative learning in the parasitoid wasp, Biosteres arisanus.We compared individual wasps that were allowed to choose hostsubstrate based on experience ("learning" wasps) to wasps thatcould only make random substrate choice ("random" wasps) inan environment where only one out of two substrates containedhost eggs. In two experiments, the average number of host eggsparasitized and offspring produced were significantly largerfor learning than for random wasps. Our results allow detailedexamination of the conditions under which learning would havepositive fitness effects in ecological systems similar to ours.These conditions include relatively long search duration forhosts; the ability to remember a learned preference over extendedperiods of interfering activities; and large mean differencesbetween alternatives, and small variances, which together allowrapid evaluation of alternatives and long duration of exploitingthe superior one.     

The role of herbivore‐ and plant‐related experiences in intraspecific host preference of a relatively specialized parasitoid

Parasitoids use odor cues from infested plants and herbivore hosts to locate their hosts. Specialist parasitoids of generalist herbivores are predicted to rely more on herbivorederived cues than plant-derived cues. Microplitis croceipes (Cresson)(Hymenoptera: Braconidae) is a relatively specialized larval endoparasitoid of Heliothis virescens (F.)(Lepidoptera: Noctuidae), which is a generalist herbivore on several crops including cotton and soybean. Using M. croceipes/H. virescens as a model system, we tested the following predictions about specialist parasitoids of generalist herbivores:(i) naive parasitoids will show innate responses to herbivore-emitted kairomones, regardless of host plant identity and (ii) herbivore-related experience will have a greater influence on intraspecific oviposition preference than plant-related experience. Inexperienced (naive) female M. croceipes did not discriminate between cotton-fed and soybean-fed H. virescens in oviposition choice tests, supporting our first prediction. Oviposition experience alone with either host group influenced subsequent oviposition preference while experience with infested plants alone did not elicit preference in M. croceipes, supporting our second prediction. Furthermore, associative learning of oviposition with host-damaged plants facilitated host location. I terestingly, naive parasitoids attacked more soybeathan cotton-fed host larvae in two-choice tests when a background of host-infested cotton odor was supplied, and vice versa. This suggests that plant volatiles may have created an olfactory contrast effect. We discussed ecological significance of the results and concluded that both plant- and herbivore-related experiences play important role in parasitoid host foraging.     

Variation between laboratory populations in the performance of the parasitoid Encarsia formosa on two host species, Bemisia tabaci and Trialeurodes vaporariorum

We tested the hypothesis that populations of the parthenogenetic parasitic wasp Encarsia formosa Gahan (Hymenoptera: Aphelinidae) differed in their ability to use two different host species, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) and Trialeurodes vaporariorum Westwood (Homoptera: Aleyrodidae). Of the three wasp populations tested, two populations had been reared for many generations on B. tabaci and one population had been reared for many years on T. vaporariorum. Performance was measured by the number of whitefly nymphs that were successfully parasitized by individual wasps, and performance on either host was measured in separate experiments. There was variation between wasp populations in their performance on the host B. tabaci, with one wasp population reared for many years on this host performing considerably better than the other two populations. There were no significant differences between populations in their use of the preferred host, T. vaporariorum. The experiments were conducted in such a way that we could distinguish heritable differences between populations from environmentally-induced conditioning differences due to the immediate host from which an individual wasp enclosed. In either experiment there were no significant effects of conditioning, although there was a trend within each population for wasps conditioned on T. vaporariorum to have higher performance than those conditioned on B. tabaci. Thirdly, we conducted a selection experiment, initiated with wasps from a single population historically reared on T. vaporariorum, to measure the effect of laboratory rearing on different hosts for 17 generations. We did not see any difference in the performance of wasps on B. tabaci after this period of rearing on either of the two hosts. In summary, populations of E. formosa do differ in their relative performance on B. tabaci. The one population that was tested further did not show any response to selection by rearing, but the ability to respond to selection on performance may not be equal for all populations. The possibility that wasp populations have differential performance on particular hosts may affect the use of this species as a biological control agent.     

Associative learning in ants: Conditioning of the maxilla-labium extension response in Camponotus aethiops

Associative learning has been studied in many vertebrates and invertebrates. In social insects, the proboscis extension response conditioning of honey bees has been widely used for several decades. However, a similar paradigm has not been developed for ants, which are advanced social insects showing different morphological castes and a plethora of life histories. Here we present a novel conditioning protocol using Camponotus aethiops. When the antennae of a harnessed ant are stimulated with sucrose solution, the ant extends its maxilla-labium to absorb the sucrose. We term this the “maxilla-labium extension response” (MaLER). MaLER could be conditioned by forward pairing an odour (conditioned stimulus) with sucrose (unconditioned stimulus) in the course of six conditioning trials (absolute conditioning). In non-rewarded tests following conditioning, ants gave significantly higher specific responses to the conditioned stimulus than to a novel odour. When trained for differential conditioning, ants discriminated between the odour forward-paired with sucrose and an odour forward-paired with quinine (a putative aversive stimulus). In both absolute and differential conditioning, memory lasted for at least 1 h. MaLER conditioning allows full control of the stimulation sequence, inter-stimulus and inter-trial intervals and satiety, which is crucial for any further study on associative learning in ants.     

Parasitoid complex of the pistachio twig borer moth,Kermania pistaciella,in Iran

The parasitoid complex of the pistachio twig borer moth, Kermania pistaciella Amsel (Lepidoptera: Tineidae), a native pest of pistachio trees, was investigated at 27 pistachio plantation sites in Kerman province, the major pistachio growing area of Iran. The present study was conducted to document the naturally established parasitoid complex and to assess the need for improving the biological control of this species. In total, 22,390 moth cocoons were collected from 186 samples collected from commercial orchards during 2006–2008 and kept singly in controlled conditions to rear immature insects. An average of 2.8% of moth cocoons had been attacked by predators at time of sampling. Of the collected cocoons, on average 46.7% completed development and emerged as adult moths, no insects emerged from 8%, suggesting that the moth or wasp died before maturing, and parasitoids emerged from the remaining 42.5%. The overall percentage of host cocoons from which wasps emerged ranged from 25.6 to 59%. Fifteen hymenopterous parasitoid species were recovered from cocoons, of which three species were primary parasitoids, two were obligatory hyperparasitoids and the remaining 10 species were facultative hyperparasitoids. The primary parasitoid, Chelonus kermakiae (Tobias) (Hymenoptera: Braconidae), was the most abundant comprising almost 85% of the total emerging parasitoids. In addition, a further four species of larval parasitoid developed within the PTBM's larval tunnels in pistachio fruit cluster-stem tissue. Conservation of these parasitoids in the pistachio growing areas is recommended since a high level of parasitized moths’ cocoons was found at the majority of experimental sites.     

Host recognition by the specialist endoparasitoidMicroplitis croceipes (Hymenoptera: Braconidae): Role of host- and plant-related volatiles

The specialist parasitoidMicroplitis croceipes Cresson can parasitize only noctuid larvae in the generaHelicoverpa andHeliothis. To be successful in their search for hosts, the ability to distinguish hosts from nonhosts feeding on the same plant is beneficial. In flight tunnel experiments, we found that prior to landing on the odor sourceM. croceipes were able to distinguish volatiles released from frass of host larvae(Helicoverpa zea Boddie) and nonhost larvae (Spodoptera exigua Hübner andSpodoptera frugiperda J. E. Smith) fed on cotton. However, an initial contact experience with frass of cotton-fed host larvae appeared to be critical for this ability. Wasps that had antennated frass of host larvae fed pinto bean diet were equally attracted to frass of host and nonhost larvae fed on pinto bean diet. In short-range walking experiments, wasps located cotton-fed host larvae faster than diet-fed larvae, regardless of their experience. Wasps that had antennated frass of cotton-fed host larvae were less attracted to cotton-fed nonhost larvae, compared to host larvae, and preferred to sting host larvae. Plant-related volatiles in host frass and larvae appear to play a major role in the successful location of host larvae.