Effect of Leaf Maturity on Host Habitat Location by the Egg-Larval Parasitoid Ascogaster reticulata.

Adoxophyes honmai, a serious pest of tea plants, prefers to lay eggs on mature tea leaves rather than young leaves. Here, we examined a hypothesis that Ascogaster reticulata, an egg-larval parasitoid of A. honmai, increases the likelihood of encountering host egg masses by searching mature tea leaves when host-derived cues are not available. In a dual-choice bioassay using a four-arm olfactometer, A. reticulata preferred odor from intact, mature leaves versus young leaves. Based on volatile analysis with gas chromatography-mass spectrometry (GC-MS), we identified 5 and 10 compounds from mature and young leaf volatiles, respectively. The 5 components in the extract from intact mature leaves included (Z)-3-hexenyl acetate, (E)-ß-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and methyl salicylate. When each individual compound, or quaternary and quintenary blends of them, ratios of which were adjusted to match those of mature leaf volatiles, were provided, parasitoids preferred the full mixture and the quaternary blend devoid of DMNT to the solvent control. Methyl salicylate, one of the components of preferred blends, was not detected among young leaf volatiles. We concluded that the volatile composition of tea leaves changes, depending on their maturity, and that this composition affects foraging behavior of the parasitoid, which is closely related to the host herbivore's oviposition preference.

Synergistic Effects of Volatiles from Host-Infested Plants on Host-Searching Behavior in the Parasitoid Wasp Lytopylus rufipes (Hymenoptera: Braconidae).

Herbivore-induced plant volatiles (HIPVs) are important cues for natural enemies to find their hosts. HIPVs are usually present as blends and the effects of combinations of individual components are less studied. Here, we investigated plant volatiles in a tritrophic system, comprising the parasitoid wasp Lytopylus rufipes Nees (Hymenoptera: Braconidae), the Oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae), and Japanese pear, Pyrus pyrifolia 'Kosui', so as to elucidate the effects of single components and blends on wasp behaviors. Bioassays in a four-arm olfactometer, using either shoots or their isolated volatiles collected on adsorbent, revealed that female wasps preferred volatiles from host-infested shoots over those from intact shoots. Analyses identified (Z)-3-hexenyl acetate (H), linalool (L), (E)-ß-ocimene (O), (E)-3,8-dimethyl-1,4,7-nonatriene (D), and (E,E)-α-farnesene (F). Among them, only F was induced by infestation with G. molesta. When tested singly, only O and D elicited positive responses by L. rufipes. Binary blends of HO and DF elicited a positive response, but that of HD elicited a negative one, even though D alone elicited a positive response. Remarkably, wasps did not prefer either the ODF or HL blends, but showed a highest positive response to a quinary blend (HLODF). These results show that synergism among volatiles released from host-infested plants is necessary for eliciting high behavioral responses in L. rufipes, enabling L. rufipes to find its host efficiently.

Oviposition Experience of Parasitoid Wasps with Nonhost Larvae Affects their Olfactory and Contact-Behavioral Responses toward Host- and Nonhost-Infested Plants.

In nature, parasitoid wasps encounter and sometimes show oviposition behavior to nonhost species. However, little is known about the effect of such negative incidences on their subsequent host-searching behavior. We tested this effect in a tritrophic system of maize plants (Zea mays), common armyworms (hosts), tobacco cutworms (nonhosts), and parasitoid wasps, Cotesia kariyai. We used oviposition inexperienced C. kariyai and negative-experienced individuals that had expressed oviposition behavior toward nonhosts on nonhost-infested maize leaves. We first observed the olfactory behavior of C. kariyai to volatiles from host-infested plants or nonhost-infested plants in a wind tunnel. Negative-experienced wasps showed significantly lower rates of taking-off behavior (Step-1), significantly longer duration until landing (Step-2), and lower rates of landing behavior (Step-3) toward nonhost-infested plants than inexperienced wasps. However, the negative-experience did not affect these three steps toward host-infested plants. A negative experience appears to have negatively affected the olfactory responses to nonhost-infested plants. The chemical analyses suggested that the wasps associated (Z)-3-hexenyl acetate, a compound that was emitted more in nonhost-infested plants, with the negative experience, and reduced their response to nonhost-infested plants. Furthermore, we observed that the searching duration of wasps on either nonhost- or host-infested plants (Step-4) was reduced on both plant types after the negative experiences. Therefore, the negative experience in Step-4 would be nonadaptive for wasps on host-infested plants. Our study indicated that the density (i.e., possible encounters) of nonhost species as well as that of host species in the field should be considered when assessing the host-searching behavior of parasitoid wasps.

Cytotoxic effects of ß-asarone on Sf9 insect cells.

ß-Asarone is the predominant component of the essential oil of rhizomes of Acorus calamus Linn ( Sweet flag). Although rhizome extracts from this plant have long been used for insect pest control, their cytotoxic effects on insect cells are not well understood. In this study, we evaluated the potency of ß-asarone as a natural insecticide by using a Spodoptera frugiperda cell line (Sf9). To assess the cytotoxic effects of ß-asarone on Sf9 cells, we observed morphologic changes in treated cells and performed a cell proliferation assay and a DNA fragmentation assay. After 24 and 48 h of treatment with ß-asarone, the proliferation of the Sf9 cells was inhibited in a dose-dependent manner, with IC50 values of 0.558 mg/ml at 24 h and 0.253 mg/ml at 48 h. Morphologic changes in ß-asarone-treated cells were typical of apoptosis and included loss of adhesion, cell shrinkage, and small apoptotic bodies. The DNA laddering present in ß-asarone-treated SF9 cells and annexin V assay confirmed that this compound can induce apoptosis in insect cells. Together, these findings suggest that apoptosis induction may be one mechanism through which ß-asarone inhibits the proliferation of insect cells and thus exerts insecticidal effects.

An Effective Chemical Permeabilization of Silkworm Embryos.

The lipid layer surrounding the vitelline membrane of insect eggs has a critical role in the waterproofing and desiccation resistance of embryos. However, this lipid layer also prevents the flux of chemicals into the embryos, such as cryoprotectants, which are required for successful cryopreservation. The permeabilization studies of silkworm embryos remain insufficient. Therefore, in this study, we developed a permeabilization method to remove the lipid layer in the silkworm, Bombyx mori, and examined factors affecting the viability of dechorionated embryos, including the types and exposure times of chemicals and embryonic stages. Among the chemicals used, hexane and heptane were effective for permeabilization, whereas Triton X-100 and Tween-80 were less effective. Regarding the embryonic stages, there were significant differences between 160 and 166 h after egg laying (AEL) at 25 °C. Consequently, we found that the treatment of 160 AEL embryos with hexane for 30 s was the best condition for the permeability and viability of embryos, in which over 62% of the permeabilized embryos grew up to the second larval instar and their moths could lay fertilized eggs. Our method can be used for various purposes, including permeability investigations using other chemicals and embryonic cryopreservation.

Different uses of plant semiochemicals in host location strategies of the two tachinid parasitoids.

Some members of the family Tachinidae (Insecta: Diptera) deposit numerous very small eggs, termed "microtype" eggs, on the food plants of their caterpillar hosts. Parasitization is successful only when the hosts ingest these eggs. To increase the chance of hosts encountering the eggs, microtype tachinid parasitoids have to choose a suitable plant that harbors hosts and lay their eggs near the hosts. In their host location process, semiochemicals emitted by host-infested plants offer the tachinids a reliable cue. We investigated the behavioral responses of two microtype tachinid parasitoids, Pales pavida and Zenillia dolosa, to maize plants infested with their caterpillar host, Mythimna separata, in a wind tunnel. P. pavida females showed a significantly higher rate of landing on caterpillar-infested plants than on mechanically wounded or intact plants, whereas Z. dolosa landed on both the caterpillar-infested and mechanically wounded plants at significantly higher rates than on intact plants. We also examined which part of a caterpillar-infested maize leaf induces oviposition. P. pavida deposited eggs on the margin of the leaf, whereas Z. dolosa preferentially laid eggs around a caterpillar-infested area or a mechanically wounded spot. P. pavida eggs retained their parasitization ability for more than 15 days after they were deposited, whereas the eggs of Z. dolosa could not survive more than 5 days after oviposition. Our results suggest that each tachinid parasitoid employs a different host location strategy to exploit semiochemicals coming from plant-herbivore interaction as cues in order to increase their parasitization success.

Effect of chemical dechorionation on silkworm embryo viability.

The chorion covering/protecting insect egg, which has some effective functions such as providing mechanical strength, protecting eggs from external environments, and keeping moisture adjustment, is one of the principal barriers to manipulation, cryopreservation, and study of insect embryos. Here we evaluated the silkworm embryo viability after dechorionation using chemical reagents. We have developed an easy and effective method for chemical dechorionation that enables embryos to develop in culture, so that the larvae could normally grow. Eggs attached to a nylon net were treated with potassium hydroxide (KOH) and sodium hypochlorite (NaClO) to remove the chorion, washed with the Grace's insect medium, and then cultured using a dry-moist method which we created. The most effective treatment with regard to embryonic development, hatching, and production of second instar larvae was 30% KOH for 7 min and 2% NaClO for 5 min at 27 °C. Embryos at later embryonic stages were more tolerant to chemical dechorionation and over 75% of embryos treated at 168 h-old (Stage 25, appearance of taenidium) survived to the second larval instar, moreover, the larvae derived from the dechorionated embryos have developed into the moths which can lay the fertilized eggs. Our method would contribute to the establishment of cryopreservation using embryos and analysis of silkworm embryogenesis and might also be applicable to other insect species.

Effects of time after last herbivory on the attraction of corn plants infested with common arymworms to a parasitic wasp Cotesia kariyai.

Females of the gregarious endoparasitoid Cotesia kariyai were attracted to a blend of volatiles released from corn plants infested with larvae of their host, the common armyworm (Mythimna separata). We investigated the effects of time after the last infestation (1-168 h) on the attractiveness of corn plants infested by host larvae by using a wind tunnel under laboratory conditions. Immediately after the removal of the larvae, parasitoids were attracted more to plants that had been infested with the larvae than to intact plants (control). This attractiveness gradually decreased with time after the last infestation. The attractiveness of the infested plants was significantly higher than that of intact plants when the time after the last infestation was within 1 day. Fifteen herbivore-induced volatiles were recorded in the headspace of infested corn plants irrespective of time. The amounts of some compounds including (Z)-3-hexen-1-yl acetate, which have already been reported to attract C. kariyai, correlated with the attractiveness. The ecological meaning of the duration of production of C. kariyai attractants is discussed.

Author Correction: Violet LED light enhances the recruitment of a thrip predator in open fields.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Silkworms suppress the release of green leaf volatiles by mulberry leaves with an enzyme from their spinnerets.

In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

Increased complexity of mushroom body Kenyon cell subtypes in the brain is associated with behavioral evolution in hymenopteran insects.

In insect brains, the mushroom bodies (MBs) are a higher-order center for sensory integration and memory. Honeybee (Apis mellifera L.) MBs comprise four Kenyon cell (KC) subtypes: class I large-, middle-, and small-type, and class II KCs, which are distinguished by the size and location of somata, and gene expression profiles. Although these subtypes have only been reported in the honeybee, the time of their acquisition during evolution remains unknown. Here we performed in situ hybridization of tachykinin-related peptide, which is differentially expressed among KC subtypes in the honeybee MBs, in four hymenopteran species to analyze whether the complexity of KC subtypes is associated with their behavioral traits. Three class I KC subtypes were detected in the MBs of the eusocial hornet Vespa mandarinia and the nidificating scoliid wasp Campsomeris prismatica, like in A. mellifera, whereas only two class I KC subtypes were detected in the parasitic wasp Ascogaster reticulata. In contrast, we were unable to detect class I KC subtype in the primitive and phytophagous sawfly Arge similis. Our findings suggest that the number of class I KC subtypes increased at least twice - first with the evolution of the parasitic lifestyle and then with the evolution of nidification.

Broadband Photoreceptors Are Involved in Violet Light Preference in the Parasitoid Fly Exorista Japonica.

Phototaxis has been described in many insects, which are often attracted to specific wavelengths of light. However, little is known about phototaxis in parasitoid insect species that are potentially useful for integrated pest management. In this study, we investigated the wavelength dependency of the phototactic behavior of the parasitoid fly Exorista japonica and its possible mechanism. Multiple-choice tests with six monochromatic stimuli revealed that the flies were specifically attracted to violet light peaking at 405 nm, which was unexpected because insects are generally attracted to ultraviolet or green light. We measured the spectral sensitivity of the compound eye, and found that the sensitivity peaked at 340 nm, as in other brachyceran flies. We used statistical modeling and optimization of the process parameters to predict the type of photoreceptor contributing to the violet preference. The analysis revealed that the wavelength preference could be explained by linear models of the quanta received by photoreceptors, including the R1-6 broadband receptors. The broadband receptors appear to contribute positively, whereas the R7-8 narrowband receptors contribute negatively to achieve the violet preference; i.e., spectral opponency might be involved.

Violet LED light enhances the recruitment of a thrip predator in open fields.

The predatory bug Orius sauteri is an indigenous natural enemy of thrips and whiteflies in Asian countries. To put these bugs to practical use in pest management, methods to attract and retain the bugs in agricultural fields are needed. We previously showed that violet light (405 nm) attracts O. sauteri selectively. Many thrips and whiteflies are attracted to UV or green light. In this study, we examined the effect of violet-LED illumination on O. sauteri in pesticide-free eggplant (Solanum melongena L.) cultivation. In three cultivation trials, the density of O. sauteri on eggplant leaves was consistently higher in the illuminated plots; at least twice that of the non-illuminated plots. Simultaneously, the density of thrips declined markedly to less than half that of the non-illuminated plots. We identified three positive effects of violet light including an "immediate-effect" on predator attraction, a "persistent-effect" on predator reproduction, and a "secondary-effect" on the food web structure. Our results showed that illumination with violet light provides a powerful tool for integrated pest management. This is the first report on the use of illumination to manipulate the behavior of natural enemies.

Visual recognition of the host in the parasitoid fly Exorista japonica.

We presented the tachinid fly Exorista japonica with moving host models: a freeze-dried larva of the common armyworm Mythimna separata, a black rubber tube, and a black rubber sheet, to examine the effects of size, curvature, and velocity on visual recognition of the host. The host models were moved around the fly on a metal arm driven by motor. The size of the larva, the velocity of movement, and the length and diameter of the rubber tube were varied. During the presentation of the host model, fixation, approach, and examination behaviours of the flies were recorded. The fly fixated on, approached, and examined the black rubber tube as well as the freeze-dried larva. Furthermore, the fly detected the black rubber tube at a greater distance than the larva. The rubber tube elicited higher rates of approach and examination responses than the rubber sheet, suggesting that curvature affects the responses of the flies. The length, diameter, and velocity of host models had little effect on response rates of the flies. During host pursuit, the fly appeared to walk towards the ends of the tube. These results suggest that the flies respond to the leading or trailing edges of a moving object and ignore the length and diameter of the object.

Attraction to herbivore-induced plant volatiles by the host-foraging parasitoid fly Exorista japonica.

Responses of the tachinid fly Exorista japonica Townsend to odors from corn plants infested with the fly's host, the larvae of the noctuid moth Mythimna separata (Walker), were examined in a wind tunnel. Naïve female flies showed a higher rate of landing on M. separata-infested corn plants from which the host larvae had been removed than on artificially damaged or intact corn plants. When paper impregnated with a solution of headspace volatiles collected from host-infested plants was attached to intact plants, females landed on the plants at a high rate. Females also responded to intact plants to which had been attached with paper impregnated with a synthetic blend of nine chemicals identified previously in host-infested plants. There was an optimum concentration of the synthetic blend for the females' landing. Of the nine chemicals identified previously, four [(E)-4,8-dimethyl-1,3,7-nonatriene, indole, 3-hydroxy-2-butanone, and 2-methyl-1-propanol] released only by host-infested plants were classified as a host-induced blend. The other five [(Z)-3-hexen-1-yl acetate, (E)-2-hexenal, hexanal, (Z)-3-hexen-1-ol, and linalool] were classified as a non-specific blend released not only by infested plants but also by artificially damaged or intact plants. In the wind tunnel, E. japonica females did not respond to intact plants to which paper containing a solution of non-specific blend or host-induced blend was attached. However, they showed a high level of response to a mixture of the non-specific and host-induced blends. These results indicate that naïve E. japonica use a combination of non-specific and host-induced blends as an olfactory cue for locating host-infested plants.

Visual control of host pursuit in the parasitoid fly Exorista japonica.

The tachinid fly Exorista japonica is a parasitoid of many kinds of lepidopterous larvae. After encountering a suitable host, the fly pursues the crawling larva on foot using visual cues to guide it. To investigate the visual control of host pursuit, we observed and videotaped pursuits of a host, the common armyworm Mythimna separata, for frame-by-frame analysis. Observation was performed in sunlight and under illumination from a fluorescent lamp. The fly pursued hosts discontinuously with a repeated stop-and-run motion. During a run, its movements consisted of rotation, forward translation and sideways translation. Rotation during a run was positively correlated with the angular position of the host's head. The direction of translation depended on the angular position of the host's head. Forward translation was negatively correlated with the visual angle subtended by the host. These results suggest that the fly orients and walks towards the leading edge of a moving target. There was little difference in the results between sunlight and illumination from a fluorescent lamp.

Learning of herbivore-induced and nonspecific plant volatiles by a parasitoid, Cotesia kariyai.

Learning of host-induced plant volatiles by Cotesia kariyai females was examined with synthetic chemicals in a wind tunnel. Wasps were preconditioned by exposure to volatiles and feces simultaneously. A blend of four chemicals, geranyl acetate, beta-caryophyllene, (E)-beta-farnesene, and indole, which are known to be specifically released from plants infested by host larvae Mythimna separata (host-induced blend), elicited a response in naive C. kariyai, but did not enhance the response after conditioning. A blend of five chemicals, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate, beta-myrcene, and linalool, which are known to be released not only from plants infested by the host larvae, but also from artificially damaged plants or undamaged ones (unspecific blend), elicited little response in naive wasps, but significantly enhanced the wasps' response after conditioning. With a blend of the above nine chemicals, wasps could learn the blend at lower concentrations than they did in the nonspecific blend. Hence, both the host-induced and nonspecific volatile compounds appear to be important for C. kariyai females to learn the chemical cues in host location.